uji imvic ; uji biokimia terhadap mikroorganisme oleh ogi nurhariDeskripsi lengkap
IMVIc Test B.K.K.K.Jinad GS/M.Sc./FOOD/36 08/08 asa
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Introduction The IMVIC tests are used to differentiate the enteric for many years. Identification of enteric bacilli is of prime importance in controlling intestinal infections by preventing contamination of food and water supplies. The groups of bacteria that can be found in the intestinal tract of humans’and lower mammals are classified as member of the familyEnterobacteriaceae. This family includes 1. Pathogens
: e.g. Salmonella and Shigella
2. Occasional pathogens
: e.g. Proteus and Klebsiella
3. Normal intestinal flora
: e.g. Escherichia and Enterobacter
IMVIC represents the first letter of each individual test within the series: 1. Indole test 2. Methyl Red test 3. Voges-Proskauer Test 4. Citrate test
5.1. Indole Test Tryptone is an essential amino acid that can undergo oxidation by the enzymes tryptophanase. Some microorganisms can oxidize tryptophan and this can be used as a biochemical marker. Tryptophanase Tryptophan
Indole + pyruvic acid + Ammonia
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The medium used for this test is tryptone broth. Indole is a component of the amino acid tryptophan. When tryptophan is broken down (the presence of indol can be detected through the use of Kovacs' reagent which is yellow, when reacts with indole and produces a red color on the surface of the test tube. Materials Cultures
: Bacterial cultures (24 to 48 hr)
Media
: SIM agar or peptone water (Tubes with tryptone/peptone broth)
Procedure Three tubes of given media were inoculated with culture. These media were incubated at 37°C for 48 hours. Then 3 ml of Kovac’s reagent was added to each tube. Results Test sample
Observation
Given bacterial culture
Deep red Colour
Control
Yellow
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Discussion According to the above results it can be seen that E.coli can convert tryptopan in to indole. This indicates the possible fecal contamination. 5.2. Methyl Red test This test is used primarily to differentiate E.Coli from E.aerogenes and a qualitative test of acid produced from oxidation of glucose iE.Coli
- produce large amounts of acid
ii .E.aerogenes
- produces neutral or non-acidic end products.
In this test the bacteria that produce stable acid end products by means of mixed acid fermentation of glucose is identified. All enteric microorganisms ferment glucose with the production of organic acids, and the formation of acids is detected by the addition of methyl red. Many gram-negative intestinal bacteria can be differentiated based on the products produced when they ferment glucose in MR-VP medium Escherichia, Salmonella and Proteus ferment glucose to produce lactic, acetic, auccinic and formic acids and CO2, H2 and Ethanol. The large amounts of acids produced lower the pH of the medium. Methyl red (a pH indicator) will turn
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red when added to the medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas. Materials Culture
: Bacterial cultures (24 - 48 hrs)
Media
: Tubes with MR-VP broth (Methyl red-vogesProskaur)
Reagent
: Methyl red indicator
Equipment
: Bunsen burner, inoculating loop, glass ware marking pencil
Procedure Three tubes of given media were inoculated with E.coli culture. Tubes containing inoculated media were kept at 37°C for 48 hours. After incubation few drops of methyl red was added each test tube.
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5.3. Voges-Proskaur Test Vogus - Proskaur test determines the capability of some microorganisms to produce non acidic or neutral end products such as acetyl methyl carbinol from the organic acid during the glycolysis. The two tests (Vogus- Proskaur and methyl red) are generally considered together because they can be performed on the same medium. Organisms those are negative in the methyl Red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products do not lower the pH as much as acids do. Enterobacter, Serratia and some species of Bacillus produce these substances. There is no satisfactory test for determining production of 2, 3 butanediol. The reagent used in this test is Barrit’s reagent. Materials Cultures
: Bacterial cultures (24 - 48 hr)
Media
: Tubes with MR-VP broth.
Reagent
: Barrit's reagent.
Equipment
: Bunsen burner, Inoculating loop, glass ware marking pencil
Procedure Three tubes of given media were taken and two of them were inoculated with E.coli culture. Tubes were incubated at 37°C for 48 hours. After the incubation period 15 drops from Barritt’s reagent A and 5 drops of Barritt’s reagent B was added to each tube. Results Test sample
Observation- MR
Observations - VP
Given bacterial culture Control
Deep red colour Yellow colour
Copper colour Copper colour
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Discussion Negative results have been obtained for the test. This indicates the absence of acetoin producing bacteria. 5.4. Citrate utilization test (Simmon" Citrate slant) When fermentable glucose or lactose is absent in the presence of citrate permease, some microorganisms are capable of using citrate as carbon source for their energy. The citrate utilization test is used to determine the ability of an organism, using the enzyme citrate permease to use citrate as its sole carbon source. Citrate agar is a medium containing sodium citrate as the sole carbon source and the ammonium ion as the sole nitrogen source. An indicator bromothymol blue is added to the medium, which changes colour based on pH, and will turn from green at neutral pH (6.9) to blue when pH higher than 7.6 is reached (basic or alkaline). Materials Culture
Procedure Three tubes of given media were taken and two of them inoculated with E.coli culture. Inoculated media were incubated at 37°C for 48 hours. After 48 hours colour change was observed. Results Test sample
