Laboratory Manual 3Rd Semester

DEPARTMENT OF MICROBIOLOGY

MICROBIOLOGY LABORATORY MANUAL 3RD SEMESTER

LABORATORY MANUAL FOR 3RD SEMESTER MICROBIOLOGY PRACTICAL –PAPER-G509.3P

1 .CATALASE TEST 2

2. OXIDASE TEST 4

3. INDOLE TEST 6

4 .METHYL RED ( MR) TEST 8

5. VOGES PROSKAUER ( VP) TEST 10

6. CITRATE TEST 13

7. UREASE TEST 15

8. GELATIN HYDROLYSIS ( LIQUEFACTION) TEST 17

9. TRIPLE SUGAR IRON TEST 19

10.β-GALACTOSIDASE ( ONPG) TEST 24

11. CARBOHYDRATE FERMENTATION TEST 26

12. ESTIMATION OF PROTEIN BY LOWRY’S METHOD 31

13. ESTIMATION OF REDUCING SUGARS BY DINTROSALICYLIC ACID (DNS) METHOD 33

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BIOCHEMICAL TESTS

EXPERIMENT NO: 1 –CATALASE TEST

INTRODUCTION AND PRINCIPLE: Catalase is an enzyme, which is produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites; H2O2. The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase enzyme. Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water. To find out if a particular bacterial isolate is able to produce catalase enzyme, small inoculums of bacterial isolate is mixed into hydrogen peroxide solution (3%) and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. Catalase-positive bacteria include strict aerobes as well as facultative anaerobes. They all have the ability to respire using oxygen as a terminal electron acceptor.

Catalase-negative bacteria may be anaerobes, or they may be facultative anaerobes that only ferment and do not respire using oxygen as a terminal electron acceptor (ie. Streptococci).

1. For routine testing of aerobes, 3% hydrogen peroxide is used.

2. 15% H2O2 solution: For the identification of anaerobic bacteria Catalase test is used to differentiate aerotolerant strains of Clostridium (catalase negative), from Bacillus species (catalase positive). 3. The superoxol catalase test used for the presumptive speciation of certain Neisseria organisms requires a different concentration of H2O2. Uses of catalase test/Catalase Test Results 1. The catalase test is primarily used to distinguish among Gram-positive cocci: Member of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcusand Enterococcus are catalase-negative. 2. Catalase test is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive. 3. Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis. 4. Catalase test can be used as an aid to the identification of Enterobacteriaceae. Members of Enterobacteriaceae family are Catalase positive. MATERIAL REQUIRED:

1.BACTERILA CULTURES 2. CAPIILARY TUBE 3.GLASS SLIDES 4.H2O2 SOLUTION 5.DROPPER 3

PROCEDURE OF CATALASE TEST (SLIDE TEST)

1. Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a capillary tube.

2. Place a drop of 3% H2O2 on to the slide and mix. 3. A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling. 4. A negative result is no bubbles or only a few scattered bubbles. 5. Dispose of your slide in the biohazard glass disposal container.

RESULTS: . Catalase Positive reactions: Evident by immediate effervescence (bubble formation) . Catalase Negative reaction: No bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide)

Precautions while performing catalase test

1. Do not use a metal loop or needle with H2O2; it will give a false positive and degrade the metal. 2. If using colonies from a blood agar plate, be very careful not to scrape up any of the blood agar as blood cells are catalase positive and any contaminating agar (Carryover of Red Blood Cells) could give a false positive. 3. Because some bacteria possess enzymes other than catalase that can decompose hydrogen peroxide, a few tiny bubbles forming after 20 to 30 seconds is not considered as positive test.

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EXPERIMENT NO: 2 –OXIDASE TEST 4

INTRODUCTION AND PRINCIPLE

The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the reagent(tetramethyl-p- phenylenediamine) to (indophenols) purple color end product. When the enzyme is not present, the reagent remains reduced and is colorless.

Note: All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration. This does NOT mean that they are strict aerobes. Bacteria that are oxidase-negative may be anaerobic, aerobic, or facultative; the oxidase negative result just means that these organisms do not have the cytochrome c oxidase that oxidizes the test reagent. They may respire using other oxidases in electron transport.

MATERIAL REQUIREMENTS: 1. Moist filter paper with the substrate (1% tetramethyl-p-phenylenediamine dihydrochloride), or commercially prepared paper disk, 2. wooden wire or platinum wire. EXPECTED RESULTS Positive: Development of dark purple color (indophenols) within 10 seconds 1. Negative: Absence of color Related posts: Modified Oxidase Test (Microdase): Principle, Procedure and Uses PROCEDURE

1. Take a filter paper soaked with the substrate tetramethyl-p-phenylenediamine dihydrochloride 2. Moisten the paper with a sterile distilled water 3. Pick the colony to be tested with wooden or platinum loop and smear in the filter paper 4. Observe inoculated area of paper for a color change to deep blue or purple within 10-30 seconds

Precaution to be taken while performing oxidase test: 1. Do not use Nickel-base alloy wires containing chromium and iron (nichrome) to pick the colony and make smear as this may give false positive results 5

2. Interpret the results within 10 seconds, timing is critical Note: The oxidase test must be performed from 5% Sheep blood agar or another medium without a fermentable sugar . Fermentation of a carbohydrate results in acidification of the medium (e.g., lactose in MacConkey Agar or Sucrose in TCBS), and a false negative oxidase test may result if the surrounding pH is below 5.1. During identification of suspected Vibrio cholerae isolate, it is not possible to perform an oxidase test directly from a TCBS culture because the acid produced by the sucrose fermenting colonies will inhibit the oxidase reaction. RESULTS Bacterial genera characterized as oxidase positive include Neisseria and Pseudomonas. Genera of the Enterobacteriaceae family are characterized as oxidase negative. Name of Oxidase positive bacteria are: Mneomoics for Oxidase Positive Organisms- PVNCH ( Its just an acronyms inspired by the famous mneomonic for Urease Positive organisms-PUNCH) 1. P: Pseudomonas spp 2. V: Vibrio cholerae 3. N: Neisseria spp 4. C: Campylobacter spp 5. H: Helicobacter spp/ Haemophilus spp. 6. Aeromonas spp 7. Alcaligens **********************

EXPERIMENT NO:3 INDOLE TEST

INTRODUCTION:

Indole test is used to determine the ability of an organism to split amino acid tryptophan to form the compound indole. Tryptophan is hydrolysed by tryptophanase to produce three possible end products – one of which is indole. Indole production is detected by Kovac’s or Ehrlich’s reagent which contains 4 (p)-dimethylamino benzaldehyde, this reacts with indole to produce a red coloured compound. Indole test is a commonly used biochemical test (eg in IMVIC test, SIM test etc). Indole test helps to differentiateEnterobacteriaceae and other genera.

