Enrique Escalera Zuñiga



, opyright © 1966 by The Williarns & Wilkins Co.
01. 45, No, 4
Printed in U.S.A.


Vol. 36, No. 3, 1966

~Departments 01 Microbiology and Medicine, University 01 Washington, School 01 Medicine,
Seattle, Washington 98105

Most clinical microbiologic laboratories in
this country now use the paper disk method
for determining susceptibility of bacteria to
antibiotics and chemotherapeutic agents. A
number of modifications of the test are employed. When this type of test was first de. veloped, only 1 disk was used for each agent
to be tested," 10 but subsequently it became
common practice to use 2 or more disks of
different potency and to [udge susceptibility
on the basis of the presence or absence of
growth around the disks. Our approach has
been to continue to develop a single disk
method based on measurement of sizes of
zones, We believe that this is rational in
theory and that it correlates better with the
results of dilution technics.
A number of reports on the technical details, experimental basis, and interpretative
standards of the single disk method have
been published.l: 4-6, 13 and recently sorne
of the theoretical aspects have been reviewed in more detail,"- 3, 11 The purpose of
the present communication is to consolidate
and update previous descriptions of the
method and provide a concise outline for its
performance and interpretation.

Rapidly Growing Pathoqens Such as Staphylococci and Enterobacteriaceae

A few colonies (3 to 10) of the organism to
be tested are picked with a wire loop from
the original culture plate and introduced
into a test tube containing 4 ml. of tryptose
phosphate or trypticase soy broth. These
tubes are then incubated for 2 to 5 hr., to
produce a bacterial suspension of moderate
cloudiness. The suspension is then diluted, if
Received, August 17, 1965.

necessary, with water 01' saline solution to a
density visually equivalent to that of a standard prepared by adding 0.5 ml. of 1 per cent
BaCb to 99.5 ml, of 1 per cent H 2S04 (0.36
N). An alternative procedure is to dilute
broth cultures overnight to the density of
the opacity standard (10- to 100-fold). For
the sensitivity plates, large (15-cm.) Petri
dishes are used with Mueller-Hinton agar
(5 to 6 mm. in depth). PIates are dried for
about 30 mino before inoculation and are
used within 4 days of preparation.
The bacterial broth suspension is streaked
evenly in 3 planes onto the surface of the
medium with a cotton swab (not a wire loop
or glass rod). Surplus suspension is removed
from the swab by being rotated against the
side of the tube before the plates are seeded.
After the inoculum has dried (3 to 5 min.),
the disks are placed on the agar with flamed
forceps or a single disk applicator and gently
pressed down to ensure contacto PIates are
incubated immediately, or within 30 mino
The large Petri dishes are spacious enough
to accommodate about 9 disks in an outer
ring, and 3 or 4 more in the center. It is advantageous to place antibiotics which diffuse
well in the outer circle and disks which produce smaller inhibition zones, such as vaneomycin and polymyxin-B, in the central area
of the plateo
After overnight incubation, the zone diameters (including the 6-mm. disk) are
measured with a ruIer on the undersurface of
the Petri dish or with calipers near the agar
surface. A reading of 6 mm. indicates no
zone, The end point is taken as complete
inhibition of growth as determined by the
naked eye, except in the case of sulfonamides, where organisms grow through several
generations before inhibition takes effe t.





