How to Test for Unknown in Proteus vulgaris. Need at least 2 tests from each cat
ID: 204045 • Letter: H
Question
How to Test for Unknown in Proteus vulgaris. Need at least 2 tests from each category (1) Morphology and Motility Determination (2) Oxidation/Fermentation Tests, (3) Hydrolysis/Biochemical Tests, and (4) Selective/Differential media. (5) specific result.
Motility test
a. Motility agar tube
b. SIM tube
i. Sulfur, Indole, Motility media tests for the presence of sulfur reducers and
motility of microorganismsii.
2. Morphology determination by Gram stain
3. Colony morphology/cultural characteristics
a. Solid media slants
b. Nutrient broth
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Category II: Oxidation/Fermentation Tests:
4. Oxygen Requirements
a. FTM media
b. Brewer’s Anaerobic agar (CDC Anaerobic 5% Sheep Blood Agar)/Anaerobic chamber
5. Sugar fermentation tests
a. Durham tubes test for fermentation of single sugars and are available for glucose,
lactose, and mannitol fermentation: acid production (color change from red to yellow) and gas
production (bubble in Durham tube)
6. Methyl Red (Mixed Acid) test
7. Voges –Proskauer (3-Hydroxybutanone) test
a. MR-VP Media and Barritt’s reagent
8. Catalase test
a. Hydrogen Peroxide
9. Oxidase test
a. Oxidase reagent
i. Detects the presence of the enzyme cytochrome c oxidase which suggests the
ability of a microorganism to utilize oxygen for ATP production via an electron
transport chain.
10. Nitrate Reduction test
a. Nitrate test reagents A and B/zinc dust
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Category III: Hydrolysis Tests/Biochemical Tests:
11. Starch Hydrolysis test
a. Starch agar plate and Gram’s iodine
12. Casein Hydrolysis test
a. Skim Milk agar
i. An agar plate containing cow’s milk. If a microorganism is capable of degrading
casein with proteases known as caseases, a clearing or halo around the
colonies will result.
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13. Fat Hydrolysis test
a. Spirit Blue agar
i. This test is designed to determine the presence or absence of lipase
production. The media contains fatty acids such as palmitic, stearic and oleic
acids and clear halos around growth are created when the microorganism
produces lipases that degrade those lipids.
14. Tryptophan Hydrolysis test
a. Tryptone broth and Kovac’s reagent
i. Determines the ability of a microorganism to degrade tryptophan into indole,
pyruvate and ammonia.
15. Urea Hydrolysis test
a. Urea slant
i. This test indicates the ability of a microorganism to hydrolyze urea, a common
constituent of urine. Hydrolysis of urea, by the enzymes urease, releases
ammonia resulting in basic conditions and a positive test result indicated by a
pink test color.
16. Hydrogen sulfide production
a. Kliger’s Iron agar slant
i. This test determines fermentation and sulfur production characteristics of
microorganisms. The media contains glucose and lactose as well as ferrous
sulfate. Sugar fermentation is determined by the indicator phenol red and
sulfur reduction is determined by the reaction between the produced H2S and
the ferrous sulfate in the media.
17. Citrate Utilization test
a. Citrate agar slant
i. Citrate can be utilized as a carbon source which typically results in basic
byproducts. The rise in pH in the media is detected by the indicator
bromothymol blue which is green at a neutral pH and blue at a pH greater than
7.8.
18. Phenylalanine Deamination test
a. Phenylalanine agar
i. Provided typically as a slant, the phenylalanine deaminase test indicates the
presence or absences of deaminases, which are enzymes capable of removing
amine groups from amino acids. The media contains ferric chloride which
detects phenylpyruvic acid (the part of phenylalanine that is left after removing
an amine) and will turn green if the test is positive.
19. Gel liquefaction
a. Gelatin tube
i. Many microorganisms contain gelatinases, which are enzymes capable of
degrading proteinaceous gelatin. Positive tests result when after incubation the
gelatin in the media remains liquid even after refrigeration.
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Category IV: Differential/Selective media:
All Differential and or Selective media require streaking for isolation on Day 1, followed by
interpretation the following lab.
20. Hektoen agar
a. Hektoen enteric agar is design to inhibit the growth of most Gram(+) bacteria with the
selective ingredient bile salts. Additionally, Hektoen can distinguish between Gram(-)
lactose fermenters (yellow to salmon colored colonies) and non-fermenters, and
Gram(-) sulfur producers (green colonies with a black center). This media is useful for
determining enteric bacterial identities.
21. Blood agar
22. MacConkey agar
23. EMB (Eosin Methylene Blue) agar
Explanation / Answer
1.Motility Test:
a) Motility agar tube: The test medium essentially comprises of semisolid agar whereby the motile bacteria swim and diffuse into the medium which is visible to the naked eye. A semisolid agar medium is prepared in a test tube and is innoculated with a straight wire thus making a stab down the middle of the medium. This is used to test the motility of the bacteria.
2.Morphology determination by gram stain:
The gram staining procedure is used to classify bacteria on the basis of their morphology i.e whether they are gram positive or gram negative in nature. This is a method of differential staining. Gram positive bacteria have a thicker outer wall made up pf peptidoglycan and hence are stained violet by crystal violet whereas gram negative bacteria has a thin outer wall of peptidoglycan and hence are not able to retain the stain and hence turns red/pink.
3.Colony morphology/ cultural characteristics:
a) Solid media slants: Used to culture micro-organisms. Agar is used in preparation of the solid media slantsalong with other media composition. When the media is hot and in a liquid form, it is poured into the culture tubes and the tubes are allowed to stand in a tilted postion till the media solidifies. This is known as slant and provides greater surface area for the growth of bacteria.
b) Nutrient broth: It is liquid in nature. It serves as a general culture medium for most bacteria. Comprises of yeast extract, beef extract, peptone, sodium chloride. It can be modified based on the requirements for the growth of a particular bacteria. It provides the bacteria all the essential conditions required for its growth. May also comprise of carbohydrates, vitamines etc.
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