Page 249 - The Veterinary Laboratory and Field Manual 3rd Edition

218 Susan C. Cork and Roy Halliwell

Gram-positive organisms (dye retained) will be drop of toluene. Put the tube in a water bath
blue/purple and Gram-negative organisms (dye at 37°C for 2–3 min. Add 0.25 ml of ONPG
4
not retained) will be pink in colour due to the (O-Nitrophenyl-Beta-D-Galactopyranoside)
counter-stain. reagent and mix. Return the tube to the water
bath at 37°C for 24 h. A yellow colour change is a
positive reaction. ONPG is a spectrophotometric
Gram stain reaction of some groups
of organisms and colorimetric substrate for the detection of
B-galactosidase activity. If the bacteria under test
Gram-positive Gram-negative contains galactosidase the ONPG reagent turns
Staphylococci Coliforms from colourless to yellow. This reaction is linked
Streptococci Neisseriae to the ability of the bacteria to ferment lactose.
Corynebacteria Campylobacter sp. (Vibrio)
Clostridia Salmonellae Oxidation fermentation test
Bacillus spp. Shigellae
Two tubes of Hugh-Liefson media are inocu-
5
Haemophilus group lated with broth containing the bacteria to be
Brucellae tested. One is covered with a layer of sterile
Pasteurellae liquid paraffin. The tubes are incubated at 37°C
overnight. This test differentiates bacteria which
Many other stains are used in microbiology (see utilize glucose by oxidation from those which
page XX for the modified Ziehl–Neelsen stain) utilize it by fermentation. The uninoculated
but will not be covered here. For more infor- medium is green (pH 7.1), if the pH becomes
mation see the bibliography at the end of the acidic, as a result of glucose utilization, it turns
chapter, see also Figure 4.9. yellow.

• Oxidation (aerobic). The colour indicator
biochemical tests turns yellow in open tubes and remains green
in covered tubes, for example, Pseudomonas sp.
Regional veterinary laboratories will usually • Fermentation (anaerobic). The colour indica-
have enough microbiology samples submitted tor turns yellow in both tubes for example,
to warrant the maintenance of supplies for a Aeromonas sp. Bromothymol blue is the indi-
5
number of routine biochemical tests. The fol- cator (pH 6.0 yellow – pH 7.6 green/blue).
lowing are a few examples of simple tests which
could also be considered for busy regional and Catalase test
district laboratories. For more detailed descrip-
tions of tests consult a text book or, preferably, For this test 3% hydrogen peroxide (H O ) is
2 2
attend a bacteriology training course. There are added to colonies taken from a pure culture of
also some useful online resources available that bacteria on a glass slide. Immediate release of
illustrate many of the following tests. bubbles of gas indicates a positive reaction. The
test can also be done on an agar slope or directly
on a plate culture.
Beta-galactosidase test
The enzyme catalase, which is produced by
Suspend a loopful of the bacterial colony to some bacteria, is responsible for the release of
be tested in 0.25 ml saline in a tube. Add one oxygen from hydrogen peroxide.

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