336 DIFCO MANUAL 



Medium Base and Bacto-Pelzer TB Medium Enrichment. The medium is poured 

 into sterile plates or flat wide-mouth bottles and allowed to solidify. A heavy 

 suspension of the organism or the clinic specimen is smeared on the surface of 

 the medium and the Bacto-Sensitivity Disks, dihydrostreptomycin or streptomycin 

 1, 10 and 100 meg., are placed on the surface about 3 cm. apart. The plates are 

 incubated for 48 hours and then sealed with either cellophane tape or melted 

 paraflin and incubation continued at 35-3 7 °C. Tubercle bacilli are frequently 

 isolated and their sensitivities determined in 10-14 days; some strains may re- 

 quire up to 6-8 weeks incubation for suitable growth. Sensitivity of the organisms 

 to the antibiotic is indicated by the presence of zones of inhibition surrounding 

 the disks. 



Interpretation 



A moderate zone of inhibition around the lowest concentration of one or more 

 antibiotics would classify the organism as being sensitive to any or all of these 

 antibiotics even though there may be a difference in zone size. Resistant organisms 

 are not inhibited in the maximum concentration of the antibiotic while very 

 sensitive strains show marked zones of inhibition even in the lowest concentra- 

 tions. In the case of very sensitive strains, one may obtain zones of inhibition 

 around the disk containing the lowest concentration nearly as large as those 

 obtained around the higher concentrations, due to the diffusion characteristics of 

 the test. 



Results of this technique cannot necessarily be expressed in terms of the tube 

 dilution method. The presence of zones of inhibition of growth around the disks 

 indicates that the organism is sensitive to the antibiotic. The diameter of the zone 

 is dependent not only upon the sensitivity of the organism but also upon the 

 diffusability of the antibiotic and other factors which may limit the availability 

 of the antibiotic to the organism in the medium. 



Hemolytic properties of microorganisms must be considered when using plates 

 of Blood Agar for the test. Growth of hemolytic organisms is characterized by a 

 lysed, cleared or discolored background with the presence of colonies, while 

 zones of inhibition leave the medium unchanged (no hemolysis). Upon longer 

 incubation the hemolysin produced by the organism may diffuse toward the disk, 

 giving a cleared zone, without growth, around the periphery of the zone of inhibi- 

 tion. The true zone of inhibition on Blood Agar includes both the unchanged 

 non-hemolytic as well as the hemolytic areas showing no bacterial growth. 



Frequently one will observe isolated colonies growing within the zone of 

 inhibition. These represent resistant variants of the strain or culture, under test. 

 The appearance of several such resistant colonies in the zone of inhibition does 

 not indicate the true resistance of the culture which is demonstrated by the zone 

 of inhibition of growth. Bacto-Sensitivity Disks thus permit the isolation of re- 

 sistant variants from a culture. 



Bacto-Sensitivity Disks are stable for one year when stored in the refrigerator 

 at 2-6°C. 



Bacto-Sensitivity Disks are available in vials of 25 disks each. They are sup- 

 plied in complete sets and as replacement packages. A complete set (B403) con- 

 sists of 1 vial each of three concentrations of the more commonly used antibiotics. 



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