Unit 5 General Techniques of Detection and Enumeration

General Techniques of UNIT 5 GENERAL TECHNIQUES OF Detection and Enumeration DETECTION AND ENUMERATION of Micro-Organisms in Food OF MICRO-ORGANISMS IN FOOD Structure 5.0 Objectives 5.1 Introduction 5.2 Microbiological Media 5.2.1 Types of Media 5.2.2 Preparation of Media 5.3 Enumeration Procedures 5.3.1 Direct Microscopic Count (DMC) 5.3.2 Standard Plate Count (SPC) 5.3.3 Spiral Plate Count 5.3.4 Dye Reduction Tests 5.3.5 Most Probable Number (MPN) Method 5.4 Pure Culture Method 5.5 Microscopic Examination of the Bacterial Culture 5.5.1 Simple Staining 5.5.2 Negative Staining 5.5.3 Gram Staining 5.5.4 Endospore Staining 5.6 Let Us Sum Up 5.7 Keywords 5.8 Suggested Further Reading 5.9 Terminal Questions 5.10 Answers To Check Your Progress Exercises 5.0 OBJECTIVES After reading this Unit, you will be able to: • identify various types of microbiological media and their method of preparation; • describe different enumeration techniques of micro-organisms; • prepare pure culture of micro-organisms from mixed population; and • identify micro-organisms on the basis of microscopic observation. 5.1 INRODUCTION Food microbiology is the branch of microbiology that deals with various types of micro-organisms occurring in the food products. These range from the beneficial micro-organisms to the harmful ones that contaminate foods and lead to its spoilage or disease out breaks by utilizing food as a vector for their spread causing food borne illness. Micro organisms may be added deliberately in the preparation of food products and in this case they are called the starter cultures that play an important role in determining the taste, texture and other properties of a food product. Various types of fermented milk products like dahi, yoghurt, cheese, kefir, kumiss etc. are prepared by addition of particular starter cultures to the milk from different sources. These cultures bring about the required changes in the milk such as lactose utilization and at times may also provide health benefits to the consumer. These benefits may be due to the changes carried out by microbes such as lactose utilization, which alleviates the symptoms of lactose intolerance or microbes may have a more direct effect when consumed in live active forms. In the latter case, these micro-organisms 5

Analytical Techniques are called ‘Probiotics’ that have been linked to several health benefits. in Microbiology Probiotics have a positive effect on individual’s health only when they are consumed in required numbers. Whether we talk about probiotics, spoilage or disease causing microbes, the detection and quantification of the microbes in the food product is very important as it points towards the quality of the food product. In case of probiotics, the functionality of the food product depends upon the cell numbers of specific probiotic strain in the food product. However, in the case of food spoilage the overall bacterial load should be taken in account. Apart from probiotics or spoilage causing microbes another category is that of pathogenic micro-organisms occurring in food products. With the production of food products at industrial scale the presence of pathogens in food can cause food borne out break of a food borne disease. So the detection of pathogens in food products is of prime importance to prevent such out breaks. Keeping all these facts in view, the contents of this book have been formulated and organized to incorporate the relevant methodologies employed in general techniques of microbiological analysis of foods, screening and enumeration of spoilage and pathogenic organisms and some rapid methods of microbiological analysis. 5.2 MICROBIOLOGICAL MEDIA Each organism needs to find in its environment all the substances required for energy generation and cellular biosynthesis. Micro-organisms are known to thrive in almost every environment where they play an important role in the bio- geochemical cycles and maintenance of food chains. They grow in particular niche and consume the nutrients existing there, increase their numbers. The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrients or nutritional requirements. The concentration of nutrients varies in the environment and this controls various microbial populations. In the laboratory, micro-organisms are grown in culture media which are designed to provide all the essential nutrients in solution for their growth. Several types of media are known and the choice of media depends upon the specific microorganism to be grown. Though there are different kinds of media but there is similarity between the media used for a particular group of micro-organisms. Some media can support the growth of several microbes while others are specific. Common media used for growing bacteria is nutrient broth which contains beef extract (carbohydrates, organic nitrogen compounds, water soluble vitamins, salts), peptone (organic nitrogen) and yeast extract (B vitamins, organic nitrogen, carbon compounds). 5.2.1 Types of Media As discussed in the above paragraph there are several hundreds of media and their modifications known for culturing bacteria. However, these media are characterized broadly into several types such as: 1. Chemically defined media/ synthetic media - In this type of media the exact chemical composition is known. It is used for the cultivation of autotrophs. Ex. Potato dextrose agar 2. Complex media; The exact chemical composition of this media is not known. It contains substances such as yeast extract, beef extract, peptones, brain heart infusion, hydrolysates, tissue extracts, blood serum etc. The exact composition of these raw materials added to the media is not known

Nutrient broth is an example of complex media. Such media are used for General Techniques of growing heterotrophs. Detection and Enumeration of Micro-Organisms in Food 3. Liquid media: This type of media are also known as broths. Broths do not contain solidifying agents and are in liquid state. They are used for routine cultivation procedures for pure culture or mixed cultures. These media are also used for production of various compounds during industrial fermentations. 4. Solid media: When a solidifying agent is added to the liquid media it is called solid media. The most common solidifying agent used for media preparation is agar. It gets solidified below 45oC and is usually added at a concentration range of 1.5-2.0%. It is inert and does not get degraded by most of the bacteria; however, a few bacteria are known to produce agarase, which play a role in the degradation of agar. Solid media is used for isolation of bacterial colonies by streaking or serial dilution methods. It is also helpful when colony characteristics of a bacterial culture are to be studied as in case of differential media. Nutrient agar is an example of solid media. 5. Semi solid media: This contains a low concentration of agar as compared to solid media. A gelly like structure is formed in this type of media. It is used for studying the motility of the bacterial cultures or for the cultivation of micro-aerophilic bacteria. 6. Selective media: This media allows the growth of only a particular type of bacteria from the mixed culture e.g. addition of antibiotics (say Penicillin) to the media renders it selective for the growth of only antibiotic resistant bacteria (Penicillin resistant strains). Selective media is used extensively for isolation of bacteria from the environmental samples and also for various screening methods in molecular biology experiments where a plasmid is carrying a drug resistance gene. 7. Enrichment media: This type of media is used for favouring a particular bacterial strain from the mixed culture. A particular compound which enhances the growth of the strain of interest is added to the media. This strain grows at an enhanced rate and soon dominates in the culture. 8. Differential media: This type of media is used for the differentiation of bacterial cultures by studying their growth and colony characteristics e.g. E. coli is known to form blue black colonies with a green metallic sheen when grown on Eosin Methylene Blue (EMB) agar. Thus this media can be used for differentiating E. coli from other bacteria. 5.2.2 Preparation of Media Most of the bacteriological media used in microbiology are available as dehydrated powders. The required amount of powder is weighed and dissolved in distilled water. The pH of the media is adjusted as per instructions provided by manufacturer. Usually for routine cultivation of bacteria nutrient broth with pH 7.0 is used, while potato dextrose medium with pH 5.0 is used for fungi. In preparation of nutrient media (broth/agar) pH is adjusted before autoclaving at 121oC for 15 min, but in case of potato dextrose agar (PDA), this adjustment is done by addition of sterilized solution of 10% tartaric acid after autoclaving the media.

Analytical Techniques At times microbiological media contain heat labile components and in this case in Microbiology stock solutions of such components are filter-sterilized by passing them through a bacteria proof membrane filter having pore size ≤ 0.2µm under positive pressure separately and added to the remaining autoclaved media to obtain the desired concentration. There are several variations in the preparation of media but all the methods follow one basic scheme depicted in the following flow chart. Weighing of Components

Dissolution in Distilled Water

Filter Sterilization of pH Adjustments by Addition of Suitable Acid or Base Heat Labile Components Boiling Addition of Agar & Boiling for mixing it

Sterilization by Autoclaving

Prepared Media

Fig 5.1: Preparation of Microbiological Media The form in which media are prepared also vary e.g. solid media can be prepared in flasks and poured into plates whenever required e.g. for Standard Plate Count (SPC) or it may be prepared as slants, deep tubes or stabs for biochemical and maintenance purposes. Care should be taken that the glassware used for media preparation is clean and plugged properly so as to avoid contamination. Use of freshly prepared media should be avoided and it should be incubated at ambient temperature to make sure that autoclaving is proper and there is no development of contaminants. #Check Your Progress Exercise 1 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) Name some defined and complex media. What is the difference between them? ……………………………………………..……………………………… ………………………………………….….……………………………… …….…………………………………………………………………….… 2) Differentiate between enriched, defined and differential media. …………………………………………………………………………… …………………………………………………………………………… ……………………………………………………………………………. 8

3) Which is the solidifying medium for microbes? General Techniques of Detection and Enumeration …………………………………………………………………………….. of Micro-Organisms in Food …………………………………………………………………………….. …………………………………………………………………………….…

4) What are probiotic micro-organisms? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….…

5.3 ENUMERATION PROCEDURES Enumeration procedures for micro-organisms can be classified into direct methods where the numbers of micro-organisms are counted as in the case of Direct Microscopic Count (DMC), Standard Plate Count (SPC), Spiral plating or indirect methods where overall microbial load is correlated to the metabolic activity of the micro-organisms as in dye reduction tests. 5.3.1 Direct Microscopic Count (DMC) Direct microscopic count is carried out to estimate the number of bacterial cells or the somatic cells in raw and processed milk. The direct microscopic count can be related to the quality of milk. Presence of a large number of bacterial and somatic cells in milk may point to the mastitic condition of the milching animal. Also, if there is reason to suspect that a food has caused food poisoning or has undergone microbial spoilage, the original product or a low serial dilution of it should be used to prepare a slide for direct microscopic examination. The DMC of sample is carried out by examining stained smear of a measure volume of sample say milk (0.01 ml) spread over a known area of glass slide (1.0 cm2 ) and counting the number of cells or clumps of cells in a microscopic field. The average number of cells per microscopic field is determined after staining 5 to 50 fields. The diameter of a field is measured with a stage micrometer for calculation of microscopic factor (MF). The total number of bacteria per ml is calculated by multiplying average number of cells with microscopic factor. The method is simple and rapid and facilitates simultaneous enumeration and observation of cells. It can be conveniently used for screening and grading of foods especially, liquid foods. However, major disadvantage is that both dead as well as cells are taken into account and hence less suitable for heat treated foods. The total number of bacteria as follows: Total number of bacteria /ml= N x MF where, N is average number of cells per field; and ⎧ A 1 ⎫ MF stands for microscopic factor ⎨MF s ×= ⎬ ⎩ m VA ⎭ As is area of smear (100 sq. mm) AM isarea of microscopic field V is volume of milk (0.01 ml) DMC=10,000/3.1416 × r2 9

Analytical Techniques (where, r is the radius of microscopic field as determined by using stage in Microbiology micrometer and 100X objective). Precautions recommended during direct microscopic count • Never dust a lens by blowing on it. Saliva will inevitably be deposited on lenses and is harmful, even in minute amounts. Never use facial tissues to clean lenses. They may contain glass filaments which can scratch lenses. Linen or chamois may be used for cleaning but may not be as convenient as lens tissue. • Never leave microscope tubes open. Always keep them closed with dust plug, eyepiece, or objective, as appropriate. • Avoid touching lenses. Even light fingerprints, especially on objectives, can seriously degrade image quality. • Avoid getting immersion liquid on non-immersion objectives; it can damage the lens mounting glue. • Use the following procedure to properly clean lenses of microscopes or other optical equipment. Crumple a piece of lens tissue to create many folds to trap dirt without grinding it into the lens. Do not touch the part of the tissue that will be applied to the lens; excessive touching transfers natural oils from the fingers to the lens tissue. Apply a small amount of lens cleaning solution to the lens tissue and blot the tissue against absorbent material to prevent fluid from entering the lens mount. Wipe the lens very lightly to remove gross dirt that was not blown away by the rubber bulb. If necessary, repeat the cleaning process with a new piece of lens tissue and with slightly more pressure to remove oily or greasy residue. 5.3.2 Standard Plate Count (SPC) Standard Plate Count is carried out to estimate the number of viable bacteria in a food sample. Bacteria growing aerobically in the mesophilic range of temperature are detected in SPC. A high SPC of the food product indicates poor sanitary conditions of the food processing plant and a low SPC indicates the good quality of the processed food in terms of bacterial load, however, SPC does not indicate the absence of pathogens in the finished food product. SPC is based on the serial dilution of the food sample in sterilized dilution blanks followed by plating on the nutrient media. The dilution of sample is required because usually food samples contain a high number of bacteria, which are difficult to count. If undiluted sample is plated, a lawn will form on the plate and the individual isolated colonies that can be counted will not be formed. After serially diluting the representative sample of food, plating is carried out by pour plate or spread plate technique. In pour plate technique, the dilution is added to the sterile petriplate followed by the addition of molten nutrient agar. The diluent and agar are mixed and plates are incubated after solidification for the growth of bacterial colonies. Pour plate technique is the preferred technique for carrying out SPC, however at times, spread plate technique which involves the spreading of the dilution on the surface of solidified nutrient media may also be used. The SPC of the food sample is reported as colony forming units per ml (cfu/ml). The term colony forming unit is used because the bacterial colony obtained on the agar plate may not be produced by a single bacteria but bacterial clumps may also give rise to the colony hence cfu/ ml and not cells/ ml is used for reporting the results.

Calculation and Results General Techniques of Detection and Enumeration For calculating SPC only those plates having the colony numbers from 25 to of Micro-Organisms in Food 250 are considered. Plates having more than 250 colonies are designated as Too Numerous To Count (TNTC). Alternatively in such cases count may be reported as estimated count. Number of colonies on valid plates (colonies ranging from 25-250) are counted. The average obtained from the duplicates at a single dilution is multiplied by the dilution factor to obtain the colony forming units per ml (cfu/ml) of the food sample e.g. if plates at 10-4 dilution are having 50 and 60 colonies the cfu/ml will be calculated as: cfu/ml = average no. of colonies at a dilution x respective dilution factor ⎛ + 6050 ⎞ ⎜ ⎟ ×104 = 5, 50000 = 55 × 104=5.5 ×105 ⎝ 2 ⎠ Precautions 1) The sample taken for analysis should be representative of the whole stock. If a sample taken for analysis is small it will give erroneous results. 2) Blending of the sample should be done carefully so as to prepare a homogeneous suspension. 3) Dilutions should be mixed properly before each transfer. 4) Molten agar should not be very hot (40-42OC is optimum temperature) at the time of pouring. 5) Plates should be incubated in an inverted position so as to avoid moisture condensation and consequently lawn formation on the surface of agar. 6) Diluent taken may differ with the type of food product. Sodium citrate (2%) may be used for fatty foods such as cheese. Normal saline, buffers or even distilled water may also be used as diluent. 5.3.3 Spiral Plate Count This method can be said to be a variation of Standard Plate Count. In this a very small volume of inoculum is plated on the surface of the plate and this gives a high dilution (10,000:1) of inoculum on a single plate. A mechanical device called Spiral Plating Machine is used for this purpose.

Fig 5.1: Spiral Plating Machine It dispenses the inoculum on the surface of rotating hardened agar plate. The plate is then incubated at the suitable incubation temperature depending on the bacterial culture plated on the plate. After incubation the isolated colonies are counted using a counting grid that covers the area of agar plate and is divided into four concentric circles and eight wedges or octants. 11

Analytical Techniques in Microbiology

Fig 5.2: Spiral counting grid The well separated isolated colonies are counted in the wedges and total number of cells present in the sample is calculated accordingly.

# Check Your Progress Exercise 2 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) Which of the counting methods is fastest: (i) DMC (ii) SPC (iii) Spiral counting 2) List a major drawback of DMC? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 3) What is a mesophile? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 4) The pouring temperature of agar in petriplates is ……………… 5) Write the formula of microscopic factor. ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 5.3.4 Dye Reduction Tests These tests are used to determine the microbiological quality of milk. These are indirect tests where the number of micro-organisms is not counted as in SPC; instead the time required for reduction of dyes (methylene blue or Rezaurin) is 12

taken into account and correlated with the milk quality. These dyes act as General Techniques of oxidation-reduction (O-R) indicators and get decolorized due to the Detection and Enumeration of Micro-Organisms in Food consumption of oxygen and decrease in the O-R potential of milk as a result of bacterial growth. A. Methylene Blue Reduction (MBR) test Methylene blue is a dye, which remains blue in its oxidized state and turns colourless on its reduction. This characteristic is put to use for estimation of bacterial load of milk and milk products. When bacteria grow in milk they release hydrogen during respiration, which is simultaneously accepted by methylene blue. As a result, it is reduced to colourless or leuco compound. The majority of bacteria, both aerobic and facultative present in milk indulge in lowering of oxidation-reduction potential of milk to such an extent that dye gets decolorized. Hence greater the number of viable cells, shorter is the time taken to reduce the dye. The result of this test is expressed in terms of time required for the colour of methylene blue to disappear as Methylene Blue Reduction (MBR) time at incubation temperature of 37oC. This test renders very useful information on general bacteriological quality of milk in a short period and requires minimum apparatus. Limitations of this technique include its suitability only for unheated milk and no indication of the type of organisms. The methylene blue reduction time also depends on the type of bacteria occurring in milk. Coliforms reduce this dye at a higher rate as compared to psychrotrophic and thermoduric bacteria occurring in milk. This is so because the incubation temperature favours the growth of mesophilic bacteria (coliforms). High numbers of lecucocytes if present also affect reduction time of methylene blue in milk samples. Requirements Milk sample, methylene blue solution (0.4 mg/100 ml of sterile distilled water), sterile tubes and stoppers, sterile pipettes, water bath. Procedure 1. Take 10 ml milk in a test tube and add 1 ml of methylene blue solution to it. 2. Insert stoppers on the tubes and mix the contents by inverting the test tubes gently. 3. Incubate the test tubes in a covered water bath at 37oC and check the tubes for decolourization after every 30 min. of incubation. 4. Remove the decolorized tubes after each reading and also invert gently the remaining tubes. 5. Monitor the tubes for decolourization till 8 h. Interpretation The quality of milk is graded according to the time taken for decolorization and is as follows: Class 1. Excellent, not decolorized in 8 h Class 2. Good, decolorized in less than 8 h but not less than 6h. Class 3. Fair, decolorized in less than 6 hours but not less than 2 h. Class 4. Poor, decolorized in less than 2 h. 13

Analytical Techniques Precautions in Microbiology • Dye solution should be prepared and kept in amber bottles without exposure to light. • Milk should be mixed properly but gently because vigorous shaking will increase the oxygen content of milk. If mixing is not proper then bacteria may arise with the cream layer and this leads to inaccurate results. • Water bath should be covered as exposure to light will decrease the methylene blue reduction time. B. Resazurin Reduction (RR) test Resazurin is also an O-R indicator and hence is liable to be reduced by bacteria. Reduction of blue dye takes place in two stages. First, the dye is irreversibly reduced to resorufin undergoing a series of color changes ranging from blue to lilac, mauve, purple and pink. During second stage, resorufin is reversibly reduced to a colourless compound, dihydroresorufin. Various colors that develop in a sequence during reduction of dye can be well compared with a standard resazurin disc with the help of a small apparatus known as resazurin comparator. Results are expressed in terms of standard resazurin disc number ranging from 6 to 0. The time taken for the reduction of dye to a specific stage (disc number) or the color change recorded on completion of incubation after a certain period can be used as a scale for measurement of bacterial activity. The test is carried out in several formats including “one-hour” and “triple reading” tests. This test finds its application in quick grading of milk (even faster than MBR test). However, reduction of dye is susceptible to light and confusion may arise in interpretation of results due to the fact that besides bacteria, this dye is also liable to be reduced by leucocytes. Requirements Milk sample, resazurin solution (11 mg/ 200 ml of hot sterile distilled water), sterile tubes and stoppers, sterile pipettes, water bath. Procedure 1. Take 10 ml milk in a test tube and add 1 ml of resazurin solution to it. 2. Insert stoppers on the tubes and mix the contents by inverting the test tubes gently. 3. Incubate the test tubes in a covered water bath at 37oC and check for the change in the colour after 1 hr of incubation in case of ‘one-hour’ test or at 1, 2 and 3 h for “triple reading” test. 4. Compare the colour with the standard resazurin disc number ranging from 6 to 0 and report the results. 5.3.5 Most Probable Number (MPN) Method Most probable number is based on the probability of the presence of bacteria in standard dilution series of the sample inoculum. Bacteria within the dilution series are detected by positive growth characteristics such as gas production or turbidity in the growth medium. An MPN procedure just like SPC detects the viable organisms and is useful when bacteria are present at low concentration (< 100/g) in food, milk, water and soil samples. However, MPN gives only the 14

estimate of organisms while plate count gives direct counts in cfu/ ml. MPN is General Techniques of used to estimate the number of coliforms in the water samples, however, this Detection and Enumeration of Micro-Organisms in Food method can be modified to estimate the number of pathogens by using specific selective culture medium for their growth. Sanitary analysis of the water sample employs the lactose broth medium with Durham’s tube (a small inverted tube added to the larger tubes containing the lactose broth) that helps in the detection of gas production by coliforms from lactose utilization. This test is also called the presumptive test for coliform detection.