1. a conventional tube method requiring overnight incubation, which identifies weak indole producing organisms and 2. a spot indole test, which detects rapid indole producing organisms PROCEDURE OF CONVENTIONAL TUBE METHOD FOR INDOLE TEST a. Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism in tryptophan broth. b. Incubate at 37°C for 24-28 hours in ambient air. c. Add 0.5 ml of Kovac’s reagent to the broth culture.

Tryptophan broth Composition:

Ingredients Gms / Litre

Meat peptone 10.000

Sodium chloride 5.000

DL-Tryptophan 1.000

Final pH ( at 25°C) 7.2±0.2

Expected results:

. Positive: Pink colored rink after addition of appropriate reagent . Negative: No color change even after the addition of appropriate reagent. e.g. Klebsiella Pneumoniae .

Indole positive organisms: Most strains of E.coli, P. vulgaris, M. morganii and Providenica are indole positive.

Point to remember: Indole test can also aid in species differentiation.

1. Klebsiella species: Klebsiella oxytoca is indole positive whereas Klebsiella pneumoniae is indole negative. 2. Citrobacter species: Citrobacter Koseri is indole positive where as Citrobacter freundii is indole negative 3. Proteus species: Proteus Vulgaris is indole positive whereas Proteus mirabilis is indole negative

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EXPERIMENT NO:4 METHYL RED ( MR) TEST

INTRODUCTION The methyl red (MR) test detects the production of sufficient acid during the fermentation of glucose and the maintenance of conditions such that the pH of an old culture is sustained below a value of about 4.5, as shown by a change in the colour of the methyl red indicator which is added at the end of the period of incubation.

Clark and Lubs developed MR-VP Broth which allowed both the MR and VP tests to be performed from the same inoculated medium by aliquoting portions to different tubes.

PRINCIPLE Some bacteria have the ability to utilize glucose and convert it to a stable acid like lactic acid, acetic acid or formic acid as the end product.These bacteria initially metabolise glucose to pyruvic acid, which is further metabolized through the ‘mixed acid pathway to produce the stable acid. The type of acid produced differs from species to species and depends on the specific enzymatic pathways present in the bacteria. The acid so produced decreases the pH to 4.5 or below, which is indicated by a change in the colour of methyl red from yellow to red.In the methyl red test (MR test), the test bacteria is grown in a broth medium containing glucose. If the bacteria has the ability to utilise glucose with production of a stable acid, the colour of the methyl red changes from yellow to red, when added into the broth culture.

The mixed acid pathway gives 4 mol of acidic products (mainly lactic and acetic acid), 1 mol of neutral fermentation product (ethanol), 1 mol of CO2, and 1 mol of H2 per mol of glucose fermented. The large quantity of acids produced causes a significant decrease in the pH of the culture medium. MEDIA AND REAGENTS COMPOSITION

Ingredients Gms / Litre

Buffered peptone 7.0

Gucose 5.0

Dipotassium phosphate 5.0 pH 6.9

Methyl red solution, 0.02% 9

a. Dissolve 0.1 g of methyl red in 300 ml of ethyl alcohol, 95%. b. Add sufﬁcient distilled water to make 500 ml. c. Store at 4 to 8 degree C in a brown bottle. Solution is stable for 1 year.

Procedure of Methyl Red (MR) Test 1. Prior to inoculation, allow medium to equilibrate to room temperature. 2. Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium. 3. Incubate aerobically at 37 degrees C. for 24 hours. 4. Following 24 hours of incubation, aliquot 1ml of the broth to a clean test tube. 5. Reincubate the remaining broth for an additional 24 hours. 6. Add 2 to 3 drops of methyl red indicator to aliquot. 7. Observe for red color immediately. RESULT INTERPRETATION Positive Reaction: A distinct red color (A) Examples: E. coli, Yersinia sps, etc.

Negative Reaction: A yellow color (B) Examples: Enterobacter aerogenes, Klebsiella pneumoniae, etc.

A weak positive is red-orange. If an orange color is seen, incubate the remainder of the broth for up to 4 days and repeat the test after further incubation. In this case it may also be helpful to set up a duplicate broth at 25C.

USES OF METHYL RED (MR) TEST Originally the paired MR-VP tests were used to distinguish between members of the family Enterobacteriaceae, but now they are used to characterize other groups of bacteria including Actinobacteria.

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EXPERIMENT NO: 5 VOGES PROSKAUER ( VP) TEST 10

AIM: To differentiate among the enteric organisms such as Escherichia coli, Enterobacter aerogenesand Klebsiella pneumoniae. INTRODUCTION AND PRINCIPLE Differentiation of the principal groups of Enterobacteriaceae can be accomplished on the basis of their biochemical properties and enzymatic reactions in the presence of specific substrates. The IMViC series of tests (indole, methyl red, voges-proskauer, and citrate utilization )can be used. The enteric organisms are subdivided as lactose fermenters and nonfermenters. Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae are lactose fermenters. Salmonella typhimurium, Shigella dysenteriae, Proteus vulgaris, Pseudomonas aeruginosa, Alcaligenes faecalis etc are non lactose fermenters. The Voges-Proskauer (VP) test has found wide acceptance in clinical laboratories as a means of classifying strains of Enterobacteriaceae, based on acetoin production.

