A Selective Differential Medium
for Histotoxic Clostridia
PAUL D. ELLNER, P H . D . , AND EDWARD D. O'DONNELL, P H . D .
Diagnostic Microbiology Service, Columbia-Presbyterian
Medical Center and Department
Microbiology, Columbia University College of Physicians and Surgeons,
New York, New York 10032
of
ABSTRACT
Ellner, Paul D., and O'Donnell, Edward D.: A selective differential medium
for histotoxic Clostridia. Amer. J. Clin. Path. 56: 197-200, 1971. A new medium
for the isolation and differentiation of the histotoxic Clostridia commonly
encountered in clinical specimens is described. The medium contains lecithin
and lactose, and is made selective by the addition of neomycin and sodium
azide. A simplified scheme for the speciation of these Clostridia is presented.
have been useful for the isolation and presumptive identification of the histotoxic Clostridia. McClung and Toabe 3 employed an egg yolk
medium for the presumptive identification
of certain species of Clostridia. Willis and
Hobbs 0 used a medium containing lactose
and milk in addition to egg yolk and were
able to group Clostridia on the basis of lactose fermentation, production of lecithinase, and hydrolysis of casein. Their medium was made selective by the addition
of neomycin sulfate. The preparation of
these media requires a supply of fresh eggs
from chickens on an antibiotic-free diet.
Furthermore, reactions on egg-containing
media are difficult to interpret, especially
when substrates such as milk and lactose
are added. Since fresh, antibiotic-free eggs
are often difficult to obtain, and since lactose fermentation and the production of
lecithinase are two of the most useful criteria for identifying Clostridia, it appeared
desirable to develop and test an egg-free
EGG YOLK-CONTAINING MEDIA
Received January 24, 1970; received revised
manuscript September 2, 1970; accepted for publication September 25, 1970.
Supported by PHS Training Grant No. A100245-07 from the National Institute of Allergy and
Infectious Diseases.
medium containing lactose and lecithin.
Willis 8 was able to substitute purified lecithin for egg yolk in media designed to
detect production of lecithinase. Vera 7
showed that fewer strains of Clostridia were
inhibited and the selective properties of
clostridial media were improved by decreasing the concentration of neomycin employed by Willis and Hobbs 0 and by adding sodium azide. Since we had used neomycin-azide in such media for a number
of years, it was decided to retain the selective advantages of this combination in the
new medium.
Materials and Methods
Lecithin-Lactose
Agar
Columbia agar base (Difco or BBL)
Lactose
Calcium-cysteine solution *
Brom-cresol purple (1.25% alcoholic
solution)
Neomycin sulfate
Sodium azide
Distilled water
46.6 Gin.
10.0 Cm.
10.0 ml.
2.0 ml.
0.15 Gin.
0.20 Cm.
1,000 ml.
Boil to dissolve, adjust pH to 6.7, and
autoclave 15 min. at 121 C. Cool to 50 C.
* Calcium-cysteine solution: dissolve 5 Gm. of
cysteine hydrochloride and 0.5 Cm. CaCU-2 H a O in
100 ml. of distilled water acidified with 1 drop o£
glacial acetic acid. Store in refrigerator.
197
198
A.J.CP.—Vol.
ELLNER AND ODONNELL
and add aseptically 20 ml. of sterile lecithin
emulsion,t mix, and pour plates.
The ability of the new medium to support growth of the commonly encountered
histotoxic Clostridia was tested by inoculating stock cultures of Clostridium perfringens, C. sordelli, C. novyi A, C. septicum,
and C. histolyticum in cooked meat medium
on plates of lecithin-lactose agar. Plates
were incubated at 35 C. in GasPak anaerobic jars (B.B.L.) for 24 hr.
The selectivity of the new medium was
tested by inoculating plates with recently
isolated strains of E. coli, Klebsiella, Serratia, Proteus sp., Pseudomonas, Bacillus
sp., Streptococcus pyogenes, enterococci,
Micrococcus sp., and D. pneumoniae. Plates
were incubated under anaerobic conditions
as described above.
Thioglycolate cultures of clinical specimens containing Gram-positive rods were
used to inoculate plates of lecithin-lactose
agar and a nonselective medium (Columbia
blood agar 1 ). Blood cultures in Columbia
Broth 4 showing Gram-positive rods were
also inoculated on plates of the new medium and of blood agar. All plates were
incubated anaerobically, as previously described.
Inoculated plates of the new medium
were examined for growth and evidence of
lactose fermentation and production of lecithinase. Gram stains were made and, when
necessary, colonies were subcultured to
Thiogel and Indole-Nitrite Media (B.B.L.).
Glucose fermentation was determined by
the addition of a few drops of phenol red
to the Thiogel culture after growth had
occurred; the culture was chilled at 5 C.
to detect gelatin hydrolysis. Motility was
determined by examining the Indole-Nitrite culture by the hanging drop method.
The ability of specific antitoxin to inhibit the production of opalescence was
f Lecithin emulsion: suspend 3.3 Gm. of egg
lecithin (Sigma L-6500) in 100 ml. distilled water.
Autoclave 15 min. at 121 C. and adjust to p H 7.0
with sterile 0.1 N NaOH.
56
tested using the half-antitoxin plate method
of Willis and Hobbs. 9 Polyvalent gas gangrene antitoxin (Wyeth) was used for this
purpose.
Results
All of the stock strains of Clostridia
tested showed growth on lecithin-lactose
agar after 24 hr., although incubation for
an additional 24 hr. was sometimes necessary for clear-cut reactions to occur in the
medium.