5.4 PURE CULTURE METHODS

The methods listed in the above sections are for determining the microbial load/ counts in the food samples; however, some of these methods such as pour/spread plating can also be used to get the pure cultures of bacteria. These methods have an advantage that they can be used both for obtaining bacterial counts and the purification of mixed cultures. Apart from these methods another method called streak plate can also be used for purification of microbial cultures. Streak plate method involves diluting the bacterial culture on the surface of agar medium so as to obtain isolated colonies of bacteria. These colonies are then differentiated on the basis of morphology (differential media) or other tests (biochemical). The main disadvantage of using this method is that you cannot count the number of bacteria as the dilution in this case is not equated. Streaking can be carried out in several formats such as continuous streaking, quadrant streaking, T streaking etc. but all involve the same principle of diluting the culture on the surface of agar plate so as to obtain isolated bacterial colonies.

Check Your Progress Exercise 3 " Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) The period of observation of MBRT is …….. hours. 2) Streaking is done to …………………. 3) What is the principle behind resazurin test technique? …………………………………………………………………………… …………………………………………………………………………… ……………………………………………………………………………

4) ……….. broth is used in MPN technique. 5.5 MICROSCOPIC EXAMINATION OF THE BACTERIAL CULTURE Bacterial cultures are examined under 100X oil immersion lens of the microscope. They may be examined as unstained cultures when the motility of the culture is to be studied or they may be stained depending upon the requirement. Several types of staining is carried out for studying bacterial culture including negative staining, grams staining, endospore staining, capsule staining, flagellar staining, acid fast staining etc. 15

Analytical Techniques 5.5.1 Simple Staining in Microbiology In this type of staining bacterial cells are stained with a single reagent. Positively charged dyes such as methylene blue, crystal violet etc. are used for this purpose. These stains are taken up by the cells and bind to negatively charged cell components (cell wall, nucleic acids). 5.5.2 Negative Staining In this method the bacterial cells do not take up the stain but the background gets stained and the cell appears as unstained transparent entity. Acidic dyes such as nigrosin are used. Negative staining is advantageous because the bacteria which do not take up the stain can be observed in this way. Another advantage of this procedure is that it does not require heat fixing of smear so cell distortion due to heat do not take place.

5.5.3 Gram Staining This is the most important staining technique for the bacterial cultures. Based on gram staining, bacteria are characterized into gram positive cells and gram negative cells depending on their capacity to retain crystal violet stain. The basis of gram staining is the difference in the cell wall structure of the two types of bacteria. Gram positive cell walls have a higher content of peptidoglycan and a lower content of lipids while gram negative cells have higher concentration of lipids and lower concentration of peptidoglycan. During gram staining, the bacterial smear is stained with crystal violet and iodine followed by destaining with alcohol. Lipids present in the cell wall of gram negative cells gets dissolved in alcohol forming pores through which crystal violet leaks out, while peptidoglycan in the cell wall of gram positive shrink due to alcohol, thus retaining the crystal violet and hence the purple colour. 5.5.4 Endospore Staining Endospores are highly refractile, heat resistant bodies that are produced by certain bacteria when the conditions are not favourable for growth. The heat resistance of endospore is an important property and allows survival of bacteria in the form of metabolically inactive endospore, which germinates on return of favourable conditions and start growing, contaminating food and spoiling it. Endospores are stained with Malachite green dye under hot conditions. Afterwards the slide is stained with saffranine (pink), the other microbes appear pink while endospores which do not stain with saffranine appear green.

5.6 LET US SUM UP

For cultivation of micro-organisms in laboratory and their enumeration, suitable media are required to be prepared and these can be classified as general purpose, selective, differential, and enrichment on the basis of application. These media are used for enumeration of microorganism by various techniques namely, DMC, SPC, spiral plate count, dye reduction methods, and MPN method. The streak plate method can be used for isolation and purification of microorganism present in food. The micro-organisms so isolated and purified can be microscopically identified and morphologically studied on the basis of staining methods such as simple, negative, gram and endospore staining.

General Techniques of 5.7 KEY WORDS Detection and Enumeration of Micro-Organisms in Food EMB : Eosin methylene blue PDA : Potato dextrose agar DMC : Direct microscopic count MF : Microscopic factor SPC : Standard plate count cfu : Colony forming unit TNTC : Too numerous to count MBRT : Methylene blue reduction test RRT : Resazurin reduction test 5.8 SUGGESTED FURTHER READING F.P. Downes and K. Ito (Editors), American Public Health Association (APHA). (2001). Compendium of Methods for the Microbiological Examination of Foods, 4th edition. Washington, D.C. American Public Health Association (APHA). (1992). Standard Methods for the Examination of Dairy Products. 16th edition, Washington, D.C. Aneja, K.R. (2003). Experiments in Microbiology In: Plant Pathology and Biotechnology. New Age International Publishers. Cappuccino, J.G. & Sherman, N. (1996). Microbiology: A Laboratory Manual, Benjamin/ Cummings Publishing Company. Gilchrist, J.E., Campbell, J.E., Donnelly, C.B., Peeler, J.T. & Delaney J. M. (1973). Spiral Plate Method for Bacterial Determination. Applied and Environmental Microbiology, 25: 244–252. Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. In: Methods in Microbiology Volume19, Colwell & Grigorova (Eds), Academic Press, pp 1-68. Pelczar, M.J, Chan E.C.S. and Krieg, N.R.(1993). Microbiology, 5th edition. Mcgraw Hill, USA. Official Methods of Analysis of AOAC International (2000), 17th ed., AOAC International, Gaithersburg, MD, USA. Hypertext source: Bacteriological Analytical Manual (1998). 8th Edition. 5.9 TERMINAL QUESTIONS 1) Define microbiological medium and describe their types. 2) Enlist various enumeration techniques and suggest their appropriate applications in various food systems. 3) How can you calculate microscopic factor? 4) How you can differentiate between gram positive and gram negative bacteria? What is the basis of gram reaction?

Analytical Techniques 5) While performing Coliform count of water by MPN method, no of positive in Microbiology tubes in 10 ml, 1.0 ml and 0.1 ml are 2,1,0 respectively. Determine the count/ ml with the help of MPN table. 6) How can you observe flagellum of a bacterial cell? Describe the protocol of a suitable method. # 5.10 ANSWERS TO CHECK YOUR PROGRESS EXERCISES Check Your Progress Exercise 1 Your answer should include following points: 1) Defined media: Potato Dextrose agar; Complex media: Ex. yeast extract, beef extract, peptones, brain heart infusion, hydrolysates, tissue extracts, blood serum etc. The exact composition of these raw materials added to the media is not known. 2) Selective media allows the growth of only a particular type of bacteria from the mixed culture and is used extensively for isolation of bacteria from the environmental samples and screening methods in molecular. Enrichment media is used for favouring a particular bacterial strain from the mixed culture. This strain grows at an enhanced rate and soon dominates in the culture. Differential media is used for the differentiation of bacterial cultures by studying their growth and colony characteristics. 3) Agar 4) Probiotic micro-organisms have been linked to several health benefits when consumed in live active forms. Check Your Progress Exercise 2 Your answer should include following points: 1) DMC 2) Even dead microbes may be counted under microscope in DMC. 3) Mesophiles are the microbes which are able to grow at room temperature. A 1 4) MF s ×= m VA

Check Your Progress Exercise 3 Your answer should include following points: 1) 8 2) isolate microbial colonies. 3) Resazurin is an O-R indicator and can be reduced by bacteria. Reduction of blue dye takes place in two stages. First, the dye is irreversibly reduced to resorufin undergoing a series of color changes ranging from blue to pink. During second stage, resorufin is reversibly reduced to a colourless compound, dihydroresorufin. The time taken for the reduction of dye to a specific stage (disc number) or the color change recorded on completion of incubation after a certain period can be used as a scale for measurement of bacterial activity. 4) Lactose 18

Screening and Enumeration of Micro- UNIT 6 SCREENING AND ENUMERATION organisms in Food OF SPOILAGE MICRO-ORGANISMS IN FOOD Structure 6.0 Objectives 6.1 Introduction 6.2 Detection and Enumeration of Spoilage Micro-organisms 6.2.1 Psychrotrophic Count 6.2.2 Thermoduric Count 6.2.3 Lipolytic Count 6.2.4 Proteolytic Count 6.2.5 Pectinolytic Count 6.2.6 Halophilic Count 6.2.7 Osmophilic Count 6.2.8 Acidophilic Count 6.3 Let Us Sum Up 6.4 Key Words 6.5 Suggested Further Reading 6.6 Terminal Questions 6.7 Answers to Check Your Progress Exercises 6.0 OBJECTIVES After reading this Unit, we shall be able to: • explain the role of food as carrier of spoilage organisms, • describe the causes of spoilage of food, • enlist major types of spoilage bacteria found in foods • identify the common source of contamination by these pathogens • specify types of media used and enumeration protocol of various spoilage bacteria 6.1 INTRODUCTION It is of common knowledge that every food item we eat is biological in origin, so knowledge of biology is important in all aspects of initial food production, as well as preparation and distribution. In particular, we expect our food to be fresh and wholesome, and not to contain any undesired added impurities (adulterants). Food can deteriorate as a result of two main factors: 1) Growth of spoilage micro-organisms - usually from surface contamination- especially important in processed food. 2) Enzymatic action - from within cells – by normal metabolic processes, e.g. respiration. It is important to note that many plants - fresh vegetables and fruit are still alive when bought and even when eaten raw, and meat from animals undergoes gradual chemical changes after slaughter. Micro-organisms enter foods from various sources on its way from production to processing, packaging, distribution and even consumption. That’s why we sometime use phrase such as “From pasture to fork” in context of total quality management. Common sources are soil, water, plant products, food utensils, gastrointestinal tract, food handlers, animal feeds, air and dust.

Analytical Techniques in "Negative" aspects of microbial growth include food deterioration and Microbiology spoilage by decay, and food poisoning, mainly caused by different and less widespread bacteria. As they grow, micro-organisms release their own extra cellular enzymes into the medium/ matrix surrounding them, and absorb the products of external breakdown. This is the main basis of microbial food spoilage, which lowers its nutritional value and decreases its shelf life. Bacteria and moulds may also produce waste products which act as poisons or toxins, thus causing the ill-effects. Growth of these organisms is facilitated by a number of intrinsic and extrinsic factors present in food such as presence of organic food (proteins, carbohydrates, fats), suitable temperature, moisture (water), animal feeds, animal hides, air, and suitable pH. 6.2 DETECTION AND ENUMERATION OF SPOILAGE MICRO-ORGANISMS

The spoilage micro-organisms on the basis of their biochemical characteristics and nutritional requirement (factors detrimental to their ability to spoil food in which they are present) can be broadly categorized into psychtrophic, thermoduric, lipolytic, proteolytic, pectinolytic, halophiles, osmophiles, acid producers etc.

6.2.1 Psychrotrophic Count

The term “Psychrotrophic” is used for the organism capable of growing at refrigerated temperatures irrespective of their optimum temperature of growth. Psychrotrophs commonly include genera of gram negative thin rods e.g. Pseudomonas, Achromobacter, Flavobacterium and Alcaligenes.

These organisms owing to their high proteolytic and lipolytic activity can cause taint production (discoloration & off-flavour production) in foods stored under refrigerated conditions for longer periods. The excessive growth of such organisms can be detrimental to the shelf-life of both raw and processed food products especially, during extended storage.

For enumeration of psychrotrophic organisms appropriate dilutions can be prepared and plated (pour or spread plate method) on non selective media such as plate count agar, tryptone glucose extract agar, Trypticase soy agar and subsequently incubated at refrigeration temperature at 7oC for 10 days. These organisms are heat labile (with few exceptions) and hence can be possibly injured or inactivated if plates are poured with agar held above 45oC. Hence due care should be taken.

6.2.2 Thermoduric Count

Thermoduric organisms differ from thermophiles in their ability only to survive heat treatment while latter can survive and grow at elevated temperature e.g. pasteurization. The species of genera viz. Arthrobacter, Bacillus, Clostridium, Enterococcus, Micrococcus and Lactobacillus belongs to these groups. Biochemical characteristics of these organisms include acid production, proteolysis and lipolysis and can cause spoilage of heat treated and canned products. These organisms are of special interest in dairy products where they are known to survive pasteurization.

These organisms along with psychrotrophs have limited ability to reduce dyes Screening and such methylene blue and resazurin and hence dye reduction tests do not Enumeration of Micro- organisms in Food correlate well with plate counts in such cases. For enumeration of thermoduric bacteria, a 5 ml volume of liquid sample in original or of decimal dilution is aseptically transferred to sterile test tube and a rack of such tubes is placed in a water bath maintained at 62.8oC. After a period of 30 min (commonly termed as laboratory pasteurization), tubes are immediately cooled in an ice water bath. Now 1.0 ml of this sample can be analyzed by plate count method using plate count agar and incubation at 37 oC for 72 h. 6.2.3 Lipolytic Count The fat rich food products are liable to be spoiled by lipolytic micro- organisms though there can be non-microbial origin of such defects. The known lipolytic organisms commonly encountered in foods include bacteria (Pseudomonas, Achromobacter, Staphylococcus), molds (Rhizopus, Geotrichum, Aspergillus, Penicillium), and yeast (Candida, Rhodotorula, Hansenula). For lipolytic count a base layer medium which comprises fat, 50.0 g; victoria blue, 1:1500 solution, 200 ml; agar, 15 g is used. Fat may consist of tributyrin, corn oil, soybean oil, cooking oil. Out of these tributyrin is the most widely used substrate and hence commercially available tributyrin agar is commonly used for lipolytic count. To such agar victoria blue is not required to be added. The plates of lipolytic medium are streaked with the dilution of sample and incubated at 20 to 25 oC for 3 to 4 days. The presence of a clear zone of hydrolysis around the colony is taken as a positive indication and inferences are drawn accordingly. 6.2.4 Proteolytic Count The proteolytic activity of micro-organisms can lead to a number of undesirable flavour defects in milk, meat, poultry and seafood. Such organisms include Bacillus, Clostridium, Pseudomonas and Proteus. Milk agar medium For enumeration of proteolytic bacteria especially from dairy products, this agar is commonly used. The hydrolysis of casein manifests into development of clear zone around the colony. The sample is poured or streaked on this medium and incubated at 21oC for 72 h. Another medium, gelatin agar medium with double layer plating technique can also be used for this purpose. A common limitation of this method is the need to flood the medium with protein precipitant (1% HCl or 10 % acetic acid solution for 1 minute to ensure that the clear zones are formed as a result of proteolysis and not due to acid production from carbohydrates. 6.2.5 Pectinolytic Count Pectinolytic enzymes capable of hydrolyzing pectic substances include pectin isomerase, pectin methoxylase, pectin methylesterase and pectate lyase. Sources of these enzymes are Achromobacter, Aeromonas, Arthrobacter, Agrobacterium, Bacillus, Clostridium, Enterobacter, Flavobacterium, Pseudomonas, Xanthomonas, yeasts, moulds, protozoa and nematodes. Medium used for detection of pectate lyase, polygalacturonase and fluorescent pectinolytic Pseudomonads are mineral pectin-7 medium (MP-7), mineral

Analytical Techniques in pectin-5 medium (MP-5), fluorescent and pectinolytic agar (FPA) Microbiology respectively. A homogeneous suspension followed by appropriate dilutions of the food sample are prepared. A volume of not more than 0.25 ml is placed and spread on surface of prepoured plates. Incubation is carried at 30oC for 48 h. Colonies which effect depressions in polypectate gel medium or are found to be encircled by a clear zone after flooding the MP-7, MP-5 or FPA plates with a pectin precipitant (1% solution of hexa-decyl tri-methyl ammonium bromide in water) are considered as pectinolytie colonies and are counted.

# Check Your Progress Exercise 1 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) Incubation temperature for psychrotrophic, thermoduric and pectinolytic micro-organisms is …………, ……………… and …………oC. 2) Most widely used substrate for lipophiles is …………. 3) Characteristic colony of proteolytic microbes on Milk Agar medium is …………………………………………………………………….. 4) List any three pectinases. ………………………………………… ………………………………………… ………………………………………… 6.2.6 Halophilic Count Halophiles are those organisms which need certain minimal salt concentrations for their growth. These organisms can be classified as slight halophiles (2 to 5% salt), moderate halophiles (5 to 20 % salt) and extreme halophiles (20 to 30 % salt). a) Slight halophiles Majority of slight halophiles are traced to marine environment and typical genera such as Acinetobacter, Flavobacterium, and Pseudomonas are known to spoil marine products and shellfish. For enumeration of these organisms, skins are removed from fish and six samples collected, three each from dorsal and ventral sides of the fish. Ten grams of skin is added to 90 ml of phosphate buffer. Sample is vigorously mixed and subsequent dilutions are prepared in phosphate buffer. A volume of 0.1 ml aliquots is dispensed on the trypticase soy agar plus 3% NaCl land evenly spread on plate using spread plate technique. Plates are incubated at 7oC for 10 days. Results are reported as cfu per gram. For light salted meat and vegetables, dilutions are prepared by blending 50 g samples with 450 ml diluent with added salt concentration equivalent to salt concentration of food sample. Rest of the procedure is same as above. b) Moderate halophiles This group includes bacteria belonging to Bacillaceae and Micrococcaceae. A 1: 10 dilution is prepared by mixing 50 g of food in 450 ml of sterile buffer 22 with added NaCl equivalent to the salt concentration prevalent in the food Screening and sample. Rest procedure for plating and incubation is same as for slight Enumeration of Micro- organisms in Food halophiles. c) Extreme halophiles They are usually found in aquatic environments of unusually high concentration and in solar evaporated sea salts. Genera like Halobacterium and Halococcus belong to this group. For isolation, the surface slime from salted fish is transferred to halophilic agar plate using a cotton or alginate swab. For enumeration 1: 10 dilution is prepared by mixing 50 g of sample with 450 ml of halophilic broth. A volume of 0.1 ml aliquot of each dilution is placed on halophilic agar and incubated at 33-35oC for 5 to 12 days in a humid incubator. Results are given as halophilic count per ml or per gram. 6.2.7 Osmophilic Count Yeasts capable of growing in high concentration of sugar happen to be the common osmophiles found in food. They are responsible for spoilage of honey, chocolate candy, jams, molasses, corn syrup, fruit juices and similar products. For their enumeration, appropriate dilutions are prepared in phosphate buffered dilution water as diluent, plated in agar (Potato dextrose agar, MY-40 agar i.e. malt yeast extract 40 % sucrose), and incubated at 30oC for 4 to 5 days. 6.2.8 Acidophilic Count Acid producing bacteria are found in various habitats e.g. soil, raw agricultural products, and processed foods. Lactic acid bacteria are well known in food industry for their ability to produce acids from sugar present in substratum and on this basis can be classified as homo-fermentative and hetero-fermentative. Besides, spore formers (Bacillus and Clostridium) and enteric bacteria (Escherichia, Enterobacter, Salmonella, Shigella) are also endowed with this ability. For detection of such bacteria, determination of titratable acidity (acidity expressed as lactic or acetic acid) and indicator microbiological media (containing pH indicator e.g. bromo cresol purple) are required. Lactic acid bacteria chiefly comprise lactic Streptococci and Lactobacilli. Lactic agar supports growth of both types of bacteria while M17 agar and MRS agar allows selective growth of former and later, respectively. Using pour plate method these media can be used for direct enumeration of lactic acid bacteria. Their identity can be further confirmed by gram staining and catalase test. These organisms are gram positive, non spore-forming, non motile and catalase negative. Measurement of titratable acidity can be used as an indirect method of estimation of acid production. It can be performed by titration of an appropriate quantity of sample (say fermented milk) with 0.1 N NaOH to the phenolphthalein end point (pH 8.3).The % acid present as lactic acid is calculated as follows: V × 009.0 % lactic acid= 2 ×100 V1 where, V1= Volume of sample/milk (in ml)

