Culture Techniques - Types

CULTURE TECHNIQUES - TYPES

Enumeration

The simplest way in which to enumerate the microorganisms that contaminate an object or liquid sample is to dilute that sample to varying degrees and inoculate the surface of a pre-dried nutrient agar with known volumes of those dilutions (see Chapter 2). Individual viable bacteria that are able to grow on the nutrients provided and under the conditions of incubation will produce visible colonies that can be counted and the numbers related back to the original sample. Such counting procedures are often lengthy and tedious; the number of colonies formed might not relate to the viable number of cells, as clumps of cells will only produce a single colony and they will only detect a particular subset of the viable bacteria present in the sample that can grow under the chosen conditions. Accordingly a variety of different media and cultural conditions are deployed to enumerate different categories of organism.

A number of techniques are currently being developed in order speed up the enumeration process, although some of these rapid enumeration techniques indirectly measure the most probable number of viable cells.

A)  Enumeration media

Enumeration media will only ever culture a subset of cells towards which the medium and incubation conditions are directed. Thus, simple salts media with relatively simple sugars as carbon sources and trace levels of amino acids are often used to enumerate bacteria associated with water (e.g. R2A medium). Such plates may be incubated under aerobic or anaerobic conditions at a range of temperatures. Different temperatures will select for different subsets of cells, therefore any description of a viable bacterial count must specify the incubation condi tions. In medical microbiology temperatures akin to the human body are often deployed because only those bacteria able to grow at such temperatures are likely to cause infection. However, psychrophilic Gram-negative bacteria (growing in water at 10 °C) can be a major source of bacterial pyrogen, so a variety of incubation temperatures are often used in monitoring pharmaceutical waters and products. Highly nutritious media, e.g. blood agar, are also used as enumeration media. This is particularly the case when looking for microorganisms such as staphylococci that are usually found in association with animals and humans. Such agar plates may be deliberately exposed to air (settle plates) and the number of colonies formed related to the bacteria content of a room. In the pharmaceutical industry microbiological monitoring will generally report the total aerobic count and, less commonly, the total anaerobic counts obtained on a moderately rich medium such as tryptone soya agar. Sometimes inhibitors of bacterial growth (e.g. Rose Bengal) can be added to a medium in order to select for moulds.

B) Rapid enumeration techniques

The detection and quantification of components of bacterial cells is considerably faster than those approaches requiring the growth of colonies, and estimates of total viable cell number can thereby be obtained within minutes rather than hours and days.

Some of the rapid methods that have been used for bacteria and other microorganisms, e.g. bio-luminescence, epifluorescence and impedance techniques, have been described in Chapter 2, but there are other rapid methods that have found more limited application; these will be considered here. In the examination of pharmaceutical waters and aqueous pharmaceutical products electronic particle counters, e.g. Coulter counters, can be used to determine bacterial concentration, although these instruments do not discriminate between living and dead cells. Similar counters are available that are able to analyse particles found in air. Other rapid techniques aim to detect microbial growth rather than to visualize individual cells and colonies. As bacteria grow in liquid culture they not only alter the conductivity of the culture (see Chapter 2), they also generate small quantities of heat. The time taken to detect this heat can be directly related to the numbers of viable cells present by means of micro-calorimeters. Once again this is a considerable improvement over conventional culture, but unlike particle counting and bio-luminescence can only detect those organisms that are able to grow in the chosen medium.

None of the rapid techniques are able to isolate individual organisms. They do not therefore aid in the characterization or identification of the contaminants.

Enrichment Culture

Enrichment cultures are intended to increase the dominance of a numerically minor component of a mixed culture such that it can be readily detected on an agar plate. Enrichment media are always liquid and are intended to provide conditions that are favourable for the growth of the desired organism and un-favourable for the growth of other likely isolates. This can be achieved either through manipulation of the pH and tonicity of the medium or by the inclusion of chemicals that inhibit the growth of unwanted species. Thus, Mac-Conkey broth contains bile salts that will inhibit the growth of non-enteric bacteria and may be used to enrich for Entero-bacteriaceae. Several serial passages through enrichment broths may be made, and after enrichment it is not possible to relate the numbers of organisms detected back to that in the original sample.

Selective Media

Selective media are solidified enrichment broths, so again they are intended to suppress the growth of particular groups of bacteria and to allow the growth of others. The methods of creating this situation are the same as for enrichment broths. Thus mannitol salts agar will favour the growth of micrococci and staphylococci, and cetrimide agar will favour the growth of pseudomonads. The use of selective media is an adjunct to characterizing the nature of contaminants. Counts of colonies obtained on selective solid media are often documented as presumptive counts, so for example, colonies formed on a MacConkey agar (containing bile salts) might be cited as a presumptive coliform count.

Identification Media (Diagnostic)

Identification media contain nutrients and reagents that indicate, usually through some form of colour formation, the presence of particular organisms. This enables them to be easily detected against a background of other species. In this fashion inclusion of lactose sugar and a pH indicator into MacConkey agar facilitates the identification of colonies of bacteria that can ferment lactose. Fermentation leads to a reduction in pH within these colonies and can be detected by an acid shift in the pH indicator, usually to red. Lactose-fermenting coliforms (Escherichia spp., Klebsiella spp.) can therefore be easily distinguished from non-fermentative coliforms (Salmonella spp., Shigella spp.). Similarly, the inclusion of egg-yolk lecithin into an agar gives it a cloudy appearance that clears around colonies of organisms that produce lecithinase (a virulence factor in staphylococci). While there are numerous types of selective and diagnostic media available, they can only be used as a guide to identification, but microscopy and biochemical or genetic characterization are much more definitive.