Biochemical tests Flashcards
What does a positive and negative test look like for oxidation-fermentation test?
-purpose?
1st 2 tubes: oxidation
2nd 2 tubes: oxidation or fermentation
3rd 2 tubes: neither
- Determines if a bacteria is able to oxidatively metabolize and/or ferment a carbohydrate (glucose):
What is the purpose of the phenol red test?
How do you distinguish from a positive or negative test?
- Determines which carbohydrates a bacteria can ferment, if gas is produced in the process of fermentation, and if amino acids are utilized as an energy source.
**
Gas production can only occur if fermentation has occurred.
Pink indicates an amino acid consumer. Produced hydroxides = more alkaline.
-Phenol red (pH indicator)
- Below 6.8 = yellow
- 6.8-7.4 = red
- Above 7.4 = pink
What is the purpose of the TSI (Triple sugar iron) test?
how do you distinguish between a postive or negative test?
- Determine if the bacteria ferments glucose; ferments lactose and/or sucrose; and/or if it reduces sulfur.
- Similar to phenol red broth, but has a 3 sugars in one slant
- Only contains a small amount of glucose (0.1%), so if the organism only ferments glucose, it will quickly run out and will resort to breaking down protein (peptone), which will increase pH and cause red coloration
- Also tells if organism reduces sulfur…Black precipitate due to the interaction of the iron compound and hydrogen sulfide gas.
- note:
If only ferments glucose, it will consume the small amount quickly and then begin to deaminate the peptone in the media, increasing the pH level (red to pink). pH change is not uniform and the butt of the slant will remain yellow.
If lactose and sucrose are fermented, large amount of acids will be produced turning it yellow.
Gas production can be detected through cracks in the media or cause the agar to rise in the tube.
Sulfur reduction only occurs under acidic conditions so fermentation must occur.
What is the purpose of the methyl red test?
What does a positive and negative look like?
- Methyl red test determines mixed-acid fermentation
- Addition of methyl red reagent gives red color for positive bacteria
- Positive test turns red (acidic)
- Negative test turns orange or yellow
What is the purpose of the vogues prokauer test?
what does a positive and negative test look like?
- Vogues Proskauer test determines if glucose fermenting bacteria utilize the butylene glycol pathway
- Addition of reagents VP-A (α-naphthol) & VP-B (KOH) gives red color for positive bacteria (react w/acetoin & 2,3-butanediol)
- Negative test is no color change or copper color
what is selective media?
What is diffrential media?
Selective Media – Chemicals added that allow certain bacteria to grow and inhibit others.
Differential Media – Allows all bacteria to grow but has chemicals to cause a change in appearance of certain bacteria.
How is the Eosin Methelyene Blue Agar (EMB) selective and differential?
–>Selective for Gram (–) bacteria:
Dyes inhibit growth of Gram (+) Bacteria
Eosin Y – Dye (selective)
Methylene Blue – Dye (selective)
–> Differential for fecal coliform bacteria by how actively it ferments the carbohydrates (rapid fermenters will produce a green metallic sheen)
Lactose – carbohydrate (differential)
Sucrose – carbohydrate (differential)
how does a EMB fast fermenter look?
metallic green sheen
How does a EMB slow fermenter look?
What is the purpose of the gelatin hydrolysis test?
how does a pos. and a negative test look like?
Gelatin is another protein
Gelatinase (enzyme) breaks down gelatin into amino acids to be taken up by bacteria
Gelatin –Gelatinase–> peptides
**Gelatin liquefies when it’s broken down
What is the purpose of the Urease test?
postive test and negative test?
Determines if your bacteria possesses the enzyme urease.
Urease is used to rapidly hydrolyze urea to ammonia and carbon dioxide.
Urea– Urease–> Ammonia + Carbon dioxideo Creates alkaline environment
Phenol red is used as an indicator
- Uninoculated media – Orange
More alkaline media – Pink
What is the purpose of the nitrate reduction test?
- How does a postive and negative test look like? (in each instance)
Determines if the bacteria possesses the enzyme nitrate reductase to reduce nitrate (NO3-) to nitrite (NO2-).
