BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

(Broth Tube Dilution Method)

The tube dilution test is the standard method for determining levels of resistance to an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration (highest dilution) of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC). At this dilution the antibiotic is bacteriostatic.

Additionally, the minimal bactericidal concentration (MBC) can be determined by subculturing the contents of the tubes onto antibiotic-free solid medium and examining for bacterial growth.

Although the tube dilution test is fairly precise, the test is laborious because serial dilutions of the antibiotic must be made and only one isolate can be tested in each series of dilutions.

Procedure:

1. Number sterile capped test tubes 1 through 9. All of the following steps are carried out using aseptic technique.
   
   
2. Add 2.0 ml of tetracycline solution (100 ug/ml) to the first tube. 3. Add 1.0 ml of sterile broth to all other tubes.
   
   
3. Transfer 1.0 ml from the first tube to the second tube.
   
   
4. Using a separate pipette, mix the contents of this tube and transfer 1.0 ml to the third tube.
   
   
5. Continue dilutions in this manner to tube number 8, being certain to change pipettes between tubes to prevent carryover of antibiotic on the external surface of the pipette.
   
   
6. Remove 1.0 ml from tube 8 and discard it. The ninth tube, which serves as a control, receives no tetracycline.
   
   
7. Suspend to an appropriate turbidity several colonies of the culture to be tested in 5.0 ml of Mueller-Hinton broth to give a slightly turbid suspension.
   
   
8. Dilute this suspension by aseptically pipetting 0.2 ml of the suspension into 40 ml of Mueller-Hinton broth.
   
   
9. Add 1.0 ml of the diluted culture suspension to each of the tubes. The final concentration of tetracycline is now one-half of the original concentration in each tube.
   
   
10. Incubate all tubes at 35^oC overnight.
    
    
11. Examine tubes for visible signs of bacterial growth. The highest dilution without growth is the minimal inhibitory concentration (MIC).

 

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