688
develops within 18 h (usually within 1 h) after the addition of the reagents. As
before with marine organisms, the medium may be prepared in MSS.
• Degradation of blood
This should be recorded within 7 days as zones of clearing around colonies on
basal medium supplemented with 5 % (v/v) defi brinated sheep’s blood.
• Degradation of gelatin
This is detected after 7 days incubation by the addition of saturated ammonium
sulphate solution to the medium of Smith and Goodner ( 1958 ). A positive result is
indicated by zones of clearing around the bacterial growth. For marine organisms,
the medium should be supplemented with MSS.
• Degradation of starch
Basal medium supplemented with 1 % (w/v) soluble starch is streaked, and incubated at 15–25 °C. After 7 days, the starch plates are fl ooded with an iodine solution
(e.g. Difco Gram’s iodine). The degradation of starch is indicated by a clear area
surrounded by a blue/black background.
• Acid production from maltose and sorbitol
The use of Andrade or phenyl red-peptone water supplemented with maltose or
sorbitol is advocated (see Cowan 1974 ). This medium contains 1 % (w/v) bacteriological peptone 0.5 % (w/v) sodium chloride (for marine organisms this amount
should be increased to 2 %), 1 % (w/v) maltose and Andrade or phenyl red indicator.
The fi lter sterilised (0.22 μm pore size porosity fi lter) maltose solution should be
added to the basal medium after autoclaving, and the completed medium dispensed
into test tubes. The production of acid is indicated by the development of a pink
colour within 48 h at 25–37 °C.
• Production of hydrogen sulphide
Many methods have been developed to detect the production of hydrogen sulphide. We have found success with triple sugar iron agar (Oxoid), which should be
prepared as slopes in test tubes. Following incubation of the inoculated media at
15–25 °C for up to 7 days, the production of hydrogen sulphide is indicated by
blackening of the agar.
• Coagulase test
We recommend a simple test using citrated plasma (of rabbit, sheep, donkey or
ox). The bacterial culture should be emulsifi ed [to form a dense suspension of
~5 × 10
8 cells/ml] in a drop of 0.9 % (w/v) saline on a clean grease-free microscope
slide. This suspension is then carefully mixed with one drop of citrated plasma. A
positive result, which is indicated by clumping of the bacterial cells, is apparent
within 2–3 min.
Most of the above-mentioned phenotypic tests have been derived from medical
microbiology. Nevertheless, careful attention to detail will generate useful data
13 Diagnosis
develops within 18 h (usually within 1 h) after the addition of the reagents. As
before with marine organisms, the medium may be prepared in MSS.
• Degradation of blood
This should be recorded within 7 days as zones of clearing around colonies on
basal medium supplemented with 5 % (v/v) defi brinated sheep’s blood.
• Degradation of gelatin
This is detected after 7 days incubation by the addition of saturated ammonium
sulphate solution to the medium of Smith and Goodner ( 1958 ). A positive result is
indicated by zones of clearing around the bacterial growth. For marine organisms,
the medium should be supplemented with MSS.
• Degradation of starch
Basal medium supplemented with 1 % (w/v) soluble starch is streaked, and incubated at 15–25 °C. After 7 days, the starch plates are fl ooded with an iodine solution
(e.g. Difco Gram’s iodine). The degradation of starch is indicated by a clear area
surrounded by a blue/black background.
• Acid production from maltose and sorbitol
The use of Andrade or phenyl red-peptone water supplemented with maltose or
sorbitol is advocated (see Cowan 1974 ). This medium contains 1 % (w/v) bacteriological peptone 0.5 % (w/v) sodium chloride (for marine organisms this amount
should be increased to 2 %), 1 % (w/v) maltose and Andrade or phenyl red indicator.
The fi lter sterilised (0.22 μm pore size porosity fi lter) maltose solution should be
added to the basal medium after autoclaving, and the completed medium dispensed
into test tubes. The production of acid is indicated by the development of a pink
colour within 48 h at 25–37 °C.
• Production of hydrogen sulphide
Many methods have been developed to detect the production of hydrogen sulphide. We have found success with triple sugar iron agar (Oxoid), which should be
prepared as slopes in test tubes. Following incubation of the inoculated media at
15–25 °C for up to 7 days, the production of hydrogen sulphide is indicated by
blackening of the agar.
• Coagulase test
We recommend a simple test using citrated plasma (of rabbit, sheep, donkey or
ox). The bacterial culture should be emulsifi ed [to form a dense suspension of
~5 × 10
8 cells/ml] in a drop of 0.9 % (w/v) saline on a clean grease-free microscope
slide. This suspension is then carefully mixed with one drop of citrated plasma. A
positive result, which is indicated by clumping of the bacterial cells, is apparent
within 2–3 min.
Most of the above-mentioned phenotypic tests have been derived from medical
microbiology. Nevertheless, careful attention to detail will generate useful data
13 Diagnosis