444
U. ULLMANN
H. Biochemical identification
The substrate for performance of biochemical tests is the thioglycollate
medium without agar and -dextrose or material of colonies. The following
methods can be used.
1. Catalase production is tested on specimen holders by application of
three to four drops of 3% solution of hydrogen peroxide into which material
is rubbed from a single colony. Bubbling occurs immediately if the isolate
is catalase positive.
2. Oxidase is proved by scraping some colonies from a pure culture and
rubbing them on a filter paper; this is soaked with two or three drops
of 1 % aqueous tetramethyl-p-phenylene diamine dihydrochloride; blue
colour within 10-20 sec indicates a positive reaction.
3. Nitrate reduction is investigated by adding 0.1% potassium nitrate to
thioglycollate medium. After four days incubation dimethyl-a-naphthylamine reagent is dropped into the culture; a red colour complex indicates
reduction to nitrite.
4. Sodium selenite reduction is examined on Brucella agar (Albimi) which
contains 0.1% sodium selenite. The test medium is inoculated with a
streak and incubated four days using the Fortner method or in an anaerobic
incubator. A positive reaction is indicated by red pigmented colonies.
5. Tween hydrolysis is tested on D.S.T. agar: 40 g D.S.T. agar (Oxoid)+
0.1 g CaCl2 * HzO + 2.0 g MgS04- 7H20 is dissolved in 990 ml of distilled
water and sterilised by autoclaving. Tween-40 (palmitic acid ester) or one
of the other Tweens are autoclaved separately and then added to the base
medium which has been cooled to 50-60°C and poured into plates, The
test medium is inoculated with a streak and incubated for four days as
described above. Hydrolysis of Tweens is revealed by speckled and cloudy
zones around the colonies (Ullmann and Blasius, 1974).
6. Hydrogen sulphide production is demonstrated on thioglycollate medium
with 0.02% cysteine; on the top of the cultures is placed a strip of filter
paper impregnated with a solution of 10% basic lead acetate; blackening
of the paper after four days indicates that the isolate produces H2S.
7. For the tolerance test 1% glycine or 3.5% NaCl are added to thioglycollate medium and multiplication is examined after four days incubation.
8. Motility is checked by the hanging drop method or in the S.I.M. medium
after four days incubation.
9. Temperature tolerance is tested by simultaneous inoculation of three
thioglycollate media; all tubes are placed in a water bath, one at 25"C, the
other at 37°C and the third at 42°C. Growth is estimated after four days
incubation.
U. ULLMANN
H. Biochemical identification
The substrate for performance of biochemical tests is the thioglycollate
medium without agar and -dextrose or material of colonies. The following
methods can be used.
1. Catalase production is tested on specimen holders by application of
three to four drops of 3% solution of hydrogen peroxide into which material
is rubbed from a single colony. Bubbling occurs immediately if the isolate
is catalase positive.
2. Oxidase is proved by scraping some colonies from a pure culture and
rubbing them on a filter paper; this is soaked with two or three drops
of 1 % aqueous tetramethyl-p-phenylene diamine dihydrochloride; blue
colour within 10-20 sec indicates a positive reaction.
3. Nitrate reduction is investigated by adding 0.1% potassium nitrate to
thioglycollate medium. After four days incubation dimethyl-a-naphthylamine reagent is dropped into the culture; a red colour complex indicates
reduction to nitrite.
4. Sodium selenite reduction is examined on Brucella agar (Albimi) which
contains 0.1% sodium selenite. The test medium is inoculated with a
streak and incubated four days using the Fortner method or in an anaerobic
incubator. A positive reaction is indicated by red pigmented colonies.
5. Tween hydrolysis is tested on D.S.T. agar: 40 g D.S.T. agar (Oxoid)+
0.1 g CaCl2 * HzO + 2.0 g MgS04- 7H20 is dissolved in 990 ml of distilled
water and sterilised by autoclaving. Tween-40 (palmitic acid ester) or one
of the other Tweens are autoclaved separately and then added to the base
medium which has been cooled to 50-60°C and poured into plates, The
test medium is inoculated with a streak and incubated for four days as
described above. Hydrolysis of Tweens is revealed by speckled and cloudy
zones around the colonies (Ullmann and Blasius, 1974).
6. Hydrogen sulphide production is demonstrated on thioglycollate medium
with 0.02% cysteine; on the top of the cultures is placed a strip of filter
paper impregnated with a solution of 10% basic lead acetate; blackening
of the paper after four days indicates that the isolate produces H2S.
7. For the tolerance test 1% glycine or 3.5% NaCl are added to thioglycollate medium and multiplication is examined after four days incubation.
8. Motility is checked by the hanging drop method or in the S.I.M. medium
after four days incubation.
9. Temperature tolerance is tested by simultaneous inoculation of three
thioglycollate media; all tubes are placed in a water bath, one at 25"C, the
other at 37°C and the third at 42°C. Growth is estimated after four days
incubation.