When a doctor sends urine, pus, sputum, blood or skin scrapings to the microbiology lab, a handful of basic techniques do most of the work: sterilisation with the autoclave, Gram staining, the KOH mount for fungi, culture and sensitivity testing, and quick biochemical tests such as the oxidase test. This guide explains the principle, basic procedure and interpretation of each, for students, lab technicians and curious patients.
Quick answer: An autoclave sterilises equipment and media with pressurised steam, typically 121°C at 15 psi for 15 to 20 minutes. Gram staining sorts bacteria into Gram-positive (purple) and Gram-negative (pink). A 10% KOH mount dissolves skin and nail keratin so fungal threads can be seen. Culture grows the organism from a sample, and sensitivity testing shows which antibiotics will work, usually within 48 to 72 hours. The oxidase test turns purple within seconds for bacteria such as Pseudomonas, Neisseria and Vibrio.
Key takeaways
- ✅ Collect samples before starting antibiotics whenever possible.
- ✅ Gram stain gives a same-day clue; culture confirms the organism.
- ✅ Sensitivity reports help doctors choose the right antibiotic and fight antibiotic resistance.
- ✅ Always handle samples as potentially infectious, and sterilise before disposal.
The autoclave: how it works
Principle: steam under pressure reaches temperatures well above boiling point. Moist heat kills microorganisms, including heat-resistant bacterial spores, by coagulating their proteins.
| Standard cycle | Temperature | Pressure | Time |
|---|
| Routine media, glassware, instruments | 121°C | 15 psi (about 1 bar above atmosphere) | 15–20 minutes |
| Flash or rapid cycle | 132–134°C | about 30 psi | 3–4 minutes |
Basic steps: add water to the required level, load items loosely so steam can reach every surface, close the lid, let steam push out all the air, allow pressure to build and hold for the set time, then let pressure fall to zero before opening. Quality control uses indicator tape or chemical indicators on each load and biological indicators with Geobacillus stearothermophilus spores periodically. Heat-sensitive items and oils are sterilised by other methods.
Gram staining
Principle: bacteria differ in cell wall structure. Gram-positive bacteria have a thick peptidoglycan layer that traps the crystal violet–iodine complex, so they stay purple after decolourising. Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane that lets the dye wash out, so they take up the pink counterstain.
- Make a thin smear, air dry and heat fix.
- Crystal violet (primary stain) for about 1 minute; rinse.
- Gram's iodine (mordant) for about 1 minute; rinse.
- Decolouriser (acetone or alcohol) for a few seconds until colour stops running; rinse at once.
- Safranin (counterstain) for about 1 minute; rinse, dry and examine under oil immersion.
| Result | Colour | Examples |
|---|
| Gram-positive cocci | Purple | Staphylococcus (clusters), Streptococcus (chains) |
| Gram-positive bacilli | Purple | Bacillus, Clostridium, Corynebacterium |
| Gram-negative cocci | Pink | Neisseria (pairs) |
| Gram-negative bacilli | Pink | E. coli, Klebsiella, Pseudomonas, Salmonella |
Over-decolourising can make Gram-positive bacteria look pink, and thick smears can make Gram-negative bacteria look purple. Mycobacteria such as TB bacilli do not stain well with Gram stain and need an acid-fast (Ziehl–Neelsen) stain.
KOH mount for fungal infections
Principle: 10% potassium hydroxide (up to 20% to 40% for nails) dissolves keratin and cells in skin, hair and nail samples, but fungal cell walls resist it, so fungal elements stand out under the microscope.
- Scrape the edge of the skin lesion, or clip the nail or pluck affected hair.
- Place the material on a slide with a drop of KOH and cover with a coverslip.
- Wait 10 to 30 minutes (longer for nails, or warm gently).
- Examine under low and then high power with reduced light.
Result: branching, septate hyphae suggest dermatophytes (ringworm, tinea); budding yeast with pseudohyphae suggest Candida; short hyphae with round spores ("spaghetti and meatballs") suggest pityriasis versicolor. A negative mount does not fully rule out fungal infection; fungal culture may be needed.
Culture and antibiotic sensitivity test
Culture grows organisms from the sample on nutrient media such as blood agar and MacConkey agar, incubated at 37°C. Colonies are identified by appearance, Gram stain and biochemical tests, or by automated systems. Sensitivity testing, most often by the Kirby–Bauer disc diffusion method or automated MIC testing, shows which antibiotics stop the organism growing.
- Sensitive (S): the antibiotic is likely to work at standard doses
- Intermediate or "susceptible, increased exposure" (I): may work at higher doses or at certain body sites
- Resistant (R): unlikely to work
Most results take 48 to 72 hours; blood cultures and slow-growing organisms take longer. Samples must be collected in sterile containers and, ideally, before antibiotics are started.
Oxidase test
Principle: some bacteria have the enzyme cytochrome c oxidase. It oxidises the reagent tetramethyl-p-phenylenediamine, producing a dark purple colour.
- Place a strip of oxidase filter paper on a clean slide, or use a commercial swab.
- Pick a colony with a platinum loop or wooden stick (nickel loops can give false positives) and rub it onto the paper.
- Watch for colour change within 10 to 30 seconds.
Positive (purple): Pseudomonas, Neisseria, Vibrio, Campylobacter, Aeromonas. Negative (no change): E. coli, Klebsiella, Salmonella and other Enterobacterales. It is a quick first step in identifying Gram-negative bacteria.
Frequently asked questions
Why should samples be taken before antibiotics?
Even one dose can stop bacteria growing in culture, giving a false negative result and making it harder to choose the right treatment.
Can a Gram stain tell which antibiotic to use?
It helps doctors choose a first antibiotic on the same day, but culture and sensitivity results are needed to confirm the best choice.
Is an autoclave the same as boiling?
No. Boiling at 100°C kills most germs but not all spores. An autoclave reaches 121°C or higher under pressure, which achieves true sterilisation.
Related reading: Tests for tuberculosis and blood tests for malaria.
Last updated: September 2026. Laboratory education only. Follow your laboratory's standard operating procedures and biosafety rules.
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