The Voges-Proskauer test determines the capability of some organisms to produce non acidic or neutral end products, such as acetyl methyl carbinol, from the organic acids that result from glucose metabolism. The reagent used in this test is Barritt's reagent, consists of a mixture of alcoholic a-naphthol and 40% potassium hydroxide solution. Detection of acetyl methyl carbinol requires this end product to be oxidized to a diacety compound. This reaction will occur in the presence of the a-naphthol catalyst and a guanidine group that is present in the peptone of the MR-VP medium. As a result, a pink complex is formed, imparting a rose color to the medium. Development of a deep rose color in the culture 15 minutes following the addition of Barritt's reagent is indicative of the presence of acetyl methyl carbinol and represents a positive result. The absence of rose color is a negative result.

MATERIALS REQUIRED:

Culture:24-48 hour tryptic soy broth culture. Media: MR-VP medium Composition Reagents: Barritt's reagents A and B. Ingredients Gms / Litre Preparation of Barritt's reagent: Buffered peptone 7.000 Dextrose 5.000 It consists of two solutions; Dipotassium phosphate 5.000 1. Solution A is prepared by dissolving 6 grams of Final pH ( at 25°C) 6.9±0.2 a-naphtholin in 100 ml of 95% ethyl alcohol. 2. Solution B is prepared by dissolving 16 grams of potassium hydroxide in 100 ml of water.

Equipments:

Bunsen burner. Inoculating loop.

PROCEDURE:

Using sterile technique, inoculate each experimental organism to the appropriately labeled tube of medium by means of loop inoculation. Incubate the cultures for 24-48 hours at 37°C. The experiment should be conducted in the LAF. Arrange the materials required for the experiment in the LAF.

1. Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot. Heat from the base of the wire first and slowly move towards the loop (tip). Heat the wire until it is red-hot. 2. From the rack, take the test tube containing the Tryptic Soy Broth(TSB) cultures that has been kept for 24 - 48 hours. 3. Remove the cap from the TSB tube and flame the neck of the tube. 4. Using aseptic technique take a loop full of the organism from the TSB (tryptic soy broth). 5. Again flame the neck of the tube and replace the cap and place the tube in the test tube rack. 6. Take two sterile MR-VP broth tubes, one named Test and the other Control. 7. Remove the cap of the MR-VP broth tube named 'Test' and flame the neck of the tube. 8. Inoculate the MR-VP broth with the inoculation loop containing the inoculum from the TSB. 9. Again flame the neck of the MR-VP tube and place it in the test tube rack. Inoculate only the broth in the tube named 'Test' using aseptic technique. Leave the broth in the tube named 'Control' uninoculated. 10. Incubate both the tubes (Test and Control) for 24 to 48 hours at 37°C. 11. Remove the broths from the incubator. 12. Remove the cap and add 10 drops of Barritt's A reagent and 10 drops of Barritt's B reagent to each broth. 13. Shake gently for several minutes. 14. Red color formation within 15 to 20 minutes is a positive result. No red color formation after 15 to 20 minutes is a negative result.

12 OBSERVATION AND RESULT:

Positive Result:

Glucose ------Glucose Metabolism------> Pyruvic Acid. Pyruvic acid ------> Acetoin. Acetoin + added alpha-naphthol + added KOH = red color.

Negative Result:

Glucose ------Glucose Metabolism------> Pyruvic Acid. Pyruvic acid ------> No Acetoin. No acetoin + added alpha-naphthol + added KOH = copper color.

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EXPERIMENT NO: 6 CITRATE TEST

INTRODUCTION

This test is among a suite of IMViC Tests (Indole, Methyl-Red, Vogues-Proskauer, and Citrate) that are used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae.

PRINCIPLE Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the sole source of nitrogen.

Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for the production of energy. Growth is indicative of utilization of citrate, an intermediate metabolite in the Krebs cycle.

When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6.

MEDIA : SIMMON’S CITRATE AGAR Composition

Ingredients gm/litre

Sodium Chloride 5.0

Sodium Citrate (dehydrate) 2.0

Ammonium Dihydrogen 1.0 Phosphate

Dipotassium Phosphate 1.0

Magnesium 0.2 Sulfate (heptahydrate)

Bromothymol Blue 0.08

Agar 15.0

6.9 +/- 0.2 at 25 Final pH degrees C. PROCEDURE 1. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony. 2. Incubate aerobically at 35 to 37C for up to 4-7 days. 3. Observe a color change from green to blue along the slant. OBSERVATION AND RESULT: Positive Reaction: Growth with color change from green to intense blue along the slant. Examples: Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, Providencia, etc.

Negative Reaction: No growth and No color change; Slant remains green. Examples: Escherichia coli.

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EXPERIMENT NO: 7 UREASE TEST

INTRODUCTION; Urea Agar was developed by Christensen in 1946 for the differentiation of enteric bacilli. The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease.

PRINCIPLE Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change ofphenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours.

MEDIA: Christensen’s Urea Agar Composition

Ingredients Grams/litre

Urea 20.0 gm

Sodium Chloride 5.0 gm

Monopotassium 2.0 gm Phosphate

Peptone 1.0 gm

Dextrose 1.0 gm

Phenol Red 0.012 gm

Agar 15.0 gm

6.7 +/- 0.2 at 250 C. Final pH

PROCEDURE 1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate slant with 1 to 2 drops from an overnight brain-heart infusion broth culture. 2. Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 48 hours to 7 days. 3. Examine for the development of a pink color for as long as 7 days.

Result Interpretation of Urease Test

Positive Reaction: Development of an intense magenta to bright pink color in 15 min to 24 h. Examples: Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicobacter pylori, Yersiniaspp, Brucella spp, etc.

