Antibiotics and Agar Diffusion - Coursework Example

Practical Antibiotics and Agar Diffusion Insert (s) Agar plate diffusion methods are increasingly being used in the study of the antibiotic effects on various microorganisms. The methods primarily involve the quantification of the ability of a particular antibiotic to inhibit bacterial growth. This practical involves three experiments which were carried out to quantitatively analyze and measure antibiotic susceptibility of various microorganisms. The first experiment investigates the effects of variables such as agar depth, presences of salt and inoculums size on the size of inhibition zones. On the other hand, experiment 2 attempts to investigate the repeatability of the essays using two different methods of antibiotic application to the discs. Lastly, experiment 3 uses gradient plates to qualitatively compare the response of new isolates to a particular antibiotic against a response of a standard strain. Practical 1: Antibiotics and Agar Diffusion Introduction Agar plate diffusion is currently one of the widely used experimental methods to determine the susceptibility of various microorganisms to antimicrobial agents. Generally the principle employed in this method is dependent on the inhibition of the microorganism reproduction on the surface of agar medium by an antimicrobial agent which gradually diffuses into the medium through a filter paper disc. In this regard, the level of susceptibility of the organisms is indicated by the size of the zone of inhibition. Typically when an antibiotic is applied to the agar medium, it moves from its region of high concentration to the surrounding areas of lower antimicrobial concentration (Cooper, 1993, p.24). Inhibition zone is the clear area of the agar plate where the growth of the microorganisms is prevented by the activities of the microbial agent. The size of inhibition is usually measured and then compared to the standardized measurements in order to determine the level of antibiotic activity on the particular medium. This is primarily because the diameter of the inhibition zone is always a function of the susceptibility of the microorganism and the amount of antibiotics on the medium. Generally there are a number of factors that may affect the antimicrobial activity and consequently determine the size of inhibition zones. Some of the likely factors include agar depth, size of the inoculums and the presence of cations on the medium. The objective of this experiment is to investigate the factors that are responsible for the variation of microbial activity of various antimicrobial drugs on different organisms. Methods To investigate the effects of variables such as agar depth, presences of salt and inoculums size on the size of inhibition zones, lawn and seeding methods were used to inoculate the plates. 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. Finally the plates were then incubated overnight at 37°C and the variations in the inhibition zone size were recorded. To determine the effect of inoculum size on the antibiotic activity, 0.5mg/ml ampillicin solution and the E.coli dilution were used.2 plates with 20 cm3 DSTA were poured into the solution. The first inoculate was then spread uniformly on the entire surface of the plate. On the second plate was also inoculated with a containing broth culture of E.coli was also inoculated. Finally 4 amplicin impregnated papers were placed on each plate before they were incubated. To investigate 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 experiment 2, the repeatability of the essays was investigated using two different methods of antibiotic application to the discs. 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. Lastly, experiment 3 uses gradient plates to qualitatively compare the response of new isolates to a particular antibiotic against a response of a standard strain with a known MIC. To determine whether amplicin or streptomycin should be used in the treatment of infections caused by three different bacterial species, 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 1. Effects of Agar depth on antibiotic activity Figure1. 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 20 1.9 1.9 30 2.0 1.8 3.0 3.0 2.45 sloped 1.3 1.3 The experimental results suggested that the variation in the depth of Agar affected the resultant zone sizes of inhibition. For example, at depth 10 cm3, the average diameter of the zone of inhibition was 3.15 while at depth 30 cm3, the average diameter for the zone of inhibition reduced to 2.45. Consequently this is a likely suggestion that the size of the inhibition decreases with the depth of agar medium. For example, if the Agar medium is thinner, antibiotic activity is likely to increase because the concentration of the antimicrobial agent is higher. On the other hand, when the agar medium is thicker, the size of the inhibition zone is reduced because of the low concentration of the effective antibiotic agent (Washington, 2004, p.124). Table 2 2. Effects of Inoculum size on microbial activity Figure3. 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 Diluted 2.2 2.3 2.2 2.4 2.275 The results tabled above clearly indicated that Inoculum depth is one of the important variables that affect the size of the zone of inhibition during susceptibility tests. For example, the average diameter of the zone of inhibition was relatively lower when undiluted Inoculum was used as compared to when diluted Inoculum was used. According to Lorian (2005, p.45), this can be explained by the fact that when heavy (undiluted) inoculum is used, the critical mass achieved is usually achieved within a shorter time and consequently the level of antimicrobial activity will be lower resulting into a smaller zone of inhibition. On the other hand, when the inoculum is diluted, it becomes lighter and the critical mass usually takes longer to develop. As a result the antibiotic agent is likely to diffuse further thereby achieving a larger zone of microbial activity. 3. Effects of Cations on the zone of inhibition Figure 4.table showing the effects of 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 2 1.0 0 S 1.7 1.2 1.45 3 0.1 0 A 1.6 1.8 1.7 4 1.0 0 A 1.5 1.3 1.4 5 0 0.1 S 0.7 0.5 0.6 6 0 1.0 S 1.2 1.0 1.1 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 The results of experiment 1 regarding the effects of potassium and calcium chlorides on antibiotic activity indicated that the zone of inhibition reduces with the increasing amounts of cations in the culture. Generally Potassium chloride produces monovalent cations (K+) while Calcium chloride produces divalent cations (Ca2+ ). Both monovalent and divalent cations have been known to have significant antagonizing effects on most gram negative bacterias such as Pseudomonas aeruginosa and E.coli. Divalent cations (Ca2+ ) however showed increased antagonism as compared to the potassium ions that are monovalent. According to Ericson (1991, p.38), consequently at lower concentrations, the antagonizing effect is lower and the antibiotic agents such as streptomycin and ampicilin retain their ability to inhibit bacterial growth leading to a large size of the inhibition zone. On the other hand, the increased amounts of the cations results in increased antagonizing thereby reducing the antibiotic activity. 4. Effects of the amount of the amount of Antibiotics on microbial activity Figure 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 As suggested by the results above, the size of zone of inhibition increases with the amount of antibiotics. For example, the average diameter of the zone of inhibition was lower when method one was used as compared to method two. This is particularly attributed to the fact that in method 1, excess liquid was allowed to drain thereby reducing the amount of the antibiotic agent used. On the other hand, the larger zone of inhibition in method 2 was caused by increased amount of antibiotic agent due to dilution. 5. Comparing the growth of microorganisms on gradient plates As the results of experiment 3 indicate, gradient plates the growth of microorganisms. This is most likely because the gradient of antibiotic concentration affects its rate of diffusion through the agar and this consequently impacts on the susceptibility and growth of the microorganisms in the culture. Lastly, the level of microbial activity varies depending on the method of applying the antibiotic to the discs as well as with different species of microorganisms For example, the growth patterns in the 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. This is most likely attributed to the fact that ampicillin is limited to certain gram-positive bacteria only, while S. aureusis already resistant to streptomycin. References Cooper, K. E. 1993. The theory of antibiotic inhibition zones. In F. Kavanagh (ed.), Analytical microbiology. New York: Academic Press, Inc. Ericson, H. M. 1991. Antibiotic sensitivity testing- report of an international collaborative study. Acta. Pathol. Microbiol. 217, pp. 18-30. Lorian,V. 2005. Antibiotics in Laboratory Medicine: Making a Difference. Philadelphia: Lippincott, Williams and Wilkins. Washington, J. A. 2004. Laboratory procedures in clinical microbiology.Boston: Little, Brown and Company. Read More