V2= Volume of NaOH used (in ml). 23

Analytical Techniques in Check Your Progress Exercise 2 Microbiology # Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) Spoilage micro-organisms in jams and pickles are called ……………… and ………………… respectively. 2) Media for plating halophiles is …………………………… 3) Formula for percent acidity in milk ………………… 4) List any two media for growing yeast ………………………………………….. ………………………………………….. 6.3 LET US SUM UP Microbes and their enzymes are major causative agents of spoilage of food. Growth and resultant biochemical activity of micro-organisms is supported by a number of intrinsic and extrinsic factors which exist in food and environment. Based on their biochemical attributes and nutritional requirement the spoilage causing microflora can be broadly grouped in to psychrotrophic, thermoduric, lipolytic, proteolytic, pectinolytic, halophiles, osmophiles, acid producers etc. P Psychrotrophic organisms can be enumerated by using media such as plate count agar, tryptone glucose extract agar, Trypticase soy agar and subsequently, incubation at refrigeration temperature at 7oC for 10 days. For thermoduric bacteria count, laboratory pasteurized sample is plated on plate count agar and incubated at 37o C for 3 days. For lipolytic and proteolytic count, tributyrin and milk agar are used respectively and plates are incubated at 20 to 25oC for 3 to 4 days. The formation of clear zone of hydrolysis around the colony is interpreted as a positive sign and conclusions are drawn accordingly. In case of pectinolytic bacteria, selective media used for assesment of activity of pectinolytic enzymes such as pectate lyase, polygalacturonase and fluorescent pectinolytic Pseudomonads (FPA medium) are mineral pectin-7 medium, mineral pectin-5 medium, fluorescent and pectinolytic agar, respectively. An appropriate dilution is placed on halophilic agar and incubated at 33-35oC for 5 to 12 days in a humid incubator for halophilic bacterial count. Paotato dextrose agar or MY-40 agar and incubation at 30 oC for 4 to 5 days are used for enumeration of osmophiles in food. Lactic acid bacteria happen to be the major acidophiles in foods and lactic agar, M17 agar and MRS agar can be used for their selective growth and enumeration. 6.4 KEY WORDS Psychrotrophs : Organisms capable of growing at refrigerated temperature Taint production : Discoloration & off-flavour production Thermoduric : Organisms able to survive pasteurization Laboratory pasteurization : Heating at 62.8oC for 30 minutes Tributyrin agar : Used for enumeration of lipolytic bacteria

Milk agar : Used for enumeration of proteolytic bacteria Screening and Enumeration of Micro- FPA Medium : Fluorescent pectinolytic Pseudomonads organisms in Food medium Halophilic Agar : Used for enumeration of halophiles MY-40 Agar : Malt yeast extract 40 % sucrose agar 6.5 SUGGESTED FURTHER READING American Public Health Association (APHA). (2001). Compendium of Methods for the Microbiological Examination of Foods (4th edition), F.P. Downes and K. Ito (Editors), Washington, D.C. James M. Jay. (2000). Modern Food Microbiology (6th. Edition), Aspen Publisher, USA. Frazier W. (1995). Food Microbiology (4th edition), Tata McGraw Hill, India 6.6 TERMINAL QUESTIONS 1. Describe the protocol for detection and enumeration of the following group of micro-organisms from the food mentioned against their name as follows: S. No. Microorganism Food 1. Psychrotrophic Ice cream 2. Thermoduric Canned peas 3. Lipolytic Cooking oil 4. Proteolytic Nugget 5. Pectinolytic Frozen vegetables rot 6. Halophilic Canned fish 7. Osmophilic Rasogulla 8. Acidophilic Curd 2. Give typical examples of each group of microorganism mentioned above. 3. Prepare a list of defects caused by these organisms in various foods with the help of literature. 4. Suggest remedial measures to control the microbial spoilage of foods. 6.7 ANSWERS TO CHECK YOUR PROGRESS EXERCISES

Check Your Progress Exercise 1 Your answer should include following points: 1) 7, 32 and 30oC 2) tributyrin 3) clear zone around the colony 4) pectin methoxylase, pectin methylesterase and pectate lyase 25

Analytical Techniques in Check Your Progress Exercise 2 Microbiology Your answer should include following points: 1) halophilic and osmophilic 2) trypticase soy agar plus 3% NaCl

3) % lactic acid= V2 x 0.009/V1 X 100 4) Potato dextrose agar, MY-40 agar

Detection of UNIT 7 DETECTION OF PATHOGENS IN FOODS Pathogens in Foods 7.0 Objective 7.1 Introduction 7.2 Detection of Bacterial Pathogens 7.2.1 Bacillus Cereus 7.2.2 Campylobacter 7.2.3 Escherichia Ccoli and Coliforms 7.2.4 Listeria Monocytogenes 7.2.5 Salmonella Species 7.2.6 Staphylococcus Aureus 7.2.7 Clostridium Perfringens 7.3 Detection of Viral Pathogens 7.3.1 Methods for Detecting Viruses Extracted from Foods 7.4 Let Us Sum Up 7.5 Key Words 7.6 Suggested Further Reading 7.0 OBJECTIVES After going through this unit you should be able to: • explain major bacterial pathogens found in foods; • identify the common source of contamination of these pathogens; • specify types of media used; and • describe the detection protocol of various pathogens. 7.1 INTRODUCTION Foods serve as important source of nutrition providing a range of valuable nutrients including essential amino acids, proteins, fats, carbohydrates, vitamins and minerals. However, prepared foods on contamination also act as carriers of harmful pathogenic bacteria such as Staphylococcus aureus, E. coli, Vibrio, Listeria, Shigella etc., which affect health and create symptoms ranging from minor food poisoning to deadly illness on consumption of such foods. In this context the ability to cause disease is known as pathogenicity and virulence is the notion applied to the degree of pathogenicity. The pathogens may exhibit pathogenicity towards both human as well as animals and hence pose a public health hazard. They cause diseases in their host either by invasion of tissues such as mucous membrane or/ and skin (infection) or by producing poisonous metabolites known as ‘toxins’. Toxins may be termed as exotoxin or endotoxin on the basis of site of their production in cell. Exotoxins are secreted outside the cell (mostly in gram positive bacteria) while Endotoxins are part of outer cell wall of gram negative bacteria. On the basis of their site of action toxins may be classified as Neurotoxins (interfere with normal nerve impulses), Cytotoxins (kill cells) and Enterotoxins (infect the cells lining the gastro-intestinal tract). Detection of these organisms is very important for the food industry from the safety point of view as they can even pose serious threat to life and are part of regulatory requirement such as Prevention of Food Adulteration Act. This task is carried out by a separate quality control unit of any food industry. It is important therefore, to have easy and effective methods of detection of these harmful bacteria with good detection limits, which can be carried out with ease in a microbiological laboratory. Hence this unit is dedicated to provide the available microbiological detection methods of important food pathogens including Staphylococcus aureus, Salmonella, Coliforms, Bacillus cereus, Listeria, Campylobacter and Clostridium in an easily comprehensible way. The detection methods for viral pathogens are also discussed. In the following sections the protocols of detection of various bacterial pathogens especially important from regulatory point of view have been described. 7.2 DETECTION OF BACILLUS CEREUS Bacillus cereus is a common cause of food poisoning. It is an aerobic gram positive rod shaped spore forming bacillus and is widely distributed in soil, vegetables and several raw and processed foods. Consumption of foods containing greater than 106 B. cereus cells may lead to food poisoning. It causes food poisoning by enterotoxin production and its excessive growth in heat treated foods especially, dairy products e.g. pasteurized milk, pasteurized cream, Ultra High Temperature (UHT) milk, dried milk results in defects such as ‘Bitty Cream’ and ‘Sweet Curdling’. Sterilization of food products either by autoclaving or ultra high heat treatment can reduce the risk of proliferation of this organism. A. REQUIREMENTS Butterfields phosphate buffered dilution water, mannitol-egg yolk-polymyxin (MYP) agar plates, trypticase soy- polymyxin broth, phenol red glucose broth, nitrate broth, reagent A, reagent B, MR-VP broth, α naphthol, potassium hydroxide, lysozyme B. METHODS OF DETECTION 7.2.1 Plate count of B. cereus 1. Weigh 50 gms of sample in a sterile blender jar. Add 450 ml of sterile Butterfields phosphate buffered dilution water and blend for 2 min. at high speed. This is the 10-1 dilution. 2. Prepare serial dilutions by adding 10 ml of 10-1 dilution to 90 ml of diluent to give 10-2 dilution and so on and so forth so as to attain a final dilution of 10-6. 3. Plate 0.1 ml of each dilution on the surface of MYP agar in duplicate and incubate the plates at 30oC for 24 h. 4. Observe for typical B. cereus colonies which will be of pink colour surrounded by a precipitate zone indicating the production of lecithinase. 5. Count the number of typical B. cereus colonies from the plates on which the colony numbers range from 15-150 colonies. Pick 5 or more representative colonies and transfer them to nutrient agar slants and incubate at 30oC for 24h. Carry out confirmatory tests from the growth obtained on nutrient agar slants, as described in the following sections. 6. Calculate the numbers of B. cereus cells per gm of sample, based on the percentage of colonies tested that are confirmed as B. cereus e.g. if average counts obtained for 10-5 dilution is 52 and all 5 out of 5 colonies tested were confirmed as B. cereus then the counts will be (52х5/5) х 106 = 52,000,000.

Fig 5.1: Growth of B. cereus on MYP agar 7.2.2 MPN for B. cereus Most probable number (MPN) is carried out for the foods where the suspected count of B. cereus is fewer than 10 cells / g of food sample. 1. Inoculate trypticase soy-polymyxin broth tubes with 1 ml of 10-1, 10-2 and 10-3 dilutions. Three tubes should be inoculated for each dilution. 2. Incubate tubes at 30oC for 48 h. Observe for dense growth. 3. Streak MYP agar plates from the tubes showing heavy growth. Incubate the plates at 30oC for 24 h and look for typical B. cereus colonies. 27

4. Confirm the isolates as B. cereus and calculate MPN of B. cereus per gram of sample (Appendix 3) based on the number of tubes that were positive for B. cereus isolates. 7.2.3 Confirmatory tests for B. cereus 1. Use the growth obtained on the nutrient agar for confirmatory tests. 2. Carry out gram and spore staining of the culture. B. cereus will appear as large gram positive bacilli in chains. The spores will be ellipsoidal, central to sub-terminal and will not swell the sporangium. 3. Prepare the suspension of bacterial cells from the slant in 0.5 ml of sterile phosphate buffered dilution water. Use this suspension to perform various tests as described. (i) Glucose utilization in anaerobic conditions: Inoculate 3 ml phenol red glucose broth and incubate anaerobically (Gas Pack system) at 35oC. Observe for colour change to yellow that indicates a positive test for B. cereus. (ii) Nitrate Reduction: The medium is evaluated for nitrate reduction by the addition of two reagents, Nitrate A Reagent (0.8% sulfanilic acid in 5N acetic acid) and Nitrate B Reagent (0.6% N, N-dimethyl-alpha- naphthylamine in 5N acetic acid), which detect the presence of a catabolic end product, and by the addition of Nitrate C Reagent, zinc dust, which detects the absence of remaining nitrate in the medium. Inoculate 5 ml of nitrate broth with a loopful of culture and incubate tubes at 35oC for 24 h. Add nitrite test reagents A and C and look for development of orange color. This indicates a positive test for nitrate reduction. (iii) VP test: Inoculate 5 ml of VP broth and incubate at 35oC for 48 h. To 1ml of this culture add 0.6 ml of α naphthol solution and 0.2 ml of 40% potassium hydroxide. Mix vigorously and add a few crystals of creatine. Observe after keeping at room temperature for 1hr. Consider the appearance of pink colour as positive test. (iv) Tyrosine decomposition: Streak the slant of tyrosine agar and incubate it at 35oC for 24 h. Observe for clearing of medium near growth, which indicates the degradation of tyrosine. (v) Growth in presence of lysozyme: Inoculate 2.5 ml of nutrient broth containing 0.001% lysozyme with a loopful of culture. Keep inoculated nutrient broth tube without addition of lysozyme as a control. Incubate tubes for 24 h at 35oC. Observe for turbidity. Tentatively identify the cultures by comparing the results produced by standard culture as given in the Table 7.1. Based on these tests it is difficult to differentiate B. cereus from other Bacillus species and some other tests such as motility, hemolysis and protein crystal formation may be carried out, but these tests too are not confirmatory for B. cereus as some strains of B. cereus are non-motile or weakly hemolytic. However, except B. thuringenesis other Bacillus species are not frequently encountered in food products so one may rely on these cultural methods as till date no other method is available for specific detection of B. cereus.

Bacillus cereus

Extraction: Sterile Butterfields phosphate buffer

MYP agar (Positive: Pink coloured colonies) NA slant MPN: TSP broth (Positive: dense growth) Streak on MYP Confirmatory: Growth from NA slants Gram staining Spore staining Anaerobic glucose utilization Nitrate reduction VP Tyrosine decomposition Lysozyme resistance

Others: Motility Hemolysis Protein crystal formation

Fig 7.2: Positive reactions of B. cereus for nitrate reduction and Catalase

Check your Progress Exercise 1 Note: a) Use the space below for your answer. b) Compare your answers with those provided at the end of this unit. Q 1. The colour of B. cereus colonies on MYP agar will be ………………. Q 2. What is the principle of precipitate zone formation around B. cereus colonies on MYP agar? …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… Q3. Describe the role of polymyxin in MYP agar? …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… Q 4. What is the role of phenol red in phenol red glucose broth? …………………………………………………………………………… …………………………………………………………………………… ……………………………………………………………………………

7.3 Detection of Campylobacter

The genus Campylobacter includes gram negative bacteria that exhibit rod shaped morphology and darting motility. Campylobacter species contaminating foods include C. jejuni, C. coli and C. lari. They are the most frequently identified agents of acute infective diarrheoa in developing countries. Detection of these bacteria starts with the enrichment at lower incubation temperatures followed by isolation on selective agars and identification by biochemical and physiological tests.

A. REQUIREMENTS Boulton broth, sodium hydroxide, Abeyta-Hunt-Bark (AHB) agar, hydrogen peroxide, oxidase reagent, carbol fuchsin, hippurate reagent, ninhydrin reagent, triple sugar iron (TSI) agar, 0.1% peptone water, nalidixic 28

Detection of acid, cephalothin, MacConkey Agar, glycine, sodium chloride, cysteine medium, nitrate broth,Pathogens reagent in Foods A, reagent B. B. METHODS OF DETECTION 7.3.1 Procedure 1. Weigh 25 g of sample in a sterile bag and add 100 ml Boulton broth to it. Shake it gently for 5 min. 2. Filter the contents of bag through a sterile cheese cloth in a sterile bag or flask. Adjust the pH of broth obtained after rinsing to 7.4 by 2N NaOH. 3. Keep the bag for pre-enrichment at 37oC for 4h under micro-aerophilic conditions (Campy gas generating system). 4. Further incubate the samples for enrichment, at 42oC for 24-48 h, using Campy gas envelope. 5. For isolation streak the enriched broth on AHB agar and incubate at 42oC for 24-48 h, anaerobically. Observe for typical colonies. Campylobacter produce thick translucent white growth to spreading, film-like transparent growth. 6. Check for motility as described in section 3.5.2. Cells picked from agar often demonstrate only "wiggly" motility, whereas those from broth swim rapidly in corkscrew motion. 7. If organism appear typical then re-streak on AHB agar without antibiotics and incubate at 42oC for 24-48 h. 8. Perform catalase test as described below (Campylobacter are positive for catalase): The enzyme catalase acts upon hydrogen peroxide and converts it to water and molecular oxygen. Hydrogen peroxide is toxic to bacterial cells and catalase serves as a mechanism for removal of this toxic compound.

2H2O2 2H2O + O2

(i) Take a drop of H2O2 on a clean dry slide.

(ii) Transfer growth from the TSA slant to the drop of H2O2. (iii) Observe the slide for the production of gas bubbles. (iv) Camylobacter is positive for catalase test. 9. Perform oxidase test by rubbing a loopful of culture on the surface of a filter paper dampened with oxidase reagent. The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. Cytochrome oxidase participates in the electron transport chain by transferring electrons from a donor molecule to oxygen. In this test, an artificial final electron acceptor (N, N, N’, N’-tetra-methyl phenylene-di-amine di-hydrochloride) is used in the place of oxygen. This acceptor is a chemical that changes color to a dark blue/purple when it takes the electron from the last element (cytochrome oxidase) in the electron transport chain. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds. All Campylobacter are positive for oxidase. 10. Carry out gram staining as described in the section 1.5.3 but with a variation. Use 0.5% carbol fuchsin as counter stain. Campylobacter are Gram negative. 11. Perform hippurate test by emulsifying a loopful of culture to 0.4 ml hippurate solution in a test tube. Incubate for 2 h at 37oC and add 0.2 ml of ninhydrin reagent. Agitate the tubes and re-incubate for 10 min. Appearance of violet colour is considered positive reaction. The test is positive only for C. jejuni. 12. Inoculate the TSI slants by stab and streak procedure. Incubate for 5 days at 37oC under micro-aerobic conditions. All Campylobacter spp. produce alkaline slant and alkaline butt. C. jejuni do not produce H2S but strains belonging to C. coli and C. lari may produce H2S. Blackening of the slant indicates H2S production. 13. Inoculate oxidation-fermentation media for glucose utilization and incubate at 37oC for 4 days under micro- aerobic conditions. Observe for colour change. Campylobacter species do not utilize glucose. 7.3.2 Biochemical tests Emulsify a colony into 5 ml of 0.1% peptone water. Use this as inocula for performing other tests such as: (i) Antibiotic sensitivity: Swab the surface of AHB agar and place antibiotic disks of nalidixic acid and cephalothin. Incubate micro-aerobically at 37 oC for 24-48 h. Observe for inhibition zones. Presence of these zones indicates the sensitivity of the culture towards the above mentioned antibiotics. (ii) Growth temperature tolerance: Streak a line of culture on the surface of 3 AHB agar plates. Four cultures may be streaked on a single plate followed by incubation of these plates at different growth temperatures (25oC, 35- 37oC and 42oC) for 3 days under micro-aerobic conditions. More growth than the initial inoculum is considered as positive test. (iii) Growth on MacConkey agar: Perform this test as described in the step above, but incubate only at 37oC for 3 days. (iv) Growth in modified semi-solid media: Inoculate surfaces of the glycine, sodium chloride, cysteine and nitrate media with 0.1 ml diluted culture. Incubate all these tubes at 35-37 oC under micro-aerobic conditions for 3 days except nitrate media which is to be incubated for 5 days. Observe for growth after the end of incubation period. Growth will be in a narrow band pattern just under the surface. • 1% glycine. Record ± growth. • 3.5% NaCl. Record ± growth.

• H2S from cysteine. Inoculate cysteine medium and hang a lead acetate strip from top, keeping cap loose. Do not let strip touch medium. Blackening of strip, even slightly, is positive reaction. • Nitrate reduction. After 5 days, add nitrate reagents (Sulfanilic acid) A and (Napthylamine) B. Red colour is positive reaction. Match the results of the above tests with the standard reactions exhibited by Campylobacter spp. as shown in Table 3.2 and identify the isolated bacteria.

Campylobacter Extraction: Sterile Boulton broth pH 7.4

Enrichment: In micro-aerophilic conditions Culturing: AHB agar/ 42oC (Positive: thick white growth to spreading film like transparent growth)

Others: Motility Catalase test positive Oxidase positive Gram negative Hippurate test: positive violet colour TSI slant: no H2S; alkaline slant Oxidation fermentation: under microaerophilic conditions (no glucose utilization) McConkey agar (37oC): positive

Check Your Progress Exercise 2 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) The nitrate test includes reagent A, B and C, what does it represent and how these reagents are involved to interpret the result? ………………………………………………………………………… ………………………………………………………………………… …………………………………………………………………………. 2) Is Campylobacter positive for catalase and oxidase test? What is the principle of catalase test? ………………………………………………………………………… ………………………………………………………………………… 29

…………………………………………………………………………. 3) What is the name of oxidase reagent and how does it help to interpret the result in oxidase test? ………………………………………………………………………… ………………………………………………………………………… …………………………………………………………………………. 4) Describe the identification of C. jejuni by hippurate test? ………………………………………………………………………… ………………………………………………………………………… ………………………………………………………………………….

5) How H2S will be identified in TSI slants? ………………………………………………………………………… ………………………………………………………………………… ………………………………………………………………………….

