The seven routine laboratory media

Suspected typhoid is a chain of decisions that begins long before a plate is opened. Salmonella enterica serovar Typhi is swallowed in contaminated water or food, crosses the intestinal mucosa, multiplies in the mesenteric lymph nodes and enters the bloodstream. The patient then develops a stepwise fever, headache, abdominal discomfort and, during the second week, rose spots on the trunk and an enlarged spleen. Because the organism is in the blood early and reaches the stool later, the specimen chosen depends on the week of illness: blood culture is most rewarding in the first week, stool or urine culture in the second and third, and bone marrow culture — uncomfortable but the most sensitive — is reserved for difficult cases. The nursing point comes first: the sample must be collected before the first dose of an antibiotic, because one dose can suppress the organism and convert a true positive into a misleading “no growth”. If transport is delayed, the stool goes into Cary-Blair medium, which keeps the organism alive without letting it proliferate. Once the stool reaches the laboratory, direct plating is usually insufficient. A gram of faeces contains hundreds of millions of bacteria and Salmonella is a tiny minority, so the specimen is first placed in an enrichment broth: classically selenite F, or tetrathionate broth, or Rappaport–Vassiliadis broth incubated at 42 °C. Selenite is reduced by the faecal flora, and the resulting products are toxic to coliforms while Salmonella survives and multiplies; after twelve to eighteen hours at 37 °C the ratio of Salmonella to its competitors has shifted enough for subculture to succeed. Over-incubation lets the competitors catch up again, which is why the broth is subcultured after a fixed interval rather than left until the next shift. The broth is then streaked onto selective and differential plates. MacConkey agar shows pale, non-lactose-fermenting colonies because Salmonella does not ferment lactose. Salmonella–Shigella agar adds bile salts and dyes that suppress Gram-positive organisms and most coliforms, and its ferric citrate reacts with hydrogen sulphide produced by Salmonella to deposit black iron sulphide at the centre of each colony; bismuth sulphite agar gives black colonies with a metallic sheen. A presumptive identification is made from the plate, and confirmation follows with biochemical tests — triple sugar iron agar showing an alkaline slant, an acid butt and blackening from hydrogen sulphide, a negative urease, a positive citrate and a negative indole — and then with serology, namely agglutination of the isolate with O somatic and H flagellar antisera according to the Kauffmann–White scheme, or detection of the Vi capsular antigen. The Widal test on the patient’s serum is no longer considered reliable. Modern laboratories add matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, or a molecular method, when the isolate is unusual or slow. Two consequences stay with the nurse. The first is public health: typhoid spreads by the faecal–oral route, so safe water, safe disposal of faeces, hand hygiene and exclusion of an infectious patient from food handling are what stop an outbreak, and every case must be notified. The second is the carrier state: a minority of patients keep the organism in the gallbladder and excrete it intermittently for years — the woman remembered as Typhoid Mary was one — and such carriers seed new outbreaks. Treatment must therefore follow susceptibility, because resistance to the older drugs is widespread, and vaccination protects the person who receives it rather than the community around them.

