Page 258 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 227

Of the three commonly used antimicrobial by comparing inhibition zone measurements
sensitivity assay types: disc diffusion, E-strip and between the sample strain and control strains.
micro-broth dilution, the E-strip and micro-broth Petri dishes are spread uniformly with an
dilution methods are quantitative, and yield MIC inoculum of the bacterial isolate to be tested,
values in mg/ml, and the disc diffusion method and after incubation at 37°C for 18–24 h, deter-
is qualitative, and yields a dichotomous result of mination of sensitivity or resistance is made
sensitive/resistant. The ability to use any of the by measuring the visibly clear area around the
three test types depends on a couple of factors: discs, which is the zone of inhibition of bacte-
commercial availability of discs or E-strips for a rial growth. The ‘zone of inhibition’ depends
particular antibiotic, availability of established on the diffusion of the antibiotic from the discs
MICs and breakpoints for specific antibiotics for into the surrounding agar, and the size depends
specific bacteria, and the physical properties of upon characteristics of the growth medium and
the antibiotic compound itself. Some antibiot- of the antibiotic compound that influence diffu-
ics require specific conditions for solubility such sion of the antibiotic and does not directly relate
as addition of methanol or specific pH of the to the degree of sensitivity or resistance of the
solution which may interfere with the assay. bacteria to the antibiotic (that is, a large zone of
Many variables may affect the quality of test inhibition does not by itself indicate resistance).
including the amount of the inoculum, age of Sensitivity disc diffusion assays can be per-
the bacterial culture, the nature and thickness formed on significant primary cultures (that is,
of the culture medium, the length of inoculation predominant or pure growth of suspect patho-
and the composition of the applied antibiotic(s). gen) from clinical specimens. The advantage is
These variables should be kept to a minimum that results can be available as early as the day
and a standard protocol developed. A basic pro- following the receipt of the specimen. However,
tocol for the most commonly used antimicrobial the disadvantage of primary culture tests is that
sensitivity testing method is outlined below. the number of bacteria in the inoculum cannot
be standardized and sometimes is so small that
results cannot be read or properly assessed; a
Disc diffusion antibiotic sensitivity
testing further test must then be done on a pure sub-
culture of the pathogen. Heavily mixed cultures
The Kirby-Bauer disc diffusion assay is simple, are not suitable for the assay. It is recommended
relatively inexpensive, reliable, and suitable that the bacterial inoculum for disc diffusion
for routine sensitivity testing. The test uses a susceptibility assays be cultured on blood agar,
selection of small discs of a standard filter paper LB, or BHI media, as some ingredients in more
containing pre-determined amounts of chosen selective media such as MacConkey media may
antibiotics. These discs are placed on large size interfere with the assay.
(140 mm) Petri dishes of culture medium. The To perform the test, it is more convenient to
choice of culture medium used in the assay will use dry antibiotic discs prepared and supplied
determine interpretation criteria used for deter- commercially. The discs are 6 mm in diameter
mining sensitivity. If Muller-Hinton agar (MH) and consist of absorbent (filter) paper impreg-
+/– 5% sheep blood, is used, the interpretation nated with a known amount of antibiotic. Each
criteria of the Clinical and Laboratory Standards disc is marked with a letter to show which anti-
Institute (CLSI) can be used. If Luria-Bertani biotic is present. There are protocols available
(LB) or Brain-Heart Infusion agar (BHI) are that describe how to make standardized antibi-
used, interpretation criteria will be determined otic filter discs in the laboratory, which may be

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