4.4.1 Routine procedures

Animal health laboratories implement highly streamlined microbiological workflows, which should adhere to the laboratory’s SOPs. Individualised analyses are only carried out in specific circumstances, such as during investigations of severe disease outbreaks.

Activity 7: Who decides what procedures to undertake?

Timing: Allow about 5 minutes

What happens in your laboratory when a specimen arrives? Who deals with the specimen first and what actions do they take?

Write in the text box and then compare your notes with the sample answer.

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Discussion

Your laboratory may operate differently, but usually a qualified staff member – ideally a veterinary microbiologist – examines the submission form to determine the most appropriate tests for the case based on the clinical information provided.

Any major discrepancy between the microbiologist’s understanding of the case and the tests requested is clarified with the submitting professional at this stage.

The main phases of bacterial isolation are illustrated in Figure 9. The process involves one to three incubation periods, depending on whether a short or long protocol is followed.

Described image
Figure 9 Steps followed in the isolation of bacteria from clinical specimens. The short pathway (blue arrows) involves a first incubation on primary solid media often followed by subculture of single colonies and a second incubation, to obtain the clinical isolate. The long pathway (red arrows) has an additional sub-culturing step onto a secondary medium before single colonies are sub-cultured on the isolation media. The choice of primary media depends on the metabolic needs of the target organisms. Blood agar supports the growth of most common organisms found in the field and is commonly used. Chocolate blood agar is another example of an enriched medium used for specific fastidious bacteria such as Haemophilus spp. Some laboratories inoculate in parallel primary selective media for Gram-negatives or Gram-positives. Modified from G. R. Carter, Diagnostic Procedures in Veterinary Bacteriology and Mycology. Fourth Edition. Charles C Thomas Publisher, Springfield, Illinois, USA
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Steps followed in the isolation of bacteria from clinical specimens.

Figure 9 Steps followed in the isolation of bacteria from clinical specimens. The short pathway (blue arrows) involves a first incubation...

Which pathway is best suited for specimens taken from sterile sites, and why?

Answer

The short pathway (blue arrows) is usually used for sterile-site specimens (for example, from pneumonia lung lesions), as direct inoculation on a primary solid medium is likely to yield a pure culture of the infecting organism. A colony can then be picked and suspended or sub-cultured onto isolation medium to obtain an isolate.

The long pathway (red arrows) is often used for the analysis of non-sterile site specimens where commensals or contaminants are present (for example, faeces), or when an enrichment of a sub-lethally injured pathogen, or a pathogen that may be present below the LoD is needed (for example, specimens collected from an animal treated with antimicrobials).

What is the purpose of the additional sub-culturing step? (See Section 4.2)

Answer

Initial growth in a selective broth enables the growth of the target organism and suppresses the growth of other bacteria present in the inoculum. This allows you to obtain visible colonies of the target organism on the secondary solid medium.

As you will learn in Section 7.3, isolation procedures for Salmonella often combine the two pathways to increase overall testing sensitivity. If salmonellosis is suspected, visceral organs can be cultured on solid primary media and selective enrichment, and faeces in selective enrichment only.

4.4 Procedures for bacterial isolation

5 Bacterial identification