Page 260 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 229

pH should be 7.2–7.5. Glucose and reducing Pasteur pipette transfer 1 or 2 ml of the dilute
substances such as thioglycollate should not be suspension to the plate, tip the plate in differ-
added to media. Blood should be added when ent directions to wet the whole of its surface.
testing Streptococcus pyogenes or Pneumococcus Remove all the excess fluid with the pipette,
spp. and heated (‘chocolated’) blood should be partially dry the plate with its lid ajar in the
used when testing Haemophilus spp. A volume incubator for up to 30 min or on the bench for
of 20 ml agar in a standard sized flat-bottomed an hour and finally apply the discs.
Petri dish 100 mm in diameter gives a uniform Spread method: The day of the test, dilute
layer of agar 3–4 mm deep. Plastic Petri dishes your liquid cultures back and grow to mid-
have a fill line. The thickness of the agar should log phase, around an OD of 0.5 at 600 nm.
be standardized for quality control. Alternatively, pick four to five colonies from the
sub-culture plate, and suspend them in sterile
broth to an OD 0.5 at 600 nm. Spread 150 µl of
Test procedure
the culture evenly throughout the plate using a
Dry the culture plate in the incubator with the sterile glass or plastic spreader, or sterile swab.
lid ajar until its surface is free from visible mois- Allow any extra liquid to dry on the plate.
ture. Do not allow the plate to become too dry as Inoculating the plate evenly is an important
this will interfere with the diffusion of the anti- quality control measure for the assay.
biotic in the medium. Inoculate the bacteria to
be tested by one of the procedures given below. Control cultures
Apply the chosen antibiotic discs at adequate
spacing (2 cm or more apart) to the surface of It is occasionally necessary to evaluate the assay
the plate with sterile fine-pointed forceps and by checking the result of the test with a standard
press gently to ensure full contact with the control organism which has a known sensitivity
medium and a resultant moistening of the disc. to specified antibiotics. For example, the Oxford
It is important to ensure full contact of the disc. strain of Staphylococcus aureus is a suitable con-
Incubate immediately for the minimum time trol for tests against most kinds of antibiotics
needed for normal bacterial growth, usually for (except polymyxin). For a ‘control’ for urinary
18–24 h at 37°C. tract cultures an ‘antibiotic-sensitive’ strain of
E. coli is recommended.

Inoculation of primary cultures direct
from specimen Reading and interpretation of results
(see Figure 4.13b)
• Swab: Rub a moist swab, well soaked in sam-
ple material, evenly over the plate. The diameters of the zones of inhibition of
• Milk or Urine: A loop full (0.95 ml) is rubbed growth (including the 6 mm diameter of the
evenly over the plate. disc itself) are measured with callipers or by
viewing the plate against a ruler or ruled screen.
The measurement of the zones of inhibition can
From pure sub-culture
be compared with those published by CSLI.
15
Flooding: this is done by using 5 ml of saline Alternately where ‘control strain’ bacteria are
containing a small amount of bacterial culture available, determination of sensitivity can be
such as an overnight broth culture, or a suspen- made by comparing the zones produced by each
sion of a few colonies in broth. With a sterile drug for the test bacteria with the zones produced

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