None of the Gram-negative isolates examined grew on the new medium, nor did
several strains of Bacillus sp. Cultures of
Gram-positive cocci often produced pinpoint colonies but no reaction or color
change in the agar except for a yellow color
that sometimes occurred around attenuated
colonies of enterococci. The scanty growth
of the Gram-positive cocci was easily distinguished from the larger clostridial colonies.
In every case when Clostridia grew on
anaerobic blood agar plates, growth was
also obtained in pure culture on lecithinlactose agar.
Lecithinase-producing Clostridia were
readily distinguished by a zone of opalescence surrounding the colonies (Fig. 1). A
yellow color around the colonies indicated
lactose fermentation. Thus, on the new medium, stock cultures and isolates of C. perfringens showed zones of opalescence and
a yellow color in and around the colonies.
Opalescence was also seen around colonies
of C. sordelli and C. novyi A, but the agar
remained purple. Colonies of C. septicum
showed the yellow color change but no
opalescence, and C. histolyticum colonies
produced no reaction or color change in
the medium. Colonies of lecithinase-producing species failed to show zones of opalescence when cultured on the new agar
containing gas gangrene antitoxin. 2
One hundred twenty-one Clostridia were
recovered from 7,534 clinical specimens
that were cultured anaerobically (including
August 1971
199
SELECTIVE MEDIUM FOR CLOSTRIDIA
6,146 blood, 64 bone marrow, 375 respiratory, 232 paracentesis, and 720 wound and
female genital specimens). Most of the isolates came from female genital, wound, or
blood cultures. One of the isolates was
identified as C. novyi A; the remainder
were C. perfringens.
Isolates showing lactose fermentation and
the production of lecithinase and which
produced the characteristic double zone of
hemolysis on blood agar were identified as
C. perfringens without further testing. The
isolate of C. novyi A was identified by the
presence of opalescence but no acid on the
new medium, and by giving the appropriate reactions according to Table 1.
Discussion
Lecithin-lactose agar provides the differential features of lactose and egg yolkcontaining media while eliminating the
need for complex materials such as milk
and eggs. The reactions are clear-cut and
easier to interpret than those on egg-containing media. The selective advantage of
neomycin-azide is retained; Gram-negative
and aerobic Gram-positive rods are inhibited, and growth of Gram-positive cocci is
markedly attentuated. The medium is easy
to prepare and is stable for at least 2 weeks
at 5 C.
Four groups of Clostridia can be distinguished on lecithin-lactose agar: leckhinase-producers fermenting lactose (C. per-
FIG. 1. Zone of opalescence surrounding the colonies of lecithinase-producing Clostridia cultured on
the medium containing lecithin, lactose, neomycin,
and sodium azide.
fringens); lecithinase-producers not fermenting lactose (C.
sordelli-bifermentans,
C. novyi); lactose-fermenters not producing
lecithinase (C. septicum, C. fallax); and
non-lactose fermenters not producing lecithinase (C. histolyticum).
Clostridium
novyi is readily distinguished from the
sordelli-bifermentans
Table 1. Scheme for Identification of Clostridia
Lecithinase
Lactose
Dextrose
Gelatinase
Motility
Indole
Nitrate reduction
* A = acid,
f V = variable.
C. perfringens
C. sordcllii
C. novyi
+
+
+
_
A*
A
—
—
+
—
—
+
C. scpticum
C. histolyticum
A
A
—
—
—
+
+
+
+
C. fallax
A
A
A
A
+
+
+
+
+
+
—
—
+
—
—
—
Vf
—
-
200
ELLNER AND O'DONNELL
complex by virtue of its ability to reduce
nitrate and its negative indole reaction.
The lecithinase reaction of C. novyi A is
inhibited by commercial gas gangrene antitoxin; that of C. novyi B is not. Clostridium sordelli differs from C. bifermentans
by giving a positive urease test. The ability
to hydrolyze gelatin differentiates C. septicum from C. fallax.
The rarity of isolation of species other
than C. perfringens in our series is not surprising. Recent clinical studies 5 have emphasized the predominance of this organism. However, the reactions described for
species other than C. perfringens were predicted from data obtained from the literature, and were confirmed with only a single
stock strain for each of the species mentioned. Thus, the value of lecithin-lactose
agar for isolation of other species of Clostridia remains to be demonstrated.
Table 1 was constructed using reactions
obtained from a recognized source.6
The use of lecithin-lactose agar permits
rapid isolation and recognition of C. per-
A.J.C.P.—Vol.
56
fringens, and should facilitate the identification of the less commonly encountered
species of Clostridia.
Acknowledgment. Dr. Konrad Hsu assisted in the
preparation of the photograph.
References
1. Ellner PD, Stoessel CJ, Drakeford E, et al.: A
new culture medium for medical bacteriology.
Amer J Clin Path 45:502-504, 1966
2. Hayward NJ: Rapid identification of Clostridium welchii by the Nagler tests in plate cultures. J Path Bact 55:285-293, 1943
3. McClung LS, Toabe R: T h e egg yolk plate reaction for the presumptive diagnosis of Clostridium sporogenes and certain species of the
gangrene and botulinum groups. J Bact 53:
139-147, 1948
4. Morello JA, Ellner PD: A new medium for blood
cultures. Appl Microbiol 17:68-70, 1969
5. Parker MX: Postoperative clostridial infections
in Britain. Brit Med J 3:671-676, 1969
6. Smith L, Holdcman L: T h e Pathogenic Anaerobic Bacteria. Springfield, 111., Charles C
Thomas, 1968, pp 192-193
7. Vera HS: Presented at the Pennsylvania Society
of Medical Technologists, York, Pennsylvania,
May 4, 1962
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9. Willis AT, Hobbs G: Some new media for the
isolation and identification of Clostridia. J Path
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