Fig 7.3: The Gram stain reactions of common microbes. E. coli, Gram negative (A), Staphylococcus epidermidis, Gram positive (B) and Bacillus cereus, Gram positive 7.4 DETECTION OF ESCHERICHIA COLI AND COLIFORMS E. coli is a member of family Enterobacteriaceae that also includes other bacterial genera such as Salmonella, Shigella and Yersinia. Although most of the strains of E. coli are non-pathogenic they may act as opportunistic pathogens and cause disease. Pathogenic strains of E. coli including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC) are also known. E. coli has also found its use as the indicator of fecal contamination of food and water. It is found in the human and animal gastro-intestinal tract and is easily identified due to its capacity to ferment lactose. However, the other genera such as Klebsiella, Citrobacter and Enterobacter are also known to ferment lactose and are difficult to distinguish from E. coli based on their phenotypic characteristics, all these genera are categorized as ‘coliforms’, which included the Gram-negative, facultative anaerobic rod-shaped bacteria that ferment lactose to produce acid and gas within 48 h at 35°C. The problem of E. coli as indicator organisms resurfaces as the other members of Coliform group are found in environments other than the gastro-intestinal tract, so by testing the lactose fermentation one cannot be sure of the contamination by E. coli or fecaes. This leads to the emergence of other related group called ‘fecal coliforms’. These included all the coliforms that ferment lactose to produce acid and gas at elevated temperatures of incubation and hence are also referred as thermo-tolerant coliforms. These coliforms include E. coli as a major group but Klebsiella is also known to ferment lactose in these conditions. Their detection is carried by lactose fermentation tests at temperatures of around 45oC. The tests for coliforms and E. coli are based on the same principle but have several variations depending upon the type of the food in which the detection has to be carried out or the format of the test. There are several formats of these tests as discussed in the coming sections. A. REQUIREMENTS Butterfield's phosphate-buffered water, lauryl sulphate tryptose (LST) broth, brilliant green lactose bile (BGLB) broth, EC broth, levine-eosine methylene blue (L-EMB) agar, plate count agar (PCA), tryptone broth, MR-VP broth, Kovacs reagent, -naphthol, potassium hydroxide, Koser’s citrate broth, creatine, 4-methylumbelliferyl -D- glucuronide (MUG), M-endo medium, LES endo agar, ColiComplete (CC) discs, Universal pre-enrichment broth (UPEB) B. METHODS OF DETECTION 7.4.1 Most Probable Number (MPN) method a) Presumptive test for detection of coliforms, fecal coliforms and E. coli 1. Weigh 50 g of sample in a sterile jar and add 450 ml of Butterfield's phosphate-buffered water. Blend it for 2 min. This gives a 10-1 dilution. 2. Prepare decimal dilutions from first dilution. 3. Mix the dilutions by shaking the bottles or by vortexing. 4. Transfer 1ml of dilution to 3 tubes of LST broth each for 3 consecutive dilutions. Transfer 1 ml dilution to 5 LST tubes if analyzing shellfish and shellfish harvest waters. 5. Incubate all tubes at 35oC for 48 h and look for gas production after 24 h and 48 h of incubation. 6. Use tubes showing positive reaction for gas production for MPN confirmed tests. b) Confirmed test for coliforms Inoculate a loopful of inoculum from positive (Gassing) LST tubes to BGLB broth and incubate them at 35oC for 48 h. Examine the tubes for gas production and calculate MPN of coliforms based on proportion of confirmed gassing LST tubes for 3 consecutive dilutions. c) Confirmed test for E. coli and fecal coliforms Inoculate a loopful from the positive LST tubes (presumptive test) to EC broth. Incubate these tubes at 45.5oC (44.5oC if analyzing shellfish) for 48 h. Observe for gas production and calculate MPN for fecal coliforms. d) MPN – Completed test for E. coli 1. Streak a loopful of culture from EC tube (positive for gas) on L-EMB agar and incubate the plates at 35oC for 24 h. 2. Observe for dark centered flat colonies with or without green metallic sheen. Only gram-negative bacteria grow on EMB agar as Gram-positive bacteria are inhibited by the dyes eosin and methylene blue added to the agar. Based on its rate of lactose fermentation, Escherichia coli produces dark, blue-black colonies with a metallic green sheen on EMB agar. The metallic sheen is due to the large amounts of acid the E. coli produces on the EMB agar. The acid causes an amide bond to form between the two dyes (eosin and methylene blue), giving rise to a sheen. 3. Transfer the suspected colonies (5 colonies) to plate count agar (PCA) slants and incubate at 35oC for 24 h. 4. Use the growth obtained on PCA for further tests such as gram staining. 5. Select gram negative and bacilli shape cultures for further testing. Confirm the reaction (gas) in LST tube from the PCA cultures. 6. Indole test: This test is positive for bacteria producing tryptophanase that leads to formation of indole from tryptophan. Indole can be detected by reaction with Kovac's reagent (para-methylaminobenzaldehyde in alcohol) to produce a red colour. Inoculate tryptone brothfrom suspected colonies and incubate at 35oC for 24 h. Add 0.3 ml Kovac’s reagent and consider the appearance of red color in upper layer as positive. 30

Detection of 7. Methyl red test: This test determines the capability of the bacterial culture to utilize glucosePathogens and in Foods produce acidic end products. Inoculate MR-VP broth with suspected culture and incubate at 35oC for 48 h. Add a few drops of methyl red indicator to this culture. Consider the appearance of red color as positive reaction. 8. Voges-Proskauer (VP test): This test determines the capability of bacterial culture to produce neutral end products by fermenting glucose. Inoculate MR-VP broth with suspected culture and incubate at 35oC for 48 h. Add 0.6 ml -naphthol solution and 0.2 ml 40% KOH. Shake the tube vigorously and then add a few crystals of creatine. Shake and let stand for 2 h. Consider the development of deep rose color as positive VP test. 9. Citrate Test: This test is used to detect citrate utilization by the bacterial culture. Inoculate Koser’s citrate broth and incubate at 35oC for 96 h. Consider the development of turbidity as positive reaction for citrate utilization.

Fig 7.4: VP and Citrate test Interpretation All gram negative rods that ferment lactose with gas production and give typical IMViC reaction as ++-- and -+-- are categorized as E. coli biotype 1 and biotype 2, respectively. Commercially available kits may also be used to perform these biochemical tests. 7.4.2 LST-MUG method for E. coli detection This method for detection of E. coli is based on the detection of enzyme β glucuronidase (GUD) that cleaves the substrate 4-methylumbelliferyl -D-glucuronide (MUG), to release 4-methylumbelliferone (MU). On exposure to 365 nm UV, MU produces bluish fluorescence. This substrate can be added to any media and fluorescence observed due to the production of GUD, which indicates the presence of E. coli. However, some other bacteria such as Shigella and Salmonella produce GUD and E. coli strain O157:H7 do not produce it. A. PROCEDURE 1. Inoculate LST-MUG broth as done in the MPN-presumptive test. 2. Incubate it at 35oC for 48 h. 3. Observe for bluish fluorescence under UV exposure. Compare with the proper negative and positive controls. 4. Streak L-EMB plates from the tubes giving positive reaction and continue with the confirmed and complete tests as indicated in the above sections. A.1. Analysis of bottled water by membrane filter method for Coliforms 1. Filter 100 ml of water sample. 2. Place the filter on the surface of M-Endo medium or LES Endo agar. 3. Incubate the plates at 35oC for 24 h. 4. Observe for the presence of typical colonies that are pink to dark red in color with a green metallic surface sheen. 5. Count the typical colonies. If they are in range of 5-10 in numbers inoculate all the colonies in LST medium and incubate at 35oC for 48 h. If the colony number is more than 10 randomly pick 10 colonies exhibiting the typical morphology. 6. Look for gas production in LST tubes and subculture from the tubes that are gas positive to BGLB. Incubate the tubes at 35oC for 48 h and again observe for gas production. 7. Gas evolution in BGLB gives a completed test. Report the result as number of coliform colonies per 100 ml of sample. 7.4.3 Analysis of E. coli in Citrus juices Detection of E. coli in citrus juices cannot be done by standard methods because the pH of these juices is acidic and interferes with the standard tests. The method for detection of E. coli in citrus juices is based on the same principles of lactose fermentation, glucuronidase and β-galactosidase, however it involves an additional step of pre- enrichment. A. Procedure 1. Add 10 ml juice to 90 ml of UPEB and incubate at 35oC for 24 h. 2. Transfer 1 ml of enriched sample to 9 ml of EC broth containing ColiComplete (CC) discs. Incubate these tubes at 44.5oC for 24h. 3. Observe the tubes in long wave length UV light. Appearance of bluish fluorescence indicates the presence of E. coli. Appearance of blue colour on the surface of discs also indicates the presence of coliforms.

Fig. 5.5: Example reactions of typical strains in fermentation broth. If a microbe does not ferment the test sugar, the indicator dye remains purple and no gas is produced (left). If fermentation does take place, acid is most often produced, lowering the pH and changing the color of the broth to yellow (middle). Some microbes produce hydrogen gas during fermentation and this will be trapped inside the inverted tube, called a Durham tube, and is observed as a bubble (right). Check your Progress: Exercise 3 Note: a) Use the space below for your answer. b) Compare your answers with those provided at the end of this unit. 1) Which color layer is formed in VP broth during VP test? ………………………………………………………………………… ………………………………………………………………………… 31

…………………………………………………………………………. 2) Which coloured colonies of E.coli are formed on EMB plate and what is the reason behind it? ………………………………………………………………………… ………………………………………………………………………… ………………………………………………………………………….

3) Which chemical represents the Kovac’s reagent? ………………………………………………………………………… ………………………………………………………………………… …………………………………………………………………………. 4) Write the name of indicator in MR test and how does it help to interpret the result? ………………………………………………………………………… ………………………………………………………………………… …………………………………………………………………………. 5) Which broth is used in citrate test? ………………………………………………………………………… ………………………………………………………………………… …………………………………………………………………………. 6) What is the principle of LST-MUG method for the detection of E. coli? ………………………………………………………………………… ………………………………………………………………………… ………………………………………………………………………….

7.5 Listeria monocytogenes Listeria (named after J. Lister) monocytogenes is a gram positive flagellated bacterium, which is a dangerous pathogen. Its infection may lead to septicemia, meningitis, encephalitis and intrauterine or cervical complications in women. Its common habitats include intestinal tracts of domestic and wild animals, fecal matter, soil, silage, sewage, house flies, ticks and human carriers and has been detected in raw milk, supposedly pasteurized fluid milk, cheeses (particularly, soft-ripened varieties), ice cream, raw vegetables, raw and cooked poultry, raw meats, raw and smoked fish. It produces haemolysin (haemolysis) and an endotoxin which causes meningoencephalitis. Listeria is killed in 3 min by boiling and in 20 minutes at a temperature of 70o C. This organism can remain viable for 7 years in dry state. Besides being able to survive high temperature and desiccation, it is also able to grow at refrigeration temperatures and fewer than thousand bacterial cells can cause disease in a susceptible person. Detection of Listeria in food samples is carried after enrichment and if the sample is positive then enumeration is carried out. A. REQUIREMENTS Buffered Listeria enrichment broth (BLEB), primaricin, Oxford agar (OXA), trypticase soy agar with yeast extract (TSAye), 0.85% saline, hydrogen peroxide, sheep blood agar (5%), nitrate broth, reagent A, reagent B, purple carbohydrate broth, dextrose, esculin, maltose, rhamnose, mannitol, xylose, Difco fluorescent antibody (FA) buffer, Tryptose broth. B. METHOD OF DETECTION 7.5.1 Plate count 1. Weigh 25 g of food sample in a sterilized jar. Add 225 ml of BLEB. 2. Blend the sample at high speeds and incubate at 30oC for 4 h. 3. Add primaricin (natamycin) at a concentration of 25 mg/l and further incubate at 30oC for 48 hours. 4. Streak this sample on OXA and incubate these plates at 35oC for 24 – 48 h. Typical Listeria colonies will be black in colour surrounded by black halo. 5. Pick 5 or more typical colonies and streak on Trypticase soy agar with yeast extract (TSAye) and incubate at 30oC. 6. Pick a typical colony from TSAye medium and prepare a thick emulsion in 0.85% saline. Put a drop of this emulsion on a coverslip and flip it on a concavity slide. Observe this preparation under phase contrast microscope and look for motile cells. Listeria spp. are thin, short rods with slight rotating or tumbling motility. 7.5.2 Biochemical tests 7. Test for catalase (Section 3.2.2.2). Listeria are positive for catalase test. 8. Carry out Grams staining of the suspected colonies. Listeria are Gram positive rods and may appear as stack of cells (palisade arrangement) in thick smears. 9. Inoculate isolates on sheep blood agar (5%) by stabbing the medium. Stab as near as possible without touching the bottom of plate. Incubate at 35oC for 24-48 h. Examine the plates with bright light and observe for haemolysis. L. monocytogenes and L. seeligeri produce a slightly cleared zone around the stab. L. innocua shows no zone of haemolysis, whereas L. ivanovii produces a well-defined clear zone around the stab. 10. Carry out nitrate reduction test for the isolates as described in section 3.2.1.2.3. Only L. grayi ssp. murrayi reduces nitrates. 11. Inoculate SIM or MTM from TSBye. Incubate for 7 days at room temperature. Observe daily. Listeria spp. are motile, giving a typical umbrella-like growth pattern. 12. Test the carbohydrate utilization pattern of the isolates by inoculating purple carbohydrate broth (having 0.5% dextrose, esculin, maltose, rhamnose, mannitol, and xylose). Incubate the tubes at 35oC for 7 days. Consider the appearance of yellow colour as a positive test. Compare the carbohydrate utilization profile with that of standard cultures as given in Table 3.3. 13. Perform Christie-Atkins-Munch-Peterson (CAMP) test. This test detects the enhanced haemolysis by Listeria sp. in the presence of S. aureus and Rhodococcus equi Streak weakly haemolytic S. aureus (ATCC 49444) and R. equi (ATCC 6939) on sheep blood agar . Streak the suspected cultures of Listeria in parallel lines between the two vertical streaks of S. aureus and R. equi Incubate the plates at 35oC for 48 h. Observe after 24 and 48 h. Haemolysis of L. monocytogenes and L. seeligeri is enhanced near the S. aureus streak; L. ivanovii haemolysis is enhanced near the R. equi streak. Other species are non-haemolytic and do not react in this test. 14. Characterize the Listeria isolates into type 1, type 4 or not type 1 or type 4. Inoculate tryptose broth and incubate at 35oC for 24 h. Inoculate tryptose agar slants (2 per culture) from this culture and incubate at 35oC for 24 h. Wash both slants in a total of 3 ml Difco fluorescent antibody (FA) buffer and transfer to a sterile 16 x 125-mm screw-cap tube. Heat in a water bath at 80°C for 1 h. Sediment cells by centrifugation at 1600 g for 30 min. Remove 2.2-2.3 ml of supernatant fluid and resuspend the pellet in the remainder of buffer. Follow manufacturer's recommendations for sera dilution and agglutination procedure. This is the serological characterization and is important for epidemiological considerations. More than 90% isolates can be typed using commercially available sera. 15. Listeria can also be detected by commercially available kits (Table 7.4 A & B).

Detection of Pathogens in Foods

Fig 7.6: CAMP test for Listeria monocytogenes: Inoculation pattern of the sheep blood agar plate. Horizontal lines represent streak inoculations of 5 test strains. Vertical lines represent streak inoculations of Staphylococcus aureus (S) and Rhodococcus equi (R). Hatched lines indicate (diagrammatically only) the locations of hemolysis enhancement regions 7.6 Salmonella

Salmonella include Gram negative bacilli that are usually found in poultry, eggs, unprocessed milk, meat and water. These bacteria have been implicated in several food poisoning instances. Members of genus Salmonella are motile and possess two antigens that play an important role in the identification of these bacteria. These antigens are flagellar H antigen and somatic O antigen. A. REQUIREMENTS Selenite cystine (SC) broth, tetrathionate (TT) broth, Rappaport-Vassiliadis (RV), bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, Hektoen enteric (HE) agar, triple sugar Iron (TSI) agar, lysine iron (LI) agar, urea broth, physiological saline, Spicer-Edwards flagellar (H) antisera, phenol red dulcitol broth, tryptone broth, polyvalent somatic (O) antisera, MR-VP broth, Koser citrate broth, -naphthol, Potassium hydroxide B. METHODS OF DETECTION 7.6.1 Plate Count 1. Sample preparation is carried out by dissolving a representative quantity of food product in the medium for pre-enrichment/enrichment purposes. The exact procedure differs for the different kinds of foods, but overall it involves adding 25 g of food product to the enrichment broth so as to have a ratio of 1:9. This step is followed by the homogenization of food sample and incubation at 35oC for 24 h. The enrichment medium and its components depend on the type of food from which the isolation of Salmonella is desired (Table 3.5). Not only the media for enrichment varies but the procedure for sample preparation also varies e.g. cellulose is added during the sample preparation of guar gum while for other foods surfactants such as triton X100 may be added. 2. Gently shake the samples after incubation. 3. Add 1ml of prepared sample to SC broth (10ml) and TT broth (10 ml). This step is to be taken for foods where the presence of S. typhi is suspected. 4. For all other foods add 0.1 ml of sample to RV medium (10 ml) and 1 ml to TT broth (10 ml). 5. Incubate the tubes of RV medium at 42oC for 24 h and tubes of TT broth at 43oC. This step is to be taken for foods with high microbial load. 6. For foods with low microbial load incubate RV tubes as in step 5 while incubate TT at 35oC for 24 h. 7. For guar gum and foods suspected for the presence of S. typhi incubate the SC and the TT tubes at 35oC for 24 h. 8. Vortex the tubes and streak 10 μl of the culture from all the tubes on BS agar, XLD agar and HE agar. Incubate the plates at 35oC for 24 h. 9. Examine the plates for the colonies that may be Salmonella i.e., yellow colonies with or without black centers (Table 3.6). 7.6.2 Biochemical tests 10. Pick at least two suspected Salmonella colonies from each agar and carry out Triple sugar iron (TSI) agar and Lysine iron (LI) agar tests. 11. Inoculate the TSI slant by stab and streak method. Without heating the loop stab the butt twice and streak the slant of LI agar. 12. Incubate the tubes at 35oC for 24 h. 13. Examine the results obtained in the TSI and LI tests. Salmonella produces an acidic butt (yellow) and alkaline slant (red). The blackening of butt may or may not occur depending on the H2S production. In LI test Salmonella produce an alkaline butt (purple) with H2S production. All the cultures giving alkaline reaction (purple butt) for LI agar are used for confirmation. Cultures giving an acidic reaction (yellow butt) in LI agar but an acidic butt and alkaline slant in TSI are also used. 14. Examine a minimum of 6 TSI cultures for 25 g of sample, for identification of Salmonella. 15. Inoculate urea broth with the presumptive Salmonella isolates and incubate the tubes at 35oC for 24 h. Keep an uninoculated tube as a control. This broth contains the pH indicator phenol red. The phenol red is peach color at a pH of 6.8 and turns a hot pink at a pH of 8.4. Discard the cultures if the colour of medium changes to purple red indicating a positive test for urease production because Salmonella is known to be negative for urease test. 16. Carry out the serological polyvalent flagellar (H) test with the urease negative cultures. Inoculate BHI broth with the cultures from TSI slant and incubate at 35oC until visible growth occurs. Add 2.5 ml of formalinized physiological saline solution to 5 ml of BHI culture. Add 0.5 ml of polyvalent flagellar (H) antisera to 10X75 mm serological test tube. To this add 0.5 ml of formalinized broth. Prepare control by adding 0.5 ml of formalinized physiological saline to the formalinized antigen. Incubate the tubes at 48 – 50oC for 1 hr. Observe for agglutination. The test is positive if agglutination is observed and negative if there is no agglutination. In some cases the test may be nonspecific where agglutination is observed both in the test mixture and in control. Repeat this test with Spicer-Edwards flagellar (H) antisera for the cultures giving a negative result. 17. Perform the additional biochemical tests on the urease negative isolates by inoculating them in Phenol red dulcitol broth in Durhams tube. Development of yellow color and gas production indicates positive reaction. Salmonella is positive for this test. 18. Inoculate the growth from TSI slants into tryptone broth tube and incubate at 35oC for 24 h. Further inoculate potassium cyanide broth and malonate broth from the tube containing tryptone broth. Keep these tubes at 35oC for 48 h. Observe for growth in potassium cyanide broth. Salmonella is not able to grow in this broth. Salmonella also give a negative test for malonate which is indicated by the green or unchanged color of the broth. 19. Transfer 5 ml of tryptone broth culture to an empty test tube and add 0.3 ml of Kovacs reagent. Development of deep red color is positive test. Salmonella is negative for this test. 20. Perform the polyvalent somatic (O) test for Salmonella by preparing an emulsion of growth obtained from TSI slant in 2 ml of 0.85% saline. Take a drop of this suspension on a clean slide and add a drop of polyvalent somatic (O) antisera. Mix them and tilt the slide back and forth for 1 min. For control add a drop of saline to the drop of culture suspension and mix as described above. Observe for agglutination. If agglutination is positive for test and negative for culture the result is taken as positive for Salmonella. If both are negative the test is also negative for Salmonella and if agglutination is positive then the test is nonspecific. 33

21. Perform additional biochemical tests including lactose fermentation, sucrose fermentation, MR-VP and citrate utilization. 22. Compare the results obtained in the above biochemical and serological tests with the standard reactions exhibited by Salmonella as shown in Table 3.7. Biochemical tests can also be carried out using commercially available kits such as API 20E, Enterotube II, Enterobacteriaceae II, MICRO-ID, or Vitek GNI for presumptive identification of food borne Salmonella.