Negative Reaction: No color change. Examples: Escherichia, Shigella, Salmonella, etc.

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EXPERIMENT NO: 8 GELATIN HYDROLYSIS ( LIQUEFACTION) TEST

INTRODUCTION: Gelatin is a protein derived from the animal protein collagen– component of vertebrate connective tissue. It has been used as a solidifying agent in food for a long time. Gelatin hydrolysis test is a great way to highlight proteolysis by bacteria.

PRINCIPLE: Gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinase (proteolytic enzyme) that liquefy gelatin. Hydrolysis of gelatin indicates the presence of gelatinases. This process takes place in two sequential reactions. In the first reaction, gelatinases degrade gelatin to polypeptides. Then, the polypeptides are further converted into amino acids. The bacterial cells can then take up these amino acids and use them in their metabolic processes.

MEDIA :Nutrient Gelatin-Composition

Ingredients Gms / Litre Peptic digest of animal tissue 5.000 Beef extract 3.000 Gelatin 120.000 Final pH ( at 25°C) 6.8±0.2

PROCEDURE

There are several methods for determining gelatinase production, all of which make use of gelatin as the substrate. The standard and most commonly employed method is the nutrient gelatin stab method.

1. Inoculate a heavy inoculum of test bacteria (18- to 24-hour-old) by stabbing 4-5 times (half inch) on the tube containing nutrient gelatin medium. 2. Incubate the inoculated tube along with an uninoculated medium at 35°C, or at the test bacterium’s optimal growth temperature, for up to 2 weeks. 3. Remove the tubes daily from the incubator and place in ice bath or refrigerator (4°C) for 15-30 minutes (until control is gelled) every day to check for gelatin liquefaction.(Gelatin normally

18 liquefies at 28°C and above, so to confirm that liquefaction was due to gelatinase activity, the tubes are immersed in an ice bath or kept in refrigerator at 4°C). 4. Tilt the tubes to observe if gelatin has been hydrolyzed.

Expected results

Negative: Complete solidification of the inoculated tube even after exposure to cold temperature of ice bath or refrigerator (4°C). Tube no:1

Positive: Partial or total liquefaction of the inoculated tube (uninoculated control medium must be completely solidified) even after exposure to cold temperature of ice bath or refrigerator (4°C). ). Tube no:2.

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EXPERIMENT NO:9 TRIPLE SUGAR IRON TEST

INTRODUCTION

Triple sugar iron (TSI) agar is a tubed differential medium used in determining carbohydrate fermentation and

H2S production. Gas from carbohydrate metabolism can also be detected. Bacteria can metabolize carbohydrates aerobically (with oxygen) or fementatively (without oxygen). TSI differentiates bacteria based on their fermentation of lactose, glucose and sucrose and on the production of hydrogen sulfide. TSI is most frequently used in the identification of theEnterobacteriaceae, although it is useful for other gram-negative bacteria.

Theory

TSI contains three carbohydrates: glucose (0.1%), sucrose (1%), and lactose (1%). TSI is similar to Kligler's iron agar, except that Kligler's iron agar contains only two carbohydrates: glucose (0.1%) and lactose (1%). Besides the carbohydrates mentioned, the medium also contains beef extract, yeast extract, and peptones which are the sources of nitrogen, vitamins and minerals. Phenol red is the pH indicator, and agar is used to solidify the medium. During preparation, tubes containing molten agar are angled. The slant of the medium is aerobic, while the deep (or butt) is anaerobic.

When any of the carbohydrates are fermented, the drop in pH will cause the medium to change from reddish- orange (the original color) to yellow. A deep red color indicates alkalization of the peptones. Sodium thiosulfate in the medium is reduced by some bacteria to hydrogen sulfide (H2S), a colorless gas. The hydrogen sulfide will react with ferric ions in the medium to produce iron sulfide, a black insoluble precipitate.

Based on carbohydrate utilization and hydrogen sulfide production, a TSI slant can be interpretted in several ways:

Glucose Fermenter. The tube reaction is alkaline over acid (K/A) signifying that only glucose is metabolized. The bacteria quickly metabolized the glucose, initially producing an acid slant and an acid butt (acid over acid; A/A) in a few hours. The Emben-Meyerhof-Parnas pathway was used both aerobically (on the slant) and anerobically (in the butt) to produce ATP and pyruvate. On the slant, the pyruvate was further metabolized to

CO2, H2O, and energy. After further incubation (18 hours) the glucose was consumed, and because the bacteria could not use lactose or sucrose, the peptones (amino acids) were utilized as an energy source aerobically, on the slant. Utilization of peptones causes the release of ammonia (NH3) increasing the pH resulting in the pH indicator, phenol red, turning from yellow to red. In the anerobic butt, the bacteria use the Embden-Meyerhof-Parnas pathway to metabolize the glucose producing ATP and pyruvate, which is converted into stable acid endproducts,

20 thus the butt remains acidic. The results would be recorded as alkaline over acid (K/A). Bacteria producing a K/A reaction with or without gas include: Citrobacter freundii* , Citrobacter koseri*, and Morganella morganii*. * = variable reactions

Glucose, Lactose and/or Sucrose Fermenter. The tube reaction is acid over acid (A/A) indicating that glucose, lactose and/or sucrose have been metabolized. The bacteria quickly metabolized the glucose, producing an acid slant and an acid butt in a few hours. The Emben-Meyerhof-Parnas pathway is used both aerobically (on the slant) and anerobically (in the butt) to produce ATP and pyruvate. On the slant, the pyruvate is further metabolized to CO2, H2O, and energy. After further incubation (18 hours) the glucose was consumed, and then the bacteria utilized lactose and/or sucrose, maintaining an acid slant. The results are recorded as acid over acid (A/A). If the medium were incubated longer, over 48 hours, the lactose and sucrose would be depleted, and the slant would revert to an alkaline pH due to metabolism of the peptones. In the anerobic butt, the bacteria convert pyruvate into stable acid endproducts, thus the butt remains acidic. The bacteria commonly producing an A/A reaction with or without gas include:Enterobacter aerogenes, E. cloacae, Escherichia coli, Klebsiella oxytoca, and K. pneumoniae.

