Laboratory Identification Approach
On this page
Direct answer
A specimen collected before antibiotics, stained before it dries and cultured on media chosen for the suspected organism identifies most pathogens within 24-48 hours. The sequence runs direct microscopy (Gram, Ziehl-Neelsen, India ink), then culture under the correct atmosphere, then identification by biochemical and serological tests, and finally antimicrobial susceptibility testing. The clinician's contribution is choosing the right specimen at the right point of illness; the laboratory can only answer the question the specimen actually asks.
What you must remember
- Gram stain timing: crystal violet 1 minute, Gram's iodine 1 minute, acetone decolourisation a few seconds, counterstain (dilute carbol fuchsin or safranin) 30 seconds; Gram-positives retain violet, Gram-negatives take the counterstain.
- Acid-fast stains: Ziehl-Neelsen decolourises with 20% sulphuric acid for tubercle bacilli; the modified stain with 1% sulphuric acid colours Cryptosporidium oocysts and weakly acid-fast Nocardia.
- Media classes with examples: enriched (blood, chocolate), selective (MacConkey, deoxycholate citrate agar), enrichment broths (selenite F, tetrathionate, alkaline peptone water at pH 8.6 for Vibrio), transport media (Stuart's, Cary-Blair) and Robertson's cooked meat for anaerobes.
- Atmosphere matters: capnophiles need 5-10% carbon dioxide (Brucella, gonococci); Campylobacter wants 5% oxygen, 10% carbon dioxide at 42 degrees Celsius; anaerobes grow in a GasPak jar.
- Sterilisation benchmarks: autoclave 121 degrees Celsius at 15 lb per square inch for 15 minutes; hot-air oven 160 degrees Celsius for one hour; membrane filtration through 0.22 micron pores for heat-labile fluids.
- Blood culture: dilute blood 1:5 to 1:10 in broth with sodium polyanetholsulphonate to neutralise complement; subculture blindly at 24-48 hours; take three separate sets when endocarditis is suspected.
- Urine thresholds: Kass's significant bacteriuria is 100,000 colony-forming units per mL of midstream urine; Stamm lowered this to 10,000 for symptomatic young women with a single organism, and any growth from a suprapubic aspirate matters.
A worked identification: suspected enteric fever
A 24-year-old presents with stepwise pyrexia of five days. Day 5 of untreated typhoid is the week of bacteraemia, so 10 mL of blood into a Castaneda biphasic bottle (broth plus agar slant) is the highest-yield sample; positivity is highest in week 1 and falls each week as antibodies appear. Blind subculture at 48 hours onto MacConkey yields non-lactose-fermenting colonies, and the laboratory follows with slide agglutination using O and H antisera.
Change the timeline and the best sample changes. In week 2 the organism localises to Peyer's patches and the biliary tree, so stool culture on deoxycholate citrate agar after selenite F enrichment and urine culture outperform blood culture. The Widal test also becomes interpretable only from the second week because it detects antibody, not the organism — a single negative Widal in week 1 excludes nothing. If this patient had instead presented with three days of profuse watery diarrhoea after the monsoon, the pathway would be alkaline peptone water enrichment at pH 8.6, subculture onto thiosulphate citrate bile salts sucrose agar, and dull yellow sucrose-fermenting Vibrio cholerae confirmed by agglutination with polyvalent O1 antiserum.
The same logic of "right specimen, right week" governs every system: pus before antibiotics for abscesses, early-morning sputum before mouthwash for tuberculosis, first-pass urine for urethral sexually transmitted infections.
Classic slips in the practical exam
Transport media and enrichment media are confused repeatedly. Stuart's and Cary-Blair keep organisms alive without multiplying them; selenite F and alkaline peptone water actively multiply Salmonella and Vibrio while suppressing commensals — opposite jobs. The second recurring error is sending anaerobes on a dry swab, where oxygen kills them within minutes; a needle aspiration in a syringe with the needle capped, or Robertson's cooked meat broth, is correct. Third, remember why Haemophilus needs chocolate agar: heating blood releases the V factor from red cells, which is why the same organism shows satellite colonies around Staphylococcus on blood agar.
Frequently asked questions
What colony count defines significant bacteriuria?
Kass's classical threshold is at least 100,000 colony-forming units per mL of a clean-catch midstream sample, but Stamm showed that 100 colony-forming units per mL of a single organism is significant in symptomatic young women with dysuria and frequency. Counts between these values usually reflect contamination or poor technique.
Which stain demonstrates metachromatic granules?
Albert's stain demonstrates the metachromatic (Babes-Ernst) granules of Corynebacterium diphtheriae, staining them black-green against a light green cell body. Loeffler's alkaline methylene blue is the older alternative.
Why is blood diluted in culture broth?
Dilution at 1:5 to 1:10 reduces the bactericidal effect of serum complement and phagocytes, while sodium polyanetholsulphonate prevents clotting and has anticomplement, antiphagocytic and anticoagulant properties that favour bacterial recovery.
Which enrichment broth is used for cholera stool?
Alkaline peptone water at pH 8.6 enriches Vibrio cholerae for 6-8 hours before subculture to thiosulphate citrate bile salts sucrose agar, where it forms yellow sucrose-fermenting colonies.
What are the standard autoclave settings?
Holding at 121 degrees Celsius under 15 pounds per square inch of pressure for 15 minutes achieves sterilisation of standard loads; longer cycles are needed for bulky or fluid loads.