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Motility tests

A motility test asks whether a bacterium can move itself, which is a flagellar property and therefore a genus-level identification clue. It separates organisms that look identical on a Gram film, such as motile Escherichia coli from non-motile Klebsiella and motile Salmonella from non-motile Shigella, and it is the reason a specimen for motility must reach the bench fresh and warm rather than sitting in a refrigerator.

The hanging drop preparation is the direct method: a drop of young broth culture on a cover slip is inverted over the cavity of a concavity slide, sealed with petroleum jelly and examined under the high-power objective with reduced light, and true motility means directional movement that changes the organism's position relative to fixed debris.
True motility must be distinguished from Brownian movement, in which the organisms vibrate in place without changing position, and from streaming caused by fluid currents under the cover slip; this is the standard viva question on the test.
The semisolid stab uses motility medium with about 0.4 percent agar: a motile organism spreads away from the stab line and clouds the whole tube, while a non-motile organism grows only along the line of the stab, and the SIM tube reads motility, indole and hydrogen sulphide production together.
Dark-ground microscopy is used for organisms too slender to see by ordinary light, chiefly Treponema pallidum from a chancre and Leptospira, while a warm saline wet mount of stool shows the directional amoeboid movement of Entamoeba histolytica trophozoites with ingested red cells.
Flagellar arrangement is itself examinable: monotrichous and polar in Vibrio cholerae and Pseudomonas aeruginosa, lophotrichous as a tuft at one end, amphitrichous with one at each end, and peritrichous all over the cell in Escherichia coli, Salmonella and Proteus.
Characteristic movements name the organism: the darting or shooting-star motility of Vibrio cholerae, the corkscrew motility of Campylobacter, the tumbling motility of Listeria monocytogenes at 25 degrees Celsius with its umbrella-shaped growth in semisolid medium, and the swarming of Proteus across a blood agar plate.
Temperature is part of the test, because Listeria is motile at 25 degrees Celsius but not at 37 and Yersinia is motile at 22 but not at 37; the important non-motile organisms to remember are Klebsiella, Shigella, Corynebacterium, Yersinia pestis and all the staphylococci and streptococci.

Tyndallization

Tyndallisation, or intermittent fractional sterilisation, is the destruction of spores by exposing a material to free-flowing steam at 100 degrees Celsius for about 20 to 30 minutes on three successive days, incubating it in between so that surviving spores germinate into vegetative forms that the next day's steaming can kill. It exists because some culture media and fluids are spoilt by the 121 degrees Celsius of an autoclave, and it is not a method for instruments.

The apparatus is a Koch or Arnold steamer, which produces steam at atmospheric pressure and therefore 100 degrees Celsius rather than the 121 degrees of an autoclave, and the schedule is one exposure a day on three consecutive days with incubation in between.
The principle is germination rather than brute force: a spore that survives the first day becomes a vegetative cell overnight, and vegetative cells are killed easily at 100 degrees Celsius, so the method fails for any material in which spores cannot germinate.
It is used for heat-labile culture media and fluids, including media containing sugars, gelatin, egg, serum or milk, and never for surgical instruments, dressings or anything that will enter sterile tissue.
The related methods must be kept separate: inspissation coagulates and sterilises egg and serum media such as Lowenstein-Jensen at 80 to 85 degrees Celsius for about 30 minutes on three successive days in an inspissator, while pasteurisation of milk by the holder method at 63 degrees Celsius for 30 minutes or the flash method at 72 degrees for about 15 seconds is only disinfection and kills the vegetative pathogens alone.
Of the moist-heat methods that work at or below 100 degrees Celsius, only tyndallisation and inspissation reach sterility, and they do so only through the repeated cycle, because a single day's steaming always leaves live spores behind.

Heterotrophs vs autotrophs

Bacteria are classified nutritionally by the source of the carbon from which they build themselves: an autotroph makes its own organic material from carbon dioxide, while a heterotroph must be supplied with ready-made organic carbon. Every bacterium of medical importance is a heterotroph, which is exactly why the laboratory can grow it on a plate, why a culture medium has to contain the right organic nutrients, and why a few pathogens cannot be grown at all.

Autotrophs use carbon dioxide as their sole carbon source: photoautotrophs such as the cyanobacteria take their energy from sunlight, and chemoautotrophs such as Nitrosomonas and Thiobacillus take it from oxidising inorganic compounds like ammonia and sulphur; they matter in soil, water and sewage treatment rather than in disease.
Heterotrophs take their carbon from organic compounds, and the medically important bacteria are all chemoheterotrophs, deriving both carbon and energy from organic molecules.
Heterotrophs are further divided into saprophytes, which live on dead organic matter, and parasites, which live on or in a living host; a facultative parasite such as Escherichia coli does both, which is why it is a harmless commensal in the gut and a pathogen in the bladder.
Nutritional requirement decides the medium: a non-fastidious organism grows on simple nutrient agar, while a fastidious one needs enrichment such as blood agar, chocolate agar for Haemophilus (which needs the X or haemin and V or NAD factors, hence satellitism around a streak of Staphylococcus aureus), Loeffler serum slope for Corynebacterium diphtheriae and Lowenstein-Jensen egg medium for the mycobacteria.
Some organisms cannot be grown on any cell-free medium because they take their nutrition and energy from within a host cell: Chlamydia and Rickettsia are obligate intracellular bacteria grown only in cell culture, embryonated eggs or animals, and all viruses are intracellular by definition.
Two important pathogens have never been grown on artificial media at all, Treponema pallidum and Mycobacterium leprae, so their diagnosis rests on microscopy and serology, and the examination expects both names.
Growth also needs the right physical conditions: temperature, with mesophiles at 37 degrees Celsius and cold enrichment at 4 degrees for Listeria and Yersinia; pH, as in alkaline peptone water at about pH 8.6 for Vibrio cholerae; oxygen, distinguishing obligate aerobes, obligate anaerobes, facultative anaerobes and microaerophiles such as Campylobacter; and carbon dioxide for the capnophiles such as Neisseria and pneumococcus grown in a candle jar.
The bedside consequence is that a report of no growth does not mean no infection: it may mean the wrong medium, the wrong atmosphere, antibiotics given before the specimen was taken, or an organism that simply does not grow on plates.

Schick, Lepromin, Frei, Dick, Widal tests

These are the classical immunological tests that recruitment papers still set as a matching item: Schick for susceptibility to diphtheria, Lepromin for the immune type of leprosy, Frei for lymphogranuloma venereum, Dick for susceptibility to scarlet fever and Widal for enteric fever. All but the Widal are now of historical interest, replaced by antibody assays and nucleic acid tests, and the Widal is the one still ordered in Indian wards, so knowing what it can and cannot prove is what stops a patient being treated for typhoid while the real illness is missed.

Schick test: 0.1 mL of a standard dilution of diphtheria toxin is injected intradermally into the flexor forearm, with heat-inactivated toxin injected into the other arm as a control; a red flush of about 10 to 15 mm read at 24 to 48 hours and persisting to the fifth to seventh day is a positive test, which means the person has too little circulating antitoxin and is susceptible, while a negative test means immunity. It tests immunity, not infection, and has been replaced by measuring serum antitoxin.
Lepromin test: 0.1 mL of lepromin is injected intradermally, and two reactions are read, the early Fernandez reaction of erythema and induration at 48 to 72 hours and the late Mitsuda reaction as a nodule at about 21 days. It is strongly positive in tuberculoid leprosy and negative in lepromatous leprosy, so it classifies the disease and gives the prognosis; it is not a diagnostic test, since it is also positive in many healthy people, and leprosy is diagnosed clinically with a slit-skin smear.
Frei test: an intradermal test using chlamydial antigen for lymphogranuloma venereum, read at 48 to 72 hours; it is obsolete and no longer available, and the disease is now diagnosed by nucleic acid amplification and serology.
Dick test: 0.1 mL of diluted streptococcal erythrogenic (pyrogenic) toxin is injected intradermally and read at 24 to 48 hours; erythema means the person lacks antitoxin and is susceptible to scarlet fever. The converse Schultz-Charlton phenomenon, in which antitoxin injected into the rash blanches it, was used to confirm a scarlet fever rash; both are obsolete.
Widal test: a tube agglutination test that measures antibody to the O (somatic) and H (flagellar) antigens of Salmonella Typhi and to the H antigens of Salmonella Paratyphi A and B. The antibodies appear only at the end of the first week and rise through the second and third weeks, so a Widal drawn in the first week of fever is commonly negative and never excludes typhoid.
Widal interpretation: a single titre is read against the baseline titre of the local population, and the meaningful result is a fourfold rise between paired sera taken 7 to 10 days apart. False positives follow past infection, typhoid vaccination and other febrile illnesses, and false negatives follow early sampling, prior antibiotics and the chronic carrier state; blood culture is the reference test in the first week, bone-marrow culture is the most sensitive, and stool and urine cultures become positive in the second and third weeks.
Every intradermal test follows the same technique: 0.1 mL injected with a 26 or 27 gauge needle, bevel upwards, at a shallow angle into the flexor surface of the forearm so that a visible wheal is raised; the site is not rubbed or massaged, it is ringed with a pen, and it is read at the stated hour. The Mantoux test for tuberculosis is the surviving example, using 5 tuberculin units in 0.1 mL and read as the transverse diameter of induration at 48 to 72 hours.
Safety and waste: the used syringe with its fixed needle goes straight into the white translucent puncture-proof container without recapping, as required by the Biomedical Waste Management Rules 2016, and adrenaline with resuscitation equipment is kept at hand whenever a foreign antigen is injected.

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