Glucose, Lactose and Sucrose Nonfermenters. The tube reaction is either alkaline over alkaline (K/K) or alkaline over no change (K/NC) indicating that all three sugars have not been metabolized. The difference between K/K and K/NC) is subtle. Some nonenteric bacteria, such as the pseudomonads, are unable to ferment glucose, lactose, or sucrose. These bacteria derive energy from peptones either aerobically or anaerobically. Utilization of peptones causes the release of ammonia (NH3) resulting in the pH indicator, phenol red, turning from pink to red. Nonglucose fermenters can produce two possible reactions. If the bacteria can metabolize peptones both aerobically and anaerobically, the slant and butt will be red (alkaline over alkaline; K/K). If peptones can only be metabolized aerobically, the slant will be red and the butt will exhibit no change (K/NC). Bacteria producing K/K or K/NC include: Acinetobacter spp. andPseudomonas spp.

Gas Production. Gas production (CO2 and O2) is detected by splitting of the agar. In some cases, so much gas is produced that the agar is pushed to the top of the tube. Bacteria commonly producing an A/A reaction with gas include: Enterobacter aerogenes, E. cloacae, Escherichia coli, Klebsiella oxytoca, and K. pneumoniae. However, some strains do not produce gas.

Glucose Fermenter and Hydrogen Sulfide Production. The tube reaction is alkaline over acid (K/A) with black precipitate. The bacteria quickly metabolized the glucose, initially producing an acid slant and an acid butt (acid over acid; A/A) in a few hours. The Emben-Meyerhof-Parnas pathway is used both aerobically (on the slant) and anerobically (in the butt) to produce ATP and pyruvate. On the slant, the

21 pyruvate is further metabolized to CO2, H2O, and energy. After further incubation (18 hours) the glucose was consumed, and because the bacteria could not use lactose or sucrose, the peptones (amino acids) were utilized as an energy source aerobically, on the slant. Utilization of peptones causes the release of ammonia

(NH3) resulting in the pH indicator, phenol red, turning from yellow to red. In the anerobic butt, the bacteria use the Embden-Meyerhof-Parnas pathway to metabolized the glucose producing ATP and pyruvate, which is converted into stable acid endproducts, thus the butt remains acidic.

The black precipitate indicates that the bacteria were able to produce hydrogen sulfide (H2S) from sodium thiosulfate. Because H2S is colorless, ferric ammonium citrate is used as an indicator resulting in the formation of insoluble ferrous sulfide. Formation of H2S requires an acidic environment; even though a yellow butt cannot be seen because of the black precipitate, the butt is acidic. The results would be recorded as alkaline over acid (K/A), H2S positive. Bacteria producing a K/A with H2S include: Citrobacter freundii*, Edwardsiella tarda, Proteus mirabilis*, and Salmonella spp*. Bacteria commonly producing an A/A with H2S include:Citrobacter freundii*, Proteus mirabilis*, and P. vulgaris*. * = variable reactions

Glucose, Lactose and/or Sucrose Fermenter and Hydrogen Sulfide Producer. The tube reaction is acid over acid (A/A) with black precipitate. The bacteria quickly metabolized the glucose, producing an acid slant and an acid butt (acid over acid; A/A) in a few hours. The Emben-Meyerhof-Parnas pathway is used both aerobically (on the slant) and anerobically (in the butt) to produce ATP and pyruvate. On the slant, the pyruvate is further metabolized to CO2, H2O, and energy. After further incubation (18 hours) the glucose was consumed, and then the bacteria utilized lactose and/or sucrose, maintaining an acid slant. The results are recorded as acid over acid (A/A). If the medium were incubated longer, over 48 hours, the lactose and sucrose would be depleted, and the slant would revert to an alkaline pH due to metabolism of the peptones. In the anerobic butt, the bacteria convert pyruvate into stable acid endproducts, thus the butt remains acidic.

The black precipitate indicates that the bacteria were able to produce hydrogen sulfide (H2S) from sodium thiosulfate. Because H2S is colorless, ferric ammonium citrate is used as an indicator resulting in the formation of insoluble ferrous sulfide. Formation of H2S requires an acidic environment; even though a yellow butt cannot be seen because of the black precipitate, the butt is acidic. The results would be recorded as acid over acid (A/A), H2S positive. Bacteria commonly producing an A/A with H2S include: Citrobacter freundii*, Proteus mirabilis*, and P. vulgaris*. * = variable reactions

Glucose Nonfermenter Hydrogen Sulfide Producer. The tube appears as alkaline over no change (K/NC) with a black precipitate (H2S). The reduction of thiosulfate in KIA and TSIA requires H+. Nonfermenters cannot produce an acid environment from the fermenation of the carbohydrates. Cysteine and perhaps

22 other organic sulfate molecules are metabolized to pyruvic acid, ammonia, and H2S. Nonfermentative H2S positive reaction is strongly suggestive of members of the genus Shewenella.

MEDIA: COMPOSITION

INGREDIENTS GMS/LITRE

Pancreatic digest of casein USP 10.0 g Peptic digest of animal tissue USP (see 10.0 g Note) Glucose 1.0 g Lactose 10.0 g Sucrose 10.0 g Ferrous sulfate or ferrous ammonium 0.2 g sulfate NaCl 5.0 g Sodium thiosulfate 0.3 g Phenol red 0 .024g Agar 13.0 g Distilled water 1,000 mL pH 7.3.

Ingredients Gms / Litre Peptone, special 10.000 Sodium chloride 5.000 Bromo cresol purple Final pH ( at 25°C) 6.8±0.2

Note: The following combination of ingredients can substitute for the first two components listed: beef extract, 3.0 g; yeast extract, 3.0 g; and peptone, 20.0 g. Combine ingredients, and adjust the Boil to dissolve the agar, and dispense into tubes. Sterilize by autoclaving at 121°C for 15 min. Cool in a slanted position to give a 2.5-cm butt and a 3.8-cm slant.

