Culture media: preparation and consistency

A pure culture is a population descended from a single organism, and separating one organism from the mixture in a clinical sample is the first step of almost every identification. A broth cannot do this, because every cell is free to drift and collide: after incubation the tube holds a blended population, and nothing in the appearance of that cloudiness reveals how many species are present. A solid surface solves the problem by physically immobilising each cell. When a dilute suspension is spread over the gel, individual bacteria settle at separate places; each divides repeatedly without moving, and its descendants accumulate into a discrete colony. If the spreading was dilute enough, every colony began as one cell, so a colony is a clone, a population of genetically identical organisms. Picking one colony with a sterile loop therefore transfers only one species into the next tube, and that transfer is called sub-culturing. The routine that produces this separation is streaking, and the technique is a dilution performed with a loop rather than with a pipette. A small volume of the specimen is spread over one sector of the plate. The loop is then flamed or replaced, so that it carries no residual organisms, and it is drawn through that first sector and across a second, picking up a fraction of the growth and distributing it over fresh agar. The procedure is repeated for a third and usually a fourth quadrant, and the loop is sterilised between sectors. Each pass deposits fewer cells than the pass before, so the last quadrant receives a concentration low enough for well-spaced colonies to develop, while the first sector is usually confluent, a continuous sheet of growth. A plate read after overnight incubation at 37 °C therefore shows isolated colonies at the end of the streak and crowded growth at the beginning, and the isolated ones are the material for identification and susceptibility testing. Two consequences follow for practice. First, sampling matters more than the medium: if the swab is rubbed over the surface of a wound instead of being taken from its depth, the plate will grow skin commensals and the clinician may treat a colonisation as an infection. Second, a culture is pure only if it was handled aseptically; an uncontaminated plate depends on a flamed loop, a sterile dish and a laminar flow cabinet, and one careless touch introduces a contaminant that will later be reported as a second organism. Robert Koch introduced solid media for exactly this purpose, and the older alternative, broth set with gelatin in a flat dish, had already shown the principle before agar replaced gelatin.

Turbidity is the cloudiness of a broth, and beginners often read it as a number. It is not a count. The instrument used to measure it, a spectrophotometer, passes light through the tube and reports optical density or absorbance, which rises as suspended cells scatter the beam. That reading reflects the total mass of particles in the light path, alive or dead, bacterial or otherwise, so a culture killed by an antibiotic can remain just as turbid as a living one. Two tubes of equal optical density may also hold very different numbers if the cells differ in size, and clumps scatter differently from single cells. Nephelometry estimates the same property from scattered light and is used to prepare a standard inoculum, commonly matched to a McFarland barium sulphate standard, so that susceptibility testing begins from a reproducible density. A viable count is a different measurement, and it needs a surface. In the colony count, the sample is diluted in a series, a known volume is spread or poured onto an agar plate, and the colonies that appear are counted after incubation. Each colony is assumed to have arisen from one living cell or one small clump, so the result is expressed as colony-forming units rather than as bacteria, which is why the two terms are not interchangeable. The plate gives the number of organisms able to multiply, which is what matters clinically; the broth reading cannot separate the living from the dead or from debris. When the number is expected to be very low, the most probable number method grows the sample in a series of broth tubes and estimates the concentration from which tubes became turbid, which remains an estimate rather than a count. So why use broth at all? Because many questions are not counting questions. Fermentation studies need a sugar and a pH indicator dissolved in the liquid, together with a Durham tube to trap gas, and the colour and the bubble are read directly; the same test on a plate gives a colour change that is harder to interpret. Biochemical identification, blood culture, the production of a large uniform biomass for antigen or vaccine work, and the growth of organisms for sub-culture all need volume and mixing rather than separation. Broth also has an important limitation that a plate does not: it cannot show purity. A contaminated broth is simply turbid, and a mixed population in liquid looks exactly like a pure one until it is plated out and the colonies are examined.

The consistency of a medium is not a laboratory habit; it is a decision that follows from the question being asked. Ask whether one organism or several are present, and the answer requires a surface, because only a solid gel can hold the descendants of a single cell apart; this is why isolation, colony morphology, purity checks and the preparation of a standardised suspension for susceptibility testing are all done with agar at 1.5–2.0 %. Ask whether an organism can move, and the answer requires a gel weak enough to swim through, which is why motility testing and the cultivation of micro-aerophiles use agar at 0.5 % or less. Ask how much biomass can be produced, or whether a sugar is fermented, and the answer requires a liquid, because a broth mixes freely, accepts dissolved reagents and can be scaled to any volume. Transport and storage media are a fourth case, but they work in opposite ways. Transport media keep organisms alive without letting them multiply, so they are usually semi-solid or liquid and deliberately poor in nutrients, which prevents overgrowth while the specimen is on its way to the laboratory. Storage media have the opposite aim: they maintain the viability of a culture for weeks or months, so they often contain nutrients — cooked meat broth, egg-saline slopes and nutrient agar stabs are the standard examples. The physical properties that make these choices possible belong to agar. Agar is a polysaccharide built from repeating galactose units, and its long unbranched chains trap water into a mesh that behaves as a reversible gel: heating breaks the mesh and the medium becomes liquid, cooling rebuilds it and the medium sets. The two temperatures are far apart, roughly 85–95 °C to melt and 40–45 °C to set, and that hysteresis is the practical gift. A medium can therefore be autoclaved at 121 °C, held molten in a water bath at about 50 °C, poured into dishes, and left to solidify on the bench; the set gel then stays firm in an incubator at 37 °C. The notes state the setting temperature wrongly at line 1138, as 37 °C, and describe the same range only approximately in section 2.3; the values above are the ones to use. Gelatin, an older alternative, fails here because it melts near 35 °C and because many bacteria secrete gelatinase, so the dish liquefies as the culture grows. Beyond the supplied notes, three further choices are made at the bench. Water activity, the availability of free water rather than the total water content, decides whether a medium supports growth, which is why salt or sugar is added to select halotolerant organisms. Buffering decides whether a fermentation can be detected, since the acid produced would otherwise kill the culture before the colour changes. And selective or indicator agents turn one gel into several different tools: a bile salt discourages Gram-positive organisms, a dye reveals a pH change, and a lysed blood layer shows haemolysis. The same base recipe can therefore be asked four different questions simply by changing what is dissolved in it, and a nurse who understands that will read the laboratory report as the answer to a specific question rather than as a fixed label.