2. Sabouraud - cycloheximide - chloramphenicol 
agar* for selective isolation of pathogenic 
fungi. 
Composition: Sabouraud dextrose agar (2% 
agar content) 
Cycloheximide** 0.5 mg./ml. 
Chloramphenicol*** 0.05 mg./ 
ml. 
Preparation: 1 liter 
a. Suspend 65 gm. dehydrated Sabouraud 
dextrose agar and 5.0 gm. agar in 1,000 
ml. distilled water. Heat to boUing. 
b. Add chloramphenicol (50 mg. suspended 
in 10 ml. of 95% alcohol) to above boil- 
ing medium. Remove quickly from heat 
and mix. 
c. Add cycloheximide solution (500 mg. in 
10 ml. of acetone). 
d. Mix well and distribute in tubes. 
e. Autoclave at 118° C. for 10 minutes — no 
longer. Slant and allow to harden. 
3. Brain heart infusion — cycloheximide-chloram- 
phenicol agar for isolation of fastidious fungi, 
for example, Histoplasma capsulatum and 
Blastomyces dermatitidis, at 25° C. or room 
temperature. 
Composition: Brain heart infusion agar (2% 
agar content) 
Cycloheximide 0.5 mg./ml. 
Chloramphenicol 0.05 mg./ml. 
Preparation: 1 liter 
a. Suspend 37 gm. dehydrated brain infusion 
agar and 20.0 gm. agar in 1,000 ml. dis- 
tilled water. Heat to boiling. 
b. Add chloramphenicol 0.05 mg./ml. to the 
boiling medium. Remove from heat quick- 
ly and mix. 
c. Add cycloheximide 0.5 mg./ml. 
d. Mix well and distribute in tubes. 
e. Autoclave at 118° C. for 10 minutes — no 
longer. Tube and slant. 
4. Chloramphenicol solution to add to sputum 
bottles for isolation of H. capsulatum 
Preparation of stock solution. 
Suspend 20 mg. chloramphenicol in 10 
ml. 95% alcohol. Add 90 ml. of dis- 
tilled water. If necessary, heat gently to 
complete solution. This is a stable solu- 
tion. 
* Two essentially similar media are available in dehy- 
drated form: Mycosel agar (BBL, Division of BioQuest); 
Mycobiotic agar (Difco). 
** Actidione (The Upjohn Co., Kalamazoo, Mich.) 
*** Chloromycetin (Parke-Davis Co., Detroit, Mich.) 
Use: Add 1.0 ml. of the stock solution to each 
sterile sputum bottle. This amount is 
ample for inhibition of contaminants in 
1 to 10 ml. sputum. If the solution dries 
in the bottle before use, its effectiveness 
is unimpaired. The concentration de- 
sired in sputum is approximately 0.2 
mg./ml. of chloramphenicol. 
5. Preparation of PVA-fixative for preservation 
of stool specimens for parasitologic diagnosis 
Add 6 gm. of polyvinyl alcohol* (PVA) 
powder to 100 ml. of Schaudinn’s fixative at 
room temperature, stirring constantly. 
Modified Schaudinn’s Fixative: 
Glacial Acetic Acid 5.0 ml. 
Glycerol 1.5 ml. 
Schaudinn’s Fixative (2 parts 
saturated aqueous solution 
of mercuric chloride and 1 
part 95% ethyl alcohol) 93.5 ml. 
Heat to about 75° C. or higher until powder 
dissolves and suspension clears. 
Cooled to room temperature, the solution 
should be clear and free of lumps. Solutions 
prepared with some lots of PVA may remain 
turbid and may exhibit some precipitate 
upon cooling. Unless these conditions are 
excessive, they will not interfere with satis- 
factory use of the solution. PVA-fixative 
remains satisfactory for several months and 
can be used either at room temperature or 
heated to 50° C. 
A quantity of specimen is thoroughly mixed 
in a vial containing three or more parts of 
PVA-fixative. Films for staining can be pre- 
pared immediately or months later by spread- 
ing two or three drops of the mixture over the 
surface of a microscope slide. The smear 
should cover about one-third of the slide sur- 
face and it should, to reduce peeling during 
staining, extend to the edge of the side of the 
slide. It is important not to have the films too 
thick and to allow them to dry thoroughly. If 
the specimen in the vial jells, it can be lique- 
fied by heating in a water bath prior to making 
the films. 
6. Chopped meat medium. 
Ground meat (fat free) 500 gm. 
Distilled water 1000 ml. 
1 normal NaOH 25 ml. 
Use lean beef or horse meat. Remove fat and 
connective tissue before grinding. Mix meat, 
* Gelvatol — available from Shawinigon Resins Co., Spring- 
field, Mass. 
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