Microbiology chapters
Culture, stains and special media
The bacterial cell (Gram-positive versus Gram-negative walls, spores, flagella, plasmids), the stains used to see bacteria (Gram, Ziehl-Neelsen and others), the bacterial growth curve with generation-time calculations, oxygen requirements, and the culture media you must match to their organisms (blood agar, MacConkey, Löwenstein-Jensen, TCBS and more).
- Gram-positive bacteria have a thick peptidoglycan wall with teichoic acids (retain crystal violet: purple); Gram-negative bacteria have a thin peptidoglycan layer plus an outer membrane containing LPS (endotoxin) and porins (decolourised, take up safranin: pink/red); organisms poorly seen on Gram stain include mycobacteria (mycolic acid: Ziehl-Neelsen), Treponema (too thin: dark-field), Mycoplasma (no wall), Chlamydia and Rickettsia (intracellular) and Legionella (silver stain).
- Spores (Bacillus, Clostridium) resist heat and disinfectants because of dipicolinic acid and need autoclaving; the growth curve has lag (adaptation), log (exponential growth; most sensitive to antibiotics), stationary (spore formation) and death phases.
- Media: blood agar (haemolysis: α green, β clear, γ none), chocolate agar (Haemophilus, Neisseria: factors X and V), MacConkey (lactose fermenters pink: E. coli, Klebsiella), Löwenstein-Jensen (TB), TCBS (Vibrio cholerae: yellow), Thayer-Martin (N. gonorrhoeae), Bordet-Gengou/charcoal agar (Bordetella), Loeffler serum and tellurite (C. diphtheriae), Sabouraud (fungi).
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Bacterial structure
Bacteria are prokaryotes: no nucleus (a single circular chromosome in the nucleoid), no mitochondria, 70S ribosomes, and (usually) a peptidoglycan cell wall. They divide by binary fission.
Cell wall
| Feature | Gram-positive | Gram-negative |
|---|---|---|
| Peptidoglycan | Thick (many layers) | Thin (one or two layers) |
| Teichoic and lipoteichoic acids | Present (adhesion, inflammation) | Absent |
| Outer membrane | Absent | Present: contains LPS (endotoxin) and porins |
| Periplasmic space | Small | Present (β-lactamases sit here) |
| Gram stain colour | Purple (violet) | Pink (red) |
| Lysozyme sensitivity | High | Low (outer membrane protects) |
| Antibiotic entry | Easier for large drugs (vancomycin) | Outer membrane blocks many drugs (vancomycin cannot cross) |
Peptidoglycan: chains of N-acetylglucosamine and N-acetylmuramic acid cross-linked by peptides (transpeptidation; target of β-lactams; D-Ala-D-Ala is the target of vancomycin). Lysozyme (tears, saliva) breaks the sugar backbone.
Acid-fast cell walls (mycobacteria, Nocardia weakly): rich in mycolic acid (waxy), resisting Gram staining and drying.
Organisms without a wall: Mycoplasma (cholesterol in the membrane; not seen on Gram stain; resistant to penicillins and cephalosporins).
Other structures
| Structure | Function and notes |
|---|---|
| Capsule | Polysaccharide (except D-glutamate in B. anthracis); antiphagocytic; basis of vaccines (pneumococcal, Hib, meningococcal, typhoid Vi). Quellung reaction (capsule swelling with antibody) |
| Flagella | Motility; H antigen (Salmonella typing); peritrichous (E. coli, Proteus: swarming), polar (Vibrio, Pseudomonas) |
| Pili (fimbriae) | Adhesion; sex pilus for conjugation |
| Spores (endospores) | Formed by Bacillus and Clostridium when nutrients run out; dormant, resistant to heat, drying and disinfectants (thanks to dipicolinic acid with calcium and a keratin-like coat); killed by autoclaving; germinate when conditions improve |
| Plasmids | Extra-chromosomal circular DNA: antibiotic resistance, toxins (E. coli LT/ST), pili |
| Inclusion granules | Metachromatic (volutin) granules in C. diphtheriae (Albert stain) |
| Cytoplasmic membrane | No sterols (except Mycoplasma); site of respiration (no mitochondria) |
Staining and microscopy
Gram stain
- Crystal violet (primary stain): all cells purple.
- Gram's iodine (mordant): forms a crystal violet-iodine complex.
- Decolouriser (acetone or alcohol): washes the dye out of Gram-negatives (thin wall, outer membrane disrupted); Gram-positives keep it.
- Safranin (counterstain): Gram-negatives turn pink/red.
Poorly seen or not seen on Gram stain:
| Organism | Reason | Alternative |
|---|---|---|
| Mycobacterium | Mycolic acid (waxy wall) | Ziehl-Neelsen (acid-fast), auramine-rhodamine (fluorescent) |
| Treponema, Leptospira | Too thin | Dark-field microscopy, silver stain, serology |
| Mycoplasma | No cell wall | Serology, PCR |
| Chlamydia, Rickettsia | Intracellular, small | Giemsa, PCR, serology |
| Legionella | Stains poorly | Silver stain, urine antigen |
"These Little Microbes May Unfortunately Lack Real Colour": Treponema, Leptospira, Mycobacteria, Mycoplasma, Ureaplasma, Legionella, Rickettsia, Chlamydia.
