Microbiology Lab Media/Test Notes

Thioglycollate Broth:

Test aerotolerance of bacteria.
Turns pink in presence of oxygen via Resazurin indicator. Alternatively, methylene blue indicator (colorless in an anaerobic environment and greenish-blue in the presence of oxygen).
Uniform growth = facultative anaerobic bacteria.
Bubbles = gas-producing bacteria.
Bottom growth = anaerobic bacteria.
Contains sodium thioglycollate, thioglycollic acid, L-cystine, methylene blue, and 0.05% agar. The sodium thioglycollate, thioglycollic acid, and L-cystine reduce the oxygen to water.

GasPak:

For incubation of anaerobic cultures in a nonreducing medium.
Generates water and CO2.
Methylene blue strip indicator is blue in presence of oxygen, and colorless in absence of oxygen.

Selective, Phenylethyl alcohol agar:

Selective, Crystal violet agar:

Selective, 7.5% NaCl agar:

Differential/Selective, MacConkey agar:

Has bile salts and crystal violet, lactose, neutral red.
Inhibit G+
Selective for G-
Contains Lactose
Contains pH neutral red which differentiates RED-Lactose-fermenting colonies; translucent-non-fermenting colonies.
Differentiate between enteric bacteria.
Coliform bacili: lactose fermenters, make acid, RED color on their surface. coli is a super fermenter and there will be pink zone surrounding growth.
Dysentery, typhoid, paratyphoid: non-lactose fermenters; TAN appearance or transparent.

Differential/Selective, Mannitol salt agar (MSA):

High salt concentration, 7.5% NaCl
Select for staphylococci, inhibit most other non-halophilic bacteria.
Contains mannitol (carbohydrate) for differential (some staphylococci can ferment).
Phenol red indicator detect acid from mannitol-fermenting staphylococci.
Yellow zone around growth is positive for mannitol fermentation; no color change is negative.

Differential/Selective, Eosin-methylene blue agar (Levine):

Has lactose and dyes eosin and methylene blue.
Partly inhibitory to G+
Promote G-
Differentiate between enteric lactose fermenters and nonfermenters.
Can identify between E. coli (blue-black w/metallic green sheen due to lots of acid).
Can identify E. aerogenes (thick mucoid, pink colonies).
Enteric bacteria that do NOT ferment lactose—colorless colonies, transparent, and appear to take on purple color of medium.

Enriched Media, Blood agar:

For cultivation of fastidious organisms (e.g. Streptococcus).
Demonstrate hemolytic properties.
Gamma: no lysis of RBC. No change in medium.
Alpha: incomplete lysis of RBC; reduction of Hb to methemoblogin results in greenish halo around growth.
Beta: lysis of RBC; results in clear zone.

Starch Hydrolysis:

Extracellular enzyme amylase to hydrolyze starch down to maltose (maltase cat.) then glucose.
Starch agar.
Flood with iodine to test: blue-black = presence of starch and NEG for starch hydrolysis; clear zone (exoenzymes present) = POS for starch hydrolysis.

Lipid Hydrolysis:

Casein Hydrolysis:

Gelatin Hydrolysis:

Test for liquifaction via gelatinase to hydrolyze protein to amino acids.
Gel deep tubes get inoculated.
After incubation, put in fridge for 30min. Cultures that remain liquified produce gelatinase and rapid gelatin hydrolysis (POS).
If solid, then re-incubate cultures for 5 more days. Put in fridge for 30min. If liquify, then they are POS for SLOW gelatin hydrolysis. If solid, then NEGATIVE.

Carbohydrate Fermentation:

*Facultative anaerobes are usu. the fermenters.
Need broth and Durham tube.
Observe w/in 48 hrs.
Add phenol red: red turns yellow = POS (no color change of indicator = NEG).
Gas = POS.
Beware that neg result does NOT mean no growth.

Hydrogen Sulfide:

Urease:

Useful to i.d. Proteus vulgaris (produces urease). Other organisms also can produce urease.
Inoculate urea broth containing indicator phenol red.
Deep pink = POS urease presence. No deep pink = NEG.

Nitrate Reduction Test:

Reduction of nitrates by some aerobic/facultative anaerobic organisms occur in absence of oxygen. Use inorganic subtrates NO3 or SO4. Some can further reduce Nitrite to ammonia.
Solution A=sulfanilic acid. Solution B is alpha-naphthylamine.
Solution A+B = cherry red = POS for reducing nitrates to nitrites.
No red gives 2 possibilities: end products were reduced even further down to ammonia; or no reduction took place.
Add zinc. No color change = no nitrates = POS. Red color change = NEG = yes nitrates are present.

IMViC (Indole, Methyl red, Voges-Proskauer, Citrate utilization):

Identification of enteric bacteria.
Indole production.
Some bacteria hydrolyze Trp to produce organic compound indole. Use SIM agar containing Trp. After incubation, add Kovac’s reagent.

Turn CHERRY RED = POS for Trp hydrolization. No cherry red = NEG.
Methyl red.
Determine if organism can ferment glucose via high-acid end-products. Differentiate between glucose-enterics (especially valuable to separate E. coli [low pH] and E. aerogenes [converts to nonacidic end products]).

Low pH (<4.4) Methy red indicator turns RED = POS. pH > 6.2 turns YELLOW = NEG.
Voges-Proskauer.
Determine if organism produces nonacidic/neutral end-products from organic acids from glucose metabolism…Glucose fermentation. Characteristic of E. aerogenes.

Barritt’s reagents A+B. Wait 15 min. Rose color = POS (for glucose fermentation). No pink rose color = NEG.

Citrate Utilization.
Differentiate enteric organisms ability to use citrate as sole source of carbon.
Growth, blue medium = POS for citrate. Green, no growth = NEG for citrate.

Catalase:

Aerobic respiration, hydrogen peroxide and superoxides are produced. Capable of producing catalase or superoxide dismutase.
Strict anaerobes don’t produce these enzymes.
Differentiate catalase-positive Staphylococci and catalase-negative Streptococci and members of Enterobacteria.
Inoculate on TSA slant/plate.
Add H2O2 (hydrogen peroxide). Bubbles = POS for catalase. No bubbles = NEG = strict anaerobe.

Oxidase:

Aerobic bacteria and some facultative exhibit oxidase activity.
Differentiate between Neisseria and Pseudomonas (both oxidase-positive) and Enterobacteria (oxidase-negative).
Test reagent alpha-aminodimethylaniline.
Pink then maroon then dark purple = POS for cytochrome oxidase production.
No color change or light pink = NEG.