290
M. A. GERENCSER
11. ISOLATION AND IDENTIFICATION O F A C T I N O M Y C E S
AND A R A C H N I A
A. Isolation
Procedures for isolation of Actinomyces and Arachnia from clinical
material have been described by Smith (1975) and by Slack and Gerencser
(1975). Briefly, pus or sputum is inspected for the presence of “sulphur
granules”. If granules are present, they are removed, washed in two or
three changes of sterile saline, and used for making smears and cultures.
If granules are not found, smears and cultures are made from well-mixed
pus or exudate or from bits of purulent material in sputum. Smears should
be stained with Gram and acid-fast stains. Cultures should be inoculated
with material from all cases of suspected actinomycosis and from other
specimens in which Gram-positive diphtheroidal or filamentous organisms
are seen in smears.
Inoculate two plates each of blood agar and brain-heart infusion agar,
and a tube of fluid thioglycollate broth containing 0*1-0.2% sterile rabbit
serum. Incubate one plate of each medium aerobically with carbon dioxide
(510%) and the second plate of each medium anaerobically with carbon
dioxide (5-10%). Plates should be examined after 24-48 h and again after
five to seven days incubation for the presence of colonies suggestive of
Actinomyces. If plate cultures are negative, the thioglycollate broth culture
is Gram stained. When Gram-positive diphtheroidal rods are seen in the
broth, they are subcultured to blood agar plates which are treated like the
primary isolation plates.
B. Identification
After pure cultures are obtained, identification is based on morphology
and biochemical tests. Actinomyces and Arachnia are Gram-positive rods
which are usually slender and irregularly staining with V, Y or T forms
present in most smears. Long filaments with or without branching may be
present. Separation of Actinomyces and Arachnia from morphologically
similar genera is usually based on oxygen requirements, end products of
glucose fermentation, and a few selected biochemical tests. Both Actinomyces and Arachnia are anaerobes or facultative anaerobes. They do not
produce indole, nor liquefy gelatin, and with the exception of A. viscosus
they are catalase negative. Actinomyces produce succinic, lactic, acetic and
formic (trace) acids from the fermentation of glucose. Arachnia propionica
produces propionic, acetic and formic acid from glucose. It is most easily
distinguished from Propionibacterium species by the catalase test.
Biochemical reactions which are useful in identifying Actinomyces and
Arachnia are shown in Table 11. Additional information on biochemical
M. A. GERENCSER
11. ISOLATION AND IDENTIFICATION O F A C T I N O M Y C E S
AND A R A C H N I A
A. Isolation
Procedures for isolation of Actinomyces and Arachnia from clinical
material have been described by Smith (1975) and by Slack and Gerencser
(1975). Briefly, pus or sputum is inspected for the presence of “sulphur
granules”. If granules are present, they are removed, washed in two or
three changes of sterile saline, and used for making smears and cultures.
If granules are not found, smears and cultures are made from well-mixed
pus or exudate or from bits of purulent material in sputum. Smears should
be stained with Gram and acid-fast stains. Cultures should be inoculated
with material from all cases of suspected actinomycosis and from other
specimens in which Gram-positive diphtheroidal or filamentous organisms
are seen in smears.
Inoculate two plates each of blood agar and brain-heart infusion agar,
and a tube of fluid thioglycollate broth containing 0*1-0.2% sterile rabbit
serum. Incubate one plate of each medium aerobically with carbon dioxide
(510%) and the second plate of each medium anaerobically with carbon
dioxide (5-10%). Plates should be examined after 24-48 h and again after
five to seven days incubation for the presence of colonies suggestive of
Actinomyces. If plate cultures are negative, the thioglycollate broth culture
is Gram stained. When Gram-positive diphtheroidal rods are seen in the
broth, they are subcultured to blood agar plates which are treated like the
primary isolation plates.
B. Identification
After pure cultures are obtained, identification is based on morphology
and biochemical tests. Actinomyces and Arachnia are Gram-positive rods
which are usually slender and irregularly staining with V, Y or T forms
present in most smears. Long filaments with or without branching may be
present. Separation of Actinomyces and Arachnia from morphologically
similar genera is usually based on oxygen requirements, end products of
glucose fermentation, and a few selected biochemical tests. Both Actinomyces and Arachnia are anaerobes or facultative anaerobes. They do not
produce indole, nor liquefy gelatin, and with the exception of A. viscosus
they are catalase negative. Actinomyces produce succinic, lactic, acetic and
formic (trace) acids from the fermentation of glucose. Arachnia propionica
produces propionic, acetic and formic acid from glucose. It is most easily
distinguished from Propionibacterium species by the catalase test.
Biochemical reactions which are useful in identifying Actinomyces and
Arachnia are shown in Table 11. Additional information on biochemical