Could reliable results be obtained if TSI agar were inoculated with a sample dir
ID: 97587 • Letter: C
Question
Could reliable results be obtained if TSI agar were inoculated with a sample directly from a fecal suspension? Explain. Summarize the chemistry of the tests that make up the IMViC series How might the IMViC series be of value in the bacteriological analysis of water? List the special procedures that must be used when working with TSI agar, and indicate why each is necessary. Which procedures in this exercise may be used to isolate Escherichia coli from a fecal suspension and verify its presence?Explanation / Answer
2. TSI agar test is not intended for primary isolation of specimens. It provides information about differentiation of Enterobacteriaceae present in the specimen based on the ability to ferment glucose, lactose, and sucrose, and their ability to produce hydrogen sulfide. The fecal suspension contains several microbes. Thus a direct culture from the fecal suspension provides a wrong test results. Only isolated organism should be used in these tests. To identify the cultured organism other biochemical tests required.
3. Chemistry of IMViC tests:
IMViC tests are performed to identify the coliform group of microorganisms. Coliform is a aerobic, gram negative, facultative anaerobic rod shaped microorganism which generates carbon di oxide gas from lactose. It is mostly found in fecal matters.
.IMViC stands for Indole , methyl red, Voges –Proskauers, and citrate test.
Indole test: Test organism grown in a peptone water broth which contains Tryptophan. Tryptophanase converts tryptophan to indole , pyruvate amd ammonium. After extraction with xylene product is subjected to the Kovac’s reagent which gives a red-pink colour upon reaction with indole molecule on top of liquid.
Methyl red and Voges proskauer test:
Test organism is grown in the same broth called MRVP broth and use for two separate assays. Methyl red assay indicates amount of acid produced during growth. Mixed acid fermentation pathway used to detect the acid content. Methyl red acts as a pH indicator, upon addition if solution turns red then mixed acid fermetaion is used thus result is positive whereas the yellow colour represents the negative result where butanediol fermentation is used.
In the VP test presence of acetylmethylcarbinol detected by -napthol and KOH. After addition of reagents the mixture is vigorously shaked which gives pink-red coloued solution when the result is positive. Acetylmethylcarbinol is a intermediate of 2,3-butanediol fermentation pathway. Positive result indicated usage of this pathway.
Citrate test: Test organism grown in Simmon's citrate agar to check whether it can use citrate as sole Carbone source or not. Agar contains citrate and ammonium ions and bromophenol blue. If citrate is used up by the organism then green colour agar turns blue.
Water is the most important thing to human body and plays a central role in the regulation of all physiological activities and metabolic activities. However, if water is fecally polluted it causes diseases in people to a great number. Coliform group of micro organism caused several diseases. Thus they can be allowed in drinking water. So these tests are used to check the quality of drinking water before supply.
4. Precautions taken at time of TSI assay:
· a. TSI agar must be treated as potentially infectious because it contains components of animal origin. Thus there is chance of presence of transmissible pathogenic agents with in it. During handing TSI agar universal blood precautions procedures must be followed.
· b. Never allow TSI agar to came in contact with skin. Never inhale or ingest it.
· c. Only adequately trained and qualified laboratory personnel should handle these agars under proper laboratory condition. Dispose of agar must follow proper guideline of biohazards disposal procedure.
5. Samples are inoculated in the MacConkey broth for enrichment. Incubate the culture for 24 hours at 37C. After that the culture streak on the MacConkey agar and again incubates for 24 hours at 37C. Pink colonies are isolated and cultures on the Eosin Methelene Blue (EMB) agar. After incubation colonies which are produce greenish metallic sheen on EMB agar were considered as E.Coli. colonies.