agent, such as fresh thioglycollate broth, at 
room temperature for a period not exceed- 
ing two hours. 
A Gram stained smear should be prepared 
on all specimens, and each type of organism 
seen in smears should be isolated in pure 
culture at the local laboratory. Pure culture 
isolates can be shipped to a reference labora- 
tory for final identification. Thioglycollate 
medium, enriched with 10% normal rabbit 
serum, is preferable for isolation of the non- 
sporeforming anaerobes, and chopped meat 
medium (Appendix II) is preferable for the 
sporeforming anaerobes. It is important to 
plate specimen material directly on fresh 
blood agar plates since some fastidious an- 
aerobes may be overgrown by other organ- 
isms in broth medium. Broth cultures are 
incubated 24 hours, preferably in an 
anaerobe jar; Gram stains are prepared, and 
a second set of blood agar plates is inocu- 
lated. Blood agar plates should be incubated 
in an anaerobe jar for a minimum of 48 
hours. Isolated colonies should then be 
picked to chopped meat or thioglycollate 
medium for isolation of pure cultures. 
For shipment of pure cultures to a reference 
laboratory, actively growing cultures in 
screw-cap tubes of chopped meat medium, 
thioglycollate medium, or semi-solid thio- 
glycollate medium with 0.5% added agar 
can be used. Prior to shipment, a one-half 
inch column of sterile 5% agar should be 
layered over the culture to act as a seal, and 
the cap should be sealed with waterproof 
tap>e. 
2. Anthrax. Anthrax is primarily an infectious 
disease of animals; however, man may be in- 
fected through contact with infected animals or 
animal products. The majority of cases are cu- 
taneous infections, but pulmonary and intestinal 
infections may be seen. “Industrial anthrax” 
occurs in workers exposed to carpet wools, 
goat hair, or skins originating in areas where 
the disease is prevalent among animals. Labora- 
tory diagnosis is made by smear examination, 
culture of the organism, animal inoculation, or 
fluorescent antibody technic. 
Specimens for laboratory diagnosis 
a. Smears may be made from sputum, blood, 
other fluids, or tissues, and then air dried 
and heat fixed. 
b. Blood, vesicle fluid, and scrapings from the 
base of the lesion, regional lymph nodes, or 
other organs, taken in as clean a manner as 
feasible, may be cultured on plain or blood 
agar or inoculated into animals. 
c. Tissue impression smears, sputum, other 
clinical material and cultures may be stained 
with fluorescent antibody for specific identi- 
fication of the organism. 
d. Aqueous extracts of soil samples, heat- 
treated to destroy vegetative cells, may be 
cultured or used for animal inoculation. 
All types of examinations should be made 
in the nearest competent laboratory because 
delay in the shipment of fluids or tissues may 
result in contamination of the specimen and 
death of the organism. 
3. Brucellosis. This disease is found primarily in 
goats, cattle, and swine. Man contracts the 
disease either by direct contact with diseased 
animals or through consumption of infected 
milk and milk products. Although the three 
organisms. Brucella melitensis, Brucella abortus, 
and Brucella suis, most often infect goats, cows, 
and pigs, they are not host specific, and all in- 
fect man as well. The disease occurs in acute, 
subacute, and chronic forms; and all three types 
are produced by any of the species of Brucella. 
Infection occurs most frequently by direct in- 
vasion of the organism through the intact in- 
testinal mucosa; but stockyard workers, farmers, 
and veterinarians are often infected through the 
skin by direct contact with living or dead tissues 
of infected animals. Laboratory diagnosis is 
made by agglutination tests, animal inoculation, 
or recovery of the organism in cultures. 
a. Specimens for isolation of the agents 
Draw 10 to 15 ml. of blood directly into a 
“B-D” blood culture medium bottle and 
send it to a local laboratory for incubation. 
Due to the intermittent appearance of or- 
ganisms in the blood stream, it may be 
desirable to take additional specimens at in- 
tervals of several hours or days, depending 
on the condition of the patient. 
b. Specimens for serologic tests 
Since an agglutination titer of about 1:100 
or higher is believed indicative of brucellosis, 
successive blood specimens taken three to 
six weeks after onset of illness are con- 
sidered sufficient for diagnostic purposes. 
7 
