3.2 Media Types, Components, and Selection

Media selection is how the laboratory turns a mixed specimen into isolated, identifiable colonies. Outline I.B asks for types (nutrient, selective, differential, enriched), components (blood, X and V factors, bile salts, antibiotics, indicators), application, and selection by specimen and organism. Specialized media for fastidious bacteria, mycobacteria, and fungi are fair game on M(ASCP). Bordetella media appear on some older lists; on the current guideline, Bordetella is SM-only in the pharyngitis cluster—know that Bordet-Gengou and Regan-Lowe exist, but do not treat pertussis plating as an M-tested pharyngitis item. BCYE for Legionella is M-tested.

Media types

Nutrient or general-purpose media (tryptic soy agar, nutrient agar) supply carbon, nitrogen, and salts for nonfastidious organisms and are rarely used as the only primary plate in clinical work. Blood agar is nutrient plus enrichment. Enriched media add extra nutrients or growth factors so fastidious pathogens can grow: chocolate agar, buffered charcoal yeast extract (BCYE), and brain-heart infusion (BHI) with blood. Selective media contain dyes, bile salts, antibiotics, or low pH that suppress part of the flora so the target can appear: Columbia CNA and PEA for Gram-positive cocci, MacConkey for Gram-negative rods, Thayer-Martin for gonococci, Campy-BAP for Campylobacter. Differential media produce a visible reaction—hemolysis on blood agar, lactose fermentation on MacConkey, H2S on HE or XLD, black colonies of Salmonella, red centers on CIN for Yersinia. Many plates are both selective and differential; MacConkey is the prototype. Do not call chocolate differential just because it is brown; it is enriched, not a biochemical indicator plate.

How the exam phrases type questions

If a stem says “inhibits Gram-positive organisms so enteric Gram-negative rods can grow,” the type is selective (MacConkey, HE). If it says “distinguishes lactose fermenters as pink colonies,” the type is differential (still MacConkey). If it says “heated blood releases factors required by Haemophilus,” the type is enriched (chocolate). You can, and should, assign more than one type to the same plate when both functions are present.

Components and what they do

Sheep blood (typically 5%) supplies nutrients and demonstrates alpha, beta, or gamma hemolysis; it is also the base for detecting CAMP reactions and pigment. Chocolate agar is blood agar heated until red cells lyse, releasing X factor (hemin) and V factor (NAD) required by Haemophilus influenzae and helping Neisseria spp. grow. Bile salts and crystal violet in MacConkey inhibit Gram-positive organisms. Antibiotics convert an enriched plate into a selective plate: vancomycin, colistin, nystatin, and trimethoprim on modified Thayer-Martin suppress flora around N. gonorrhoeae; cefsulodin, irgasan, and novobiocin define CIN for Yersinia enterocolitica; cycloheximide on Mycosel or mycobiotic agar suppresses saprophytic molds so dermatophytes can grow; chloramphenicol in inhibitory mold agar reduces bacterial overgrowth. Indicators include phenol red, neutral red, and bromothymol blue for fermentation; ferric salts for H2S; charcoal in BCYE to bind inhibitors; and the pH indicator in dermatophyte test medium (DTM) that turns red when dermatophytes grow.

Ingredient-to-organism pairs

X and V disks or a Staphylococcus streak on blood agar still demonstrate Haemophilus satellitism when chocolate is not the primary plate. TCBS uses a high pH, bile, and sucrose to select Vibrio and differentiate V. cholerae (yellow, sucrose positive) from many V. parahaemolyticus isolates (green). Malachite green in Lowenstein-Jensen is the selective dye against contaminating bacteria. L-cysteine and soluble iron in BCYE are growth factors, not antibiotics; a BCYE plate poured without cysteine is the negative control that fails to grow Legionella. Know the component that makes the plate work, not just the brand name.

Selection by specimen

Primary plating is protocol-driven. A CSF or other sterile fluid is inoculated to blood agar, chocolate agar, and usually a broth (thioglycollate or BHI); MacConkey is added if a Gram-negative rod is seen or if the laboratory’s sterile-fluid setup includes it. Sputum and other lower-respiratory specimens receive blood agar, chocolate, and MacConkey (or another enteric plate); add CNA or PEA when mixed oral flora is expected. Urine is plated to blood agar and MacConkey (or a biplate) with a calibrated loop. Stool for bacterial culture uses a battery: MacConkey or EMB, HE or XLD, Campy-BAP, and often CIN or SMAC depending on season and orders; TCBS is added for suspected Vibrio (seafood, cholera-endemic exposure, watery rice-water stool). Genital specimens for N. gonorrhoeae go to Thayer-Martin, NYC, or Martin-Lewis, incubated in CO2. Wound and tissue setups combine blood, MacConkey or EMB, CNA/PEA, and anaerobic media when the source is valid for anaerobes. Blood-culture broth is a liquid enrichment; positives are subcultured to blood and chocolate, with MacConkey and anaerobic plates as indicated by the Gram stain.

Specialized media the M exam names

Chocolate agar is the primary plate for Haemophilus, pathogenic Neisseria from sterile sites, and many fastidious respiratory organisms. BCYE with L-cysteine and iron is required for Legionella; charcoal binds fatty acids that otherwise inhibit growth. Thayer-Martin and New York City (NYC) agar recover N. gonorrhoeae from genital and other contaminated sites. Campy-BAP (blood agar with cephalothin or cefoperazone, vancomycin, and an antifungal) is incubated at 42°C in a microaerophilic atmosphere for Campylobacter jejuni/coli. CIN yields bull’s-eye red colonies of Y. enterocolitica, often after cold enrichment. TCBS is the Vibrio plate.

Bordet-Gengou (potato-blood-glycerol) and Regan-Lowe (charcoal-cephalexin) recover Bordetella pertussis. Flag them as SM-only on the current pharyngitis list: M candidates should recognize the names so they are not confused with BCYE, but pertussis media are not an M-tested pharyngitis requirement. Legionella/BCYE remains in M scope.

Mycobacteria and fungi

Mycobacteria require egg-based Lowenstein-Jensen (LJ), agar-based Middlebrook 7H10/7H11, and liquid Middlebrook 7H9 in MGIT or similar automated bottles. Contaminated specimens reach those media only after NALC-NaOH digestion (Section 3.1). Fungi are plated to Sabouraud dextrose agar (SDA), inhibitory mold agar (IMA), Mycosel or DTM for dermatophytes, and BHI with blood for dimorphic fungi and Cryptococcus from sterile sites. Cycloheximide helps dermatophyte recovery but can inhibit Cryptococcus and some significant molds, so a pair of media—with and without cycloheximide—is the usual mycology setup. Do not send a CSF fungal request to DTM; DTM is a dermatophyte plate for skin, hair, and nails.

If you can map those rows and name the key ingredient (X/V, L-cysteine, bile salts, malachite green, cycloheximide), you can answer the media cluster on M(ASCP).