Stains, Culture Media & Growth Requirements
How the lab sees, grows and sorts bacteria
When the Gram stain fails
The procedure. Crystal violet stains every cell, iodine locks it into a complex, alcohol washes the complex out of thin gram-negative walls, and safranin counterstains the decolorized cells pink while thick gram-positive walls stay purple. The method assumes a peptidoglycan wall and an organism large enough to see, so some bacteria slip through.
- Too thin to resolve: Treponema and Leptospira.
- No cell wall: Mycoplasma.
- Waxy mycolic acid: mycobacteria.
- Mostly inside host cells: Legionella, Rickettsia, Chlamydia (which also lacks classic muramic acid-containing peptidoglycan), Bartonella and Ehrlichia.
Special stains
| Stain | Organisms | Notes |
|---|---|---|
| Giemsa | Chlamydia inclusions, Borrelia, Plasmodium, trypanosomes | Standard for blood-smear parasites and spirochetes |
| PAS | Tropheryma whipplei | Fills foamy lamina propria macrophages in Whipple disease |
| Ziehl-Neelsen | Mycobacteria | Carbolfuchsin held despite acid-alcohol: red rods on blue |
| Modified acid-fast | Nocardia; Cryptosporidium, Cyclospora, Cystoisospora oocysts | Weaker decolorizer reveals partial acid-fastness |
| India ink, mucicarmine | Cryptococcus | Clear capsular halo in CSF; red capsule in tissue |
| Silver | Pneumocystis, Legionella, H. pylori | Organisms stain black; methenamine silver for Pneumocystis cysts |
| Dark-field, fluorescent antibody | Treponema pallidum; Leptospira (dark-field) | Motile corkscrews in chancre exudate |
Practical points. Fluorescent auramine-rhodamine staining is the faster, more sensitive screen for acid-fast bacilli. Cryptococcal antigen testing of CSF or serum is more sensitive than India ink. Syphilis is usually diagnosed by serology: nontreponemal RPR/VDRL and treponemal FTA-ABS or TP-PA. Whipple disease causes malabsorption, arthralgia and eventually dementia.
Mycobacterium avium complex in AIDS also fills intestinal macrophages with PAS-positive material but is acid-fast; T. whipplei is PAS positive and not acid-fast.
Culture media and growth factors
X and V factors. H. influenzae needs hematin (factor X) and NAD+ (factor V). Chocolate agar, made from heat-lysed red cells, releases both. On sheep blood agar the organism grows only as satellite colonies beside a streak of S. aureus, whose hemolysis frees X and which secretes NAD+.
| Medium | Organism | Key feature |
|---|---|---|
| Chocolate agar | H. influenzae (also Neisseria) | Supplies X and V factors |
| Thayer-Martin | N. gonorrhoeae, N. meningitidis from nonsterile sites (genital, throat) | Chocolate base plus vancomycin (gram-positives), colistin (other gram-negatives), nystatin (fungi), trimethoprim (Proteus swarming) |
| Bordet-Gengou, Regan-Lowe | Bordetella pertussis | Potato-based; charcoal-based |
| Tellurite, Löffler | Corynebacterium diphtheriae | Black colonies; metachromatic granules |
| Löwenstein-Jensen | M. tuberculosis | Egg-based; growth takes weeks |
| Eaton | Mycoplasma pneumoniae | Supplies cholesterol for its sterol membrane |
| Buffered charcoal yeast extract | Legionella | Iron and cysteine required |
| MacConkey | Gram-negative rods | Bile salts and crystal violet block gram-positives; lactose fermenters pink |
| Eosin-methylene blue | E. coli | Green metallic sheen |
| Sorbitol MacConkey | E. coli O157:H7 | Does not ferment sorbitol (colorless) |
| TCBS | Vibrio cholerae | Yellow colonies from sucrose fermentation |
| Sabouraud | Fungi | Acidic, glucose-rich |
Lactose fermentation. MacConkey agar is both selective and differential: lactose fermenters (E. coli, Klebsiella with mucoid colonies, Enterobacter) turn pink, while nonfermenters stay colorless: Salmonella, Shigella, Proteus (swarming) and Pseudomonas (oxidase positive, unlike the Enterobacterales).
Oxygen and intracellular lifestyle
Obligate aerobes. Nocardia, Pseudomonas aeruginosa and M. tuberculosis depend on aerobic respiration and cannot grow without oxygen; Pseudomonas is accordingly a nonfermenter. M. tuberculosis reactivates in the lung apices, where oxygen tension is highest.
Obligate anaerobes. Clostridium, Bacteroides, Fusobacterium and Actinomyces lack catalase and/or superoxide dismutase, so oxygen radicals kill them. Normally resident in the gut, mouth and genital tract, they invade devitalized tissue or aspirated material and produce foul-smelling, often gas-forming abscesses. Aminoglycosides are useless against them because drug uptake requires oxygen-dependent transport; metronidazole, clindamycin or a β-lactam–β-lactamase inhibitor is used instead.
Obligate intracellular. Rickettsia (needs host CoA and NAD+), Chlamydia (an energy parasite that uses host ATP) and Coxiella cannot grow on routine media; diagnosis relies on serology, PCR or cell culture.
Facultative intracellular. Salmonella, Listeria, Legionella, Brucella, Francisella, Mycobacterium and Yersinia pestis grow on media but survive inside macrophages, so their clearance depends on Th1 cell-mediated immunity and IFN-γ-activated macrophages, often forming granulomas.
Enzyme and pigment clues
Catalase and CGD. Catalase converts H2O2 to water and oxygen; staphylococci are catalase positive and streptococci negative. In chronic granulomatous disease, NADPH oxidase deficiency leaves phagocytes unable to make superoxide, so they rely on H2O2 produced by the microbe itself. Catalase-positive organisms destroy that peroxide, which is why S. aureus, Burkholderia cepacia, Serratia marcescens, Nocardia and Aspergillus cause recurrent abscesses and pneumonias in CGD.
Urease. Urease hydrolyzes urea to ammonia and CO2. Proteus and Klebsiella alkalinize urine and precipitate struvite (magnesium ammonium phosphate) staghorn calculi; H. pylori buffers gastric acid, the basis of the urea breath test; Cryptococcus, Ureaplasma and Nocardia are also urease positive.
Pigments. S. aureus makes golden staphyloxanthin, a carotenoid that also resists neutrophil oxidants; P. aeruginosa makes blue-green pyocyanin and pyoverdine; Serratia marcescens makes red prodigiosin; Actinomyces israelii forms yellow sulfur granules of tangled filaments in draining sinus pus.