Antibiotics for Agar
Adding antibiotic to agar - Agar Agar
Antibiotic Activity in the Presence of Agar
Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Historically and in a modern context, it is chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium for microbiological work. The gelling agent is an unbranched polysaccharide obtained from the cell walls of some species of red algae, primarily from the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii.
Agar has been shown to interfere with the activity of some antibiotics against Staphylococcus aureus. This interference has been observed as an increase in the minimal inhibitory concentration and in the diameter of the zone of inhibition. Purifying the agar with water extractions substantially reduced this adverse effect.
Previous studies in this laboratory have shown that the presence of agar in a test medium can seriously interfere with the inhibitory activities of antimicrobial agents (13, 24, 25). In an effort to overcome this interference, agar was extracted with water prior to use in the medium. The washed agar medium was found to be less antagonistic for these inhibitors, and the minimal inhibitory concentration in this medium often approached those in nutrient broth.
It was felt that this antagonistic effect might also extend to the antibiotics. If this were true, it could be an important factor in both the detection and use of these agents. It is well known that in the search for new antibiotic-producing organisms the initial screening tests are routinely performed in an agar medium, and quite often the antibacterial spectrum of a new antibiotic is determined by the agar plate dilution technique (2, 18). In addition, a semisolid medium is routinely used in sensitivity tests to establish the best antibiotic for use in clinical therapy.
This investigation was therefore carried out to determine whether the presence of agar in the test medium had any adverse effect upon the activities of antibiotics, and whether this antagonism could be reduced by the use of washed agar.
MATERIALS AND METHODS
Concentrated aqueous solutions of the antibiotics were filter-sterilized through an ultrafine Morton fritted-glass filter, and were aseptically diluted to the desired concentration in sterile, distilled water. The washed agar cubes (3% Difco agar) were prepared in the same manner as described in previous studies (13, 24). The cubes were melted under flowing steam in the autoclave prior to use in the test medium; 3% aqueous solutions of unwashed Difco agar were melted in a similar manner.
Double-strength nutrient broth was prepared, and was divided into three portions. One portion was diluted to single strength with melted, washed agar; the second portion was diluted to single strength with melted, unwashed agar; and the third portion was diluted to single strength with deionized, distilled water. In each of the semisolid media, the final agar concentration was 1.5%.
A 5-ml amount of each medium was pipetted into a series of screw-cap tubes and was autoclaved for 15 min at 121 C. The tubes were then placed in a water bath and were brought to a temperature of 46 C. The tubes were removed individually from the water bath, and 0.1 ml of the antibiotic solution was added aseptically. The tube was then vigorously shaken on a Vortex Jr. mixer. A 0.1-ml amount of the standardized cell suspension was then added, and the tube was again shaken. When the agar had solidified, the tubes were incubated at 37 C for 48 hr. At the end of this period, the tubes were examined under 1OX magnification for growth.
The approximate inhibitory concentration for the organism was first determined by a screening test in which concentrations of 0.01, 0.1, 1.0, 10, 100, and 1,000 ppm were used. Once the inhibitory range was determined, it was divided into 10 increments. For example, if the range was 0.1 to 1.0, the increments in this range were 0.1 ppm. Two additional experiments were performed with the use of the smaller increments. All data from these experiments are given in parts per million (ppm) of antibiotic (w/v). The media for the agar plates were prepared in the same manner as for the tubes. They were autoclaved, cooled to 45 C, and seeded with the standardized suspension of S. aureus, and 10 ml was pipetted into sterile petri plates. Antibiotic sensitivity discs (Difco) of intermediate concentration were placed on the solidified agar, and the plates were refrigerated at 4 C for 2 hr. They were then incubated at 37 C for 48 hr and were examined.
When the sensitivity of S. aureus to the tetracyclines obtained in nutrient broth was compared with that found in nutrient broth solidified with unwashed Difco agar, it was noted that the minimal inhibitory concentrations (MIC) were only slightly higher in the agar medium (Table 1). A comparison of the washed and unwashed agar media reveals that use of the washed agar medium reduced the MIC by one-half. This difference is statistically significant.