IX. IDENTIFICATION OF MYCOPLASMAS
42 1
individual precipitation lines in the often complex patterns are better
resolved. The macro-test is therefore preferred for the final tests in any
comparative investigation.
1. Equipment
For the micro-test, a length of 4.5 cm is marked off with a diamond in
the centre of a 7.5 x 2.5 cm microscope slide. Slides are soaked in chromic
acid for 48 h, rinsed in distilled water, air-dried (at 37-50°C), and stored in
acetone.
For the macro-test, 8 x 8 cm slide cover glasses are similarly treated
except for diamond marking. On these plates, agar is confined to a circular
area approximately 6.4 cm in diameter by means of a metal ring.
For the micro-test, the template is drilled to give wells 3 mm in diameter
with centres 7 mm apart, and for the macro-test 6 mm in diameter and
12 mm apart.
2. Reagents
(a) Gelling agent. The following formula for an agar gel has proved satisfactory for most purposes:
Sodium chloride (Analar)
4.5 g
Difco Noble Agar
5.0 g
Sodium azide
0.325 g
Deionised water buffered
to pH 7.0 with 0.5 M NaZHP04 500 ml
The sodium chloride is dissolved in the buffered water in a 1-litre
conical flask and heated to 98°C on a magnetic stirrer. The agar is added and
allowed to dissolve at this temperature, which is not exceeded. Sodium
azide is added just before the flask is removed from the heat. Universal
containers are filled with agar, screwed down tightly and stored at 4°C.
If the agar is not calcium-free, phosphate ions from the buffer may precipitate and produce turbid gels. This can be overcome by adding diaminoethane tetra-acetic acid (disodium salt) at 30 mg/litre to chelate Ca2+ ions
(Feinberg, 1957).
If a very clear gel is required, specially purified agar can be prepared by
the method of Feinberg (1956) or that of Crowle (1961). Alternatively,
agarose (Miles Laboratories Ltd, Stoke Poges, England) may be substituted
for agar (Lemcke et al., 1965; Kenny 1969).
(b) Stains and decolourising solution, Amidoschwarz or Ponceau fuchsin are
prepared as follows:
42 1
individual precipitation lines in the often complex patterns are better
resolved. The macro-test is therefore preferred for the final tests in any
comparative investigation.
1. Equipment
For the micro-test, a length of 4.5 cm is marked off with a diamond in
the centre of a 7.5 x 2.5 cm microscope slide. Slides are soaked in chromic
acid for 48 h, rinsed in distilled water, air-dried (at 37-50°C), and stored in
acetone.
For the macro-test, 8 x 8 cm slide cover glasses are similarly treated
except for diamond marking. On these plates, agar is confined to a circular
area approximately 6.4 cm in diameter by means of a metal ring.
For the micro-test, the template is drilled to give wells 3 mm in diameter
with centres 7 mm apart, and for the macro-test 6 mm in diameter and
12 mm apart.
2. Reagents
(a) Gelling agent. The following formula for an agar gel has proved satisfactory for most purposes:
Sodium chloride (Analar)
4.5 g
Difco Noble Agar
5.0 g
Sodium azide
0.325 g
Deionised water buffered
to pH 7.0 with 0.5 M NaZHP04 500 ml
The sodium chloride is dissolved in the buffered water in a 1-litre
conical flask and heated to 98°C on a magnetic stirrer. The agar is added and
allowed to dissolve at this temperature, which is not exceeded. Sodium
azide is added just before the flask is removed from the heat. Universal
containers are filled with agar, screwed down tightly and stored at 4°C.
If the agar is not calcium-free, phosphate ions from the buffer may precipitate and produce turbid gels. This can be overcome by adding diaminoethane tetra-acetic acid (disodium salt) at 30 mg/litre to chelate Ca2+ ions
(Feinberg, 1957).
If a very clear gel is required, specially purified agar can be prepared by
the method of Feinberg (1956) or that of Crowle (1961). Alternatively,
agarose (Miles Laboratories Ltd, Stoke Poges, England) may be substituted
for agar (Lemcke et al., 1965; Kenny 1969).
(b) Stains and decolourising solution, Amidoschwarz or Ponceau fuchsin are
prepared as follows: