Unit 1: Culture and staining technique
Systemic Bacteriology notes · PTU syllabus (BMLS201-18)
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Unit summary
Culturing and staining are the first steps in identifying bacteria from clinical samples. This unit covers instruments for seeding culture media, culture procedures, and the principle, procedure and interpretation of simple, Gram, Ziehl–Neelsen, Albert's and capsule staining.
After this unit you can
- Use instruments to inoculate culture media
- Perform streak, lawn, stab and pour cultures
- Perform and interpret simple, Gram and Ziehl–Neelsen stains
- Perform Albert's and capsule stains
PTU syllabus topics
- Instruments for seeding culture media
- culture procedures
- staining techniques — principle
- procedure
- significance and interpretation of simple staining
- Gram stain
- Ziehl-Neelsen staining
- Albert's stain and capsule staining
- 1Crystal violet
1 min: primary stain
- 2Gram's iodine
1 min: mordant
- 3Decolourise
Acetone or alcohol, a few seconds
- 4Safranin
Counterstain
- 5Read
Gram-positive purple, Gram-negative pink
Topic 1
Instruments for seeding culture media
- Nichrome or platinum loop
- Streaking plates; usual loop delivers about 0.001 or 0.01 mL
- Straight wire (needle)
- Stab cultures, picking single colonies
- Sterile swabs
- Collecting and inoculating samples
- Pasteur pipettes and micropipettes
- Liquid inocula
- Spreader (L-shaped rod)
- Lawn and spread plates
- Bunsen burner or incinerator
- Sterilising loops by red heat
- Flame the loop until red hot, let it cool before touching the sample, and flame again after use; flame the mouths of tubes.
Topic 2
Culture procedures
- 1
Label the plate on the base
- 2
Spread the inoculum on a small area (primary inoculum)
- 3
Flame and cool the loop; streak from the first area into the second
- 4
Repeat into the third and fourth areas
- 5
Incubate inverted at 37 °C for 18–24 hours
- 6
Pick isolated colonies for identification
- Lawn culture
- Even growth over the plate for antibiotic sensitivity
- Stab culture
- Needle into semisolid agar for motility
- Pour plate
- Diluted sample mixed with molten agar for colony counts
- Semi-quantitative urine culture
- Calibrated loop; colonies × 1,000 = CFU/mL
- Liquid culture
- Blood culture bottles; broth enrichment
- Colony morphology: size, shape, margin, elevation, surface, colour, haemolysis on blood agar, lactose fermentation on MacConkey.
Topic 3
Simple staining
- Principle: a single basic dye (methylene blue, crystal violet) binds negatively charged bacterial cells; shows shape and arrangement. Procedure: fix smear, apply stain 1 minute, wash, dry, observe under oil immersion.
Topic 4
Gram staining
- 1
Heat-fix a thin smear
- 2
Crystal violet (primary stain) — 1 minute
- 3
Gram's iodine (mordant) — 1 minute; forms crystal violet–iodine complex
- 4
Decolourise with acetone or alcohol — a few seconds
- 5
Safranin (counterstain) — 1 minute
- 6
Wash, dry, examine under oil
- Principle: Gram-positive cells have a thick peptidoglycan wall that traps the dye complex when dehydrated by alcohol → purple. Gram-negative cells have a thin wall and an outer lipid membrane dissolved by alcohol → decolourised → pink/red with safranin.
- Significance: first step in identification and helps guide early antibiotic choice (e.g., Gram-positive cocci in clusters in blood culture suggest Staphylococcus).
Exam tip
Over-decolourisation makes Gram-positive organisms appear negative; old cultures may stain Gram-variable. Always use known control slides.
Topic 5
Ziehl–Neelsen (acid-fast) staining
- 1Fix the smear
- 2Flood with strong carbol fuchsin; heat until steaming for 5 minutes (do not boil)
- 3Decolourise with 20% sulphuric acid (3% acid-alcohol for some methods)
- 4Counterstain with methylene blue for 1 minute
- 5Wash, dry, examine under oil
- Principle: mycolic acids in the cell wall of Mycobacterium hold carbol fuchsin even after acid decolourisation → acid-fast bacilli appear red on a blue background.
| AFB seen | Fields examined | Report |
|---|---|---|
| None | 100 | Negative |
| 1–9 per 100 fields | 100 | Scanty (record number) |
| 10–99 per 100 fields | 100 | 1+ |
| 1–10 per field | 50 | 2+ |
| More than 10 per field | 20 | 3+ |
- Modifications: Kinyoun (cold) method; 5% sulphuric acid for M. leprae; 0.5–1% for Nocardia (weakly acid-fast).
Topic 6
Albert's staining
- Purpose: demonstrates metachromatic (volutin) granules of Corynebacterium diphtheriae. Procedure: Albert's stain (toluidine blue and malachite green) 3–5 minutes, then Albert's iodine 2 minutes. Result: green bacilli with bluish-black granules at the poles, arranged in V and L shapes ("Chinese letters").
Topic 7
Capsule staining
- Negative staining (India ink or nigrosin): the background darkens while the capsule appears as a clear halo — used for Cryptococcus neoformans in CSF.
- Hiss's method: crystal violet with heat, washed with 20% copper sulphate; capsule appears light blue around a dark purple cell.
- Significance: capsules are virulence factors (Pneumococcus, Klebsiella, Haemophilus influenzae type b).
Key terms
- Streak plate
- Method spreading organisms to obtain isolated colonies
- Mordant
- Substance fixing a stain (Gram's iodine)
- Acid-fast
- Resisting decolourisation by acid after carbol fuchsin staining
- Metachromatic granules
- Granules staining a different colour from the dye
- Negative staining
- Staining the background, not the cell
Quick revision
- Loops, needles, swabs, spreaders; flaming technique.
- Streak, lawn, stab, pour; semi-quantitative urine culture; colony features.
- Gram stain steps and principle; Gram-positive purple, Gram-negative pink.
- ZN stain steps, principle, grading; Kinyoun.
- Albert's stain for C. diphtheriae; India ink and Hiss's capsule stain.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Why is the plate incubated inverted?
- Q2.What is the role of iodine in the Gram stain?
- Q3.Why do Gram-negative bacteria lose crystal violet?
- Q4.What gives acid-fastness to mycobacteria?
- Q5.What do Albert's stain granules look like?
- Q6.Which organism is detected by India ink?
Long-answer questions
- Q1.Describe culture procedures and the instruments used.
- Q2.Explain the principle, procedure and interpretation of the Gram stain.
- Q3.Explain the Ziehl–Neelsen stain and grading of smears.
- Q4.Describe Albert's and capsule staining.
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