Page 250 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 219

Citrate test Hydrogen sulphide production
For this test inoculate a tube of Koser citrate Inoculate a tube of TSI (triple sugar iron) agar
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medium with the test bacterium and incubate with test bacteria by stabbing the butt and
at 37°C for 96 h. Growth will only occur when streaking the slope; incubate for 24–48 h and
a bacterium can use a citrate compound as a observe for blackening due to the production
carbon and energy source and an ammonium of hydrogen sulphide (H S), see Figure 4.11c.
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compound as a source of nitrogen. These two TSI agar contains three sugars, glucose (0.1%),
chemicals, but no other sources of carbon and lactose (1.0%) and sucrose (1.0%). Phenol red
nitrogen, are in Koser citrate medium. Growth is the pH indicator. Unreacted and alkaline TSI
is assessed by the degree of turbidity. slopes are red, and an acid pH results in a yellow
colour change. Ferrous sulphate or ferric ammo-
Coagulase test nium citrate with sodium thiosulphate react
to form a black deposit to indicate hydrogen
The enzyme coagulase is produced by the vast sulphide production.
majority of pathogenic Staphylococci and its
presence therefore is taken as an indication of
pathogenicity. Coagulase has the ability to clot Methyl red (MR) reaction
or coagulate blood plasma. Inoculate MR-VP medium (glucose phosphate
Emulsify a colony of the bacteria to be peptone water) with test bacteria and incubate
tested in sterile distilled water on a slide and at 35–37°C for 2 days. Add two drops of methyl
add a loopful of rabbit plasma. A positive test red solution, shake and examine. A red colour
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is indicated by clumping or agglutination of the change indicates a positive reaction and a yellow
bacteria. Occasionally a bacteria that is coagu- colour indicates a negative reaction. A positive
lase positive will be negative to this test so reaction occurs when glucose is fermented pro-
‘negative’ tests should be confirmed by a tube ducing acidic conditions (pH < 4.5) which result
test. To do this, dilute rabbit plasma 1 in 10 with in a change of colour of MR dye in the medium.
normal saline. Place 0.5 ml of diluted plasma and
five drops of an overnight broth culture of test
bacteria in tubes approximately 80 × 12 mm in Voges-Proskauer (VP) reaction
size. Incubate at 37°C and examine for clotting After completion of the MR test, add 0.6 ml of
at intervals up to 5 h. 5% α-naphthol solution and 0.2 ml of 40%
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Note: Use a known coagulate positive potassium hydroxide (KOH) aqueous solution
Staphylococcus sp. as a positive control and use the to the sample, shake, slope the tube and examine
diluent without the organism as a control for auto- after 15 min (or up to 4 h) later. A positive reac-
agglutination. Human (but not bovine) plasma tion is indicated by the development of a pink
can be used if rabbit plasma is not available. colour. To intensify and speed up the reaction
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add a few crystals of creatine or two drops of
0.5% creatine solution. The pink/red colour is
Gelatine liquefaction
due to the formation of acetyl methyl carbinol.
Inoculate a tube of nutrient gelatine medium
with test bacteria using a straight wire stab.
Incubate at room temperature for up to 7 days Nitrate reduction test
and observe for liquefaction. Liquefaction occurs Inoculate nitrate broth with test bacteria and
due to breakdown of protein in the medium. incubate for 24 h. Add two drops of sulfanilic

Vet Lab.indb 219 26/03/2019 10:25