Microbiology - Lab Final

Front 1

Triple Sugar Iron agar slant

Back 1

Glucose fermentation – yellow butt, red slant (pH begins to be lowered after 12 hrs)
Lactose and/or sucrose fermentation – yellow throughout (higher quantities of these sugars)
H2S production – cysteine desulfurase and/or thiosulfate reductase presence both tested in medium. Tubes may need to be reread after 24 hours in order to look at sulfur production.
Gas production can also be detected from presence of bubbles in butt.

Front 2

Methyl Red

Back 2

tests conversion of mixed-acid byproduct from glucose fermentation. Methyl red (5 drops) is added as indicator of such byproduct presence. (Red below pH 5; yellow/orange above pH 5)

Front 3

Voges Proskauer

Back 3

tests conversion of acid byproduct butanediol from glucose fermentation. Specifically tests for intermediate product acetoin, immediate precursor to butanediol, using reagent A (15 drops) & reagent B (5 drops) (15 min. waiting time).

Front 4

Simmons Citrate slant

Back 4

indicator is bromthymol blue, testing for using citrate as sole carbon source. If organism uses citrate, solution will be blue, indicating basic pH.

Front 5

Lysine Decarboxylase & Orinthine decarboxylase

Back 5

Indicator is bromcresol purple (yellow = acid, grey = neutral, purple = basic). Substrate is amino & also glucose is in broth. Mineral oil is added to top to keep air out & force fermentation of small amount of glucose. Yellow color indicates acid/no decarboxylation; purple color indicates decarboxylation.

Front 6

Cytochrome C oxidase test

Back 6

Differentiates Pseudomonas from Enterobacteriae as strict aerobes that produce cytochrome C oxidase. Indicator reagent added to filter paper w/culture will turn culture purple immediately if oxidase is present. Timing is of the essence as eventually filter paper will turn purple anyway when it reacts with oxygen in environment (usually after 1 min)

Front 7

Nitrite/N2 reduction from nitrate broth

Back 7

First test uses 2 reagents (5 drops each) to test for nitrate to nitrite; second test uses zinc powder to test for nitrate to N2 gas.

Front 8

Pyocyanin agar

Back 8

Tests for blue color (production of pyocyanin, a water soluble pigment). Indicator of Pseudomonas vs. one of the Enterobacteriae.

Front 9

Tests for Pseudomonas vs. Enterobacteriae?

Back 9

Catylase, Cytochrome-C oxidase, Pyocianin production, Nitrate reduction, Glucose fermentation. Thioglycolate medium

Front 10

RPR

Back 10

Rapid Plasma Reagent, a serological test for Treponema pallidum (agent of syphilis). Indirect agglutination assay. Tests for Ab (reagin) formed in response to lipid antigen (cardiolipin) released by cells damaged early in disease.
1) Test subject serum placed in circles on cards
2) Control serum placed on same test card
3) All samples spread with stirrers
4) A drop of cardiolipin+charcoal is added to each sample
5) Cards rotated for 8 minutes at 100 RPM
6) Observation for agglutination as compared to positive and negative cards.

Since antigen is not directly from Treponema pallidum, false positives posible w/ other health conditions. Other tests for Treponema pallidum include VDRL tests.

Front 11

SIM media - Sulfur?

Back 11

SIM media is an agar deep.
Sulfur is present if there is a reduction of H2S gas, detected by the indicator ferric citrate, that will turn the precipitate black. One of two enzymes is responsible for this breakdown: thiosulfate reductase or cysteine desulfurase.

Front 12

SIM media - Indole?

Back 12

SIM media is an agar deep.
Detects catabolism of tryptophan into indole + pyruvic acid + NH3, which is tested for by adding Kovac's reagent to the medium after incubation. If the top of the medium turns red, we can determine that the bacteria break down tryptophan.

Front 13

PD agar

Back 13

Phenylalanine deaminase agar slant. Phenylalanine, if metabolized, will be broken down into phenyl pyruvic acid and NH3. The addition of ferric chloride to the medium will turn a dark green color if phenyl pyruvic acid is present.

Front 14

Catalase production?

Back 14

Detected production of catalase enzyme by the addition of H2O2 to a cultured growth medium, which will bubble if the bacteria indeed contain catalase. Bubbles are indicative that the organism is breaking down the catalase.

Front 15

Acid fast stain

Back 15

1) Steam + carbolfuschin for 5 min, to penetrate thick cell walls with waxy material (mycolic acid) contained within along with peptidoglycan layers.
2) H2O wash, and then acid alcohol is applied as a decolorizer.
3) H2O wash, and then counterstain of methylene blue is applied.