the colonies are regarded as significant growth. and the results are reported to the clinician. The technic has proved satisfactory for sulfonamide susceptibility testing of all oro ganisms examined except Group A ¡3-hemo· lytic streptococci.Q. 5 per cent sheep or human blood may be added to the medium. 30 ¡. If several colonies are seen within a zone of inhibition.¡g. with zone sizes in .¡g. 30 ug. 10 ¡.¡g. have been less completely studied. § Tentative standard. 10 ¡. such as Hemophilus. 10 unit~ 30 ¡. 5 ¡. Slight growth (80 per cent or more inhibition) with sulfonamides is therefore disregarded.¡g.¡g. sulfonamide disks may be used with the same standards of zone interpretation. 10 units 300 units 10 ¡. When they are needed. 30 ¡.¡g. 45 BAUER ET AL. For testing such organisms. 300 ug. If they are still present.¡g. 300 ¡.¡g. t Zone sizes not applicable when blood is added to medium.¡g.¡g. Intermediate 20 or 11 or 8 or 14 or 12 or 8 or 13 or 13 or less less less less less less less less 21-28 12-13 9-12 11P17 13-17 9-10 14-17 14-17 9 or 13 or 12 or 14 or 17 or 11 or 20 or 8 or 11 or 12 or 14 or 9 or less less less less less less les s less less less les s less 10'--13 14-18 13-16 15-16 18-21 12-16 21-28 9-11 12-14 13-16 15-18 10-11 Sensitive 29 or 14 or 13 or 18 or 18 or 11 or 18 or 18 or 17 or 14 or 19 or 17 or 17 or 22 or 17 or 29 or 12 or 15 or 17 or 19 or 12 or more more more more more more more more more more more more more more more more more more more more more * Standards apply to urinary tract infections only.¡g. this is not a common occurrence. for which the test is highly satisfactory. Enrique Escalera Zuñiga 494 Vol. which may also be "chocolatized" when indicated. zone diameters may be read after incubation for 6 to 8 hr. and a veil of swarming into an inhibition zone is also ignored. aureus Al! other organisms Bacitracin Cephalothin Chloramphenicol Colistin Erythromycin \ Kanamycin Lineomycin § Methicillin N alidixic acid* Neomycin Nitrofurantoin* N ovobiocin] Oleandomycin Penicillin-G Polymyxin-B Streptomycin Sulfonamides] Tetracycline Vancomycin 10 ¡. these do not show olearcut-zones and the method should not be used for the organismo Slower Growing and Fastidious Organisms The standards in Table 1 have been developed for rapidly growing pathogens. 15 ¡." Swarming of Proteus strains is not inhibited by all antibiotics.or 250-¡.¡g. 30 ¡. but zone sizes are somewhat larger for an equivalent minimum inhibitory concentration than they are with rapid growers. 15 ug. Standard control organisms of known susceptibility should be employed at least once a week as a check on the activity of the disks and on the reproducibility of the test.¡g. Organisms of this type.B. 30 ¡. 30 ug. The zone diameters are recorded and interpreted according to Table 1. 30 ¡. and the margin of heavy growth read to determine the zone size. TABLE 1 ZONE SIZES AND THEIR INTERPRETATION FOR FREQUENTLY USED CHEMOTHERAPEUTICS Inhibition Zone Diameter to Nearest Millimeter Antibiotic or Chemotherapeutic Agent Disk Potency Resistant Ampicillin S. the strain should be checked for purity and retested. t Any of the commercially available 300. 2 ¡.¡g. More slowly growing organisms.F.¡g.

but separated sensitive and resistant popula. given pathogen. such as the type of practice of theIaboratory and the local preferehce for a particular agent. For most Gram-positive organisms. around a 30-. chloramphenicol.Q.also because the diffusion and solubility tions. of the border. DISCUSSION with added nitrofurantoin for urinary tract infections. various species. The test cannot be regarded as more than very rough guide for organisms requiring ore than 24 hr. ampicillin. kanamycin. sulfamethizole and ampicillin. The choice of antibiotics to be tested depends on a number of factors. scribed.ug.which are tested and certified for potency by .properties of the drugs-in Mueller-Hinton . occasionally.of 26 mm. thus Group A {3In the case of nitrofurantoin and nalidixic hemolytic streptococci and pneumococci acid.ug. streptomycin. distribution curves show 2 rather clearlY'nqt only because the disk potencies vary. Sorne organisms may be ests and by relating these to blood levels tested only against antibiotics to which reound with frequently used dose schedules. cline. kanamycin. and cephalothin. a strain of The technic should be used exactly as de. because although it has considerable around a 30-. the disk producing aureus faH into the interrnediate range with the largest inhibition zone does not necestetracycline. chloramphenicol.Escherichia coli shows a zone of 22 mm. For example. 495 erly standardized inoculum. the clinician may select the listed in the tableo Undiluted overnight one most appropriate for therapy of the parbroth cultures should never be used as an ticular case.! With many species from It will be noted that the interpretative which resistant mutants have emerged. it should be disk. not more:than 3 medium are different and characteristic for to 5 per cent of all strains of Staphylococcus each agent. levels in the urinary tract have been may need to be tested only against tetracytaken into account in establishing standards. method should be confluent with a prop. but diluted at least lO-fold. Enrique Escalera Zuñiga ANTIBIOTIC SUSCEPTIBILITY TESTING he resistant range. streptomycin. e between sensitive and intermediate ould be regarded as of uncertain susceptiility and tested by a dilution method if one indicated. methicillin. They should be stored . Growth by this taken into account in making this selection. Gram-negative rods are tested with chloramphenicol.ards has also been obtained from curves the Food and Drug Administration have . erythromycin. tetracycline. all that can be said is that the strain is particularly stressed that the zone sizes in susceptible to both compounds. with few strains falling in the inter. and. sulfamethizole. sistance has developed. Obviously.A report of "intermediate" or "resistant" . Pharmacologic and toxicologic inoculum. It should not be used for sensitivities n N eieseria gonorrhoeae or for sulfonamide ensitivities on meningococci until further ata become available.B. Mueller-Hinton medium and to disk contents given organism. chloramphenicol bine to produce inaccuracies. Those giving zones in the interediate range or within 2 mm. the disks routinely used in our laboratories are penicillin-G.mediate zone. and the test must be repeated if this is not achieved. tetracycline. for instance. may be safely reported resistant. such zone diameters are different for each agent. or erythro.F.sarily indicate the antibiotic of choice for a mycin.showing the distribution of susceptibilities proven reliable. Disks from first line supply houses Confirmation of the validity of these stand. or properties. of large numbers of individual strains of exactly as recommended. as well as the bacteriostatic or preferably to a density equivalent to the bactericidal actions of the drugs. From the the table are only applicable to the use oj agents reported as being effective against a . will be barium sulfate standard. changes in conditions may com. to yield macroscopic coloies. tetracycline disk and a zone flexibility. If. Only 1 penicillinase-resistant penThe numerical values in the table have icillin and 1 of the tetracyclines are tested een established by comparing zone sizes routinely because of essential oross-resistance ith a large series of tube or plate dilution within these groups. polymyxin-B.