Fig 7.7: Antibody reactions of salmonella. Note the grainy appearance of the positive reaction

Salmonella Extraction: Lactose broth

Enrichment: SC broth (in case S. typhi is suspected) or RV broth (42oC) Tetrathionate broth

Culturing: BS agar XLD agar HE agar TSI slant and stab [Acidic butt (yellow) and alkaline slant (red)] Lysine iron slant [Acidic butt and H2S production (black colour)] Urea broth (Salmonella if present yields no purple colour) Others: Serological polyvalent flegellar (H) test BHI broth inoculated from TSI + slant TSI – slant: Spicer-Edwards flagellar antisera Phenol red dulcitol (positive: yellow) Tryptone broth KCN broth Malonate broth Kovac’s reagent Polyvalent somatic (O) test Biochemical tests: Lactose fermentation Sucrose fermentation MRVP and Citrate utilization

7.8 Staphylococcus aureus

S. aureus is spherical shaped, facultative anaerobe, non spore forming, coagulase positive, gram positive bacterium. Some strains are capable of producing a highly heat-stable protein toxin that causes illness in humans. Staphylococcus aureus is responsible for pyogenic infections. It is carried in the nasopharynx region of 50-75% of healthy individuals. S. aureus is known to produce several toxins including haemolysins, fibrinolysin, coagulase, leucocidin, hyaluronidase, DNAase, epidermolytic toxins, enterotoxins and toxin shock syndrome toxin (TSST-1). These toxins play an important role in the pathogenesis of these bacteria. S. aureus have been identified as the causative agent in various food poisoning outbreaks and their count of the magnitude 102 cfu/g or more in the ready to eat foods deems them unsatisfactory for consumption. Staphylococcal food poisoning (staphyloenterotoxicosis; staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S. aureus produce. Staphylococcus aureus is a common cause of mastitis in dairy cattle and are frequent contaminants of raw milk and are found in dairy products. They are present in nasopharyngeal cavity and on skin and there are chances of contamination with staphylococci every time the food is handled. They cause staphylococcal food poisoning. The presence of these bacteria in the foods points towards the poor sanitary conditions of the processing plant. As these bacteria are readily inactivated by heat and sanitizer treatments, their presence indicates post processing contamination of the food product. Pasteurisation, low temperature storage, proper hygiene and sanitation are some of the control measures. A. REQUIREMENTS Baird Parker medium, trypticase soy agar (TSA), brain heart infusion (BHI) broth, carbohydrate utilization medium, rabbit plasma with EDTA, toluidine blue DNA agar, 0.02 M phosphate-saline buffer with 1% NaCl, Butterfield's phosphate-buffered dilution water, sterile paraffin oil, Lysostaphin, hydrogen peroxide.

B. METHOD OF DETECTION 7.8.1 Isolation, identification and enumeration of S. aureus Isolation of S. aureus from food products is carried out by direct plate count method as follows: 1. Weigh 50 g of food sample in a sterile jar and add 450 ml of Butterfield's phosphate-buffered dilution water to it. Blend it at high speed. This is 10-1 dilution of the food sample. 2. Further prepare the decimal dilutions by adding 10 ml of previous dilution to 90 ml of sterile diluent. Mix the dilutions properly. 3. Transfer 1ml of each dilution aseptically on 3 plates of Baird Parker agar. 1 ml of inoculum should be equitably distributed on three plates. 4. Spread the inoculum evenly on the surface of the media by sterile glass spreader. 5. Incubate these plates at 35oC for 45-48 h in inverted position. 6. Consider the plates having typical S. aureus colonies (circular, smooth, convex, moist, 2-3 mm in diameter, gray to jet-black, frequently with light-coloured (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency (when touched with inoculating needle). Some other bacteria may also form colonies similar to that of S. aureus but these colonies lack opaque and clear zones as formed by S. aureus colonies. Rough colonies with dry texture may also be produced by S. aureus in certain cases. Plates having typical S. aureus colonies and counting in the range of 20 – 200 colonies (including other bacterial colonies) are used for determining cfu, however, when typical colonies are present at lower dilutions only, then plates with > 200 colonies may be used. In this case, colonies showing typical morphology of S. aureus are counted for determining cfu. Select >1 colony of each counted colony type and test for coagulase production. 7. Count all the colonies of particular types that give positive coagulase test. Add all such colonies obtained on triplicate plates and multiply with the sample dilution factor to report the cfu of S. aureus / g of sample.

Detection of Pathogens in Foods

Fig 7.8: Staphylococcus aureus on Baird parker agar 7.8.2 Coagulase Test 1. Transfer the suspect colonies to narrow tubes containing 0.3 ml of BHI broth and emulsify. Inoculate TSA medium with a loopful of BHI suspension and incubate them (BHI tubes and TSA slants) at 35oC for 24 h. 2. Add 0.5 ml of reconstituted plasma with EDTA to BHI suspension and incubate at 35oC for 6h. Examine periodically for clot formation during the incubation period. 3. Consider the tubes exhibiting firm clot as positive for S. aureus. 4. Keep the positive and negative controls for the coagulase test. 5. Carry out gram staining for all the suspect cultures and observe microscopically for presence of gram positive cocci in bunches.

Staphylococcus aureus Extraction: Sterile Butterfields phosphate buffer Direct plate count: Baird Parker agar [circular, smooth, convex, moist, 2-3 mm in diameter, gray to jet-black, frequently with light-coloured (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency (when touched with inoculating needle] Coagulase test: positive (Incubation on TSA medium in presence of BHI broth + Plasma with EDTA) Gram staining: positive; cocci in bunches Supportive tests: Thermostable nuclease test Catalase test Lysostaphin sensitivity Anaerobic utilization of glucose and mannitol (positive: yellow colour)

7.8.3 Supportive Tests These tests are also carried out for identification of S. aureus and its differentiation from S. epidermidis and Micrococci. a) Thermostable Nuclease Test S. aureus produces a heat stable DNase that hydrolyzes DNA to oligonucleotides. This test is as specific as coagulase test and is often used as a supportive test for strains giving weak coagulase test (where firm clot is not formed). 1. Prepare micro-slides by adding toluidine blue – deoxyribonucleic acid agar on the surface of microscopic slide. 2. Allow agar to solidify and cut wells (2mm diameter) on its surface. 3. Take the broth culture and heat it for 15 min. in the boiling water bath. 4. Add 10 μl of heated broth sample. 5. Prepare a moist chamber by adding a layer of wet cotton to the Petri plate. 6. Incubate the slide in this chamber at 35oC for 4 h. 7. Appearance of bright pink halo around the wells indicate the positive reaction. b) Catalase test (as per Section 7.3.1.B) c) Lysostaphin sensitivity Lysostaphin is an endopeptidase which cleaves the pentaglycine cross linkages occurring in the Staphylococcus peptidoglycan. Sensitivity of Staphylococcal strains against Lysostaphin can be used to differentiate it from micrococci. 1. Transfer and emulsify an isolated bacterial colony from agar plate to 0.2 ml of phosphate saline buffer. 2. Transfer 0.1 ml of this suspension to another tube. 3. Add 0.1 ml lysostaphin solution (25 μg/ ml of lysostaphin dissolved in 0.02 M phosphate saline buffer containing 1% NaCl) to one tube (test) and 0.1 ml of phosphate saline buffer to second tube (control). 4. Incubate both the tubes at 35oC for 2 h. 5. Test is considered positive if the turbidity clears (due to cell lysis) within 2 h. d) Anaerobic utilization of glucose and mannitol 1. Inoculate the tubes of carbohydrate fermentation medium containing glucose (0.5%). Add a layer of sterile paraffin oil on the surface of the fermentation medium. Incubate the tubes at 37oC for 5 days. 2. Repeat step 1 with media containing 0.5 % mannitol. 3. Observe for appearance of yellow colour throughout the tube and note it as positive test for S. aureus. Biochemical activities of S. aureus and its comparison to S. epidermidis and micrococci are depicted in Table 3.8. 7.9 Clostridium perfringens Clostridium perfringens continues to be a major food safety concern to the food industry. This organism has been implicated as the cause of food-borne illness in roast beef, turkey, meat-containing foods, and other meat dishes. Foods contaminated with large numbers of vegetative cells of Clostridium perfringens can give rise to illness characterised by diarrhoea and abdominal pain which may be accompanied by vomiting. C. perfringens produces spores which can survive normal cooking processes and cause a hazard in meat products that are left at ambient temperatures for a long time after cooking. C. perfringens spores may survive cooking and receive sufficient heat activation to germinate and produce cells that may subsequently multiply in cooked foods if the rate and extent of cooling are not sufficient. C. perfringens generation time is as rapid as 7.4 min in autoclaved ground beef with an optimal growth range of 37 to 45 oC, and growth has been reported at temperatures as low as 68 oC. Other sulphite- reducing clostridia are implicated in food spoilage, especially of poorly processed canned food.

Fig 7.9: Anaerobic culture of Clostridium perfringens on blood agar. The characteristic double zone of clear beta- hemolysis around a colony is clearly seen (arrow) 7.9.1 Cultural Methods for Enumeration and Identification of Clostridium perfringens in Foods Specified volumes of dilutions of the sample are mixed with a tempered molten selective culture medium in a sterile Petridish. An overlay of the same medium is added and the plates are incubated anaerobically at 37oC for 20 h. The number of black colonies is counted and the number of C. perfringens colonies determined following results of confirmation tests. C. perfringens is provisionally identified as a non-motile, Gram-positive bacillus which produces black colonies in TSC agar, reduces nitrates to nitrites, produces acid and gas from lactose, and liquefies gelatin within 48 h. Some strains of C. perfringens exhibit poor sporulation in sporulation medium or weak lecithinase reactions on TSC agar containing egg yolk. For dilution, a 10-1 homogenate is prepared in either peptone saline diluent (PSD) or buffered peptone water (BPW) and further decimal dilutions as required in PSD. Starting with the highest dilution 1 mL of each dilution is transferred to a sterile Petri dish and 15 to 20mL of tryptose sulphite cycloserine agar (TSCA), tempered to 45 oC, is poured into each petri dish. After proper mixing (rotating each dish five times clockwise, anti-clockwise and sideways in each direction), plates are allowed to set and further a 10 mL of TSCA is added as an overlay and allowed to solidify. Plates are incubated under anaerobic conditions in an incubator at 37C for 20 ± 2 h. On completion of incubation, the plates are examined for presence of black colonies and typical colonies are enumerated and recorded. 7.9.2 Confirmatory Tests 5-10 black colonies are transferred to two blood agar (BA) plates. Incubate one plate aerobically and the other anaerobically in an incubator at 37C for 18 - 24 h. Examine the plates for the presence or absence of growth and for purity. Perform confirmatory tests for C. perfringens on colonies that fail to grow aerobically. Anaerobic cultures with a diffuse spreading morphology are considered motile, and therefore confirmatory tests are not carried out. Using pure non spreading cultures from the anaerobic blood agar subculture plates following media are incubated: 1. Motility-nitrate medium Immediately prior to use, heat the medium in boiling water for 15 minutes and then cool rapidly to set. Inoculate by stabbing into the medium and incubate under anaerobic conditions at 37+1 oC for 20 - 24 h. After incubation, examine the medium for growth along the stab-line. Motility is evident as diffuse growth out into the medium away from the stab line.

Fig 7.10: Motlity tests: (a) Positive for B. cereus (b) negative for Staphylococcus epidermidis

Mix equal volumes of nitrite reagent A and B just before use and test for the presence of nitrite by adding 0.2 to 0.5 mL of this mixture to each tube of motility-nitrate medium. The formation of a red colour confirms the reduction of nitrate to nitrite. If a red colour does not develop within 15 minutes add a small amount of zinc dust and allow standing for 10 minutes. If a red colour develops after the addition of zinc dust no reduction of nitrate has taken place. 2. Lactose gelatine medium Immediately prior to use heat the medium in boiling water for 15 minutes and then cool rapidly. Inoculate the medium and incubate anaerobically at 37 oC for 20 - 24 h. Examine the tubes for the presence of gas and for a yellow colour indicating the production of acid. Chill the tubes for 1 hour at 2 to 8oC and check for gelatine liquefaction. If the medium has solidified re-incubate for an additional 24 h and again check for liquefaction of the gelatine. lostridium perfringens xtraction: Peptone saline diluent/ peptone diluent buffer irect plate count: Tryptose Sulphite Cycloserine agar (black colonies) thers: Nitrate reduction: positive Lactose fermentation: with acid and gas Gelatin liquefication: positive onfirmatory tests: Blood agar incubated aerobically and anaerobically (non spreading culture used for further tests) otility-nitrate medium: diffuse growth and red colour (if not initially present add zinc dust and wait for 10 min) actose gelatine medium: (under anaerobic conditions) gas and yellow colouration; Chilling used to check for gelatin liquefication

7.10 VIRAL PATHOGENS Detection of viruses in food is most likely to be undertaken when an outbreak has occurred. It would be helpful, however, if some routine testing methods were available to apply to foods, such as shellfish, that often serve as vehicles for viruses. Outbreak investigation and routine monitoring present very different sets of priorities. When people have already been made ill, the relatively high costs of attempting to detect viruses in food samples may be acceptable, especially if litigation is anticipated. However, as was mentioned earlier, an outbreak of hepatitis A affecting several people is likely to be recognized only 4 weeks or more after the contaminated food was consumed, so pertinent food samples most likely will be unavailable for testing. With other viral diseases that have shorter incubation periods, clinical histories and diagnostic samples of feces and blood serum may afford some indication of which virus is to be sought in the food, which could prove very helpful. In contrast, testing foods for virus contamination in the hope of preventing human illness presents special problems. Costs are likely to outweigh demonstrable benefits. In addition, since almost all foodborne viruses are transmitted by fecal-oral cycles, one virus is as likely as another to occur in food, subject to fecal contamination. As ingenious as the methods are that have been devised for detecting food-borne viruses, none could be considered routine. A fundamental problem is that the viruses of greatest concern, hepatitis A viruses and the Norwalk like gastroenteritis viruses, replicate slowly and in apparently or not at all in laboratory cell cultures. If food-borne viruses cannot be detected on the basis of their infectivity, alternate basis include their morphology (as seen by electron microscopy), their antigenic specificity (as demonstrated by reactions with homologous antibody), their genetic specificity (as demonstrated with complementary probes or polymerase chain reaction [PCR] primers), or combinations of these. These methods may be less sensitive than tests based on infectivity, and by their nature all carry some risk of yielding a positive result with virus that has been inactivated (no longer infectious). The final detection method to be used must be considered when the food sample is being processed for testing. An additional problem 36

Detection of encountered with the hepatitis A virus is that the incubation period of the illness is so longPathogens (average in Foods 4 weeks) that pertinent food samples are unlikely to be available once the disease has been recognized.

Most foods other than milk, water, and a few others require liquefaction as a first processing step. Addition of liquid for this purpose is usually kept to a minimum because virus that is present in food will be diluted, to the detriment of the sensitivity of the method. Solid food samples are shaken, comminuted, or otherwise dispersed in a diluent that has been selected to encourage dissociation of virus from the food solids, which will be removed by centrifugation, filtration, or other means. Additives may be used to encourage separation of the food solids from the liquid suspension containing the virus. Because foods suspected of viral contamination are likely to contain bacteria from both the food and feces, a step to remove or kill bacteria in the suspension is usually included. Because the detection methods available can usually accommodate only very small volumes of sample, as much water as possible is removed from the food sample extract before testing begins. Applicable concentration methods include adsorption-elution, differential precipitation, ultra-centrifugation, and ultra-filtration. METHODS FOR DETECTING VIRUSES EXTRACTED FROM FOODS The detection methods are based on the morphology, antigenic specificity, or genetic specificity of the viral particle. 7.10.1 Electron Microscopy Morphology of viral particles affords an important basis for their classification. However, many viruses not of human origin are indistinguishable by simple electron microscopy from viruses suspected as food contaminants. One way to demonstrate that the viruses seen in the electron micrograph are indeed the suspected type is to attach them to the grid or to each other with homologous antibody (immune electron microscopy.), which is a combination of serologic and morphologic criteria. When the serological type of the virus is unknown, an alternative is to compare immune electron micrographs done with acute and convalescent serum from a patient, assuming that paired samples are available. If the convalescent phase serum reacts with the virus and the acute phase serum does not, this is likely to have been the agent that caused the illness. Unfortunately, the method will not detect the small numbers of viral particles that are capable of causing disease.

7.10.2 Immunoassay Antigenic specificity of the viral coat protein can serve directly as a means of detection by enzyme immunoassay or by radioimmunoassay. These methods have been of considerable use in diagnostic virology, where they are applied to fecal samples containing levels of virus often exceeding a million particles per gram. Because contaminated foods are likely to contain only imperceptible levels of feces, the quantities of virus present in food samples are likely to be much smaller, and immunoassay tests are seldom adequately sensitive. Therefore, serologic reactions between virus and antibody are most often applied in combination with electron microscopy or with nucleic acid-based tests. 7.10.3 PCR Methods Nucleic acid tests are generally based on the specific interactions of portions of the viral genome with complementary probes, PCR primers, or both. PCR and other genetic amplification methods afford highly sensitive means of detecting small quantities of virus. Because almost all known food-borne viruses are RNA agents, the viral nucleic acid must be extracted and reverse-transcribed to complementary DNA before amplification by PCR can begin. Short complementary probes bracketing a selected region of the viral genome are introduced with a polymerase that functions at high temperature, and many successive cycles of replication, denaturation, and annealing are carried out by a programmed thermal cycler. The result is millions of short copies of the selected region of the viral genome. The specificity of these copies is demonstrated on the basis of their appropriate length (in terms of number of nucleotide bases), their reaction with a complementary probe that is directed at a portion of the amplified segment, or both. Probes used for this purpose are labeled with a radionuclide, an enzyme, or some other means of expressing their presence in association with the PCR-amplified product. In addition to the challenges of performing the demanding PCR and probe-specificity tests themselves, testing food samples has been found to present some special problems. Certain food components interfere with reverse transcription or with PCR amplification. 7.10.4 Combined Methods Combined methods consist typically of a serologic method followed by another detection procedure, as with the immune electron microscopy approach described above. Virus has also been captured from sample extracts with homologous antibody for later detection by PCR. This method seems to obviate some of the problems with food inhibitors of reverse transcription and PCR, and it allows RNA to be released from the viral particle simply by heating. It seems that detection methods combining nucleic acid tests and morphologic tests have not been devised. Check Your Progress Exercise 4 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) What coloured colonies of Listeria monocytogenes are formed on oxford agar? ……………………………………………………………………………… ……………………………………………………………………………… 2) What is CAMP test? …………………………………………………………………………… …………………………………………………………………………… ………………………………………………………………………….… 3) How Salmonella will be identified by TSI slant and LF slant test? …………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 4) Which coloured colonies of Salmonella are formed on BSA, XLD, and HE agar? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 5) What is the name of indicator in urea broth? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 6) What coloured colonies of Staphylococcus aureus are forms on Braid Parker medium? ..……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 7) Name the substrate used for coagulation by staphylococcus aureus in coagulase test? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 8) What is the name of the media used in thermostable nuclease test and which indicator is used in this test? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 9) Why we are heating Staph ylococcus aureus sample for 15 min during this TNase test? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 10) What colour zone is formed in TNase test by staphylococcus aureus? ……………………………………………………………………………… 37

……………………………………………………………………………… 11) Is staphylococcus aureus positive for mannitol fermentation? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 12) Is staphylococcus aureus sensitive to lysostaphin? ……………………………………………………………………………… ……………………………………………………………………………… ……………………………………………………………………….… 7.11 LET US SUM UP

Owing to their high nutritive value, foods both raw and processed are ideal medium for growth of microorganisms including pathogens. Pathogens manifest pathogenecity either through invasion of host tissues or by production of toxins. The pathogens found in foods broadly belong to bacteria, fungi and viruses. Bacterial pathogens are the predominant group and include Bacillus cereus, Campylobacter, Escherichia coli, Listeria monocytogenes, Salmonella species, Staphylococcus aureus etc. Most of the pathogens are heat labile and are destroyed by heat treatment. However, most of the toxins produced by them being heat resistant survive processing and can pose public health hazards. Detection of such pathogens is mandatory from public health and regulatory point of view. They have specific requirement for growth media and cultivation and are identified on the basis of their typical morphological and biochemical characteristics. Specific growth media, detection protocol and interpretation of results with respect to above mentioned food pathogens have been discussed in this unit. The information rendered in Table 3.9 can be practically used as a kind of ready reckoner for enumeration and detection of pathogens in different foods. 7.12 KEYWORDS Pathogenicity : The ability to cause disease Virulence : The degree of pathogenicity Exotoxins : Toxins secreted outside the cell Endotoxins : Toxins as part of outer cell wall of bacterial cell Neurotoxins : Toxins which interfere with normal nerve impulses Cytotoxins : Toxins which kill cells Enterotoxins : Toxins infecting the cells lining of Gastrointenstinal tract EEC E. coli : Enterovirulent E. coli that cause gastroenteritis MYP : Agar Mannitol-egg yolk-polymyxin agar Salmonellosis : Gastroenteritis caused by Salmonella spp. Campylobacteriosis : Gastroenteritis caused by Campylobacter spp