-Reduction is either the addition of an electron or removal of an oxygen molecule. Which is happening here? NO3- → NO2-
This process can be used to supply oxygen as a final hydrogen receptor during anaerobic respiration (fermentation).
- A positive test is red (has nitrate reductase) after the addition of reagents Sulfanilic Acid (Nitrate A) and α-naphthylamine (B)
- A negative test is clear after the addition of reagents A and B
- A positive test is clear after the addition of zinc
- A negative test us red after the addition of zinc
What is the purpose of the decarboxylase test?
- how do we distinguish positive from negative?
to determine if your bacteria posses the enzyme lysin decarboxylase to remove the carboxyl group from the amino acid lysine.
- Must have fermentation to create acidic environment to activate the enzyme decarboxylase (therefore we create and anaerobic enviornment with oil)
pH Indicator: Bromcresol purple
Positive test = purple to yellow to purple
Negative test = purple to yellow
what is the purpose of the phenylalanine deaminase test? What is the reagent
- how do you distinguish a positive from a negative test?
to determine if the bacteria posses the enzyme phenylalanine deaminase to remove the amine group from the amino acid phenylalanine. Ferric chloride is the reagent.
- positive: green surface after ferric acid added
- negative: not green (no color change)
What does SIM stand for? What is the SIM test purpose?
- How are positive results for each portion of the SIM test determined?
Sulfur Indole Motility. The purpose of this test is to SIM determines
- Sulfur reduction (black precipitate): Sulfur reacts with hydrogen sulfide gas to produce black precipitate.
- Indole production from tryptophan (red color with Kovac’s reagent)
- Motility (radiating growth)
- 1) reduces sulfur if black percipitate formed
- 2) Produces indole if pink surface is ontop (after adding kovac’s reagent)
- 3) it is motile if the bacteria spread from the stab (funnel cloud)
What is the purpose of the Citrate test?
- how do you distinguish positive from a negative test?
Tests to see if bacteria can use citrate as its carbon source and ammonium phosphate as nitrogen source…Citrate is the only form of carbon in this media. Must have citrate-permease to transport it into the cell and citrase to convert it into pyruvic acid, which will then form a variety of carbon containing products that can be utilized in the Kreb’s Cycle for energy production. Carbon dioxide generated will react with sodium and water to form alkaline sodium carbonate…Ammonium phosphate is the only source for nitrogen so citrate positive bacteria can convert this into ammonia and ammonium hydroxide which further increases the pH.
- Bromthymol blue turns blue when pH increases (becomes more alkaline)
- Green at pH 6.9
- blue at pH 7.6
What are the IMViC tests?
The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative,
- Indole test
In this test, the organism under consideration is grown in peptone water broth. It contains tryptophan, which under the action of enzyme tryptophanase is converted to an Indolemolecule, pyruvate and carbon dioxide. The indole is then extracted from the broth by means of xylene. To test the broth for indole production, Kovac’s reagent is added after incubation. A positive result is indicated by a pink/red layer forming on top of the liquid.
- Methyl red and Voges–Proskauer test
These tests both use the same broth for bacterial growth. The broth is called MRVP broth. After growth, the broth is separated into two different tubes, one for the methyl red (MR) test and one for the Voges-Proskauer (VP) test.
The methyl red test detects production of acids formed during metabolism using mixed acid fermentation pathway using pyruvate as a substrate. The pH indicator Methyl Red is added to one tube and a red color appears at pH’s lower than 4.2, indicating a positive test (mixed acid fermentation is used). The solution remaining yellow (pH = 6.2 or above) indicates a negative test,
The VP test uses alpha-naphthol and potassium hydroxide to test for the presence of acetylmethylcarbinol (acetoin), an intermediate of the 2,3-butanediol fermentation pathway.
- Citrate test
This test uses Simmon’s citrate agar to determine the ability of a microorganism to use citrate as its sole carbon source. The agar contains citrate and ammonium ions (nitrogen source) and bromothymol blue as an indicator. The citrate agar is green before inoculation, and turns blue as a positive test indicator, meaning citrate is utilized.
