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APPLI MICROBIOLOGY, May 1969, p. 707-709 Vol. 17, No. 5
Copyright @ 1969 American Society for Microbiology Printed in U.S.A.
Tube Dilution Antimicrobial Susceptibility Testing:
Efficacy of a Microtechnique Applicable
to Diagnostic Laboratories
LAWRENCE A. CHITWOOD
Department ofPediatrics, Children's MemorialHospital, University ofOklahoma Medical Center, Oklahoma City,
Oklahoma 73104
Received for publication 7 March 1969
Amicrotechnique for determining antibiotic susceptibilities by the serial dilution
method was evaluated in a clinical diagnostic microbiology laboratory. As com-
pared with the standard tube method, an agreement of 94% was achieved for de-
termining minimal inhibitory concentration with i onetube dilution as the criterion
of comparison. The experience with this system suggests that it could easily be in-
corporated into diagnostic laboratories as a routine procedure.
In recent years, the increasing availability and weighed on an analytical balance, diluted in glass-
types ofantimicrobial agents have added appreci- distilled water to a concentration of 1,000 Ag/mlI
ably to the problems confronting the diagnostic sterile filtered, and stored as 1 ml of frozen stock. ml
laboratory. In many clinical situations, the tube Macromehd. Foreach antibiotic examined, 0.2
dilution method of determining antimicrobial Of 1,000 pg/ml was added to 1.6 ml of Brain Heart
susceptibilities is generally recognized as provid- Infusion broth (BHI), and nine serial twofold dilu-
ing more accurate and useful information as c6m- tions were made in 0.9 ml of broth. Subsequently,
pared with results obtained by the disc method. 0.1 ml of a 0I dilution of an 18-hr broth culture of
from a of view, cost, the organism was added to each tube. One tube with-
However, practical point out antibiotic served as the organism control. After
time, and personnel factors frequently require 18 hr of aerobic incubation at 37 C, tubes were ex-
that the tube dilution method be used only with amined for evidence of turbidity or sediment. The
isolates from the problem patient. tube exhibiting no visible growth and containing the
A number of papers (1, 4, 9) have appeared least amount ofantibiotic was considered the minimal
which question disc antibiotic susceptibility test- inhibitory concentration (MIC). V
ing procedures. Discrepancies frequently arise Micromethod. Disposable transparent plates,
when one compares the results obtained with the (Cooke Engineering Co. or Linbro Chemical Co.),
disc and tube dilution methods. containing 96 cups arranged in 8 rows of 12 cups,
were sterilized by being rinsed in ethyl alcohol,
These studies were undertaken to evaluate the drained in an inverted position overnight, and placed
feasibility of an in vitro susceptibility testing under a germicidal ultraviolet source for 1 hr. Work-
technique in a clinical laboratory which is re- ing solutions of antibiotics were prepared weekly
producible, accurate, and reliable, but lacks the by diluting the frozen stock to 200 pug per ml of
disadvantages ofbeing time-consumingexpensive, BHI. Each week a disposable plate containing 12
and tedious. different antibiotics was prepared. Each cup, in a
For this purpose, the Microtiter (Cooke vertical row of 12 cups, was filled with a different
Engineering Co.) system, as used routinely in antibiotic and stored at 4 C. At the end of a week,
many serological laboratories (10), was investi- the working stock standards were discarded and
fresh solutions were prepared. For the microtitration
gated as a possibility to resolve the inherent procedure, 0.025 ml of BHI was added to each well
difficulties in the standard accepted method. with a calibrated pipette dropper. A heat-sterilized,
0.025-mil multi-microdilutor was utilized for diluting
MATERIALS AND METHODS 12 antibiotics simultaneously (Fig. 1), by placing
the microdiluter in the plate containing the working
Stock standards of antibiotics generously provided stock of antibiotics and transferring to the first row
by pharmaceutical companies, including cephalothin, of the titration plate, mixing, and transferring to
ampicillin, colistin, neomycin, tetracycline, kanamycin, subsequent rows until the titration was complete.
polymyxin B, streptomycin, chloramphenicol, gent- The complete twofold serial titration of 8 dilutions
amicin, cephaloridine, linocomycin, erythromycin, of 12 different antibiotics usually took less than
penicillin G, carbenicillin, and cloxacillin, were 1 min. An inoculum of 0.025 ml of an 18-hr BHI
707
708 MICROBIOL.
CHITWOOD APPL.
broth culture diluted 10 was added to each cup. TABLE 1. Comparative evaluation of antibiotic
After inoculation, the plate was covered with trans- susceptibility testing with standard
parent sealing tape, a small pin-hole was made at macromethod-and micromethod
the center of each cup, and the plate was incubated
at 37 C for 18 hr. The end point of the titration was Series No. agreeing ± one tube
determined by examining the plate, with transmitted dilution/no. examined
light against a dark background, for growth in the 1 55/71 (78%)
first cup (50 jg/ml) and each succeeding cup (Fig. 2). 2 90/113 (89%)
RESULTS 3 168/179 (94%)
Isolates of various gram-negative rods, includ-
ing Pseudomonas, Salmonella, Shigella, Klebsiella, were compared, a correlation of 78% was
Enterobacter, and Escherichia, gram-positive achieved initially (series 1). However, after tech-
organisms such as coagulase-negative and nical familiarization with the technique was
coagulase-positive Staphylococcus, and other established (series 2), a correlation of 89% was
rapidly growing organisms were examined against noted. Subsequently, after the technique was
a variety of antibiotics. As noted in Table 1, if firmly established (5 months), a correlation of
one accepts -+ one tube dilution as the criterion 94% was achieved between the two methods.
for comparison between methods, an excellent
correlation was achieved. When the methods DISCUSSION
Variabilities (4, 7, 9, 11) encountered in anti-
biotic disc susceptibility testing have been attrib-
uted to diffusibility of the antibiotics, size and
distribution of the inoculum, moisture content of
the agar, type of medium used, interpretation of
narrow zones, failure to set the disc properly, and
other variables. The multidisc technique, two
discs, or the use of a single high- or low-concen-
tration disc are additional variations of the tech-
nique which tend to cause confusion in the interp-
retation of results from a clinical laboratory.