Ziehl-Neelsen (acid-fast) stain
- Carbol fuchsin with heating (heat softens the waxy mycolic acid so the dye penetrates).
- Decolourise with acid-alcohol (20–25% sulphuric acid for TB): acid-fast bacilli resist and stay red.
- Counterstain with methylene blue: background and other cells blue.
Acid-fast organisms: M. tuberculosis, M. leprae (weakly: 5% acid), Nocardia (weakly, modified ZN), Cryptosporidium oocysts, bacterial spores, Isospora.
Kinyoun stain is a cold modification (more phenol, no heating).
Other stains
| Stain | Use |
|---|---|
| India ink (negative stain) | Cryptococcus neoformans capsule in CSF |
| Giemsa | Malaria, Borrelia, Chlamydia inclusions, Leishmania, Trypanosoma, Rickettsia |
| Silver stains (Warthin-Starry, GMS) | Legionella, H. pylori, Treponema, fungi (Pneumocystis: methenamine silver) |
| Albert / Neisser stain | Metachromatic granules of C. diphtheriae |
| PAS | Fungi; Tropheryma whipplei (Whipple disease) |
| KOH mount | Fungal hyphae in skin and nail scrapings |
| Spore stain (malachite green) | Bacterial spores |
Bacterial growth
Growth curve (in a closed culture)
| Phase | What happens |
|---|---|
| 1. Lag | Bacteria adapt: enzymes and ribosomes made; no increase in number |
| 2. Log (exponential) | Rapid doubling at a constant rate; cells most uniform and most sensitive to antibiotics (especially cell wall drugs) |
| 3. Stationary | Nutrients run out and wastes build up: growth = death; spores and many exotoxins form |
| 4. Decline (death) | Viable numbers fall exponentially |
Generation (doubling) time: E. coli about 20 minutes in ideal conditions; M. tuberculosis about 15–20 hours (cultures take weeks); M. leprae about 2 weeks.
N = N₀ × 2ⁿ, where n = time ÷ generation time
E. coli, generation time 20 min, start 1,000 cells, 2 hours:
n = 120 ÷ 20 = 6 generations
N = 1,000 × 2⁶ = 1,000 × 64 = 64,000 cells
M. tuberculosis, generation time about 18 h, 1 cell → visible colony needs about 10⁶ cells:
2ⁿ = 10⁶ → n ≈ 20 generations (2²⁰ ≈ 1,048,576)
time ≈ 20 × 18 h = 360 h ≈ 15 days (cultures take 2–8 weeks)
Oxygen requirements
| Group | Examples |
|---|---|
| Obligate aerobes | M. tuberculosis (favours lung apices), Pseudomonas, Nocardia, Bordetella, Bacillus (most) |
| Obligate anaerobes (lack catalase/superoxide dismutase) | Clostridium, Bacteroides, Actinomyces, Fusobacterium, Peptostreptococcus: foul-smelling abscesses, gas; resistant to aminoglycosides |
| Facultative anaerobes | Most bacteria: E. coli, staphylococci, streptococci, Salmonella |
| Microaerophilic | Campylobacter, H. pylori, Treponema |
| Capnophilic (need extra CO₂) | Neisseria, Haemophilus, Brucella (candle jar) |
Temperature: most pathogens are mesophiles (optimum 37 °C); Listeria and Yersinia grow in the refrigerator (4 °C: cold enrichment); Campylobacter jejuni grows at 42 °C.
