STEPTRACKERMICROBIOLOGY | BACTERIAL BASICS | HIGH-YIELD REVIEW

Stains, Culture & Lab Identification

Which stain shows which bug, which medium grows it, and what its oxygen and nutrient needs tell you.

FOCUS ON
Giemsa: Chlamydia, Borrelia, Rickettsia, Trypanosoma, Plasmodium. PAS: glycogen — Whipple (T whipplei). Ziehl-Neelsen: acid-fast (Mycobacteria, Nocardia — modified, Cryptosporidium oocysts). India ink: Cryptococcus capsule. Silver: Pneumocystis, Legionella, Helicobacter, fungi. Media: chocolate + V and X (Haemophilus), Thayer-Martin (Neisseria), Bordet-Gengou/Regan-Lowe (Bordetella), charcoal yeast + iron/cysteine (Legionella), Löwenstein-Jensen (TB), Eaton (Mycoplasma).
STAIN · MEDIUM
SHOWS · GROWS
HOW IT WORKS
CLUE
Giemsa
Chlamydia, Borrelia, Rickettsia, Trypanosomes, Plasmodium
Romanowsky stain of blood/tissue
Blood-smear and intracellular organisms
PAS
Tropheryma whipplei (in macrophages), glycogen, mucopolysaccharides
Periodic acid–Schiff stains carbohydrates
Foamy PAS⁺ macrophages in small bowel
Ziehl-Neelsen (carbol fuchsin)
Mycobacteria; modified for Nocardia; Cryptosporidium oocysts
Acid-alcohol fails to decolorize waxy walls
Auramine-rhodamine = fluorescent screen
India ink · mucicarmine
Cryptococcus neoformans capsule
Negative stain (halo) · capsule stains red
Latex agglutination/antigen test more sensitive
Silver stains
Fungi (Pneumocystis), Legionella, Helicobacter pylori, Bartonella
Methenamine/Warthin-Starry
Pneumocystis cysts on GMS
Chocolate agar + factors V, X
Haemophilus influenzae
Lysed RBCs supply NAD⁺ (V) and hematin (X)
Satellites around S aureus on blood agar
Thayer-Martin
Neisseria (gonorrhoeae, meningitidis)
Chocolate agar + vancomycin, colistin, nystatin, trimethoprim
Selective — kills competitors
Bordet-Gengou / Regan-Lowe
Bordetella pertussis
Potato, charcoal, blood
PCR now preferred
Buffered charcoal yeast extract
Legionella
Needs iron and cysteine
Urine antigen (serogroup 1) for diagnosis
Löwenstein-Jensen
Mycobacterium tuberculosis
Egg-based; slow growth (weeks)
Liquid culture faster
MacConkey · EMB
Lactose-fermenting gram-negatives turn pink (MacConkey) / E coli green sheen (EMB)
Bile salts select gram-negatives
Non-fermenters: Salmonella, Shigella, Proteus, Pseudomonas
Sabouraud · Eaton · tellurite · TCBS
Fungi · Mycoplasma (cholesterol) · C diphtheriae (black colonies) · Vibrio
Selective additives
Löffler medium: C diphtheriae metachromatic granules
Oxygen requirements
Obligate aerobes: Nocardia, Pseudomonas, M tuberculosis, Bacillus · obligate anaerobes: Clostridium, Bacteroides, Fusobacterium, Actinomyces
Anaerobes lack catalase/superoxide dismutase
Anaerobes: foul abscesses, gas, aminoglycoside-resistant

Mechanism — why some bugs won't Gram stain

Too thinTreponema, Leptospira → darkfield
No wallMycoplasma, Ureaplasma
Mycolic acidsMycobacteria → acid-fast
IntracellularRickettsia, Chlamydia, Legionella, Bartonella, Anaplasma, Ehrlichia, Coxiella

Organisms that Gram stain poorly: Treponema, Mycobacteria, Mycoplasma, Legionella, Rickettsia, Chlamydia (+ Bartonella, Ehrlichia, Anaplasma).

Intracellular lifestyles

TYPE
ORGANISMS
WHY
Obligate intracellular
Rickettsia, Chlamydia, Coxiella
Can't make their own ATP
Facultative intracellular
Salmonella, Listeria, Legionella, Mycobacterium, Brucella, Francisella, Yersinia
Survive in macrophages
Encapsulated
S pneumoniae, H influenzae b, N meningitidis, Salmonella typhi, Klebsiella, GBS, E coli K1
Need opsonization + spleen

How it's tested

COMMON CONFUSIONHaemophilus needs both V (NAD⁺) and X (hematin) — S aureus hemolysis releases V, hence satellite growth on blood agar.
THE TRAPA gram stain showing many neutrophils but no organisms suggests something that doesn't Gram stain (Chlamydia, Mycoplasma, Legionella).
YOUR APPROACHOrganism suspected → stain to see it → medium to grow it → rapid alternative (antigen, PCR).

If you see… think…

Foamy PAS⁺ macrophages in the duodenum → Whipple disease
Halo around yeast in CSF ink prep → Cryptococcus
Weakly acid-fast branching rods → Nocardia
Needs factors V and X → Haemophilus influenzae
Grows on charcoal yeast extract with iron/cysteine → Legionella
Black colonies on tellurite → Corynebacterium diphtheriae
Pink colonies on MacConkey → lactose fermenter (E coli, Klebsiella, Enterobacter)
Needs cholesterol in medium → Mycoplasma (Eaton agar)
Silver stain cysts in BAL fluid → Pneumocystis
Spirochete seen only on darkfield → Treponema