Staphyloenterotoxicosis : Staphylococcal food poisoning Staphyloenterotoxemia : Staphylococcal food poisoning 7.13 SUGGESTED FURTHER READING American Public Health Association (APHA). (2001). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. F.P. Downes and K. Ito (Editors), American Public Health Association, Washington, D.C. American Public Health Association (1998). Standard Methods for the Examination of Water and Wastewater, 20th edition, APHA, Washington, DC. AOAC INTERNATIONAL (1995). Official Methods of Analysis, 15th ed., sec. 987.09, AOAC INTERNATIONAL, Arlington, VA. AOAC INTERNATIONAL (1995). Official Methods of Analysis, 16th edition, sec. 975.55, AOAC INTERNATIONAL, Arlington, VA. AOAC INTERNATIONAL (2000). Official Methods of Analysis, 17th edition, Sec. 967.25-967.28, 978.24, 989.12, 991.13, 994.04, and 995.20. AOAC INTERNATIONAL, Gaithersburg, MD. AOAC Official Method 993.09 (2000). Listeria in dairy products, seafoods, and meats. Colorimetric deoxyribonucleic acid hybridization method (GENE-TRAK Listeria Assay). Chapter 17.10.04, pp. 147-150 In: Official Methods of Analysis of AOAC International. 17th edition. W. Horwitz (Editor). Volume 1. Agricultural Chemicals, Contaminants and Drugs. AOAC International, Gaithersburg, MD. AOAC Official Method 994.03 (2000). Listeria monocytogenes in dairy products, seafoods, and meats. Colorimetric monoclonal enzyme-linked immunosorbent assay method (Listeria -Tek). Chapter 17.10.05, pp. 150-152 In: Official Methods of Analysis of AOAC International. 17th edition. W. Horwitz (ed.). Volume 1. Agricultural Chemicals, Contaminants and Drugs. AOAC International, Gaithersburg, MD. AOAC Official Method 995.22 (2000). Listeria in foods. Colorimetric polyclonal enzyme immunoassay screening method (TECRA Listeria Visual Immunoassay [TLVIA]). Chapter 17.10.06, pp. 152-155 In: Official Methods of Analysis of AOAC International. 17th edition. W. Horwitz (Editor). Volume 1. Agricultural Chemicals, Contaminants and Drugs. AOAC International, Gaithersburg, MD. AOAC Official Method 996.14 (2000). Assurance Polyclonal Enzyme Immunoassay Method. Chapter 17.10.07, pp. 155-158 In: Official Methods of Analysis of AOAC International. 17th Edition. W. Horwitz (Editor). Volume 1. Agricultural Chemicals, Contaminants and Drugs. AOAC International, Gaithersburg, MD. AOAC Official Method 997.03 (2000). Visual Immunoprecipitate Assay (VIP). Chapter 17.10.08, pp. 158-160 In: Official Methods of Analysis of AOAC International. 17th edition. W. Horwitz (Editor). Volume 1. Agricultural Chemicals, Contaminants and Drugs. AOAC International, Gaithersburg, MD Bacteriological Analytical Manual Online (www.cfsan.fda.gov) http://www.bact.wisc.edu:

7.14 ANSWERS TO CHECK YOUR PROGRESS EXERCISES"

Check Your Progress Exercise 1 Your answer should include following points: 1) Pink colour surrounded by a precipitate zone indicating the production of lecithinase. 2) The presence of precipitate zone around colonies of B. cereus indicates that lecithinase is produced. B. cereus colonies are usually a pink color which becomes more intense after additional incubation. 3) Polymyxin B is added in MYP agar to suppress the growth of other bacteria able to grow at such a high salt concentration, e.g. Proteus spp. 4) Phenol red is an acid-base indicator. Phenol red changes from red to yellow when the medium has a pH < 6.8 (more acidic) and from red to fuchsia when the medium has a pH >7.4 (less acidic). Check Your Progress Exercise 2 Your answer should include following points: 1) The medium is evaluated for nitrate reduction by the addition of two reagents, Nitrate A Reagent (0.8% sulfanilic acid in 5N acetic acid) and Nitrate B Reagent (0.6% N, N-dimethyl-alpha-naphthylamine in 5N acetic acid), which detect the presence of a catabolic end product, and by the addition of Nitrate C Reagent, zinc dust, which detects the absence of remaining nitrate in the medium. 2) Yes, the enzyme catalase acts upon hydrogen peroxide and converts it to water and molecular oxygen. Hydrogen peroxide is toxic to bacterial cells and catalase serves as a mechanism for removal of this toxic compound. 38

Detection of 2H2O2 2H2O + O2 Pathogens in Foods The production of gas bubbles indicates a positive catalase test. 3) The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. Cytochrome oxidase participates in the electron transport chain by transferring electrons from a donor molecule to oxygen. In this test, an artificial final electron acceptor (N,N,N’,N’-tetramethyl phenylenediamine dihydrochloride) is used in the place of oxygen. This acceptor is a chemical that changes color to a dark blue/purple when it takes the electron from the last element (cytochrome oxidase) in the electron transport chain. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds. 4) Perform hippurate test by emulsifying a loopful of culture to 0.4 ml hippurate solution in a test tube. Incubate for 2 h at 37oC and add 0.2 ml of ninhydrin reagent. Agitate the tubes and re-incubate for 10 min. Appearance of violet colour is considered positive reaction.

5) H2S production is indicated by blackening of slant.

Check Your Progress Exercise 3 Your answer should include following points: 1) The appearance of pink colour indicates positive results for test. 2) Only gram-negative bacteria grow on EMB agar as Gram-positive bacteria are inhibited by the dyes eosin and methylene blue added to the agar. Based on its rate of lactose fermentation, Escherichia coli produces dark, blue-black colonies with a metallic green sheen on EMB agar. The metallic sheen is due to the large amounts of acid the E. coli produces on the EMB agar. The acid causes an amide bond to form between the two dyes (eosin and methylene blue), giving rise to a sheen. 3) Kovac's reagent is para-methylaminobenzaldehyde in alcohol. 4) Methyl Red. It is an acid base indicator. Its colour changes from red (at pH 4.4) yellow at pH > 6.2. 5) Koser’s citrate broth 6) Enzyme β glucuronidase (GUD) cleaves the substrate 4-methylumbelliferyl -D-glucuronide (MUG), to release 4-methylumbelliferone (MU). On exposure to 365 nm UV, MU produces bluish fluorescence. This substrate can be added to any media and fluorescence observed due to the production of GUD, which indicates the presence of E. coli.

Check Your Progress Exercise 4 Your answer should include following points: 1) Typical Listeria colonies will be black in colour surrounded by black halo. 2) This test detects the enhanced haemolysis by Listeria sp. in the presence of S. aureus and Rhodococcus equi. Haemolysis of L. monocytogenes and L. seeligeri is enhanced near the S. aureus streak; L. ivanovii haemolysis is enhanced near the R. equi streak. Other species are non-haemolytic and do not react in this test. 3) Salmonella produces an acidic butt (yellow) and alkaline slant (red). The blackening of butt may or may not occur depending on the H2S production. In LI test Salmonella produce an alkaline butt (purple) with H2S production. All the cultures giving alkaline reaction (purple butt) for LI agar are used for confirmation. Cultures giving an acidic reaction (yellow butt) in LI agar but an acidic butt and alkaline slant in TSI are also used. 4) BSA: Green colonies with little or no darkening of the surrounding medium; XLD: Yellow colonies with or without black centers; HE: Yellow colonies with or without black centers 5) This broth contains the pH indicator phenol red. The phenol red is peach color at a pH of 6.8 and turns a hot pink at a pH of 8.4. 6) circular, smooth, convex, moist, 2-3 mm in diameter, gray to jet-black, frequently with light-coloured (off- white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency. 7) reconstituted plasma with EDTA to BHI suspension 8) The medium used is toluidine blue - DNA agar (TDA). 9) S. aureus produces a heat stable DNase that hydrolyzes DNA to oligonucleotides 10) Appearance of bright pink halo around the wells indicate the positive reaction. 11) yes 12) Yes

able 7.1 Differential characteristics of large-celled group I Bacillus species

Feature B. cereus B. thuringiensis B. mycoides B. anthracis B. megaterium Gram reaction +(a) + + + + Catalase + + + + + Motility +/-(b) +/- -(c) - +/- Reduction of + +/ + + -(d) nitrate Tyrosine + + +/- -(d) +/- decomposed Lysozyme- + + + + - resistant Egg yolk + + + + - reaction Anaerobic + + + + - utilization of glucose VP reaction + + + + - Acid produced - - - - + from mannitol Haemolysis + + + -(d) - (Sheep RBC) Known produces endotoxin rhizoidal pathogenic pathogenicity(e)/ enterotoxins crystals growth to animals characteristic pathogenic to and humans insects a +, 90-100% of strains are positive. b +/-, 50% of strains are positive. c -, 90-100% of strains are negative. d -, Most strains are negative.

Table 7.2 Biochemical tests of Campylobacter spp

Characteristics C. jejuni C. jejuni C. C. C. fetus C. hyo- "C. upsali subsp. coli lari subsp. intestinalis doylei fetus Growth at 25°C - ± - - + D -0 Growth at 35-37°C + + + + + + + Growth at 42°C + ± + + D + + Nitrate reduction + - + + + + + 3.5% NaCl ------H2S, lead acetate strip + + + + + + + (c) H2S, TSI - - D - - + - Catalase + + + + + + - Oxidase + + + + + + + MacConkey's agar + + + + + + - Motility (wet mount) +(81%) + + + + + + Growth in 1% glycine + + + + + + + Glucose utilization ------Hippurate hydrolysis + + - - - - - Resistance to naladixic S(d) S S R R R S acid Resistance to cephalothin R R R R S(e) S S a Symbols: +, 90% or more of strains are positive; -, 90% or more of strains are negative; D, 11-89% of strains are posi resistant; S, susceptible. b Proposed species name. c Small amount of H2S on fresh (<3 days) TSI slants. d Nalidixic acid-resistant C. jejuni have been reported. e Cephalothin-resistant C. fetus subsp. fetus strains have been reported.

Detection of Table 7.3 Differentiation of Listeria species Pathogens in Foods Species Acid produced from - Mannitol Rhamnose Xylose Haemolysisa L. monocytogenes + - + -

L. ivanoviib + - - +

L. innocua - - Vc -

L. welshimeri - - Vc +

L. seeligeri + - - +

L. grayid - + Vc - a Sheep blood agar stab. bRibose fermenting strains are classified as L. ivanovii subsp. ivanovii and ribose non-fermenters as L. ivanovii subsp. londiniensis. c V, variable biotypes d Includes two subspecies - L. grayi subsp. murrayi reduces nitrate; L. grayi subsp. grayi does not reduce nitrate.

Table 7.4 A. Listeria genus detection test kits prescribed for regulatory screening

1. AOAC Official Method 993.09. 2000. Listeria in dairy products, seafoods, and meats. Colorimetric deoxyribonucleic acid hybridization method (GENE-TRAK Listeria Assay). 2. AOAC Official Method 994.03. 2000. Listeria monocytogenes in dairy products, seafoods, and meats. Colorimetric monoclonal enzyme-linked immunosorbent assay method (Listeria Tek). 3. AOAC Official Method 995.22. 2000. Listeria in foods. Colourimetric polyclonal enzyme immunoassay screening method (TECRA Listeria Visual Immunoassay [TLVIA]). 4. AOAC Official Method 996.14. 2000. Assurance (Polyclonal Enzyme Immunoassay Method). 5. AOAC Official Method 997.03. 2000. Visual Immunoprecipitate Assay (VIP). 6. AOAC Official Method 999.06. 2000. Enzyme Linked Immunofluorescent Assay (ELFA) VIDAS LIS Assay Screening Method.

Table 7.4 B. Test kits useful in confirming Listeria isolates as Listeria monocytogenes or not*

1. AccuProbeTM Listeria monocytogenes culture confirmation test (Gen-Probe, Inc, San Diego, CA). 2. GeneTrak Listeria monocytogenes test kit (Neogen, Lansing, MI). 3. Probelia Listeria monocytogenes test kit (BioControl, Seattle, WA). 4. VIDAS Listeria monocytogenes test kit (BioMerieux). 5. Transia Plate Listeria monocytogenes (Diffchamb SA, Lyon, France) 6. FDA, SRL application of Niederhauser et al. method for PCR detection and identification of L. monocytogenes 7. BAX Listeria monocytogenes test. (Qualicon, Inc., Wilmington, DE)

* These kits are in various stages of validation and when suitably validated can also be used to screen enrichments for L. monocytogenes. Presently, FDA only prescribes validated kits that screen for all Listeria species.

Table 7.5 Enrichment of Salmonella Product Media k, dried egg whites, dried whole eggs, liquid Lactose broth lk, 2% fat milk, whole, and buttermilk), sodium net casein, soy flour, egg-containing products rolls, macaroni, spaghetti), cheese, dough, ds (ham, egg, chicken, tuna, turkey), fresh, d fruits and vegetables, nut meats, crustaceans crayfish, langostinos, lobster), and fish, meats, es, meat by-products, animal substances, ducts, and meals (fish, meat, bone), gelatin, guar quid whole eggs(Homogenised) Trypticase soy broth supplemented with ferrous sulfate ggs (chicken, duck, and others), dried yeast Trypticase soy broth active yeast, black pepper, white pepper, celery , chili powder, cumin, paprika, parsley flakes, ame seed, thyme, and vegetable flakes nstant, dry whole milk Brilliant green water orange juice (pasteurized and unpasteurized), Universal pre-enrichment asteurized and unpasteurized), and apple juice broth opping mixes Nutrient broth onion powder, garlic flakes Trypticase soy broth with potassium sulfite ndy coating (including chocolate) Reconstituted non fat dry milk d food coloring substances Tetrathionate broth Buffered peptone water

Detection of Pathogens in Foods Table 7.6 Colony morphology of Salmonella on various selective media Culture media Typical Salmonella Atypical Salmonella colonies colonies Bismuth sulfite (BS) Brown, gray, or black Green colonies with agar colonies; sometimes little or no darkening of they have a metallic the surrounding medium sheen. Surrounding medium is usually brown at first, but may turn black in time with increased incubation. Xylose lysine Pink colonies with or Yellow colonies with or desoxycholate (XLD) without black centers. without black centers agar Colonies may be glossy with black centers or may appear as almost completely black. Hektoen enteric (HE) Blue-green to blue Yellow colonies with or agar colonies with or without without black centers black centers. Colonies may be glossy with black centers or may appear as almost completely black

Table 7.7 Biochemical and serological reactions of Salmonella r substrate Result Salmonella Positive Negative species reaction(a) yellow butt red butt + ecarboxylase purple butt yellow butt +

blackening no blackening + LIA) purple-red colour no color change - ecarboxylase purple colour yellow colour +

ed dulcitol yellow colour and/or no gas; no colour +(b) gas change oth growth no growth - e broth blue colour no colour change -(c) st violet colour at surface yellow colour at surface - ent flagellar test agglutination no agglutination + ent somatic agglutination no agglutination + red lactose yellow colour and/or no gas; no colour -(c) gas change red sucrose yellow colour and/or no gas; no colour - gas change Proskauer pink-to-red colour no colour change -

red test diffuse red colour diffuse yellow colour + ns citrate growth; blue colour no growth; no colour v change more positive in 1 or 2 days; -, 90% or more negative in 1 or 2 days; v, variable. of S. arizonae cultures are negative. of S. arizonae cultures are positive.

Detection of Table 7.8 Typical characteristics of S. aureus, S. epidermidis and micrococci(a) Pathogens in Foods

Characteristic S. aureus S. epidermidis Micrococci Catalase activity + + +

Coagulase production + - - Thermonuclease production + - - Lysostaphin sensitivity + + - Anaerobic utilization of glucose + + - mannitol + - - a +, Most (90% or more) strains are positive; -, most (90% or more) strains are negative.

Table 7.9: Evaluation of microbiological quality of foods with special reference to pathogens Food Microbiological test • Cereal products • Enumeration of micro-organisms at 30ºC • Nuts and nuts products • Enumeration of micro-organisms by spiral plating at 30ºC • Enumeration of Staphylococcus aureus • Enumeration of E. coli at 44ºC • Enumeration of Bacillus cereus • Enumeration of Clostridium perfringens • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species

• Dairy products • Enumeration of micro-organisms at 30ºC • Enumeration of micro-organisms at 37ºC • Enumeration of micro-organisms by spiral plating at 30ºC and 37ºC • Enumeration of Staphylococcus aureus • Enumeration of E. coli at 44ºC • Enumeration of Bacillus cereus • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Detection of thermo-tolerant Campylobacter species and identification of C.jejuni, C.coli • Enumeration of Enterobacteriacae • Enumeration of coliforms at 30ºC • Enumeration of coliforms at 37ºC • Detection and enumeration of Enterobacteriaceae by MPN

• Meat and meat • Enumeration of micro-organisms at 30ºC products • Enumeration of micro-organisms at 37ºC • Poultry and poultry • Enumeration of micro-organisms by spiral products plating at 30ºC and 37ºC • Enumeration of Staphylococcus aureus • Enumeration of E. coli at 44ºC • Enumeration of Bacillus cereus • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Detection of thermotolerant Campylobacter species and identification of C.jejuni, C.coli • Enumeration of Enterobacteriacae • Enumeration of coliforms at 30ºC • Enumeration of coliforms at 37ºC • Detection and enumeration of Enterobacteriaceae by MPN

• Eggs and egg products • Enumeration of microorganisms at 30ºC Detection of Pathogens in Foods • Enumeration of microorganisms by spiral plating at 30ºC and 37ºC • Enumeration of Staphylococcus aureus • Enumeration of E. coli at 44ºC • Enumeration of Bacillus cereus • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Detection of thermo-tolerant Campylobacter species and identification of C.jejuni, C.coli • Enumeration of Enterobacteriacae • Detection and enumeration of Enterobacteriaceae by MPN

• Fish crustaceans and • Enumeration of micro-organisms at 30ºC molluscs • Enumeration of micro-organisms at 22ºC • Enumeration of micro-organisms by spiral plating at 30ºC and 22ºC • Enumeration of Staphylococcus aureus • Enumeration of E.coli in shellfish using the MPN technique and detection of Salmonella • Enumeration of Bacillus cereus • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Edible fats/oils • Enumeration of microorganisms at 30ºC • Enumeration of microorganisms by spiral plating at 30ºC • Enumeration of E.coli at 44ºC • Enumeration of Staphylococcus aureus • Enumeration of Clostridium perfringens • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Heat processed foods • Enumeration of microorganisms at 37ºC in sealed containers • Enumeration of microorganisms by spiral plating at 37ºC • Enumeration of Staphylococcus aureus • Enumeration of E. coli at 44ºC • Enumeration of Bacillus cereus • Detection of Salmonella species • Enumeration of Clostridium perfringens • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Detection of thermo-tolerant Campylobacter species and identification of C.jejuni, C.coli • Enumeration of Enterobacteriacae • Enumeration of coliforms at 30ºC • Enumeration of coliforms at 37ºC • Sugar products honey • Enumeration of microorganisms at 30ºC and confectionery • Enumeration of microorganisms by spiral plating at 30ºC • Enumeration of E.coli at 44ºC using membranes • Enumeration of Staphylococcus aureus • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Enumeration of coliforms at 30ºC • Enumeration of coliforms at 37ºC • Enumeration of Bacillus cereus • Detection of thermotolerant Campylobacter species and identification of C. jejuni, C. coli • Beverages • Enumeration of microorganisms at 30ºC • Fruit juice and • Enumeration of microorganisms by spiral concentrates plating at 30ºC • Alcoholic beverages • Enumeration of E.coli at 44ºC • Enumeration of Staphylococcus aureus • Detection of Salmonella species Enumeration of coliforms at 37ºC • Bottled waters • Enumeration of coliforms and E. coli • Enumeration of Enterococci • Colony count by pour plate method • Enumeration of Clostridium perfringens by • Enumeration of Pseudomonas aeruginosa • Enumeration of coliforms and E. coli • Surfaces (factory • Enumeration of microorganisms at 30ºC hygiene) • Enumeration of microorganisms by spiral plating at 30ºC • Enumeration of E.coli at 44ºC using membranes • Enumeration of Staphylococcus aureus • Detection of Salmonella species • Detection of Listeria monocytogenes • Enumeration of Listeria monocytogenes and Listeria species • Enumeration of coliforms at 37ºC • Detection of thermo-tolerant Campylobacter species and identification of C.jejuni, C.coli • Enumeration of Clostridium perfringens