Culture media
Types
| Type | Purpose | Examples |
|---|---|---|
| Basal/simple | Supports non-fastidious organisms | Nutrient agar, peptone water |
| Enriched | Extra nutrients for fastidious organisms | Blood agar, chocolate agar, Loeffler serum |
| Selective | Inhibits unwanted organisms | Thayer-Martin, LJ, TCBS, Wilson-Blair, tellurite, deoxycholate citrate agar |
| Differential (indicator) | Shows differences between organisms (colour) | MacConkey (lactose), blood agar (haemolysis), CLED, TCBS (sucrose) |
| Enrichment (liquid) | Boosts the wanted organism among many others | Selenite F and tetrathionate broth (Salmonella from stool), alkaline peptone water (Vibrio) |
| Transport | Keeps organisms alive without growth | Stuart, Amies, Cary-Blair (stool: Vibrio, Salmonella, Shigella), Venkatraman-Ramakrishnan (Vibrio) |
| Anaerobic | Low oxygen | Robertson's cooked meat broth, thioglycollate broth |
Media you must know
| Medium | Organism or use |
|---|---|
| Blood agar (5–10% sheep blood) | General; shows haemolysis: α (partial, green: S. pneumoniae, viridans), β (complete, clear: S. pyogenes, S. agalactiae, S. aureus), γ (none: Enterococcus often) |
| Chocolate agar (heated blood: releases factors X (haemin) and V (NAD)) | Haemophilus influenzae, Neisseria |
| MacConkey agar (bile salts + lactose + neutral red) | Gram-negative enteric bacilli; lactose fermenters → pink (E. coli, Klebsiella (mucoid), Enterobacter); non-lactose fermenters → pale (Salmonella, Shigella, Proteus, Pseudomonas) |
| Löwenstein-Jensen (LJ) (egg, glycerol, malachite green) | Mycobacterium tuberculosis (buff, rough, "breadcrumb" colonies after 2–8 weeks); liquid MGIT culture is faster |
| TCBS (thiosulphate-citrate-bile salts-sucrose) | Vibrio cholerae (sucrose fermenter: yellow colonies) |
| Thayer-Martin (chocolate agar + vancomycin, colistin, nystatin, trimethoprim) | Neisseria gonorrhoeae (and meningitidis) from contaminated sites |
| Bordet-Gengou (potato-blood-glycerol) / Regan-Lowe (charcoal) | Bordetella pertussis ("bisected pearl"/mercury drop colonies) |
| Loeffler serum slope | C. diphtheriae (rapid growth, granules) |
| Potassium tellurite agar (Tinsdale, McLeod) | C. diphtheriae (black colonies) |
| Buffered charcoal yeast extract (BCYE) (cysteine, iron) | Legionella |
| Skirrow / Campy-BAP (42 °C, microaerophilic) | Campylobacter |
| Deoxycholate citrate agar (DCA), XLD, SS agar | Salmonella, Shigella (Salmonella black colonies from H₂S) |
| Wilson-Blair (bismuth sulphite) | Salmonella Typhi (black, metallic sheen) |
| Sabouraud dextrose agar (acidic, with antibiotics) | Fungi |
| EMB (eosin methylene blue) | E. coli: green metallic sheen |
| CLED | Urine culture (prevents Proteus swarming) |
| Cell culture / animals | Viruses, Chlamydia, Rickettsia (obligate intracellular); M. leprae (armadillo, mouse footpad: cannot be cultured in media) |
| Blood culture bottles | Septicaemia, typhoid (first week), endocarditis |
Key identification tests
- Catalase: separates staphylococci (+) from streptococci (−).
- Coagulase: S. aureus (+) vs coagulase-negative staphylococci (S. epidermidis, S. saprophyticus).
- Oxidase (+): Pseudomonas, Neisseria, Vibrio, Campylobacter, H. pylori (Enterobacteriaceae are oxidase-negative).
- Urease (+): H. pylori (breath test), Proteus (struvite stones), Klebsiella, Cryptococcus, Ureaplasma.
- Optochin sensitive + bile soluble: S. pneumoniae (viridans: resistant). Bacitracin sensitive: S. pyogenes. CAMP test (+), hippurate: S. agalactiae. Bile esculin (+), grows in 6.5% NaCl: Enterococcus.
- H₂S: Salmonella, Proteus. Indole: E. coli.
Expect: Gram-positive vs Gram-negative walls, peptidoglycan and teichoic acid, organisms not seen on Gram stain and their alternatives, Mycoplasma lacking a wall, spores and dipicolinic acid, Gram stain steps, Ziehl-Neelsen principle and acid-fast organisms, India ink and Giemsa uses, growth curve phases and antibiotic sensitivity, generation times, oxygen requirements, media types with examples, chocolate agar (X, V), MacConkey lactose fermenters, LJ, TCBS, Thayer-Martin, Bordet-Gengou, tellurite, and catalase, coagulase, oxidase and urease tests.
Quick revision
- Gram-positive: thick peptidoglycan, teichoic acid, purple. Gram-negative: thin + outer membrane (LPS), pink.
- Not on Gram stain: mycobacteria (ZN), Treponema (dark-field), Mycoplasma (no wall), Chlamydia, Rickettsia, Legionella (silver).
- ZN: heat + carbol fuchsin; acid-fast stays red.
- Spores: Bacillus, Clostridium; dipicolinic acid; autoclave.
- Growth curve: lag, log (antibiotic-sensitive), stationary (spores), death.
- Blood agar haemolysis; chocolate (X, V) for Haemophilus; MacConkey pink lactose fermenters; LJ for TB; TCBS for Vibrio; Thayer-Martin for gonococcus; tellurite for diphtheria.
- Catalase: staph + / strep −. Coagulase: S. aureus.
Written by The House Job from standard microbiology references (Levinson's Review of Medical Microbiology and Immunology).
FAQ
Why are mycobacteria not stained by the Gram method?
Their cell walls are rich in waxy mycolic acid, which keeps dyes out; they are stained with the Ziehl-Neelsen method using heat.
In which phase of growth are bacteria most sensitive to antibiotics?
The log (exponential) phase, when they are dividing rapidly.
Which medium is used to grow Mycobacterium tuberculosis?
Löwenstein-Jensen medium (or liquid systems such as MGIT); growth takes 2–8 weeks.
What does MacConkey agar show?
It grows Gram-negative enteric bacilli and separates lactose fermenters (pink colonies, such as E. coli) from non-lactose fermenters (pale colonies, such as Salmonella and Shigella).