Problems in the Use of the in Vitro Toxigenicity
Test for Corynebacterium diphtheriae
SUSAN T . BICKHAM, B.S., AND WALLIS L. JONES, P H . D .
Center for Disease Control, Health Services and Mental Health Administration,
Public Health Service, U. S. Department of Health, Education, and Welfare,
Atlanta, Georgia 30333
ABSTRACT
Bickham, Susan T., and Jones, Wallis L.: Problems in the use of the in Vitro
toxigenicity test for Corynebacterium diphtheriae. Amer. J. Clin. Path. 57:
244-246, 1972. A method for the in vitro toxigenicity test for C. diphtheriae
that has given accurate results when compared with tests in vivo is reported
in detail. The major factors, such as the antitoxin, serum, and basal ingredients, that are known to cause erroneous results in the in vitro test are discussed. Antitoxins obtained from five sources were found to produce nonspecific lines of precipitation with known nontoxigenic strains when used at
the previously advocated concentration of 500 antitoxin units (AU) per ml.
At a concentration of 100 AU per ml. of the only antitoxin currently commercially prepared in the United States, all 115 strains tested gave the correct
reaction.
IN RECENT MONTHS, the Diphtheria Laboratory of the Bacteriology Section at the
Center for Disease Control has been concerned with discrepancies reported by other
laboratories in the in vitro test for toxigenicity of Corynebacterium
diphtheriae
isolates. Some of the reporting laboratories
have been unable to demonstrate a positive reaction with known toxigenic strains,
whereas other laboratories have found
known nontoxigenic strains to give a precipitin reaction. T h e method used in our
laboratory that has given accurate results
for many years when compared with tests
in vivo is reported in detail. In an effort
to define the difficulties encountered with
the in vitro procedure, we will review and
Received February 22, 1971; received revised
manuscript March 22, 1971; accepted for publication March 30, 1971.
244
discuss the major factors reported to cause
erroneous results.1*8
The agar base used in this laboratory is
a modification of Elek's original formula. 1
First, 20 Gm. of proteose peptone (Difco),
3.0 Gm. of maltose, 0.7 ml. of lactic acid,
and 1.5 ml. of a 40% NaOH solution are
added to 500 ml. of distilled water. This
mixture is heated to boiling and constantly
shaken, then filtered through Whatman
No. 12 filter paper. The filtrate is added
to 500 ml. of a solution of 3 % agar and
1% NaCl in distilled water. After the pH
is adjusted to 8.0, 10 ml. volumes of the
agar are dispensed into screw-capped tubes
and autoclaved at 115 C. for 10 min. The
base can be stored at room temperature for
as long as 6 months.
T h e following procedure is used to prepare the test plate: 2.0 ml. of sterile serum,
February 1972
IN VITRO
TOXIGENICITY T E S T FOR
1.0 ml. of a sterile solution of 0.3% potasium tellurite, and a tube of melted agar
base (60 C.) are placed in a flat, scratch-free
plastic or glass petri dish. The contents
are mixed thoroughly by gentle rotation.
A strip of sterile filter paper (Whatman
No. 3, 70 by 15 mm.) is saturated with diluted diphtheria antitoxin. A dilution containing 100 antitoxin units per ml. (AU
per ml.) is recommended for most antitoxins currently available. The excess antitoxin is allowed to drain, then the strip
is centered on the agar with sterile forceps
just before the agar has completely hardened. The plate is allowed to sit with the
top slightly open for at least an hour to
allow excess moisture to evaporate; it must
be inoculated within 2 hr. by streaking
each test culture perpendicular to and on
either side of the paper strip. Care should
be taken not to touch the strip with inoculum. The plate is incubated in an inverted position at 35 to 37 C. and read after
18 to 48 hr. The potassium tellurite may
be omitted if pure cultures are used.
A positive reaction is indicated by a
toxin-antitoxin (primary) line that forms a
45-degree angle with the inoculum streak.
Between adjacent positive cultures these
lines will form an arc of identity. A toxigenic control should be included on every
plate and should be strongly positive
within 36 to 48 hr. A nontoxigenic control
should not produce any precipitin lines
under optimal conditions.