PROCEDURE:

1.Use a straight inoculating needle to pickup an isolated colony.

2.Inoculate the TSI slant by first stabbing the butt down to the bottom, withdraw the needle, and then streak the surface of the slant. Use a loosely fitting closure to permit access of air.

Read results after incubation at 37°C for 18 to 24 h. Three kinds of data may be obtained from the reactions.

OBSERVATION AND RESULT

Sugar fermentations Acid butt, alkaline slant (yellow butt, red slant): glucose has been fermented but not sucrose or lactose. Acid butt, acid slant (yellow butt, yellow slant): lactose and/or sucrose has been fermented. Alkaline butt, alkaline slant (red butt, red slant): neither glucose, lactose, nor sucrose has been fermented.

Gas production

Indicated by bubbles in the butt. With large amounts of gas, the agar may be broken or pushed upward.

Hydrogen sulfide production

Hydrogen sulfide production from thiosulfate is indicated by a blackening of the butt as a result of the reaction of H2S with the ferrous ammonium sulfate to form black ferrous sulfide.

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EXPERIMENT NO:10 β-GALACTOSIDASE ( ONPG) TEST

INTRODUCTION

On hydrolysis, through the action of the enzyme β-galactosidase, ONPG cleaves into two residues, galactose and o-nitrophenol. ONPG is colorless compound: O-nitrophenol is yellow, providing visual evidence of hydrolysis. Lactose fermenting bacteria posses both lactose permease and β-galactosidase, two enzymes required for the production of acid in the lactose fermentation test. The permease is required for the lactose molecule to penetrate the bacterial cell where the β-galactosidase can cleave the galactoside bond, producing glucose and galactose. Non-lactose fermenting bacteria are devoid of both enzymes and are incapable of producing acid from lactose. Some bacterial species appear to be non-lactose fermenters because they lack permease, but do possess β- galactosidase and give a positive ONPG test. So called late lactose fermenters may be delayed in their production of acid from lactose because of sluggish permease activity. In these instances, a positive ONPG test may provide a rapid identification of delayed lactose fermentation.

ONPG Test results Vs. Lactose fermentation: 1. Lactose fermenter (ONPG Positive): E.coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and permease 2. Late lactose fermenter (ONPG Positive): Citrobacter spp, Arizona spp produce only β-galactosidase so they slowly ferment lactose. 3. Non lactose fermenter (ONPG Negative): Salmonella spp; Shigella spp; Proteus spp;Providencia spp and Morganella spp do not produce β-galactosidase so can not ferment lactose. MEDIA AND REAGENTS 1. Sodium phosphate buffer, 1 M, pH 7.0 2. O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M 3. Physiologic saline 4. Toulene PROCEDURE Bacteria grown in medium containing lactose (to induce the production of the galactosidase enzyme) , such as Kligler iron agar (KIA) or Triple sugar Iron (TSI) agar, produces optimal results in ONPG Test. Note: β-galactosidase enzyme (inducible enzyme) is made ONLY in the presence of the lactose substrate 1. A loopful of bacterial growth is emulsified in 0.05mL of physiologic saline to produce a heavy suspension

25 2. One drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the enzyme for bacterial cells. 3. An equal quantity of buffered ONPG solution is added to the suspension. 4. The mixture is placed in a 37oC water bath 5. When Using ONPG Tablets 6. A loopful of bacterial suspension is added directly to the ONPG substrate resulting from adding 1mL of distilled water to a tablet in a test tube. 7. This suspension is also placed in a 37oC water bath

Results and Interpretations:

The rate of hydrolysis of ONPG to o-nitrophenol may be rapid for some organisms; producing a visible yellow color reaction within 5 to 10 minutes. Most tests are positive within 1 hour; however, reactions should not be interpreted as negative before 24 hours of incubation.

The yellow color is usually distinct and indicates that the organism has produced o-nitrophenol from the ONPG substrate through the action of β-galactosidase

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EXPERIMENT NO: 11 CARBOHYDRATE FERMENTATION TEST

INTRODUCTION AND PRINCIPLE

Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate. Fermentation patterns can be used to differentiate among bacterial groups or species (. For example, all members of the Enterobacteriaceae family are classified as glucose fermenters because they can metabolize glucose anaerobically . Within this family however, maltose fermentation differentiates Proteus vulgaris(positive) from Proteus mirabilis ( negative).

While fermentation tests can be performed on microorganisms other than bacteria, this protocol only addresses fermentation of carbohydrates by bacteria. Theory

During the fermentation process, an organic substrate serves as the final electron acceptor . The end-product of carbohydrate fermentation is an acid or acid with gas production.Various end-products of carbohydrate fermentation can be produced. The end-product depends on the organisms involved in the fermentation reaction, the substrate being fermented, the enzymes involved, and environmental factors such as pH and temperature. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen .

Fermentation reactions are detected by the color change of a pH indicator when acid products are formed. This isaccomplished by adding a single carbohydrate to a basal medium containing a pH indicator. Because bacteria can also utilize peptones in the medium resulting in alkaline by-products, the pH changes only when excess acid is produced as a result of carbohydrate fermentation .

Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests because most of the endproducts of carbohydrate utilization are organic acids . However, other pH indicators such as bromocresol/bromcresol purple, bromothymol/bromthymol blue, and Andrade’s can be used (Table 1).

