Biochemical Tests for Streptococcus Organisms

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BACKGROUND

Streptococcus organisms are Gram positive, microaerophilic and non-motile bacteria. There exist several sources of streptococcus organisms that include humans as well as many animals where they mostly form colonies in the mucosal surface of the mouth, pharynx, etc. When they are found in drinking water then the water is aid to be contaminated with fecal matters. Some of the food materials that may be infected with streptococcus are milk, egg, ground ham etc. Some of the foods which are also left for a long time at room temperature before eating are also infected with streptococcus organisms.

So, the basic objective of this test is to study about various biochemical tests for Streptococcus organisms.

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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

           DN  ase 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

Hemolysis

Take TSA plate with 5% sheep blood and streak it with pure and isolated colonies of the organisms. Then incubate the plate at 35oC for about 24 hours. If a clear zone is formed around the colony then it beta-hemolytic. A streptococcus organism is beta-hemolytic.

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. Staphylococcus 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 28^oC, 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. Staphylococcus is gelatin positive.

H₂S production

Take a labelled tube containing SIM medium and inoculate the organism by stabbing.¹ Then incubate these tubes at 37^oC 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. Staphylococcus 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 37⁰C.¹ 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. Staphylococcus 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 37^oC 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. Staphylococcus 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 0.6ml 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^. Staphylococcus 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 37^oC for about 24 hours.¹ Then 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. Staphylococcus is citrate positive.²

Urease test

Prepare urea agar slant and streak in its surface with isolated colonies of the staphylococcus 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. Staphylococcus 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 37^oC 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. Staphylococcus 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. Staphylococcus organism gives a positive reaction for nitrate reduction test.

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CONCLUSION

The above biochemical test can help us in easy detection or identification of streptococcus organisms.

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REFERENCES

1. Adams, S. E., A. J. Theobald, N. M. Jones, M. G. Brading, T. F. Cox, A. Mendez, D. M. Chesters, D. G. Gillam, C. Hall, and J. Holt. The effect of a toothpaste containing 2% zinc citrate and 0.3% Triclosan on bacterial viability and plaque growth in vivo compared to a toothpaste containing 0.3% Triclosan and 2% copolymer. Int. Dent. J. 2003;53:398-403.
   
2. Ajdic, D., W. M. McShan, R. E. McLaughlin, G. Savic, J. Chang, M. B. Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Lin, Y. Qian, S. Li, H. Zhu, F. Najar, H. Lai, J. White, B. A. Roe, and J. J. Ferretti. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA 99:14434-14439.