Microbiology · Short notes

Microbial Culture and Identification: Short Notes & Cheat Sheet

Gram and acid-fast staining, culture media, haemolysis patterns, the bacterial growth curve, oxygen requirements and key biochemical tests.

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Short notes

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The Gram stain

If decolourisation is too long, Gram-positives look pink; too short, and Gram-negatives look purple.

Staining

Gram stain: crystal violet (primary stain), iodine (mordant), alcohol or acetone (decolouriser — the critical step), then safranin (counterstain).
Gram-positive bacteria stay purple because of their thick peptidoglycan wall. Gram-negatives turn pink; they have a thin wall and an outer membrane containing lipopolysaccharide.
Ziehl–Neelsen (acid-fast) stain uses hot carbol fuchsin, acid–alcohol and methylene blue; mycobacteria stay red because of their mycolic acid.
Other stains: India ink for capsules (Cryptococcus), malachite green for spores, Albert's stain for the granules of Corynebacterium diphtheriae, and KOH mounts for fungi.

Culture media

Enriched media: blood agar and chocolate agar (for fastidious organisms such as Haemophilus and Neisseria).
Selective media: MacConkey (bile salts inhibit Gram-positives), Löwenstein–Jensen (TB), TCBS (Vibrio cholerae), Sabouraud dextrose agar (fungi), Thayer–Martin (gonococci) and mannitol salt agar (S. aureus).
Differential media: on MacConkey, lactose fermenters (E. coli, Klebsiella) form pink colonies and non-fermenters (Salmonella, Shigella) pale ones.
Blood agar haemolysis: α (partial, green — S. pneumoniae, viridans streptococci), β (complete, clear — S. pyogenes, S. aureus) and γ (none — Enterococcus).
Transport media (Stuart's, Amies, Cary–Blair) keep organisms alive; enrichment broths (selenite F) favour Salmonella.

Growth and identification

Growth curve: lag phase, log (exponential) phase, stationary phase and death phase. Cells are most sensitive to antibiotics in the log phase. E. coli divides about every 20 minutes.
Obligate aerobes (Mycobacterium, Pseudomonas), obligate anaerobes (Clostridium, Bacteroides), facultative anaerobes (E. coli, Staphylococcus) and microaerophiles (Campylobacter, H. pylori).
Catalase separates staphylococci (positive) from streptococci (negative). Coagulase identifies S. aureus.
Oxidase is positive in Pseudomonas, Neisseria and Vibrio but negative in Enterobacterales. Urease is positive in Proteus, Klebsiella and H. pylori.
Optochin sensitivity identifies S. pneumoniae; bacitracin sensitivity identifies S. pyogenes. IMViC for E. coli is ++−−.
Modern laboratories also use MALDI-TOF mass spectrometry and PCR.

Cheat sheet

High-yield facts in one place. Cover the right-hand column and test yourself.

Medium → organism

Medium	Used for
Löwenstein–Jensen	Mycobacterium tuberculosis
TCBS	Vibrio cholerae
Sabouraud dextrose agar	Fungi
Chocolate agar	Haemophilus, Neisseria
Thayer–Martin	Neisseria gonorrhoeae
Mannitol salt agar	Staphylococcus aureus
MacConkey	Gram-negative enterics; lactose fermentation
Selenite F broth	Salmonella enrichment

Quick identification tests

Test	Positive
Catalase	Staphylococci (streptococci negative)
Coagulase	S. aureus
Oxidase	Pseudomonas, Neisseria, Vibrio
Urease	Proteus, Klebsiella, H. pylori
Optochin sensitive	S. pneumoniae
Bacitracin sensitive	S. pyogenes
Acid-fast	Mycobacteria, Nocardia (weakly)

Memory aids

Purple Positive, Pink negative: Gram-positive bacteria stain purple; Gram-negative bacteria stain pink.

Exam traps

Decolourisation, not the primary stain, is the step that separates Gram-positive from Gram-negative.
β-haemolysis is complete (clear); α-haemolysis is partial (green).
Enterobacterales are oxidase-negative; Pseudomonas is oxidase-positive.
Antibiotics that act on cell walls work best on actively dividing (log phase) bacteria.

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