2 Basic tests used to isolate and identify bacterial pathogens

We turn now to a description of the laboratory tests used to identify pathogens.

2.1 Culture media

Growing isolates from clinical samples in the laboratory is a useful part of diagnosing the cause of an infectious disease and is an essential step for determining the antibiotic sensitivities of bacteria.

All artificial media comprise a mineral base, a nitrogen source, a carbon source and an energy source. Supplements of organic compounds may be added to encourage or suppress the growth requirements of particular microorganisms. Media may be in liquid (broth) or solid form.

Most clinical samples are inoculated directly onto solid, agar-based culture media on a plate. When grown at optimal temperatures in suitable conditions, this process enables the laboratory technologist to view individual colonies and select them for additional work according to their appearance (colonial morphology) and relative abundance. Table 1 provides details of the main media types used routinely in microbiology laboratories. Note that media may fall into more than one of the categories listed, for example it may be both selective and indicator.

Table 1 Media types used in diagnostic microbiology
Media Use
Basic Standard nutritional content for non-fastidious organisms. Also used as a base for other media.
Selective Contains substances (antibiotics, bile salts etc.) which suppress certain organisms. Used for specimens where a lot of competing commensal flora are likely to be found.
Enriched Contains extra nutrients (blood, vitamins etc.) for fastidious organisms which have special growth requirements.
Indicator Contains substances which change colour, for example when carbohydrate fermentation leads to acid production by certain organisms.
Chromogenic Contains chromogenic substrates that change colour in response to the presence of bacterial enzymes. The colour change is species specific, therefore organisms can be identified by colony colour. Allows rapid, basic identification but is relatively expensive and requires well-controlled storage conditions.

Activity 6: Culture media in your workplace

Timing: Allow 10 minutes

Use Table 2 to list the types of agar-based media you have in your laboratory and what you use them for. Note which category of media described in Table 1 each comes into. What considerations govern your laboratory’s choice of media?

Table 2 Media used routinely in your workplace
Name of medium Use Category
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Discussion

You may use many of the media in the example provided or use others not on the list. The media available to your laboratory will depend on factors such as local suppliers, transport and logistics, preferences in your country/region, and cost.

There may be more up-to-date media than the ones you are using as the technology continues to advance. These media save a lot of time, but may be too costly or complicated to use for many district laboratories or for those processing small numbers of specimens. Ready-made media save time in pouring, sterilising, and QC, but are not always available or practical outside of big cities.

Name of agar medium Use Category
Nutrient General purpose; supports growth of a wide range of non-fastidious organisms Basic
Blood For fastidious and non-fastidious organisms, most swabs, subculture of blood cultures Enriched
Chocolate Growing the most fastidious organisms Enriched
CHROMagarTM Orientation urines Identify common pathogens quickly Chromogenic
CLED (cysteine- lactose- electrolyte-deficient) Urine samples: only culture those samples with positive microscopy. This medium has characteristic colony appearance and colour for the common pathogens. It also stops Proteus from swarming over the plate. Selective Indicator
MacConkey Isolate Gram-negatives Selective Indicator
Modified New York City (MNYC) Isolate Neisseria gonorrhoeae Selective Enriched
Mueller-Hinton (M-H) Antimicrobial Susceptibility Testing (AST) – can add supplements for some species Enriched
Xylose Lysine Deoxycholate (XLD) agar Isolate Salmonella and Shigella species Selective Indicator

1.3 Transport, labelling and processing of samples

2.1.1 Processing blood cultures