The serial tube method of susceptibility testing
is considered the common method of reference,
but the tedious nature of the macromethod has
1. Simultaneous dilution of 12 antibiotics with prevented universal usage on routine isolates.
FIG. The obvious advantage of determining discrete
multi-microdilutor. end point susceptibilities which can be correlated
with therapeutic levels obtainable in the patient is
~lo'- thatthephysicianisaffordedamoresophisticated
A0i;
i_
_s;SlingtR: approach in the management of bacterial infec-
-o tions. Although this approach is generally rec-
K''w'''0~ts "S^w.If ognized as ideal, tube dilution susceptibility test-
ing usually is relegated to the research oriented
laboratory. An initial modification of the macro-
technique utilizing spot depression plates (3) was
reported from this laboratory in 1965. Although
the spot plate compared favorably with the tube
technique, the time required and the glassware
used remained significant problems to be re-
solved. Subsequently, commercially available
microtitration equipment and disposable plates
became available and provided an alternate
FIG. 2. Completed antimicrobial susceptibility methodology which could overcome inherent
titration ofan organism against 12 antibiotics. Row H problems in manual pipetting procedures.
contains 50 ,gg ofantibiotic per ml, row G contains 25 Thesedataand otherreports (8; L. A. Chitwod,
,ug ofantibiotic per ml, etc. S. Med. J. 60: 1358; J. D. MacLowry and H. H.
VOL. 17, 1969 ANTIMICROBIAL SUSCEPTIBILITY TESTING 709
Larsh, Intersci. Conf. Antimicrob. Agents ACKNOWLEDGMENT
Chemother., 8th, 1968, New York) suggest that The author acknowledges the fine technical assistance pro-
an excellent correlation exists between the micro- vided by Vi Patnode during these studies.
andmacrotechniques and that the microtechnique LITERATURE CITED
can be incorporated into a routine diagnostic 1. Bauer, A. W., C. E. Roberts, and W. M. Kirby. 1960. Single
laboratory. of the disc versus multiple disc and plate dilution techniques for
Tke most time-consuming procedure each antibiotic sensitivity testing. Antibiot. Ann.-1959, p.
technique is the addition of the diluent to 574-580.
cup. However, with the use of an automatic 2. Bauer, A. W., M. M. Kirby, J. C. Sherris, and M. Turck.
dispensing instrument (Cooke Engineering Co.), 1966. Antibiotic susceptibility testing by a standardized
automation of the technique becomes a possi- single disc method. Amer. J. Clin. Pathol. 45:493-496.
bility. 3. Beargie, R. A., E. C. Bracken, and H. D. Riley, Jr. 1965.
Micromethod (spot-plate) determination of in vitro anti-
Decreased activity of antibiotics, usually by biotic susceptibility. Appl. Microbiol. 13:279-280.
two tubes, was noted when the 10-3 dilution of 4. Branch, A., D. H. Starkey, and E. E. Power. 1959. The inter-
the test organism was inadvertantly allowed to national situation with regard to the use of discs for anti-
remain at room temperature for several hours be- biotic sensitivity tests. Antibiot. Ann.-1958, p. 833-835.
5. Branch, A., D. H. Starkey, and E. E. Power. 1965. Signifi-
fore being added to the antibiotics. Obviously, cance of the occurrence of nonreacting discs in antibiotic-
the size of the inoculum requires standardization. sensitivity tests. Antimicrob. Agents Chemother.-1964,
Although a 103 dilution was empirically estab- p. 395-399.
lished for most rapidly growing organisms, a 1O-2 6. Goss, W. A., and E. B. Cimijotti. 1968. Evaluation of an
automatic diluting device for microbiological applications.
dilution was used for more fastidious organisms Appl. Microbiol. 16:1414-1416.
such as beta-and alpha-hemolytic streptococciand 7. Greenberg, L., K. M. Fitzpatrick, and A. Branch. 1957. The
pneumonococci. In addition, with these and sim- status of antibiotic disc in Canada. Can. Med. Ass. J.
ilar organisms, it was found advantageous to add 76:194-198.
8. Harwick, H. J., P. Weiss, and R. F. Fekety. 1968. Applica-
5% fresh sheep blood as an indicator of growth. tion of microtitration techniques to bacteriostatic and
Triphenyl tetrazolium chloride in the media has bactericidal antibiotic susceptibility testing. J. Lab. Clin.
been used as a visible indicator of growth in the Med. 72:511-516.
9. Petersdorf, R. G., and J. C. Sherris. 1965. Methods and
micotechnique (6). significance of in vitro testing of bacterial sensitivity to
Occasionally, spurious results do occur which drugs. Amer. J. Med. 39:766-779.
have been attributed invariably to a mixed culture 10. Sever, J. L. 1962. Application of a micro technique to viral
and serological investigations. J. Immunol. 88:320-329.
for the inoculum; however, by maintaining a log 11. Truant, J. P., J. E. Bolin, and J. Mullins. 1965. Evaluation of
oftube dilution susceptibilities by organism, such susceptibility testing procedures w.t"th single and multiple
errors are detected easily. antibiotic discs. Antimicrob. Agents Cherother.-1964,
p. 384-394.
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