BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

Description of BSCI 424 Laboratory Media

Alkaline Peptone Broth or Alkaline Peptone Water: enrichment medium for increasing the numbers of target bacteria that can multiply under alkaline conditions (e.g., Vibrio cholerae), while inhibiting the growth of contaminating flora that cannot multiply at an elevated pH

Bile Esculin Agar (BEA): selective medium for the detection of fecal streptococci (group D) and enterococci; tests ability of the organism to hydrolyze esculin to esculetin. Brownish-black colonies surrounded by a black zone are positive.
      Oxgall (bile) is inhibitory agent.
      Ferric citrate is indicator.

Blood Agar (BAP): consists of a basal medium such as TSA enriched with 5% defibrinated sheep blood or in some locations, horse blood. This is the most commonly used medium, and supports the growth of most of the common fastidious organisms, as well as, all of the less fastidious organisms (e.g., coliforms).
      BAP will be used to practice streaking technique to obtain isolated colonies; to observe differences in colony morphology; to determine various patterns of hemolytic activity; to perform CAMP test for Group B streptococci.

Brilliant Green Agar: selective medium for the isolation of Salmonella, except S. typhi.
      Brilliant green is the selective agent.
      Phenol red is the pH indicator.

Campy-blood agar (Campy-BAP): Brucella agar supplemented with sheep blood and vancomycin (inhibits Gram-positives), trimethoprim (broad spectrum) , and cephalothin (inhibits streptococci)

Cary-Blair Transport Medium: semi-solid alkaline medium used to transport sensitive specimens to the laboratory when the specimen cannot be cultured immediately

Chopped (Cooked) Meat Medium: used for cultivation and maintenance of Clostridium and to evaluate proteolysis.

Chocolate Agar: blood agar prepared by heating blood to 56^o C until medium becomes brown or chocolate in color. Heating the blood releases both X and V growth factors and also destroys the inhibitors of V factor. These factors are required for the growth of most species of Haemophilus and also Neisseria gonorrhoeae.
      For clinical gonococcal specimens, isolates are subcultured from the selective primary medium to a noninhibitory enrichment medium, e.g., chocolate agar supplemented with 1% IsoVitaleX to obtain a pure culture of the isolate.

Coagulase Test Medium: citrated rabbit plasma which clots in the presence of the enzyme coagulase.

Cystine (Cysteine) Tellurite Blood Agar: both a differential and selective medium for the isolation of C. diphtheriae; however, a few strains of streptococci and staphylococci are able to grow on this medium; C. diphtheriae (also Staphylococcus) produces gray to black colonies because the tellurite is reduced intracellularly to tellurium

Cystine Trypticase Agar (CTA): carbohydrate-supplemented CTA medium dispensed in tubes is used to detect fermentation of the various carbohydrates and can be used for detemination of motility.
      Phenol red is pH indicator.

Cystine Trypticase Agar (CTA) Sugar Fermentation Media: CTA supplemented with either glucose, maltose or sucrose; tests for utilization of carbohydrates.

Glucose Broth: for anaerobic fermentation (overlaid with mineral oil after inoculation).
      Phenol red is the pH indicator.

Loeffler (Loeffler's) Medium: primary isolation medium for Corynebacterium diphtheriae; distinctive colonial morphology is observed

Lysine Decarboxylase Medium: a yellow color indicates a low pH and that the test is negative (failure to produce an amine by decarboxylation of lysine).
      Bromcresol purple is the pH indicator.

MacConkey Agar: an inhibitory and differential medium used to distinguish lactose-fermenting Gram-negative organisms from nonfermenters.
      Crystal violet, bile salts and neutral red are inhibitory agents.
      Neutral red is the pH indicator.

Mannitol Salt Agar (MSA): for selective isolation of coagulase-positive, mannitol-fermenting Staphylococcus. Mannitol fermentation by pathogenic staphylococci is indicated by a yellow halo surrounding the colonies.
      Sodium chloride is the inhibitory agent.
      Phenol red is the pH indicator.

Motility-Indole-Ornithine Agar: motility is indicated by the character of the growth in the butt of this tube. Motile organisms will produce a general clouding of the medium or a fuzzy stab line. Non-motile organisms will give a sharply delineated stab line. The ornithine reaction is indicated by the color in the butt of the tube. Yellow indicates a negative test (failure to decarboxylate ornithine); purple is a positive test(decarboxylation of ornithine). Kovac's reagent is added to the tube to determine the indole reaction. Red indicates a positive reaction (indol production); yellow is a negative test (failure to produce indol from tryptophan).

Mueller Hinton Agar: a rich medium consisting of 30% beef infusion, 1.75% acidicase peptone, 0.15% starch and 1.7% agar that supports the growth of most microorganisms. It is commonly used for antibiotic susceptibility testing: disk diffusion antibiotic susceptibility; antibiotic serum level measurements; MBC determination.

Mueller-Hinton Broth: culture medium for broth tube antibiotic MIC assay

Nitrate Broth: some bacteria (e.g., Pseudomonas aeruginosa) have respiratory enzyme systems that can use nitrate as a terminal electron acceptor. The product of the reaction is nitrite. Some of the organisms that reduce nitrate to nitrite will then reduce the nitrite further. In the scheme below, first test for nitrite by a colorimetric test. If this test is negative, it can mean either that nitrite was not reduced, or that it was reduced beyond the nitrite stage. This can be resolved by the addition of zinc dust; if nitrate is still present, the zinc will reduce it chemically to nitrite, which will then by revealed by the colorimetric reaction.