• Potable water • Enumeration of coliforms and E. coli • Environmental water • Enumeration of Enterococci • Colony count by pour plate method • Enumeration of Clostridium perfringens • Detection of Salmonella • Enumeration of coliforms and E. coli

Rapid Detection UNIT 8 RAPID DETECTION TECHNIQUES Techniques for Food FOR FOOD MICRO-ORGANISMS Micro-organisms

8.0 Objectives 8.1 Introduction 8.2 Need for Rapid Detection techniques 8.3 Biochemical Kits 8.4 Immunological Methods 8.4.1 Latex Agglutination Assay 8.4.2 Immuno-diffusion Format 8.4.3 Enzyme Linked Immunosorbent Assay (ELISA) 8.4.5 Immuno-magnetic Separation (IMS) 8.5 Genetic Methods 8.5.1 Nucleic Acid Probes 8.5.2 Polymerase Chain Reaction 8.5.3 DNA Chips and Micro-Arrays 8.6 Flow Cytometry 8.6.1 Principle 8.6.2 How Does It Work? 8.7 Impedance 8.8 Biosensors 8.8.1 Applications 8.8.2 Types of Biosensors 8.9 Other Methods 8.10 Limitations 8.11 Let Us Sum Up 8.12 Key Words 8.13 Some Useful Books 8.14 Terminal Questions 8.15 Answers to Check Your Progress Exercises 8.0 OBJECTIVES After studying this Unit, we shall be able to: • appreciate the importance of rapid techniques; • enlist commercially available biochemical kits; • specify immunological methods and kits used specially for quick detection of food borne pathogens; • explain genetic methods employed for detection and enumeration of micro-organisms; • describe working principles and working of fluorescence, impedance methods e.g. flow cytometry; and • state the role of biosensors 8.1 INTRODUCTION Traditional culture techniques are the most common methods of microbiological examination of foods, but these are fraught with a number of disadvantages such as being labour intensive, time consuming and failure to isolate viable but non-culturable organisms. On the other hand, total microscopic count methods are relatively fast, but limitations of these techniques include operator fatigue from prolonged use of microscopes and inability to discriminate between living and dead bacteria. To alleviate problems, associated with culture-based detection systems and direct microscopic methods, various other methods have been developed. These 71

Analytical include colorimetric assay methods based on liberation of dyes from Techniques in substrates by enzymes, dye reduction tests, and ATP bio-luminescence. The Microbiology rapid techniques have the futuristic potential to usher in an era of real time microbiology and have wider applications in industry such as food- cosmetic and pharmaceutical industry, control laboratories, biotechnology and environmental testing and research laboratories. 8.2 NEED FOR RAPID DETECTION TECHNIQUES It is impossible to completely eliminate pathogens from the food supply; some pathogens (e.g., Bacillus cereus) are common in soil and on vegetation, and food handlers often carry others (e.g., Staphylococcus aureus), even if they follow standard hygiene practices. Lapses in worker hygiene or sanitation in food processing plants results in a dramatic expansion of the range of bacterial pathogens, because of the broad distribution of pathogenic bacteria and viruses. This ubiquity makes it essential that the food industry have access to efficient diagnostic tools that allow detection and identification of pathogens. These tools are also important for clinicians to help diagnose food borne illnesses, which is usually characterized by gastro-enteritis. Identifying the culprit is important because the effective treatments vary among the pathogens.

Accurate identification of the cause of food-borne illness is also useful to public health officials attempting to identify the source of an outbreak, and for epidemiologists interested in long-term trends, such as the increasing frequency of E. coli O157:H7 infections. Public health officials identify sources by testing the stools of affected people for the presence of pathogens. Once a pathogen has been identified, investigators carefully interview victims and attempt to uncover commonalities. For example, all the victims may have eaten at the same restaurant the previous day or may have attended a recent family reunion. In some cases, it is essential to identify all the people affected by an outbreak, so that they can be closely monitored for the eruption of severe symptoms. For example, children under the age of 5 are at risk for developing potentially life-threatening kidney failure after infection by E. coli O157:H7; it is essential to identify this pathogen as soon as possible.

Diagnostic speed is also essential in food processing plants. If pathogen contamination can be detected before a product lot leaves the plant, it may be possible to avert an outbreak. However, if enough time elapses before the contamination is detected; it may be too late to prevent an outbreak, depending on the nature of the product and its distribution system. Current methods of microbial identification often require 2 to 3 days, which increases the risk that contamination of food by pathogens will be undetected until it is too late.

Food companies have traditionally relied on end product inspection and testing to ensure that the food that leaves the factory does not contain an excessive load of non-pathogenic and pathogenic microbes. Theoretically, 100% of products can be visually inspected, but human frailties (e.g. distractability, boredom) decrease the efficiency of inspection. Furthermore, many microbial defects cannot be detected by visual inspection. Hence, destructive sampling of end products is often required. In principle, this is a 72

simple process – a defined proportion (subset) of products is removed at the Rapid Detection end of the production line and the level of microbial contamination is Techniques for Food Micro-organisms assessed. Typically, microbes are put into suspension by grinding each sample in a StomacherTM or similar apparatus. A dilution series of this suspension is then plated onto an agar medium, and after a suitable period (24 h at 37oC), bacterial of fungal colonies are counted. In some cases, enrichment and selective media are used to detect specific pathogens (e.g. Salmonella). This technology can be quite powerful; in some cases, cultural methods can distinguish closely related strains. For example, E. coli O157:H7 can be distinguished from non-pathogenic strains through the use of selective and differential media (differential media produce a detectable change in appearance/colour of either the agar medium or the colonies when the target bacterium is present).

Cultural methods such as this are a reliable indicator of microbiological quality in food, but there are many disadvantages of end product microbial analysis. Financial concerns sometimes make it difficult to test an adequate number of samples, because cultural methods are labour and materials intensive. If too few samples are assessed, the risk of missing contaminated product increases. This risk also increases if contamination occurs sporadically, rather than on a regular basis. Also, cultural methods are slow, particularly if the aim is to identify specific pathogens. For example, conventional detection of Salmonella requires that a food sample be incubated in three successive media for a total time of 72 h. Additional tests that confirm the presence of Salmonella are then required. This confirmation can be biochemical; the colonies that are presumed to be Salmonella are typically inoculated into a series of media containing a range of different carbon sources (e.g., glucose, sucrose, mannitol, proline, etc.). The bacteria respiring or fermenting (depending on the diagnostic system) these carbon sources produce a ‘pattern of utilization’ that can be used to identify the bacteria. Unfortunately, the growth period required for these tests further adds to the delay in assessment of contamination. Antibody-based tests, in contrast, can confirm the presence of Salmonella immediately. However, if negative, they do not provide any other information that could be used for identification, unlike biochemical tests.

The methods for the rapid detection of food borne micro-organisms, both spoilage and pathogens can be broadly classified into biochemical, immunological and genetic approaches. However, several methods for detection involve the use of more than one approach for identifying micro- organisms. The advantages of these methods are that they are less labour intensive and produce quick results. Most of the rapid methods are available as kits that are very easy to handle and can be carried to the sampling site. However, the main problem faced in the rapid detection of food borne microbes is the presence of inhibitors that interfere with particular detection method. This problem does not occur in the traditional methods because they involve enrichment of the stressed and injured cells from the food sample and this in turn leads to the dilution of inhibitors. Hence in comparison with rapid methods, traditional methods are better if accuracy of detection is to be considered. Food regulatory agencies suggest the use of rapid methods to screen the food samples for the presence of pathogens. In such tests negative, reaction is taken as an absence of pathogen in the food product however, the positive reaction for the presence of a pathogen is considered as presumptive 73

Analytical and the sample is tested by traditional method to confirm the result obtained Techniques in with a rapid method. Various rapid methods for identification can be Microbiology categorized as described in following sections. 8.3 BIOCHEMICAL KITS These kits are designed on the same principles as the conventional methods such as the use of specific or differential media and biochemical tests for the identification of bacteria. However, the format of test is modified. These kits are made up of a disposable device containing an array of different media and substrates used for biochemical identification of bacteria. It will not be wrong to say that these are the miniaturized version of the biochemical tests carried out conventionally for the identification of food borne microbes especially, pathogens. These tests are classified under rapid tests because these kits are commercially available and one does not have to go through the time consuming and cumbersome procedure of preparing the media and test reagents as in the case of conventional methods. However, most of the kits are not truly rapid and require an incubation period of around 24 h to give the results. The accuracy obtained with such kits is around 90-99% as compared to conventional methods. Several such kits are available for the detection of food borne bacteria e.g. API kits developed by bioMerieux for Enterobacteriaceae, Listeria, Staphylococcus, Campylobacter, non-fermenters and anaerobes. With the advancement of the technology, the automation of such miniaturized kits have been made possible. Such automated systems automatically monitor the biochemical changes and prepare a phenotypic profile of the tested organism which is then matched with the computer database for identification purposes e.g. Vitek automated system developed by bioMerieux for identification of Enterobacteriaceae, Gram negatives and Gram positives. # Check Your Progress Exercise 1 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit.

1) Enlist the need for rapid detection techniques of micro-organisms in food? ……………………………………………………………………………… ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 2) What are biochemical kits? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….…

8.4 IMMUNOLOGICAL METHODS Immunological methods of rapid detection are based on the highly specific antigen-antibody reactions and involve the use of monoclonal antibodies for detection of bacteria in the food samples. These methods comprise the largest 74

group of methods used for detection purposes. The versatility and specificity Rapid Detection of these reactions have prompted the production of immunological kits in Techniques for Food Micro-organisms various formats such as: 8.4.1 Latex Agglutination Assay This involves the antibodies tagged with the colored latex beads or colloidal gold for the detection purposes e.g. Campyslide, Microscreen for the detection of Campylobacter, RIM, Prolex, Ecolex O157, Wellcolex O157 for E. coli O157:H7, Microscreen, Listeria Latex for Listeria. 8.4.2 Immuno-diffusion Format This involves the diffusion of antigen through a gel impregnated with antibody. The appearance of precipitation line indicates the presence of specific antigen and is considered a positive test. An example of the kit based on the immunodiffusion is 1-2 test for the detection of Salmonella. 8.4.3 Enzyme Linked Immunosorbent Assay (ELISA) This is the most popular rapid method for the detection of food borne pathogens. This is also based on the specificity of antigen-antibody reaction. In most of the immunological kits, sandwich format for ELISA is used. In this format antibody is coated on the surface of microtitre well. This antibody is known as primary antibody. Bacterial antigen obtained from the food sample is added to the antibody coated wells. Antigen-antibody reaction is allowed to occur followed by washing the wells to remove the unbound antigen. After this, a secondary antibody tagged with an enzyme is added to the microtitre plate. This antibody is also specific for the antigen to be detected but it recognizes a different epitope on the surface of antigen. This step is again followed by washing to remove the unbound antibody, followed by the addition of substrate for the enzyme. This substrate is usually colourless and a coloured product is formed by the action of antibody bound enzyme. The colour development is taken as a positive reaction and even the intensity of the colour can be measured using ELISA reader. This intensity when compared with standard controls can be used for quantification of the antigen or the results may be reported as positive and negative depending on the instructions given with the specific kit. If specific antigen is not present in food then the primary antibody will not bind to the added antigen, which will be removed during washing step. In the absence of specific antigen the secondary antibody will also not bind and will be washed from the well. The absence of enzyme conjugated secondary antibody from the well will effect the formation of coloured product from the substrate, thus giving a negative signal. Several enzymes such as horseradish peroxidase, alkaline phosphatase and p-nitrophenyl phosphatase have been used to tag the secondary antibody. A few examples of some ELISA based kits for the detection of pathogens include EHEC-TEK, Assurance, HECO157, TECRA, Premier O157, E. coli Rapitest, Transia Card E. coli O157 for E. coli O157:H7. Several such kits are also available for other pathogens. 8.4.4 Immuno-precipitation This format is also a sandwich procedure but it does not make use of enzyme conjugated antibody for the detection purposes, instead it relies on the antibody conjugated with the latex beads or colloidal gold for the detection purposes. These assays are simple and require no washings or manipulations and take very less time to be completed. Examples of the tests based on 75

Analytical immunoprecipitation include PATH-STIK, Reveal, Clearview, for Techniques in Salmonella, choleraSMART, bengalSMART for Vibrio cholerae. Microbiology 8.4.5 Immuno-Magnetic Separation (IMS) This is basically an enrichment step for the bacteria occurring in food matrix. This test utilizes the antibodies coupled with the magnetic particles. The specific bacteria bind to the antibody and is separated from the food matrix under the influence of magnetic field. It is similar to the enrichment using selective media. This enriched bacterial culture can then be detected by any of the rapid methods being discussed in this chapter. 8.5 GENETIC METHODS Genetic methods for identification of micro-organisms are based on the detection of the specific gene sequences in the genotype of the organism. These sequences may be selected to detect particular group, genus, species or even strain of the micro-organism e.g. suppose a kit has to be developed for the detection of particular bacterial genus then the DNA sequence occurring only in the members belonging to that genus is to be targeted i.e. the gene sequence should occur in all the species belonging to that genus so that false negative reactions could be avoided, but if a kit for a particular species is to be designed then a DNA sequence occurring only in the members of this species should be targeted. This sequence should not occur in other species belonging to the same genus. The specificity of the detection process totally depends upon the targeted sequence, which is the most important part of designing an assay system. Several genes have been targeted for the detection purposes but rDNA is preferred because it is universally present in all bacteria and also shows required variation for designing the specific assay. Another advantage of targeting this gene is the availability of sequences of rDNA due to the development of databases. Other genes such as those involved in toxin production may also be targeted for this purpose. A genetic method for pathogen detection can be developed in various formats but the two most popular formats are the use of gene probes and polymerase chain reaction (PCR). Both these formats are designed to obtain a signal if the specific gene sequence is present. 8.5.1 Nucleic Acid Probes For a diagnostic test to achieve widespread use in the food industry, it must also be easy to use. Probe-based tests have achieved this goal through immobilizing probes to inorganic supports (dipsticks) that allow the user to easily manipulate the probe (e.g., wash off unhybridized DNA) without damaging or losing it. This is referred to as solid-phase hybridization systems (e.g. solution-based) are also possible. The principle behind the use of DNA probes is quite simple. Short single strands of DNA that are complementary to genes present in a pathogenic microbe are synthesized. The food sample must then be treated so that any microbial cells are lysed, releasing their DNA. Microbial DNA is then treated to convert it from double strands to single strands, and the probe is added. Hybridization (annealing of complementary strands) then occurs between the single-stranded DNA probe and single-stranded DNA released from pathogenic microbes present in the food. Probe DNA that has not hybridized must then be removed, usually by washing the sample, and the presence of hybridized DNA probes can then be detected. 76

Stomach food samples Rapid Detection Techniques for Food ↓ Micro-organisms Enrichment of Salmonella ↓ Lyse cells and convert ds DNA to ss DNA ↓

Add capture probe AAAAA

and detector probe

Transfer dipstick

AAAAA AAAAA Salmonella DNA

Solution containing enzyme AAAAA AAAAA

Look for colour change

Salmonella DNA

Fig 8.1: The use of a gene probe to detect a pathogen in food. The Gene TrakTM system for the detection of Salmonella is used as an example. The capture and detection probes anneal to different regions of the ribosomal DNA gene of Salmonella. The dipstick is used to remove the capture probe- Salmonella DNA-detection probe complex. The complex is then placed in the appropriate solution that will reveal the presence of the detection probe. In earlier versions of this system, detection was based on fluorescein in the probe binding to anti-fluorescein antibodies, which in turn bind to enzyme-linked antibodies. The enzyme then catalyzes formation of a coloured end product. The principle advantage of this system is the reduced time required foe positive identification of Salmonella. Another advantage is the assay’s 77

Analytical specificity; several bacteria found in food (Citrobacter, Enterobacter, Techniques in Escherichia, Klebsiella, and Proteus are very closely related to Salmonella Microbiology and are often able to grow in media that are “selective” for Salmonella. Furthermore, some isolates of Salmonella are atypical and do not produce the usual colony characteristics of Salmonella on differential media. The main problem with the Gene Trak system is that enrichment of Salmonella is still required, making “instant” identification of Salmonella impossible. However, enrichment is in a sense useful, because it makes it unlikely that deal Salmonella will be detected, unless they are present in high numbers. This is important, because dead Salmonella do not pose a health hazard, and the detection of deal Salmonella constitutes a false positive that may be wasteful of a company’s resources. Because it is still possible that dead bacteria could lead to a false positive. Formats using gene probes are based on the hybridization of the labelled DNA sequence (probe) to its complementary sequence. These probes may be labelled with radioactive or non radioactive reporter molecules. If the desired targeted sequence is present then the signal is obtained due to the hybridization of the labelled probe. If the targeted sequence is absent then probe will not bind and no signal will be obtained. gene probes can be used in dot blot hybridization assays or other formats. Examples of some kits based on gene probes are AccuProbe for Campylobacter, GENE-TRAK for Campylobacter, E. coli, Listeria, Salmonella, Staphylococcus aureus and Yersinina enterocolitica. 8.5.2 Polymerase Chain Reaction This test is also based on the hybridization of the oligonucleotide primer pair followed by amplification of the DNA stretch spanning these primers. First step to carry out PCR is the isolation of DNA from the food matrix or enriched sample. This DNA provides the target for the hybridization of specific oligonucleotide primer and is called the template DNA. In addition to template DNA and primer pair, PCR amplification mix contains four dNTPs (Deoxynucleoside triphosphates, the building blocks from which the DNA polymerases synthesize a new DNA strand), Taq enzyme and Taq buffer. If a primer is having a complimentary DNA sequence it will hybridize with it. This step is termed as annealing. This DNA stretch between the two primers will be synthesized due to the addition of corresponding dNTPs by the action of Taq, which have its optimum activity at 72oC. Once the synthesis is complete the DNA is denatured by heating the mixture to 95oC and cooled again for primers to anneal. The annealing temperature is dependent on the primer sequence. This cycle (Fig. 8.2) is repeated several times in a machine called thermocycler to obtain the amplification of DNA. The amplified DNA is visualized by agarose gel electrophoresis, followed by Ethidium bromide (EBr) staining. Several formats are available for carrying out PCR such as Real time PCR where the amplification of DNA is monitored while the reaction is being carried out, Multiplex PCR where more than one primer pair is used for the simultaneous detection of more than one type of bacteria but all these formats have not been converted to commercially available kits. Examples of PCR based kits used for pathogen diagnosis are Probelia for Clostridium botulinum, E. coli O157:H7, Salmonella and BAX for E. coli O157:H7, Listeria and Salmonella.

