Antibiotics and Agar Diffusion - Coursework Example

BRIEF SUMMARY This experiment showed that aside from the sensitivity of a particular bacterial species on the antibiotic being tested, other factors such as agar depth, inoculum size, volume of antibiotic, and presence of cations, all affect bacterial growth, as reflected by the zones of inhibition in agar plate diffusion assays. Agar depth, volume of antibiotic on disk and presence of cations each can modify the effective antibiotic concentration to which the plated bacteria are ultimately exposed to. On the other hand, differences in inoculum size vary considerably the amount of bacteria loaded onto the plate. Since these factors significantly change the diameters of zones of inhibition used both qualitatively and quantitatively in determining the antimicrobial potential of a substance, then these should be standardized during experiments. INTRODUCTION The agar plate diffusion methods are assays used to observe and quantify the antimicrobial property of tested substances. Briefly, the newly inoculated agar is placed with a small piece of paper soaked with the agent. After incubation, antimicrobial substances should produce a sterile ring surrounding the soaked paper. The diameter of the ring is an indication of the strength of the substance’s antimicrobial property. The aim of this experiment was to determine the effects of agar depth, inoculum size, presence of salts, and method of applying antibiotic on inhibition zones. METHODS In determining the effect of agar depth on the result of agar plate diffusion, four plates containing 10 cm3, 20 cm3, 20 cm3 and 30 cm3 agar were first swabbed with Eschericia coli 10418 (1:20 dilution). All plates were oriented horizontally except for the third one, which was slanted. Meanwhile, 2 plates each containing 20 cm3 DSTA were inoculated with 100 µl of either 1:20 E.coli dilution or undiluted broth culture. 4 discs each containing 20 µl 0.5 mg/ml ampicillin were placed in each of the plates. The plates were incubated overnight at 37°C. For determining the effects of potassium or calcium ions on zone sizes, 0.1 or 1.0 ml of 2M KCl or CaCl2 were added onto their corresponding plates each containing 20 ml DSTA, which would be inoculated with 0.1 ml Pseudomonas aeruginosa (1: 50). 4 discs each with 20 µl of either 20 mg/ml ampicillin (A) or 1 mg/ml streptomycin (S) were then placed on the agar plates. The plates were incubated overnight at 4°C In observing the difference between two methods of antibiotic application, 8 discs were soaked with 0.5 mg/ml ampicillin, while another 8 were each added with 20 µl of the antibiotic solution. The discs were distributed equally to four DSTA plates inoculated with Staphylococcus aureus. Next, gradient plates containing 0.1 ml of either 0.5 mg/ml ampicillin or 1 mg/ml streptomycin were inoculated with the standard strain (S. aureus (for ampicillin-containing agar) or E. coli (for streptomycin containing agar)) and three other isolates. Results were observed after overnight incubation at 37°C. RESULTS AND DISCUSSION Zones of inhibition are ways to visualize the effect of a material on bacterial growth. If present, the solution in the disk contains antibacterial property. Otherwise, it has no significant effect on preventing bacterial growth. It can also be used as quantitative measure of effectiveness, as a diameter similar to or greater than that obtained using a standard antibiotic is a good candidate for an antimicrobial product. However, there are factors, aside from the solution used and the bacterial species it is being tested against, that affect the sizes of zone of inhibition. Measuring the average zone of inhibition plotted in fig. 1, there is a noted decrease of zone of inhibition with increased depth of agar. Such change has been noted by several studies already (Lorian, 2005, p. 33). This is because zone inhibition experiment using higher agar volume has a lower effective antibiotic concentration as compared to that conducted in a lower agar volume. As a result, bacterial growth is greater and zone of inhibition is smaller in the former (Anon., n. d.). However, the values obtained in this study were not significantly different, taking into account the standard deviation. For the sloped agar, although the inhibition seemed to be decreased, this cannot be verified because of lack of replicates used in the study (Fig, 1). Table 1. Effects of Depth of Agar on Zone of Inhibition of E. coli with 20 µl 0.5 mg/ml ampicillin Agar depth (cm3) Zone of inhibition 1 2 3 4 Average 10 2.8 3.6 3.2 3.0 3.15 ± 0.34 20 1.9 1.9 30 2.0 1.8 3.0 3.0 2.45 ± 0.64 sloped 1.3 1.3 Looking at the effects of inoculum size on