Nontoxigenic strains may be interpreted
incorrectly as being in vitro positive when
antitoxin that produces nonspecific (secondary) lines is used.1- 2>7 These lines are
usually located close to the antitoxin strip,
are less intense, and do not form lines of
identity with the primary line. Elek 1 recommended using the antitoxin at a concentration of 1,000 AU per ml., but King and
associates5 found 500 AU per ml. to be
equally satisfactory. Freeman 2 noted that
C.
DIPHTHERIAE
245
these secondary lines were produced with
certain brands of antitoxin at a dilution
containing 500 AU per ml. and that the
lines could be eliminated by reducing the
antitoxin concentration.
We tested antitoxin obtained from four
commercial sources and from two state
health departments. One of the commercial
antitoxins was a prepared presoaked strip.
All six antitoxins produced secondary lines;
the liquid products were tested at 500 AU
per ml. Several dilutions in sterile distilled
water of the only commercial antitoxin
presently prepared in the United States
(National Drug Company) were tested. At
a dilution containing 100 AU per ml., all
of 94 toxigenic strains tested gave strongly
positive reactions and all of 21 nontoxigenic strains gave clearly negative reactions. T h e secondary lines were almost
completely eliminated at this concentration.
When antitoxin known to give secondary
lines is used, we recommend that a maximum of seven inoculations be placed on
a plate and that a toxigenic strain be
placed adjacent to each test strain in order
to observe the primary line of identity. The
100 Au per ml. concentration should not
be used after storage at 4 C. for more than
2 weeks. A stock dilution containing 500
AU per ml. stored at either 4 C. or —10 C.
for as long as 6 months may be used to
prepare the 100 AU per ml. concentration.
False negative reactions with toxigenic
strains will occur if conditions are not optimal for the inocula to produce a sufficient
quantity of toxin to form a precipitate with
the antitoxin. Schubert and Blank 8 found
Elek's base to be superior to both the King
modification e (which omits the maltose and
lactic acid) and a dehydrated commercial
medium prepared according to the King
formula. They found that more toxigenic
strains were positive with Elek's base than
with the other two bases.
246
BICKHAM AND JONES
Many workers have reported that the
use of unsatisfactory serum will lead to erroneous results. 1 - 8 - 8 ' 8 Any serum (rabbit,
monkey, horse, sheep, or human) is satisfactory in the test if the toxigenic control
produces a strong precipitin line after
being incubated for a maximum period of
48 hr. Rabbit serum (Hyland Laboratories)
has been used in this laboratory for the
past few years with excellent results, with
the exception of one lot out of five lots
tested. The serum may be stored at —10 C.
for an indefinite period.
Elek 1 and King and co-workers8 found
the type of peptone to be another factor
which caused false negative results. When
they used proteose peptone in the base,
earlier and more numerous positive precipitin reactions were recorded than when
other peptones were used. Later workers 8
found that there were discrepancies in results with different lots of proteose peptone; therefore, each lot must be checked
with toxigenic controls. The dehydrated
A.J.CP.—Vol.
57
proteose peptone, if tightly sealed and
properly stored, will remain in satisfactory
condition for an extended period.
References
1. Elek SD: The plate virulence test for diphtheria.
J Clin Path 2:250-258, 1949
2. Freeman VJ: Influence of type and concentration of antitoxin on the in vitro toxigenicity
test for C. diphtheriae. Public Health Rep 65:
875-882, 1950
3. Hermann GJ, Moore MS, Parsons EI: A substitute for serum in the diphtheria in vitro toxigenicity test. Amer J Clin Path 29:181-183,
1958
4. Hook JT, Parsons EI: Use of human serum in
in vitro test for virulence of Corynebacterium
diphtheriae. Amer J Clin Path 21:979-981, 1951
5. King EO, Frobisher M, Parsons EI: The in vitro
test for virulence of Corynebacterium diphtheriae. Amer J Public Health 39:1314-1320,
1949
6. King EO, Frobisher M, Parsons EI: Further
studies on the in vitro test for virulence of
Corynebacterium diphtheriae. Amer J Public
Health 40:704-707, 1950
7. Ouchterlony O: In vitro method for testing the
toxin-producing capacity of diphtheria bacteria.
Acta Path Microbiol Scand 26:516-524, 1949
8. Schubert JH, Blank BE: A comparison of media
for the in vitro toxigenicity test. Public Health
Lab 23:170-175, 1965