It is too early to tell if the food industry will embrace PCR-based diagnostics. Rapid Detection Considerable expenses are involved in the acquisition of thermal cyclers and Techniques for Food Micro-organisms the training of personnel in their use. Also, these techniques need to be

Denaturation of dsDNA

Annealing of Primer

Extension

Newly formed DNA strands

Fig 8.2: Steps in a PCR cycle extensively validated, through comparison of their ability to detect low levels of contamination in food to those of conventional culture-based methods. The ability of PCR to detect dead organisms is a problem. Tiny amounts of DNA released from pathogens killed by heat treatments, for example, would be amplified by PCR. This is less of a problem if enrichment precedes PCR, but that extends the procedure to at least 1 d. In some situations the presence of live or dead pathogens is an important indicator of food safety. For example, if PCR detects Clostridium botulinum in a food sample, it is a cause for concern whether the bacterium is alive or dead, because dead bacteria may have produced botulinum toxin before dying. The toxin could then persist in the food, causing a potential for serious illness. One final note on PCR: several alternative amplification systems are actively under development. For example, nucleic acid sequence based amplification (NASBA®) uses three viral enzymes to amplify either RNA or DNA targets. The main advantage of this technique is that it is isothermal (occurs at a constant temperature), avoiding the need for expensive thermal cyclers. So far, NASBA diagnostics have mainly been aimed at identification of viruses, but applications for the identification of Campylobacter and L. monocytogenes are also under development. 8.5.3 DNA Chips and Micro-arrays Few techniques have caused as much excitement among microbiologists as DNA chips and micro-arrays. Both are intrinsically miniaturized extensions of conventional tests of nucleic acid hybridization. A sample of DNA is placed onto the membrane; labelled probe is then added; and hybridization (if present) is detected. Now consider a slightly different scenario: a number of oligonucleotides (each representing a different gene) are 79

Analytical immobilized onto separate points of a membrane. A bacterial culture is then Techniques in exposed to a chemical that results in labelled messenger RNA (mRNA) Microbiology transcripts. The bacteria are lysed and then placed on each oligonucleotide on the membrane. After washing, hybridization between labelled mRNA and immobilized oligonucleotides can be detected. This micro-array of oligonucleotide probes allows simultaneous detection of expression of a number of genes. Now imagine a similar array of immobilized oligonucleotides on a 1 x 1 cm square on a glass microscope slide. Further miniaturization can be achieved with tiny wells etched into a circuit board. Oligonucleotides are then immobilized onto these wells, and the pattern of probe immobilization (i.e , which probes are loaded into which wells) can be controlled electronically. These are often referred to as DNA chips or micro-arrays (arrays on glass slides are also commonly referred to as micro-arrays). The great advantage of these systems is that they require only small amounts of resources. DNA chips can also be developed into laboratories in a chip, wherein an experimental routine, perhaps involving heating of reagents and mixing of several different chemicals, or even electrophoresis, can occur at a micro scale. If successfully applied to diagnostic testing, micro-arrays embedded into silicon chips could allow efficient testing of large numbers of samples and could even be used to amplify sample DNA using PCR. Theoretically, with one test the investigator could detect the presence of DNA of many different pathogens in a food sample. Micro-arrays are currently commercially available for assessment of global gene expression (the total pattern of gene expression by a cell). Up to 8000 genes can be simultaneously tested for hybridization, allowing investigators to dissect changes in gene expression after different experimental treatments. This type of research has many potential applications in food microbiology (e.g. determining pattern of gene expression in a pathogenic bacterium triggered by exposure to acidic preservatives). Micro arrays also have great potential as diagnostic systems, but this is currently in the research and development phase.

# Check Your Progress Exercise 2 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) What are the principles behind immuno-precipitation techniques? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 2) What is a DNA probe? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 3) List the drawbacks of using PCR? ……………………………………………………………………………… ……………………………………………………………………………… …………………………………………………………………………….… 80

Rapid Detection 8.6 FLOW CYTOMETRY Techniques for Food Micro-organisms 8.6.1 Principle

Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5µm to 40µm diameter. Lasers are most often used as a light source in flow cytometry. One unique feature of flow cytometry is that it measures fluorescence per cell or particle. This contrasts with spectrophotometry in which the percent absorption and transmission of specific wavelengths of light is measured for a bulk volume of sample. Scattered and emitted light from cells and particles are converted to electrical pulses by optical detectors (Fig. 8.3). Collimated (parallel light waveforms) light is picked up by confocal lenses focused at the intersection point of cells and the light source. Light is sent to different detectors by using optical filters. For example, a 525 nm band pass filter placed in the light path prior to the detector will only allow “green” light into the detector. The most common type of detector used in flow cytometry is the photo-multiplier tube (PMT). The electrical pulses originating from light detected by the PMTs are then processed by a series of linear and log amplifiers. Logarithmic amplification is most often used to measure fluorescence in cells. This type of amplification expands the scale for weak signals and compresses the scale for “strong” or specific fluorescence signals.

Figure 8.3: Working of a flow cytometer

After the different signals or pulses are amplified they are processed by an Analog to Digital Converter (ADC) which in turn allows for events to be plotted on a graphical scale (One Parameter, Two parameter Histograms).

Flow cytometry data outputs are stored in the form of computer files using the FCS 2.0 or 3.0 standards. Data corresponding to one sample can be stored as a listmode file and/or histogram file. 81

Analytical 8.6.2 How does it work? Techniques in Microbiology The tissue sample is broken up into single cells and held in a test tube, which is placed into the flow cytometer. The liquid containing the cells is drawn up from the test tube and pumped into the flow chamber.

1. Flow chamber – Cells flow through the flow chamber one at a time very quickly, about 10,000 cells in 20 seconds or 500 cells per second. 2. Laser – A small laser beam of very bright light hits the cells as they pass through the flow chamber. The way the light bounces off each cell gives information about the cell’s physical characteristics. Light bounced off at small angles is called forward scatter. Light bounced off in other directions is called side scatter. 3. Light detector - The light detector processes the light signals and sends the information to the computer. Forward scatter tells you the size of the cell. Side scatter tells you if the cell contains granules. Each type of cell in the immune system has a unique combination of forward and side scatter measurements, allowing you to count the number of each type of cell. 4. Filters – The filters direct the light emitted by the fluorochromes to the colour detectors. 5. Colour detectors - As the cells pass through the laser, the fluorochromes attached to the cells absorb light and then emit a specific colour of light depending on the type of fluorochrome. The fluorochromes on the cells act like the bar code on groceries as the cashier passes them over the scanner. Any one cell can have one, both or none of the markers on its surface. The colour detectors collect the different colours of light emitted by the fluorochromes. The fluorochrome data signal is also sent to the computer. 6. Computer – The data from the light detector and the colour detectors is sent to a computer and plotted on a graph called a histogram.

The possible application of flow cytometry to the analysis of foods for their content of micro-organisms is by staining bacteria with fluorescent dyes, one of which binds preferentially to DNA rich in guanine-cytosine (G-C) while the other binds to adenine-thymine (A-T)-rich DNA. Thus, the rapid identification of bacteria in food based on their specific A-T/G-C ratios is possible. In addition to the simultaneous measurement of protein and DNA in cells and the enumeration of cells based on G-C and A-T content, flow cytometry has been used to distinguish between living and dead cells by dual staining; to determine the ploidy of yeast cells; to differentiate between spores and vegetative cells in Bacillus spp.; and to separate pathogenic and non pathogenic amoebae. It has been used to identify Listeria monocytogenes present in naturally contaminated milk. Flow cytometry (FCM) is extremely sensitive, avoids the need for culturing or enrichment procedures, and can be both qualitative and quantitative. Use of fluorescent stains or fluorogenic substrates in combination with FCM allows the detection and discrimination of viable culturable, viable non culturable, and non viable organisms. Furthermore, there is the possibility that numerous (or even rare) microbial cells could be detected against a background of other bacterial or non bacterial particles by combining FCM and specific fluorescently-labeled antibodies or oligonucleotide probes. A flow cytometry method for rapid detection and enumeration of total bacteria in milk has been developed by Gunasekera et al. (2000). 82

Rapid Detection 8.7 IMPEDANCE Techniques for Food Micro-organisms Impedance can be simply defined as the resistance to flow of an alternating current as it passes through a conducting material. To elaborate it further, when two metal electrodes are immersed in a conductive medium the test system behaves as a resistor and capacitor in series. Considering the case where the system is treated as a series combination then application of an alternating sinusoidal potential will produce a resultant current which is dependent on the impedance of the system which in turn is a function of its resistance, capacitance and applied frequency.

When micro-organisms grow in culture media, they metabolize substrates of low conductivity into products of higher conductivity and thereby decrease the impedance of the media. Simple examples include the conversion of glucose from a non ionized substrate to two molecules of lactic acid with a corresponding increase in conductivity. Further metabolism will take the lactic acid and three oxygen molecules to produce carbonic acid resulting in three ion- pairs, including the smaller, more mobile bicarbonate ion which is a more effective electrical conductor than the lactate ion. The impedance of a system is a function of its resistance, capacitance and the applied frequency. It is usually the resistive element which is measured and this is most frequently recorded as changes in conductance. These changes occur in the bulk electrolyte solution (culture medium) due to metabolism of uncharged or weakly charged substrates which are converted to highly charged end products e.g. proteins to amino acids. Capacitance is related to the behaviour of ions at the surface of the electrode and can be monitored separately from conductance. Several instruments which monitor changes in impedance are available and include the Bactometer 123 (Bactomatic Ltd.), Malthus 2000 (Malthus Instruments Ltd) and the Rapid Automated Bacterial Impedance Technique (RABIT) (Don Whitley Scientific Ltd). Detection of bacteria can be by direct conductimetry which is achieved by monitoring changes in the growth medium or by indirect conductimetry which monitors changes due to evolution of CO2 produced by the metabolism of substrates in the culture medium. When the impedance of broth cultures is measured, the curves are reproducible for species and strains, and mixed cultures can be identified by the use of specific growth inhibitors. The technique has been shown capable of detecting as few as 10 to 100 cells and cell populations of 105-106/ml can be detected in 3-5 h and 104-105/ml in 5-7 h. Impedance has been evaluated by a large number of investigators as a means of monitoring the overall microbial quality of various foods.

The redox potential is one of the most complex indicators of the physiological state of microbial cultures and its measurement could be a useful tool for the qualitative and quantitative determination of the microbial contamination. During the bacterial growth, the redox potential of the medium decreases. The shape of the redox potential curve is characteristic of the type of microorganism, and the rate of the change (dE/dt) is proportional to the living cell concentration. The time required to reach a significant change in redox potential is defined as Time to Detection (TTD). Similarly to the impedimetric measurements, a strict linear correlation could be established between the TTD and the logarithm of the initial concentration of micro-organisms. On the basis of this calibration curve, the determination of living cell concentration could be simplified. 83

Analytical The applications of this technique remains to be fully exploited though it has Techniques in already been shown to be a powerful tool for working with strains of Aerobic Microbiology mesophilic micro-organisms, Psychrotrophic micro-organisms, Thermophilic micro-organisms, gram negative bacteria, Enterobacteria, Enterococci, Lactobacilli, Coliforms, E. coli, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Beer spoilers, Clostridia, Aerobic spore formers, Bacillus cereus, Yeasts and Molds. 8.8 BIOSENSORS Biosensors have a biological sensor that is connected to a transducer. A transducer is a device capable of converting signals from the biological sensor into signals (usually electrical) that can be easily recorded and stored. For example, a number of biosensors use the specificity of antibody-antigen binding to detect pathogens in food samples. When the pathogen is present, it binds to the antibody. The key event follows: binding of antigen to antibody produces an electrical signal that can be detected and recorded. Biosensors, then, are an example of what science fiction authors describe as: ‘cybernetics’– the fusion of organic matter to electronic circuitry. 8.8.1 Applications Biosensors have many applications in clinical settings (e.g. diagnosis of food borne pathogens from stool and other samples) and in maintenance of food safety (e.g., assessment of microbial loads or detection of specific pathogens in food). For example, it is theoretically possible to design biosensors that are sensitive (e.g. , able to detect one pathogen in 25 g of food), selective (able to discriminate specific pathogens from a large background of non-pathogenic microbes), fast 9real time), automated, portable, and inexpensive. To date, this potential has not been realized, but research in biosensor development is highly active and steady improvements in design are predicted. Continuous monitoring is also useful for many food safety or spoilage applications, particularly in the processing of liquids (milk, beer, etc.), where it is desirable to monitor microbial numbers in line in piping systems used to transfer liquids from one vessel to another or to packaging processes). For example, post-pasteurization contamination of beverages and foods is a significant cause of spoilage and has been implicated in outbreaks of food- borne illness. Contamination of processing equipment is often difficult to eradicate. A continuous monitoring system that detects microbial growth in the lines is much better than monitoring based on examination of discrete samples or equipment swabs. If microbial growth could be immediately detected in transfer lines, the process could be stopped and the contamination eliminated, thus avoiding the production of large amounts of contaminated product. Conventional diagnostic systems based on enrichment and selective culture are not adaptable for continuous monitoring. Instead, they require the collection of discrete sampling units (batch samples). Each sample is then cultured in the appropriate media. Continuous monitoring using a culture-based system would require an infinite (or at least very large) number of sampling units, whereas biosensors can continuously monitor without the collection of discrete samples. Biosensors can also be used for batch sampling. One important application of biosensors is to speed up pathogen identification using culture-based methods. 84

For example, spoilage of fresh meat is an economically important problem and Rapid Detection is often linked to the presence of high levels of a variety of spoilage microbes. Techniques for Food Micro-organisms Conventional monitoring is done through plate counting (total bacterial count) after incubation on non-selective media that allow a wide range of bacteria to grow. However, this requires at least 24 h – not ideal for preventing meat spoilage. Biosensors have been designed that assess levels of microbial contamination after short (1 h or less) incubation of meat samples. It is also often desirable to continuously monitor physical processes in the food industry. Physical such as temperature can easily be monitored continuously, allowing immediate adjustment if the temperature strays outside a defined range, and also providing a record of temperature changes. A continuous record can be useful if product quality declines; deviations in the temperature of the process could be a causal factor. Biosensors can be used to monitor some physical – chemical processes (e.g., CO2 production). 8.8.2 Types of Biosensors Biosensors can be classified according to the type of sensor, the transduction strategy, and the directness of the assay. Affinity-based biosensors rely on specific recognition between the sensor and the target. Antibodies are most commonly used, but nucleic acid hybridization, similar to that used in gene probe assays, and receptor-ligand interactions (e.g. insulin binding to insulin receptor molecules) are also used to create affinity-based biosensors. Antibody-based biosensors have been developed for most of the major food- borne pathogens (e.g. Salmonella, E. coli O 157:H7, and L. monocytogenes).

Improvements in out ability to miniaturize electronic components and to develop “laboratories in a chip” are also expected to lead to more frequent application of biosensors in the food industry. Applications are currently not common, mostly because of low sensitivity, interference by compounds in food matrices, and difficulties regenerating electrodes. The potential benefits, through, are significant enough to justify continued research and development of these elegant diagnostic systems.

# Check Your Progress Exercise 3 Note: a) Use the space below for your answer. b) Compare your answers with those given at the end of the unit. 1) What is a biosensor? ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… 2) Enlist the components of Flow Cytometry? ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… 3) How can impedance be used to detect microbes? ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… 85

Analytical Techniques in 8.9 OTHER METHODS Microbiology These methods include the use of fatty acid profiling e.g. MIS Management Information System for the identification of Enterobacteriaceae, Listeria, Bacillus, Staphylococcus and Campylobacter, use of specific media with the incorporated flourogenic or chromogenic compounds for the detection of enzymatic activities of bacteria. Another important rapid method for detection of total bacterial counts in food samples is Bioluminiscence, which can be defined as conversion of chemical energy to light by living organisms. In these assays enzyme luciferase present in fire fly is used, which in presence of ATP and luciferin forms oxy-luciferin that emits light. The fluorescence obtained by this method is measured by luminometer and is proportional to the concentration of ATP which in turn is related to the total bacteria occurring in the sample. The kits for obtaining rapid bacterial counts in the food samples are Enliten, Profile-1, Biotrace and Lightning. 8.10 LIMITATIONS The main disadvantage of rapid methods is that the results may vary with the variation in the food products. This is because some food products may contain inhibitors for various reactions that form the very backbone of the rapid detection methods. This is observed in case of PCR because it is a very sensitive reaction and presence of very minute quantities of interfering agents may inhibit the reaction. On the other hand some food components may react with the added chemicals and lead to false positive results in the rapid tests. 8.11 LET US SUM UP Rapid enumeration and detection of food borne micro-organisms both spoilage and pathogens can be accomplished by biochemical, immunological and genetic methods. Broadly, these methods can be categorized in to Biochemical methods, Immunological methods, Genetic methods, Flow Cytometry, Impedance method and miscellaneous methods are commercially available Kits to facilitate the hassle free performance of these methods. The above mentioned methods have been described in this unit along with their working principle, procedure (wherever possible), kits and automated equipment. 8.12 KEY WORDS API Kits : Biochemical kit used for detection of pathogens ELISA : Enzyme Linked Immunosorbent Assay PCR : Polymerase chain reaction PMT : Photomultiplier tube ADC : Analog to Digital Converter FCM : Flow cytometry RABIT : Rapid Automated Bacterial Impedance Technique TTD : Time to Detection dNTPs : Deoxynucleoside triphosphates, the building blocks from which the DNA polymerases synthesize a new DNA strand 86

Rapid Detection 8.13 SOME USEFUL REFERENCES Techniques for Food Micro-organisms N.A. Cox, D.Y.C. Fung, J.S. Bailey, P.A. Hartman, and P.C. Vasavada (1987). Miniaturized kits, immunoassays and DNA hybridization for recognition and identification of food borne bacteria. Dairy Food Sanit. 7: 628-631.

D'Aoust, J.Y., A.M. Sewell, and P. Greco 1991. Commercial latex agglutination kits for the detection of food borne Salmonella. J. Food Prot. 54: 725-730.

Dziezak, J.D. 1987. Rapid methods for microbiological analysis of foods. Food Technol. 41(7): 56-73.

Feldsine, P.T., R.L. Forgey, M.T. Falbo-Nelson and S. Brunelle. (1997). Escherichia coli O157:H7 Visual Immunoprecipitation assay: a comparative validation study. J. AOAC 80: 43-48.

Feng, P. 1997. Impact of molecular biology on the detection of food borne pathogens. Mol. Biotech. 7: 267-278.

Hill, W.E. 1996. The polymerase chain reaction: application for the detection of food borne pathogens. CRC Crit. Rev. Food Sci. Nutrit. 36:123-173.

Johnson-Green, Perry (2002). Diagnostic Systems. In Introduction to Food Biotechnology, CRC Press, Florida.

Reichart, O.K. Szakmár, Á. Jozwiak, J. Felföldi and L. Baranyai. (2007). Redox potential measurement as a rapid method for microbiological testing and its validation for coliform determination. International Journal of Food Microbiology, 114( 2) , 143-148

Silley, P and S. Forsythe, (1996). Impedance microbiology - a rapid change for microbiologists-A review. Journal of Applied Bacteriology, 80: 233-243. www.stemcell.umn.edu

8.14 TERMINAL QUESTIONS

1. What is ELISA method? How does it work? 2. Describe various steps in a PCR cycle. 3. Describe the principle and working of flow cytometry. 4. How does Impedance technique function? Give names of instruments available for its commercial application as a rapid method for detection of bacteria. 5. Describe the functioning of biosensors. 6. Rapid detection methods have the potential of ushering in a new era of Real Time Microbiology. Give your comments.

Analytical Techniques in 8.15 ANSWERS TO CHECK YOUR PROGRESS " Microbiology EXERCISES

Check Your Progress Exercise 1 Your answer should include the following points:

1 Required by: food industry for in-line detection; Clinicians for diagnosis; Public health officials

2. These are the miniaturized version of the biochemical tests carried out conventionally for the identification of food borne microbes. Check Your Progress Exercise 2 Your answer should include the following points: 1. An antibody is coated on the surface of microtitre well. This antibody is known as primary antibody. Bacterial antigen obtained from the food sample is added to the antibody coated wells. Antigen-antibody reaction is allowed to occur followed by washing the wells to remove the unbound antigen. After this, a secondary antibody tagged with an enzyme is added to the microtitre plate. This antibody is also specific for the antigen to be detected but it recognizes a different epitope on the surface of antigen. This step is again followed by washing to remove the unbound antibody, followed by the addition of substrate for the enzyme. This substrate is usually colourless and a coloured product is formed by the action of antibody bound enzyme. The colour development is taken as a positive reaction and even the intensity of the colour can be measured using ELISA reader. 2. Immobilizing probes having nucleic acid strands specific to the pathogen bound to inorganic supports (dipsticks) that allow the user to easily manipulate the probe (e.g., wash off unhybridized DNA) without damaging or losing it. 3. (i) Considerable expenses are involved in the acquisition of thermal cyclers and the training of personnel in their use. (ii) These techniques need to be extensively validated, through comparison of their ability to detect low levels of contamination in food to those of conventional culture-based methods. (ii) The ability of PCR to detect dead organisms is a problem. In some situations the presence of live or dead pathogens is an important indicator of food safety. For example, if PCR detects Clostridium botulinum in a food sample, it is a cause for concern whether the bacterium is alive or dead, because dead bacteria may have produced botulinum toxin before dying. The toxin could then persist in the food, causing a potential for serious illness. Check Your Progress Exercise 3 Your answer should include the following points: 1) Biosensors have a biological sensor that is connected to a transducer. A transducer is a device capable of converting signals from the biological

sensor into signals (usually electrical) that can be easily recorded and Rapid Detection stored. Techniques for Food Micro-organisms 2) Flow chamber; Laser; Light detector; Filters; Colour detectors; Computer 3) When micro-organisms grow in culture media, they metabolize substrates of low conductivity into products of higher conductivity and thereby decrease the impedance of the media. Simple examples include the conversion of glucose from a non ionized substrate to two molecules of lactic acid with a corresponding increase in conductivity. The impedance of a system is a function of its resistance, capacitance and the applied frequency. It is usually the resistive element which is measured and this is most frequently recorded as changes in conductance. These changes occur in the bulk electrolyte solution (culture medium) due to metabolism of uncharged or weakly charged substrates which are converted to highly charged end products e.g. proteins to amino acids. Capacitance is related to the behaviour of ions at the surface of the electrode and can be monitored separately from conductance.