10. A. C. Enrique Escalera Zuñiga 496 Vol.B. Shulman. Sc. Ericsson.. 9. Bauer. 466. 1-24.. Am. Bauer. aureus with ampicillin are different from those of other organisms. pp. Roberts. G.: A simple method of testing the sensitivity of wound bacteria to penicillin and sulfathiazole by use of impregnated blotting paper discs. 1. 574-580. G.. Perry. M. BADER ET AL.> which is sponsoring further studies towards the possible establishment of internationally acceptable standards.: The significance of bacterial inhibition zone diameters. A. In Antibiotics Annua!. D.. R.: Determination of bacterial sensitivity in vitro and its clinical evaluation. 1959-1960. A. E. 5. Similar emphasis on standardization has been made by Eriesson''. 1947. J. Antibiotica et Chemotherapia 6: 41-74 1959..: The determination of sulfonamide susceptibility of bacteria. Arch. R. B. we do not recommend routine direct application of disks to plates seeded with clinical material because of problems of inoculum control and mixed cultures. W. M. 57: 379-382. 1965. M. 484.. Anderson. M. and of great 'alue as a guide to therapy. M.. M.. A. 12: (Supp1. can only be obtained from comparisons of the results of dilution technics with anticipated chemotherapeutic levels at the sites of infection. J. and Dietz.. Inc. although they should be regarded as clinicdlly resistant. 12. No. W. and Sherris. which is always then confirmed with the standard method. Chemotherapia.1961. J. The importance of standardizing test conditions as far as possible in practice cannot be overstressed. Ericsson. J. For this reason. Bondi. Morley. R. Useful predictions of the efficacy of such treatment. p. 1964.. N.. In the interim.: Single disc antibiotic sensitivity testing of staphylococci. Turck. It will also be seen that the sensitivity criteria given for S. W.: Effeetiveness of ampicillin against Gram negative bacteria. and Sherris.. p. C. Clin.. Bauer. 3. 50): 1-55. New York: Medical Encyclopedias. 213: 221-225. 6. 13. J. Stuttgart: IIIrd Internationa! Congress of Chemotherapy. W. W. and Petersdorf.: Comparison of single dise and tube dilution techniques in determining antibíotic sensitivities of Gram negative pathogens. 7. J.. A. Such standards are highly desirable and may lead us to sorne modifications of our method. 8. 1964. No. W. Path. 1964. 2.. D. and Kirby. 58: 56-65. Bauer. Standardization of Methods for Conducting Microbie Sonsitivity Tests. 1960. 1959. G. Plorde J. W. REFERENCES 1. Petersdorf.. Spaulding. R. & Lab.. All nonpenicillinase-producing staphylococci have zones similar to those seen with penicillin.. In emergency situations. 210. E. M. Kennedy. 1945. 11. and Petersdorf.: Methods and significance of in vitro testing of baeterial sensitivity to drugs. A. does not necessarily imply that treatment may not be successful with unusually high dosage.. M. 187: 555-561.: Rational use of antibiotics in hospitals.. reproducible. 1963. E. Int. J. D.. Med. Lindemeyer. P.: The two definitions of bacteria! resistance. Bauer. pp. 1964.: Single disc versus multiple dise and plate dilution techniques for antibiotie sensitivity testing.. A. such as those in which direct smears of sorne cerebrospinal fluid and urine specimens indicate that a pure culture may be anticipated. A. Med. B. we make direct tests and issue a tentative report. Ann. Smith. K. G. we have found the technic to be easily performed. Int. and has been stressed in the Second Report of the Expert Committee on Antibiotics of the World Health Organisation. B. J.. Second Report of the Expert Committee on Antibiotics. because sorne weakly penicillinase-producing strains of staphylococci may give zone sizes of more than 14 mm. however. R. Geneva World Bealth Organization Teehnica! Report Series. C. C. Med. 4.. A. however. and Svartz-Malmberg. Stuttgart: IIIrd International Congress of Chemotherapy. C. 9: 1-19. 7. 39: 766-779. Am. A. Invest.F. Scandinav.9 in his description of an excellent single disk technic which is widely used in Scandinavia. 1960. 104: 208-216. J. . and Kirby. & Bact. A.: A routine method for the rapid determination of susceptibility to penicillin and other antibiotics. C. M.Q.