DIAGNOSTIC REAGENTS 335 



assay method suitable for an antibiotic screen test using a large variety of test 

 organisms by Joslyn and Galbraith.^^ 



Performance of the Test 



Bacto-Sensitivity Disks are sterile and should be removed from the vials using 

 aseptic technique. Antibiotics are differentiated by the color of the disk and 

 concentrations are identified by the markings on each disk. The vials should be 

 tightly stoppered when not in use, and stored in the refrigerator at 2-6° C. 



The sensitivity test may be performed on plates of medium inoculated with 

 either the isolated organism or the pathological material containing these 

 organisms as suggested by Bondi, Spaulding, Smith and Dietz.^ Wood-^ suggested 

 the inoculation of duplicate plates with the specimens, placing the sensitivity 

 disks on one and using the second plate for the primary isolation of the etiological 

 agent. With either of these procedures, often the antibiotic of choice can be 

 determined prior to the isolation and identification of the organism. 



Plates of Bacto-Heart Infusion Agar, with or without added blood, are pre- 

 pared. These are inoculated either by smearing or streaking with a heavy sus- 

 pension of the test organism or clinic specimen containing the causative organism, 

 to obtain confluent or solid growth. Excessive moisture is to be avoided. Im- 

 mediately following the inoculation of the medium, Bacto-Sensitivity Disks are 

 placed on the surface spaced about 3-4 cm. apart. Use a flamed tweezer in re- 

 moving the disks from the vial so as not to contaminate the remaining disks. 

 Plates are incubated at 35-3 7 °C. for 12 hours or until growth develops, and 

 then examined for zones of inhibition of growth. It is recommended that in any 

 case the etiological agent be isolated since mixed cultures may not always give 

 dependable sensitivities. 



Heavy inoculation of plates permits more rapid sensitivity determinations 

 with the possibility of earlier treatment. Experience has demonstrated that with 

 some organisms plates so inoculated have shown definite zones of inhibition 

 within 5-6 hours after inoculation. Generally 5-6 sensitivity disks can be placed 

 on a regular 95 mm. petri plate. Some laboratories may prefer to use large size 

 petri plates (140 mm.) permitting the testing of the sensitivity of an organism 

 to a larger number of antibiotics or therapeutic agents. Crowding of disks is to be 

 avoided. 



If desired, a base and "seed layer" technique may be employed. For this pur^ 

 pose, pour 15 to 20 ml. of sterile Bacto-Heart Infusion Agar in sterile 95 mm. 

 plates and allow to solidify (use 60 ml. for 140 mm. plates). The inoculum is 

 incorporated in sterile melted Bacto-Heart Infusion Agar with or without blood 

 at 45 °C. This inoculated seed agar is poured on the base layer and distributed 

 evenly over the surface by tilting the plate to give a thin "seed layer" inoculation. 

 The disks are placed on the surface of this layer after it has solidified. 



The sensitivity of tubercle bacilli can be determined using Bacto-Sensitivity 

 Disks in combination with Bacto-Peizer TB Medium Base and Bacto-Peizer TB 

 Medium Enrichment. The medium used in this test is prepared according to the 

 formula described by Peizer and Schecter.-^ This procedure has been compared 

 with other methods and found to be as accurate and less time consuming than 

 other techniques presently in use. 



The method for determining the sensitivity of the tubercle bacillus to thera- 

 peutic agents using Bacto-Sensitivity Disks is essentially similar to the determina- 

 tion of sensitivities of other organisms. Either the pure culture or the clinic 

 specimen, such as treated sputum, spinal fluid, urine, etc., provided these contain 

 a moderate number of acid-fast organisms, Gaffky III or IV, may be used for 

 the inoculation. The complete medium is prepared from Bacto-Peizer TB 