  Procedure: To the nitrate broth, after 48 hours of incubation, add 0.2 ml of acid reagent (Solution A), a mixture of acetic acid and sulfanilic acid, and then 0.2 ml of dimethyl-alpha-napthylamine reagent (Solution B). If nitrite is present you will get a red color: this is a positive test for nitrate reduction. If there is no color, pick up some zinc dust on the end of an applicator stick, and add it to the tube. ZINC DUST SUSPENDED IN AIR CAN BE EXPLOSIVE; KEEP AWAY FROM FLAMES! If you get a red color at this stage, what can you conclude? What if no color is obtained?

Nutrient Agar: contains 0.5% gelysate peptone, 0.3% beef extract, and 1.5% agar, and will support the growth of many organisms which are not nutritionally fastidious (e.g., staphylococci, and enterics). (Note: Agar is a substance which melts at 100^o C and solidifies at about 42^o C; it has no nutritional benefits, but is only a stabilizer to allow for solidification of the medium.)

OF Glucose Medium: to detect fermentation or oxidation of glucose (see above).
      Bromthymol blue is pH indicator.

OF Maltose Medium: to detect fermentation or oxidation of maltose (see above).
      Bromthymol blue is pH.

Phenylethyl Alcohol Agar (PEA): for the isolation of Staphylococcus and inhibition of Gram-negative bacilli (particularly Proteus).
      Phenylethyl alcohol is the inhibitory agent.

Serum Tellurite Agar: isolation medium for Corynebacterium diphtheriae; C. diphtheriae (also Staphylococcus) produces gray to black colonies because the tellurite is reduced intracellularly to tellurium

SF Broth: (Streptococcus [Enterococcus] faecalis broth): selective medium for the detection of fecal streptococci (group D) and enterococci from water, milk and other materials of sanitary importance. Growth of all other cocci is inhibited. Fermentation of glucose is indicated by a color change in the broth.
      Sodium azide is the inhibitory agent.
      Bromcresol purple is the indicator.

Simmons Citrate Agar: utilization of citrate as the sole source of carbon is indicated by the medium turning a deep blue color because of an alkaline reaction. Non-utilizers will leave the green color of the slant unchanged.
      Bromthymol blue is the indicator.

Skirrow's agar: peptone and soy protein base agar supplemented with lysed horse blood and vancomycin (inhibits Gram-positives), polymyxin B (antifungal), and trimethoprim (broad spectrum)

Sugar Utilization Medium: supplemented with glucose (with Durham tubes to determine gas production), sucrose, mannitol, or lactose.
      Phenol red is the pH indicator.

TCBS Agar (Thiosulfate-Citrate-Bile salts-Sucrose agar): Differential and selective plating medium for Vibrio adjusted to pH 8.6 (alkaline); Vibrio cholerae colonies appear yellow; other Vibrio spp. colonies appear green

Thayer-Martin Agar: chocolate agar supplemented with antibiotic inhibitors for selective isolation of pathogenic Neisseria
      Vancomycin inhibits Gram-positive organisms.
      Colistin inhibits Gram-positive enteric organisms.
      Nystatin inhibits yeast.

Triple Sugar Iron Agar (TSI): this is a key medium for use in beginning the identification of a Gram-negative bacilli of the enteric group. It contains glucose (0.1%), lactose (1%), sucrose (1%) and peptone (2%)as nutritional sources. Sodium thiosulfate serves as the electron receptor for reduction of sulfur and production of H₂S. Detects fermentation of sucrose, lacotse, glucose, as well as production of hydrogen sulfide and/or gas.
      Phenol red is pH indicator; ferric ammonium citrate is H₂S indicator.
      Click here to view Explanation of TSI Reactions.

Trypticase Lactose Agar: to determine motility and lactose fermentation by anaerobes.
      Phenol red is pH indicator.

Trypticase Nitrate Broth: to determine indole production and nitrate reduction.

Trypticase Salicin Agar: to determine motility and salicin fermentation by anaerobes.
      Phenol red is pH indicator.

Trypticase Soy Agar (TSA): enriched medium containing 1.5% trypticase peptone, 0.5% phytone peptones, 0.5% NaCl, 1.5% agar that supports the growth of many of the more fastidious organisms: e.g. streptococci, and some members of the genera Neisseria, Brucella, Corynebacterium, Listeria, Pasteurella, Vibrio, Erysipelothrix, etc.
      To be used to practice streaking technique and to observe differences in colony morphology, as well as, for recovery of microorganisms from skin; to perform catalase test; to test requirements for X and/or V factors.

Trypticase Sucrose Agar: to determine motility and sucrose fermentation by anaerobes.
      Phenol red is pH indicator.

Urease Broth or Urea Agar Slant: Prompt hydrolysis of urea by Proteus species is indicated by a deep pink color appearing in the medium within eight hours. At 18 hours, this color will have spread throughout the whole tube. Many strains of Klebsiella, Enterobacter and Citrobacter will yield a positive reaction, but usually the pink color will be limited to the slant in 24 to 48 hours. Do not reincubate tubes that show any evidence of color change.

XLD Agar (Xylose lysine desoxycholate agar): Selective media for the isolation of Shigella and other enteric pathogens.

XLT-4 Agar: Selective media for the isolation of Salmonella developed at and patented by investigators at the University of Maryland and the USDA. It contains peptone, yeast extract, lysine, lactose, sucrose, xylose as nutritional sources. Sodium thiosulfate acts as electron receptor for reduction of sulfur to H₂S. The medium is highly inhibitory for non-salmonellae. Tergitol-4 also may be inhibitory to S. typhi and S. choleraesuis.
      Tergitol-4 (surfactant) is the inhibitory agent.
      Phenol red is the pH indicator and aluminum-iron (III) citrate is the H₂S indicator.

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