THE MERCK VETERINARY MANUAL
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Clinical Microbiology

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In-house microbiology can be a valuable asset to practitioners, providing quick results with minimal investment. Expensive equipment and materials are not usually necessary for the recovery of common aerobic or facultatively anaerobic bacterial pathogens, such as Staphylococcus spp, Streptococcus spp, and coliforms. While microbiologic media is not difficult to prepare, it may be more convenient to purchase from a scientific supply house. Most bacteria will grow readily on standard media (blood agar and MacConkey agar plates) when incubated aerobically. Basic equipment should include an incubator, refrigerator, Bunsen burner or portable gas torch, and microscope with low, high, and oil immersion objective lenses. Materials should consist of inoculating loops, prepared microbiologic media, microscope slides, Gram-stain reagents, 3% hydrogen peroxide, oxidase reagent, microbial identification systems, and a current veterinary microbiology textbook.

Although it is not always easy to obtain optimal specimens when working with animals, certain practices can ensure the best possible specimen under the circumstances. Application of the following principles should result in acceptable specimens that produce high-quality microbiology results: 1) Specimens must be obtained aseptically from a site that is representative of the disease process. 2) Swabs are the most common specimen collected, but they are generally not the specimens of choice, as they may become contaminated with commensals during the collection process, and they provide a small sample volume. Swabs are most useful for obtaining specimens from skin pustules, ears, conjunctiva, deep within draining tracts or wounds, soft tissue infections, or the reproductive tract. 3) A sufficient quantity of material should be collected to permit adequate examination. 4) Specimens must be collected at the proper time in the disease process and prior to the initiation of antimicrobial therapy to maximize pathogen recovery. 5) If cultures are not immediately initiated after collection, specimens should be refrigerated.

Microbiologic testing should include both direct, microscopic examination and culture of the specimen. Gram-stained smears should be examined using oil immersion in order to determine the correct reaction. Generally, both solid (agar) and liquid (broth) media should be inoculated. Solid media permit colony isolation, rough bacterial quantitation, and selection or differentiation of normal flora from potential pathogens, while broth media allow for the recovery of small numbers of organisms.

Clinical specimens should be inoculated onto both general purpose and selective media to maximize bacterial recovery. Plates containing trypticase/tryptic soy agar with 5% sheep blood are the most widely used types of general-purpose media. Selective and/or differential media include MacConkey agar (gram-negative bacteria), mannitol salt agar (staphylococci), and phenylethyl alcohol agar (gram-positive bacteria). Microbiologic media should be stored in the refrigerator, but should be allowed to warm to room temperature prior to inoculation.

Transfer of specimens to plated media depends on the type of specimen. Liquid specimens are inoculated by use of a sterile syringe or pipette. Swabs are generally plated directly by rolling the swab over an area ∼2 cm in diameter. Feces are inoculated by dipping a swab into the specimen. Surgical biopsy specimens may be touched directly to the agar surface.

Bacterial identification methods depend on obtaining isolated colonies. The most common technique to achieve isolation is to streak plates using a wire loop. After the specimen has been inoculated onto a plate, a wire loop is flamed, cooled, and passed at a 90° angle several times through the initial area of inoculation. The plate is rotated 90°, the loop is flamed and cooled, and the process is repeated 3 more times. This results in 4 quadrants on the plate, which enable quantitation of the relative numbers of bacteria present in a given sample after colonies appear.

After inoculation, plates and tubes should be labeled and placed in an incubator set at 35–37°C. Plates are incubated with their lids down to prevent condensed water from dropping down from the lid onto the agar surface, which can result in confluent bacterial growth.

The first step in culture evaluation is the visual examination of plated media. Most bacteria produce visible colonies in 24 hr, although some require 48–72 hr. Inspection includes examination of colonial morphology, noting both the types and numbers of colonies and any hemolytic reactions on blood-based agar. Further classification is based on the presence or absence of growth on differential or selective media.

After the evaluation of plated media, examination of Gram stains made from each different colony type is performed. Those reactions, combined with colonial morphology, may allow for the presumptive identification of organisms. If more than one colony type is present, subcultures of each are made. A single colony is streaked to a plate of nonselective medium. This will ensure a pure culture of the unknown organism, which is required for biochemical characterization and identification.

Several microbial identification systems are commercially available. Systems may be manual or automated. Each usually contains a complete package for the identification of a particular group of organisms. There are specific systems for staphylococci, streptococci, corynebacteria, nonfermenting gram-negative rods, and Enterobacteriaceae. All are useful for conducting a wide variety of biochemical tests simultaneously. Most manual systems consist of plates or strips made up of a series of wells or cups that contain test substrates. A pure culture of the unknown organism in suspension is added to the test wells. The strips are incubated aerobically at 37°C for 24–48 hr. The wells are viewed for colorimetric changes and a biocode is generated from scoring the test results. This numeric code is compared to those in the system database to obtain an identity for the test organism. Most of the systems are adequate for the identification of common, rapidly growing aerobic to facultatively anaerobic veterinary pathogens.

There are 2 common procedures for determining the antimicrobial susceptibility of bacteria that are able to grow aerobically within 24 hr. Both procedures have been accepted by the Clinical and Laboratory Standards Institute (CLSI). The first procedure is qualitative and is based on the agar disk diffusion method of Bauer and Kirby. The second procedure is quantitative and involves dilutions of antimicrobial agents, which are tested either in liquid or solid agar media. The latter is referred to as the minimal inhibitory concentration test.

The agar disk diffusion method is more widely used for testing the common, rapidly growing aerobic to facultatively anaerobic pathogenic bacteria. The determination of antimicrobial susceptibility of many fastidious bacteria, such as Haemophilus parasuis or Pasteurella spp, remains to be standardized. The Bauer-Kirby method is based on the diffusion of an antimicrobial agent impregnated within a paper disk through an agar medium. Briefly, a suspension of actively growing test organism is standardized to a turbidity equivalent to 0.5 on the McFarland scale. Within 15 min of standardization, a sterile swab is dipped into the bacterial suspension and a dry Müller-Hinton agar plate is inoculated by streaking the swab over the entire surface 3 times. To ensure an even distribution of the inoculum, the plate is rotated ~60° each time. Antimicrobial disks are placed on the plate and are gently pressed down to ensure their close contact with the agar surface. Inverted plates are placed in an incubator at 35°C. Plates are examined after 18 hr of incubation. Zones of complete inhibition are measured in millimeters using a ruler. The zone sizes around each drug are compared to those published by the CLSI in document M31-A3 in order to make an interpretation of susceptible, intermediate, or resistant for each agent tested.

Reliable antimicrobial susceptibility testing results can only be obtained by following standardized procedures. Any procedural deviation may lead to erroneous results. It is imperative to properly store antimicrobial susceptibility test disks and test media and to ensure quality control for each test. Any clinic that does not regularly perform testing on a weekly basis may wish to use a veterinary diagnostic laboratory for this procedure instead.

Last full review/revision March 2012 by Karen W. Post, DVM, MS, DACVM

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