0 DEPARTMENT OF MICROBIOLOGY MICROBIOLOGY LABORATORY MANUAL 3RD SEMESTER 1 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 ************** 2 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. ************** 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 ********************** 6 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 7 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 ********************* 8 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