Table 1 pH indicators for carbohydrate fermentation media

pH Uninoculated Acid Alkaline indicator media (fermentation) (negative) pH Color pH Color pH Color

7.1- 12.0- Yellow, Andrade’s Light pink 5.0 Pink-red 7.2 14.0 colorless

Bromocresol Deep 26 7.4 5.2 Yellow 6.8 Purple

purple purple Deep Bromothymol 7.0 Green 6.0 Yellow 7.6 Prussian blue blue

Reddish- Phenol red 7.4 6.8 Yellow 8.4 Pink-red orange

Fermentation tubes or Durham tubes are used to detect gas production (6, 8, 9). These small, slender test tubes (6 by 50 mm) are inserted upside down inside larger (13 by 100 mm) test tubes. After sterilization, Durham tubes become filled with the media. If gas is produced, it will be trapped inside the Durham tube and is evident by the presence of a visible air bubble.

Three characteristic reactions can be observed from the fermentation of a specific carbohydrate. Based on these reactions, bacteria are classified as: . Fermenter with acid production only . Fermenter with acid and gas production . Nonfermenter

28 RECIPE Phenol red carbohydrate broth Trypticase or proteose peptone no. 3 10 g Sodium chloride (NaCl) 5 g Beef extract (optional) 1 g Phenol red 0.018 g (7.2 ml of 0.25% phenol red solution) Distilled and deionized water 1,000 ml Carbohydrate 10 g

Prepare broth medium by mixing all ingredients in 1,000 ml of distilled and deionized water; heat gently to dissolve Use a single carbohydrate for each batch of medium prepared. Fill -100-mm test tubes with 4 to 5 ml of phenol red carbohydrate broth . Insert an inverted fermentation tube or Durham tube to detect gas production. Sterilize media by autoclaving for 15 minutes at 116 to 118°C (8, 13). The sterilization process will also drive the broth into the inverted fermentation tube or Durham tube. When using arabinose, lactose, maltose, salicin, sucrose, trehalose, or xylose, autoclave at 121°C for only 3 minutes as these carbohydrates are subject to breakdown by autoclaving . Broth medium will be a light red color. The final pH should be 7.4 ± 0.2.

Alternatively, mix all ingredients except carbohydrate in 800 ml of distilled and deionized water to prepare the base broth. Heat while mixing. Fill 100-mm test tubes with 4.5 ml of base broth. Insert an inverted fermentation tube or Durham tube to detect gas production. Sterilize base broth by autoclaving for 15 minutes at 121°C (8, 13). Cool sterilized base broth in a 42 to 50°C water bath before adding carbohydrates. Prepare carbohydrate solution by dissolving 10 grams of desired carbohydrate in 200 ml of distilled and deionized water and sterilize by filtering the solution through a bacteria-retaining membrane filter with a 0.45-µm pore size . Aseptically add 0.5 ml of filtrate to each tube of sterilized and cooled broth. Shake gently to mix. Broth medium will be a light red color. The final pH should be 7.4 ± 0.2. Cool all media before use. Store prepared media at 4 to 10°C (4). Refrigerated media has a shelf life ofapproximately 6 to 8 weeks (8). Run controls to check for possible breakdown of carbohydrates. BROMOCRESOL PURPLE CARBOHYDTRTE BROTH Ingredients Gms / Litre Peptone, special 10.000 Sodium chloride 5.000 Bromo cresol purple 0.020 Final pH ( at 25°C) 6.8±0.2

29 **Formula adjusted, standardized to suit performance parameters Directions Suspend 15.02 grams in 1000 ml distilled water. Add 5 - 10 grams of the carbohydrate to be tested. Heat if necessary to dissolve the medium completely. Dispense in tubes, containing inverted Durhams tubes as desired and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Alternatively sterilize the basal medium prepared using 900 ml distilled water and add 100 ml separately sterilized 5 - 10% solution of the desired carbohydrate to it. Recipe notes Generally, the carbohydrate concentration used for carbohydrate fermentation testing is 0.5% or 1.0%. The recipe provided will produce a 1.0% carbohydrate broth. It is preferred to use a 1.0% carbohydrate concentration to prevent the reaction from reverting due to the rapid depletion of the carbohydrate by some microorganisms (3).

A modified Andrade’s pH indicator is recommended instead of the phenol red pH indicator when prolonged incubation is required (Streptococci and Enterococci) or when testing aerobic Gram-negative bacilli such as those in the Enterobacteriaceae family (6, 8).

Add NaCl for a final concentration of 2% to 3% when preparing fermentation media for fermentation studies on halophilic Vibrio species (13).

For fermentation studies on Neisseria species enrich broth by adding 5% sterile rabbit serum (3) or use 28 cystine-tryptic agar (a semisolid medium with only 0.25% agar) for the base broth (10, 14). Due to the semisolid nature of cystine-tryptic agar medium, the entire medium may not turn yellow. Therefore, the appearance of any yellow color is indicative of a positive reaction. Please note that the color change may only occur at the top of the medium. If no color change is evident at 24 hours, recheck results at 48 hours and then again at 72 hours.

PROTOCOL

A. Inoculation of media

Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop. Make sure to avoid the Durham tube, if present. Swirl the tube gently to mix contents. Avoid contact of liquid with tube cap (8).

Alternatively, inoculate each test tube with 1 to 2 drops of an 18- to 24-hour broth culture of the desired organism (6, 8). Examples of broth media that could be used include brain heart infusion, nutrient agar, and tryptic soy agar.

B. Incubation

Incubate tubes at 35 to 37°C for 18 to 24 hours (6, 8). Longer incubation periods may be required to confirm a negative result (6, 8).

C. Interpretation of results

1. Fermentation results

When using phenol red as the pH indicator, a yellow color indicates that enough acid products have been produced by fermentation of the sugar to lower the pH to 6.8 or less. A delayed fermentation reaction may produce an orange color. In such cases, it is best to reincubate the tube (8). Refer to Table 1 for other commonly used pH indicators and their corresponding colors.

2. Gas production results

Bubbles trapped within the Durham tube indicate the production of gas. Even a single bubble is significant and denotes evidence of gas production (6, 8). No bubbles within the Durham tube indicate a non-gas-producing or anaerogenic organism (8).