the zone of inhibition, the 1:20 dilution of inoculum resulted to a decrease in bacterial growth, as seen in about 10% increase in the diameter of zone of inhibition (fig. 2). According to Lorian (2005, p. 34), greater inoculation size will enable the reaching of the critical population to be done at a sooner possible time. A very dense inocula may result to a very profuse bacterial growth that will result to the absence of zone of inhibition. Table 2. Effects of Inoculum size on Zone of Inhibition of E. coli with 20 µl 0.5 mg/ml ampicillin Inoculum size Zone of inhibition 1 2 3 4 average Undiluted 2.0 2.0 2.1 2.0 2.025 ± 0.05 Diluted 2.2 2.3 2.2 2.4 2.275 ± 0.01 Fig. 2. Plot of effects of Inoculum Size on Zone of Inhibition of E. coli with 20 µl 0.5 mg/ml ampicillin In looking at the effects of the presence of cations on zone of inhibition, addition of 0.1 ml 2M KCl was noted to decrease bacterial growth and subsequently increase zone of inhibition by ampicillin and streptomycin. However, when the volume added was increased to 1 ml, the zones of inhibition for the two antibiotics were smaller than that measured from agar with 0.1 ml 2M KCl (fig. 3). KCl, like other salts such as NaCl, has long been recognized as antimicrobial because it causes dehydration, enzyme weakening interference, weakening of DNA structure (Parish, 2006). In contrast, the effect of CaCl2 was different for ampicillin and streptomycin. For the former, bacterial growth decreased with the addition of the solution, as seen with the increase in zone of inhibition. On the other hand, the zones of inhibition measured from disks with streptomycin were considerably lower, meaning an increase in bacterial growth with the presence of the cation (fig. 4). This is so because divalent cations, such as Ca2+, inhibit the activity of aminoglycosides, such as streptomycin (Lorian, 2005, p. 9). Table 3. The effects of the presence of cations on the zone of inhibition 2M KCl (ml) 2M CaCl2 (ml) antibiotic 1 2 average 1 0.1 0 S 2.0 2.3 2.15 ± 0.21 2 1.0 0 S 1.7 1.2 1.45 ± 0.35 3 0.1 0 A 1.6 1.8 1.7 ± 0.14 4 1.0 0 A 1.5 1.3 1.4 ± 0.14 5 0 0.1 S 0.7 0.5 0.6 ± 0.14 6 0 1.0 S 1.2 1.0 1.1 ± 0.14 7 0 0.1 A 1.6 1.6 1.6 8 0 1.0 A 1.6 1.6 1.6 9 0 0 S 1.5 1.5 1.5 10 0 0 A 0.4 0.6 0.5 ± 0.14 Fig. 3. Effects of increasing volume of 2M KCl to zone of inhibition by streptomycin and ampicillin Fig. 4. Effects of increasing volume of 2M CaCl2 to zone of inhibition by streptomycin and ampicillin In determining whether disk soaked in a solution of antibiotics, thus supposedly containing more antibiotics, had wider zones of inhibition than those added with a certain amount, it was seen that the disk soaked had a narrower zone of inhibition than that added with 20 µl antibiotics, although the difference was only around 0.03. Table 4. The effects of amount of antibiotics on zone of inhibition Effects of amount of antibiotics Zone of inhibition 1 2 3 4 Average Soaked 1.8 1.6 1.4 1.6 1.6 added 1.8 1.8 1.8 Fig. 5. Growth of E. coli on gradient plates streaked with unknown cultures A, B, and C as well as standard culture (SA). Comparing the growth on gradient plates, culture C is more likely to be the standard culture, because of the similarities in growth pattern. Gradient plates are used to determine the relative susceptibility of the culture to the antibiotic. The growth patterns in this experiment reveal that culture B is most sensitive to the antibiotic, followed by A. On the other hand, SA and C were least sensitive to the bacteria (Liu, et al., 2011, p. 953). This is as expected, since ampicillin is limited to certain gram-positive bacteria only, while S. aureus is already resistant to streptomycin (Carter, et al., 2000, p. 340). References Carter, A. P., Clemons, W. M., Brodersen, D. E., Morgan-Warren, R. J., Wimberly, B. T., and Ramakrishnan, V., 2000. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature, 407(6802), pp. 340–348. Liu, Y., Li, J., Du, J., Hu, M., Bai, H., Qi, J., Gao, C., Wei, T., Su, H., Jin, J., Gao, P., 2011. Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate. Sci. China Life Sci., 54(10), pp. 953-960. Lorian, V., 2005. Antibiotics in Laboratory Medicine: Making a Difference. Philadelphia: Lippincott, Williams and Wilkins. Oxoid Limited, n. d. Antibiotic Susceptibility Testing Best Practice. [online]. Available at: < http://www.oxoid.c9om/UK/blue/techsupport/its.asp?itsp=faq&faq=tsfaq021&cat=antibiotic+sensitivity+testing&lang=EN&c=UK> [Accessed 09 November 2012] Parish, M., 2006. How do salt and sugar prevent microbial spoilage? [online]. Available at: < http://www.scientificamerican.com/article.cfm?id=how-do-salt-and-sugar-pre> [Accessed 10 November 2012] Read More