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A. SARAGEA KT AL.
transfer the yolk aseptically to a sterile stoppered bottle, containing glass
beads. The next day after having checked the sterility of the egg yolks, all
the above mentioned ingredients are mixed with the sterile egg yolks, by
stirring them thoroughly with the help of glass beads. The final medium
should be passed through a funnel with gauze and again checked for
sterility using both aerobic and anaerobic media. This medium may be
stored for 1-2 months at 4°C.
2. Tinsdale medium (modified by Meitert and Saragea, 1967)
(a) Tinsdale basic agar formula
Labemco meat extract (Oxoid) or any other beef extract 1 g
Sodium chloride
0.5 g
Distilled water
100 ml
Adjust to pH = 7-4 and autoclave at 120°C for 20 min. Filter through
cotton, divide into suitable bottles, in known quantities and autoclave at
115°C for 30 min. The basic medium can be stored in the refrigerator at
+4"C for 3 4 months.
(b) Enrichment. T o 100 ml of the basic medium melted and cooled to 55°C
is added strictly in the following order (thoroughly stirring after each
ingredient) :
(i) Normal bovine or horse serum
15 ml
(ii) Defibrinated sheep blood, formalised (per each 100 ml
sheep blood: 0.125 formalin)
(iii) Sodium hydroxide N/10
6 ml
(iv) L-Cystine 0.4% solution in N/IO HCI, pasteurised for
30 min in a waterbath at 60°C
6 ml
(v) Potassium tellurite, 1% solution in distilled water,
sterilized for 15 min at 115 "C
3 ml
(vi) Sodium thiosulphate 2-5 g/100 ml solution in sterile
distilled water pasteurised at 60"C, for 30 min
1.7 ml
Proteose peptone (Difco No. 3)
Bacto agar powder (or equivalent)
1 g
1.5 g
0.3 ml
All solutions are stored at +4"C. The formolised sheep blood can be stored
indefinitely in glass-stoppered bottles. At most five Petri plates can be
prepared for 100 ml base.
I n designing this medium, the suitability of different available peptones
was checked. With some, the brown halo developed very late. Witte
peptone, Difcok Neopeptone and Richter's Peptone gave good results in
contrast to Difco Tryptose, Bactopeptone and Merck Peptone, which are
therefore not recommended.
A. SARAGEA KT AL.
transfer the yolk aseptically to a sterile stoppered bottle, containing glass
beads. The next day after having checked the sterility of the egg yolks, all
the above mentioned ingredients are mixed with the sterile egg yolks, by
stirring them thoroughly with the help of glass beads. The final medium
should be passed through a funnel with gauze and again checked for
sterility using both aerobic and anaerobic media. This medium may be
stored for 1-2 months at 4°C.
2. Tinsdale medium (modified by Meitert and Saragea, 1967)
(a) Tinsdale basic agar formula
Labemco meat extract (Oxoid) or any other beef extract 1 g
Sodium chloride
0.5 g
Distilled water
100 ml
Adjust to pH = 7-4 and autoclave at 120°C for 20 min. Filter through
cotton, divide into suitable bottles, in known quantities and autoclave at
115°C for 30 min. The basic medium can be stored in the refrigerator at
+4"C for 3 4 months.
(b) Enrichment. T o 100 ml of the basic medium melted and cooled to 55°C
is added strictly in the following order (thoroughly stirring after each
ingredient) :
(i) Normal bovine or horse serum
15 ml
(ii) Defibrinated sheep blood, formalised (per each 100 ml
sheep blood: 0.125 formalin)
(iii) Sodium hydroxide N/10
6 ml
(iv) L-Cystine 0.4% solution in N/IO HCI, pasteurised for
30 min in a waterbath at 60°C
6 ml
(v) Potassium tellurite, 1% solution in distilled water,
sterilized for 15 min at 115 "C
3 ml
(vi) Sodium thiosulphate 2-5 g/100 ml solution in sterile
distilled water pasteurised at 60"C, for 30 min
1.7 ml
Proteose peptone (Difco No. 3)
Bacto agar powder (or equivalent)
1 g
1.5 g
0.3 ml
All solutions are stored at +4"C. The formolised sheep blood can be stored
indefinitely in glass-stoppered bottles. At most five Petri plates can be
prepared for 100 ml base.
I n designing this medium, the suitability of different available peptones
was checked. With some, the brown halo developed very late. Witte
peptone, Difcok Neopeptone and Richter's Peptone gave good results in
contrast to Difco Tryptose, Bactopeptone and Merck Peptone, which are
therefore not recommended.