Counting bacteria and growth factors

Microbiologists have exact words for the two states described above. A prototroph is a strain that can grow on a minimal medium — one containing only a carbon source, inorganic salts and water — because it can synthesise every organic compound it needs. An auxotroph is a strain that cannot grow on that same minimal medium because it has lost the ability to make one particular compound and must be supplied with it. The word auxotroph comes from the Greek for additional nourishment, which is precisely what it needs. The mutation that creates an auxotroph usually damages a single gene, and the commonest kinds of damage are a base substitution that changes one amino acid in the enzyme, a deletion or an insertion that shifts the reading frame, or the insertion of a transposable element into the middle of the gene. The enzyme may then be absent, malformed or catalytically dead; in every case one step of the pathway stops, and whatever the pathway makes after that step is the compound that must now be supplied. Microbiologists exploit this deliberately. If a candidate compound is added to the medium and the mutant then grows, that compound is either the missing end product or a later intermediate of the pathway — a technique called auxanography, or growth-factor screening, which was used to work out the order of steps in many biosynthetic pathways long before genes could be sequenced. Auxotrophy is also the basis of the Ames test for mutagens and of many industrial fermentation strains, which are deliberately made auxotrophic so that they cannot survive if they escape the fermenter. Two practical points follow. First, auxotrophy is a property of a strain, not of a species: a wild-type isolate of a species is usually a prototroph, while a laboratory mutant of the same species may be an auxotroph. Second, an auxotroph can revert, because a second mutation can restore the damaged gene and the strain then grows on minimal medium again; this is why stock cultures must be checked and preserved carefully rather than endlessly subcultured.

The routine viable count is a chain of dilutions followed by plating, and every step exists to make the final colonies countable. A measured volume of the sample — usually 1 mL or 0.1 mL — is added to a known volume of sterile diluent, such as physiological saline or a buffered peptone solution, and mixed thoroughly. From that first dilution the same volume is transferred into a second tube, and so on. A tenfold series is the commonest: 1 mL of sample into 9 mL of diluent gives 10⁻¹, 1 mL of that into 9 mL gives 10⁻², and so on. Mixing between steps is not optional; a vortex mixer, or twenty-five strokes of a pipette, is what breaks up clumps and makes each dilution truly represent the one above it. A fixed volume of the chosen dilutions, usually 0.1 mL, is then spread over the surface of a solid medium with a sterile spreader, or 1 mL is mixed with molten agar that has been cooled and poured into a plate. The plates are incubated at the appropriate temperature, 37 °C for clinical material, and the colonies are counted once they are visible. Only plates carrying a countable number are used: below about 30 colonies a single contaminant or a stray pipetting error can distort the estimate, and above about 300 colonies crowding prevents accurate counting and neighbouring colonies may merge. The number obtained is then corrected for the dilution and for the volume plated, so that the answer is expressed per millilitre of the original sample rather than per plate. Duplicate plates are usually prepared and the two counts averaged; if they disagree wildly, the sample must be mixed better and the count repeated. The procedure looks simple, which is exactly why it is so easily got wrong: an uncalibrated pipette, a tube that was not mixed, a spreader used on unevenly dried agar, or a delay before incubation can all change the answer without any error being visible on the bench.

A viable count is reported in colony-forming units, abbreviated CFU, because a colony is not the same thing as a cell. A CFU is the smallest unit that can produce one visible colony: it may be a single cell, but it may equally be a pair of cells still joined after division, a chain of streptococci, a cluster of staphylococci, a clump of cells stuck together by protein or by biofilm, or a filament of a mould. Whatever its composition, the whole unit gives rise to exactly one colony, so the plate counts units of growth rather than individual cells. Correcting the count back to the original sample is what gives the number its meaning. If a plate was inoculated with 0.1 mL of a 10⁻³ dilution and 42 colonies grew, then that 0.1 mL contained 42 CFU, and 1 mL of the undiluted sample, a thousand times more concentrated, contained 4.2 × 10⁵ CFU. As a formula, CFU per millilitre equals the number of colonies divided by the product of the dilution factor and the volume plated. Without that correction the figure is meaningless to a clinician, because the same 42 colonies could stand for anything from 42 to several million organisms per millilitre. The same reasoning explains why a total count can grossly overestimate the living population. A direct microscopic count in a counting chamber sees every particle with the shape and the staining properties of a bacterium, including cells killed by antibiotics or by a disinfectant, cells damaged by heat or by drying, and cells that are alive but cannot grow on the medium offered. Turbidity, read as optical density in a spectrophotometer or nephelometer, has an additional weakness: it estimates total biomass, cannot distinguish living from dead at all, and detects a suspension only once it is dense — roughly above 10⁶ to 10⁷ cells per millilitre — so a sample containing a small but clinically decisive number of living pathogens may read as completely clear. The two approaches therefore answer different questions, and a laboratory that reports a total count without saying so invites the reader to overestimate both the number and the danger of the organisms present.

A growth-factor requirement is a stable, testable property of a strain, so it is recorded and preserved rather than rediscovered in every laboratory. A culture collection — also called a type culture collection or a standard collection — is a repository that acquires, authenticates, preserves and distributes reference strains. Well-known examples are the American Type Culture Collection, whose catalogue numbers begin with ATCC, the National Collection of Type Cultures in the United Kingdom, and the Deutsche Sammlung von Mikroorganismen und Zellkulturen in Germany. When a new species is described, one strain is designated the type strain — the permanent reference for all later isolates — and deposited in at least two collections in different countries, so that it remains available even if one laboratory closes. The collection checks that the deposit is what it claims to be — its identity, its purity and its properties, including any growth-factor requirement — and preserves it by freeze-drying or in liquid nitrogen so that it does not drift through repeated subculture. Catalogues list them plainly, for example “requires nicotinic acid” or “grows on minimal medium”. A documented auxotrophy tells the next laboratory exactly which medium will grow the organism — essential when a strain is used to test an antibiotic, validate a batch of medium, prepare a vaccine or act as a quality-control organism in an accreditation scheme. It also prevents a silent failure in which a laboratory blames the specimen for an organism that did not grow, when the medium simply lacked the one compound the strain cannot make. Growth-factor requirements matter equally in practice: they are why Haemophilus influenzae needs blood-derived factors in its medium, and they are engineered into production strains so that the organism cannot escape the fermenter. Depositing a strain is also a legal requirement for a patent on a biological invention under the Budapest Treaty. For all these reasons, the ability or inability to synthesise a particular compound belongs in an organism’s formal description, alongside its shape, its staining reaction and its oxygen requirements.