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BACKGROUND
Enteric bacteria are generally found in the intestines of human and many animals. Sometimes it may be harmless and sometimes harmful.
The objective of this test is to study the biochemical property of enteric bacteria.
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REQUIREMENTS
Media: Nitrate Broth with upturned Durham’s tube
MR-VP Broth
Nutrient gelatin medium
SIM media- 20 g pancreatic digest of casein+ 6.1 g peptic digest of animal tissue+ 3.5 g Agar+0.2 g
Fe(NH4)2(SO4)2·6H2O + 0.2 g Na2S2O3·5H2O
Simmon citrate agar
DNase Agar
Urea Agar
Trypticase soy agar plates containing 5% sheep blood
Water
Reagents: Sulphalinic acid reagent,
Alpha naphthylamine reagent
Zinc dust
Baritt reagent A – 5G Alpha naphthol in 100ml absolute ethyl alcohol
Baritt reagent B – Potassium Hydroxide 400gm in 1000ml Distilled water
Methyl red reagent
3% hydrogen peroxide
Miscellaneous: Inoculating loop
Burner
Dropper
Test tubes
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PROCEDURE
Motility
Prepare a broth culture of the organisms and put it in the slide. Then put a cover slip over it and invert it and then observe it under microscope. The enteric bacteria show flagellar motility.
Catalase test
Take about 4-5 drops of 3% hydrogen peroxide in a test tube. With the help of inoculating tube, take a little amount of 24 hours culture of test organism and dip it in the test tube. Then keep this test tube in a dark background and see if there is any bubble formation in the tip of inoculation tube. If bubble is formed then it is catalase positive and if no bubble formation then it is catalase negative. Enteric organisms gives negative test for Catalase test.1
Gelatin hydrolysis
Take nutrient gelatin medium in a tube and to it add the test bacteria to it by stabbing. In another tube take nutrient gelatin medium and leave it uninoculated for standard.1 Then incubate both in a incubator for about 2 weeks. Up to then everyday bring out the tubes and keep it in ice bath or refrigerator for about 15-30 min for checking gelatin liquefaction. The purpose of keeping it in refrigerator is because in general gelatin liquefies at 28oC, therefore to confirm that it is liquefying because of the bacteria it is kept at a low temperature in a refrigerator. You can confirm hydrolyzation by tilting the tubes a bit.2 So, if there is complete or partial liquefaction in the tube even after keeping in refrigerator, then gelatin hydrolysis is confirmed. Enterics are gelatin positive.
H2S production
Take a labelled tube containing SIM medium and inoculate the organism by stabbing.1 Then incubate these tubes at 37oC for about 24-48 hours. Then observe for the formation of black precipitation. If there is blackening of medium then, positive result and if no blackening of the medium then negative result. Enteric bacteria gives a negative reaction for H2S production.
Indole
Take the bacterial sample and inoculate it carefully in tryptone broth. Then incubate it at a temperature of 370C1.To this add about some drops of the Kovac reagent. Be careful while observing without shaking the tube, as it may disturb the experiment. If a red colour or pink colour ring is formed in the top then it is taken as indole positive and if there is no colour change after adding Kovac’s reagent, then the bacterium is indole negative. Enteric is indole negative.
Methyl red test
In two test tubes take freshly prepare MR-VP medium. To one of the test tube inoculate the sample microorganism, with the help of an inoculating loop. The other test tube containing the broth is used as a standard. Then keep both the tubes in incubator at 37oC for about 24-48 hours for proper growth of organism. After incubation, bring both tubes and to it add about 4-5 drops of Methyl Red reagent. Now leave for some time undisturbed and observe for the colour change. In the standard test tube if there is no colour change, then there is no contamination and vice-versa. If red colour is formed then it is MR positive. If no colour change then MR negative. Enteric is MR positive.
Vogues Proskauer test
Take the pure culture of the test organism and inoculate it in MR-VP broth. Then keep it for incubation for about 24 hours at a temperature 350C. After bringing out the inoculated broth from incubator, take some clean test tubes and to it add 1ml of broth.1 Then to it add 0.6 ml of 5% alpha naphthol, then 0.2 ml of 40% KOH. Then shake the test tube well and then allow the tube to remain stagnant for about 10-15 min. If after 15 min red colour appears, it indicates the presence of diacetyl and if no colour change then negative test. Enteric is VP negative.
Citrate test
Prepare Simmons citrate agar medium and autoclave it. Then after it becomes little cool, prepare the slant. One for control and another for inoculating organism. Then in the test tube for inoculation takes small amount of organism in the inoculating loop and streak it in the slant agar. Then incubate it at 37oC for about 24 hours. 1Then after incubation observe for the colour observation. If the colour changes to blue colour then it is citrate positive, if no colour change then it is citrate negative. Enteric is citrate positive.2
Urease test
Prepare urea agar slant and streak in its surface with isolated colonies of the Enteric organism. Incubate the slant in a incubator for about 48 hours at a temperature of 350C to 370C. Then after observe for colour changes. If the colour changes to pink colour then it is urease positive and if there is no colour change then it is urease negative. Enteric gives a negative reaction for urease.2
DNase test
Prepare DNase agar plate and inoculate it with a loop of test organism in a small area in the plate. Incubate this plate in incubator at a temperature of 37oC for about 18-24 hours. If the medium around the inoculum becomes colorless then it is DNase positive and if there is no colour change around the inoculum then it is DNAse negative. Enteric organism gives a positive reaction for DNase test.2
Nitrate reduction test
Prepare nitrate broth in test tube and inoculate the test organism in this under aseptic conditions. Then incubate it at required temperature for about 24-48 hours. Then to broth add each sulfanilic acid 1 drop and alpha-naphthylamine one drop. If at this stage there is formation of red coloration, then it is nitrate reduction test positive and you can stop the test here. If there is no formation of red colour, then you can go to next step. Take small amount of zinc and add to each broth. Addition of zinc catalyzes the reduction of nitrate to nitrite. If red colour is formed then nitrate reduction test positive and no colour change then negative test results. Enteric organism gives a positive reaction for nitrate reduction test.
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CONCLUSION
This tests forms a strong base for identification of enteric bacteria.
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REFERENCES
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Michael j. Pelczar., ECS Chan, Noel R. Krieg. McGraw company New York Fifth edition.1986: 597.
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Isenberg H. D., and L. H. Sundheim. Indole reactions in bacteria. J. Bacteriol.1958; 75:682–690.