Neisseria gonorrhoeae is a Gram-negative diplococcus that lives inside the polymorphonuclear leukocytes of purulent discharge. It causes urethritis with dysuria and discharge in men, cervicitis that is often silent in women and can ascend to pelvic inflammatory disease and infertility, and, in the newborn delivered through an infected cervix, ophthalmia neonatorum. In the laboratory it is oxidase-positive and extremely fastidious, and the reason is nutritional rather than merely environmental: the organism cannot synthesise several compounds it needs, so it must find them preformed in the medium. Its requirements are precise. It needs haemoglobin as an iron source, and it needs nicotinamide adenine dinucleotide, the coenzyme also called V factor, together with cystine or cysteine and other amino acids. It also needs a humid atmosphere enriched in carbon dioxide, usually five per cent, which is why plates are incubated in a carbon dioxide incubator or a candle jar, and a temperature held at 35 to 37 °C. Plain nutrient agar supplies none of this, and ordinary blood agar is often not enough either, because the red cells are intact and keep their intracellular cofactors inside them. Chocolate agar solves the problem by a physical step rather than a chemical one. The blood added to the base is heated to about eighty degrees Celsius until the red cells lyse, or break open, releasing their haemoglobin and their intracellular nicotinamide adenine dinucleotide into the medium. The result is not chocolate-coloured because of chocolate but because the lysed blood turns brown; the name is a laboratory nickname for heated blood agar. It is an enriched medium, not a selective one: it has no inhibitor, so anything that can grow on it will grow on it. That distinction matters in practice, because a gonococcal specimen rarely contains only the gonococcus. A urethral swab from a man may be almost pure, but an endocervical, rectal or pharyngeal swab carries a dense normal flora, and on plain chocolate agar that flora would overgrow the pathogen. The laboratory therefore uses a selective variant, such as Thayer–Martin or New York City medium, which is chocolate agar with vancomycin to suppress Gram-positive organisms, colistin to suppress Gram-negative organisms other than Neisseria, nystatin for yeasts and trimethoprim for Proteus. This plate is both enriched and selective, which is exactly the overlap the seven categories warn about. For the nurse, the practical consequences are concrete. The swab should reach the plate quickly, plates are brought to room temperature before inoculation, and the specimen is transported in a carbon dioxide atmosphere; refrigeration is avoided because cold kills the organism, which is why a gonococcal swab is never placed in the refrigerator overnight. Culture remains valuable because it gives a susceptibility result, but nucleic acid amplification testing on first-void urine or a self-taken vaginal swab is now more sensitive for diagnosis and is often used first. Treatment has become harder as resistance has accumulated, first to penicillin through penicillinase-producing strains, then to fluoroquinolones, and now with reduced susceptibility to the cephalosporins, so guidelines change and partner notification and prevention counselling matter as much as the prescription.

Selectivity is a chemical trick played on a mixed population, and each inhibitor works in a different way. Bile salts and sodium deoxycholate are surface-active molecules that disrupt the membranes of Gram-positive organisms, which is why MacConkey agar, Salmonella–Shigella agar and deoxycholate agar can be used on faeces, a specimen in which Gram-positive organisms are irrelevant. Crystal violet is a dye that penetrates the thinner Gram-positive wall and blocks growth. Malachite green, the inhibitor in Lowenstein–Jensen medium, is a dye to which Mycobacterium tuberculosis is comparatively resistant, so it suppresses the contaminants of a sputum sample while the tubercle bacillus survives. Tellurite, used in Monsur’s taurocholate and in potassium tellurite media for diphtheria, is reduced by many bacteria to a toxic metallic deposit. Antibiotics are used the same way in Thayer–Martin medium, where vancomycin, colistin, nystatin and trimethoprim each remove one class of competitor. A high salt concentration works by osmosis rather than by poisoning, and an alkaline pH does the same job chemically: alkaline peptone water favours Vibrio because vibrios tolerate alkalinity that suppresses much of the faecal flora. A selective plate, however, only helps if the organism is present in detectable numbers. This is where an enrichment broth earns its place, and the reason is a ratio rather than a total count. Faeces contain perhaps a hundred million organisms per gram, and in typhoid the Salmonella may be a few hundred of them — a ratio of roughly one in a million. A selective plate can suppress the competitors, but it cannot multiply the target, so the few Salmonella colonies that appear may still be missed or overgrown. An enrichment broth changes the ratio itself: in selenite F the competitors are inhibited and the Salmonella divides, so that after a subculture interval the ratio may be closer to one in a thousand, which a selective plate can then handle. The same logic explains why the broth is subcultured after twelve to eighteen hours rather than left for a day: over-incubation allows the surviving competitors to multiply and the advantage is lost. The clinical translation is a lesson in reading a culture as a hypothesis rather than a fact. A selective plate answers the question it was asked, and no more: mannitol salt agar will grow Staphylococcus aureus, but it will also grow coagulase-negative staphylococci from the skin, so a yellow colony is not yet a diagnosis. MacConkey agar will grow Salmonella, but it will also grow any other non-lactose-fermenting Gram-negative rod from the gut. This is why the laboratory moves in steps — presumptive identification from the plate, then biochemical confirmation, then serology or a molecular method — and why the nurse should never treat a plate result in isolation from the Gram stain, the site the specimen came from and the patient’s own condition. A medium is a tool for asking a question, and every tool has a limit.