Broth Dilution Method Determining Minimum Inhibitory Concentration

In certain instances of life-threatening infections such as bacterial endocarditis, or infections caused by highly or multiple resistant organisms, the physician may require a quantitative assessment of microorganism susceptibility rather than the qualitative report of S, I, or R. The laboratory then tests the susceptibility of the organism to varying concentrations of one or more appropriate antimicrobial agents. Twofold dilutions of each antimicrobial agent are prepared over a range of concentrations that are achievable in the patient’s bloodstream or urine (depending on the infection site) when standard doses of the drug are administered. In some cases, rather than preparing a full series of twofold dilutions, the organism is tested in only two or three antimicrobial concentrations. In this breakpoint dilution method, the concentrations tested are chosen carefully to discriminate between susceptible and resistant organisms.

The antimicrobial dilutions may be prepared in a broth medium, or each concentration of antimicrobial agent to be tested can be incorporated into an agar medium. In this agar dilution method many organisms (up to 32) can be tested on a single agar plate although several plates, each containing a different antimicrobial concentration, are needed to perform the assay. When only a single organism is tested, the broth dilution method is more rapid and economical to perform. Many laboratories now use commercially available microdilution plates. These consist of multiwelled plastic plates prefilled with various dilutions of several antimicrobial agents in broth (see colorplate 15). Using a multipronged device, the microwells are inoculated simultaneously with a standardized suspension of the test organism. Thus the susceptibility of an organism to many antimicrobial agents may be readily tested.

Regardless of the dilution method chosen, the results are interpreted in the same manner. After 18 to 24 hours of incubation at 35°C, the broths or plates are examined for inhibition of bacterial growth. For each antimicrobial agent, the lowest concentration that inhibits growth is referred to as the minimum inhibitory concentration, or MIC. As with the disk agar diffusion method, in order to obtain accurate information, variables such as inoculum size, phase of organism growth, broth or agar medium used, and antimicrobial agent storage conditions must be rigidly controlled.

Purpose	To learn the broth dilution method for antimicrobial susceptibility testing
Materials	Nutrient broth (Mueller-Hinton if available) Sterile tubes Broth containing 128 g of ampicillin per ml Sterile 1- and 5-ml pipettes Bulb or other aspiration device for pipette Tubes of sterile saline (5.0 and 9.9 ml per tube) Overnight plate culture of Escherichia coli

Procedures

1. Place nine sterile tubes in a rack and label them:
   
   

   Tube No.:	1	2	3	4	5	6	7	8	9
   Label:	64	32	16	8	4	2	1	Growth Control	Sterility Control



2. With a 5-ml pipette add 0.5 ml of sterile broth to each tube.
3. Add 0.5 ml of the ampicillin broth to the first tube (fig. 15.1a). Discard the pipette. The concentration of ampicillin in this tube is 64 µg per ml.
4. Take a fresh pipette, introduce it into the first tube (64 µg per ml), mix the contents thoroughly, and transfer 0.5 ml from this tube into the second tube (32 µg per ml). Discard the pipette.
5. With a fresh pipette, mix the contents of the second tube and transfer 0.5 ml to the third tube (16 µg per ml).
6. Continue the dilution process through tube number 7. The eighth and ninth tubes receive no antibiotic.
7. After the contents of the seventh tube are mixed, discard 0.5 ml of broth so that the final volume in all tubes is 0.5 ml.
8. From the plate culture of E. coli prepare a suspension of the organism in 5 ml of saline equivalent to a McFarland 0.5 standard (see Experiment 15.1).
9. With a sterile 1-ml pipette, transfer 0.1 ml of the E. coli suspension into a tube containing 9.9 ml of saline. Discard the pipette.
10. With a fresh pipette, mix the contents of the tube well. Add 0.1 ml of this organism suspension to the antibioticcontaining broth tubes 1 through 7 and to the growth control tube (fig. 15.1b).
11. Shake the rack gently to mix the tube contents and place the tubes in the incubator for 18 to 24 hours.

Figure 15.1 Broth dilution technique. (a) The ampicillin-containing broth is serially diluted in tubes that have been filled with 0.5ml of a nutrient broth. The growth and sterility control tubes receive no antibiotic. (b) After the antimicrobial dilutionsare completed, 0.1 ml of the appropriately diluted organism suspension, in this case E. coli, is added to all exceptthe sterility control tube. The number on each tube is the final concentration of ampicillin in that tube (µg/ml).

Results
Examine each tube for the presence or absence of turbidity. Record the results in the chart and indicate the MIC of ampicillin for the E. coli strain tested.