3. Negative results

A reddish or pink color indicates a negative reaction (8). In negative tubes, the presence of turbidity serves as control for growth. A reddish or pink color in a clear tube could indicate a false negative. 29 Picture-1:Peptone media with bromocresol purple indicator. From left to right: uninoculated tube; E. coli, glucose fermenter with gas production (visible air bubble in the inverted Durham tube); S. sonnei, glucose fermenter without gas production (no visible air bubble in the inverted Durham tube); P. aeruginosa, nonfermenter.

Picture:2: Peptone media with phenol red indicator. From left to right: uninoculated tube; Escherichia coli, a glucose fermenter with gas production (visible air bubble in the inverted Durham tube); Shigella sonnei, a glucose fermenter without gas production (no visible air bubble in the inverted Durham tube); Pseudomonas aeruginosa nonfermenter.

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EXPERIMENT NO 12 -ESTIMATION OF PROTEIN BY LOWRY’S METHOD

Aim: To estimate the amount of protein in the given sample by Lowry’s method.

PRINCIPLE: The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction of the FolinCiocalteay phosphomolybdic phosphotungstic acid to heteropolymolybdenum blue by the copper- catalyzed oxidation of aromatic acids [Dunn, 13]. The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained at 10 - 10.5. The Lowry method is sensitive to low concentrations of protein. Dunn [1992] suggests concentrations ranging from 0.10 - 2 mg of protein per ml while Price [1996] suggests concentrations of 0.005 - 0.10 mg of protein per ml. The major disadvantage of the Lowry method is the narrow pH range within which it is accurate. However, we will be using very small volumes of sample, which will have little or no effect on pH of the reactionmixture. A variety of compounds will interfere with the Lowry procedure. These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents [Dunn, 1992]. Price [1996] notes that ammonium ions, zwitter ionic buffers, nonionic buffers and thiol compounds may also interfere with the Lowry reaction. These substances should be removed or diluted before running Lowry assays.

REAGENTS

A. 2% Na2CO3 in 0.1 N NaOH

B. 1% NaK Tartrate in H2O

C. 0.5% CuSO4.5 H2O in H2O

D. Reagent I: 48 ml of A, 1 ml of B, 1 ml C

E. Reagent II- 1 part Folin-Phenol [2 N]: 1 part water BT 0413 – Bioseparation f.BSA Standard - 1 mg/ ml

PROCEDURE:

1 2 3 4 5 6 7 8 Volume Conc. of Vol. of Alkali Folin Read OD of stock protein water Reagent Ciocalteau @660nm Protein ( µg) Reagent Solution in ml BLANK 00 1ml 5ml KEEP ALL 0.5ml 0.2ml 40 µg 0.8ml 5ml TUBES 0.5ml KEEP TUBE 0.4ml 80 µg 0.6ml 5ml AT 0.5ml AT ROOM ROOM 0.6ml 120.0 µg 0.8ml 5ml 0.5ml TEMP.FOR TEMP 0.8ml 160 µg 0.2ml 5ml 0.5ml 30 MINUTES 1ml 200 µg 00ml 5ml FOR 10 0.5ml MINUTES

1. Set up the tubes of varying concentration of protein solution as shown in the column 1 of the table above.

2. Make the total volume 1ml with water.

3. Add 5ml of alkali reagent to all tubes (Column 4).

4. Maintain tubes at room temperature for 10 minutes.

5. Add 0.5ml of Folin reagent (Column 6). To all tubes

6. Keep all tubes at room temperature for 30 minutes.

7.Measure the absorbance at 660 nm against reagent blank.

8. Plot a graph of protein concentration on x axes and absorbance at y axes. This is standard calibration curve.

9. Determine the value of glucose concentration in unknown samples using the standard calibration curve.

CALCULATIONS: Result: The amount of protein present in the given sample was found to be …………

EXPERIMENT NO:13 ESTIMATION OF REDUCING SUGARS BY DINTROSALICYLIC ACID (DNS) COLORIMETRIC METHOD

Aim: To Estimate the Amount of Sugar Colorimetrically By DNSA Method.

THEORY: This method tests for the presence of free carbonyl group (C=O), present in the so called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5- dinitrosalicylic acid (DNS) is reduced to 3-amino,5- nitrosalicylic acid under alkaline conditions which is red brown in colour with absorbance maxima at 540 nm oxidation aldehyde group ------> carboxyl group reduction 3,5-dinitrosalicylic acid ------> 3-amino,5- nitrosalicylic acid

REQUIREMENTS:

Equipment and glassware: Test tubes, pipette, Colorimeter

Reagents: Dinitrosalicylic Acid Reagent Solution: Mix 100 ml of 5% (w/v) 3,5-dinitrosalicylic acid in 2 M NaOH with 250 ml of 60% (w/v) sodium potassium tartrate and make the total volume up to 500 ml with distilled water.

PROCEDURE:

I 2 3 4 5 6 7 Sugar Conc Water DNS OD @540nm Water solution Blank 00 1ml 1ml KEEP TUBE 5ml IN BOILING 0.2ml 200 µg 0.8ml 1ml WATER 5ml BATH FOR

0.4ml 400 µg 0.6ml 1ml 10 5ml MINUTES 0.6ml 600 µg 0.4ml 1ml 5ml 0.8ml 800 µg 0.2ml 1ml 5ml 1ml 1000µg 00 1ml 5ml

1. Prepare 100ml glucose stock solution of 10mg/ml concentration. 2. Set up tubes of varying concentration of glucose as shown in the column 1 of the table above. 3. Make the total volume to 1ml with water(Column 3). 4. Add 1 ml DNA reagent to all tube. 5. Keep tubes in boiling water bath for 10 minutes.

6. Cool the tubes and add 5ml of water to all tubes (Column 6). 7. Measure the absorbance at 540 nm against reagent blank.

8. Plot a graph of glucose concentration on x axes and absorbance at y axes. This is standard calibration curve.

9. Determine the value of glucose concentration in unknown samples using the standard calibration curve.

Precautions: DNS reagent is corrosive, so handle it with care.

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