Analytical Microbiology Laboratory MCR 222L (2011-2012) Experiment #3 3MB2 Lee, Stephanie, Nuez, Karen, Oracion, Donna Faye,

, Pean, Janey, Saada, Therese Joy Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) Date of submission: July 12, 2011 Abstract
The tube dilution technique was used in the experiment to determine levels of resistance of certain bacteria to Streptomycin. Serial dilutions of the antibiotic were made in a liquid medium which was inoculated with a standardized number of organisms (using McFarland Standard) and incubated for 24 hours. The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC). At this dilution, the antibiotic is known to be bacteriostatic. Additionally, the minimal bactericidal concentration (MBC) was determined by subculturing the contents of the tubes onto antibiotic-free solid medium and was examined for bacterial growth. The results from this experiment indicate that tube no. 3 was the MIC.

I.

Introduction

Streptomycin is an antibiotic synthesized by the soil organism Streptomyces griseus. The drug acts by interfering with the ability of a microorganism to synthesize certain vital proteins. It was the first antimicrobial agent developed after penicillin and the first antibiotic effective in treating tuberculosis. It is also used in combination with penicillin for treating infections of heart valves (endocarditis) and with tetracyclines in the treatment of plague, tularemia, and brucellosis. The most commonly employed methods for determining levels of resistance to an antibiotic are the tube dilution method and agar dilution methods. Test products that are not clear or precipitate the growth media are tested by agar and dilution method which is about same as tube dilution method except dilutions are plated on agar. The tube dilution test is the standard method for determining levels of resistance to an antibiotic. This is often used to determine the smallest amount of antibiotic necessary to inhibit a test organism. This amount is known as the minimum inhibitory concentration (MIC). The lowest concentration

(highest dilution) of test agent preventing appearance of turbidity (growth) is considered to be the MIC. At this dilution the test agent is bacteriostatic. The MIC or minimum inhibitory concentration test determines antimicrobial activity of a material against specific bacteria. On the other hand, the minimal bactericidal concentration (MBC) of an antibacterial which is defined as the maximum dilution of the product that will kill a test organism can be determined by subculturing last clear MIC tube onto growth medium and examining for bacterial growth. Serial dilutions are made of the products in bacterial growth media. 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. II. Materials and Methods
A. Determination of MIC Using the Broth Dilution Method.

B. Determination of the Minimum Bactericidal Concentration (MBC). I.

Determination of the MIC Using the Plate Dilution Method.

II.

Results and Discussion

Table 1: Results of Tube Dilution Method, MIC and MBC Tube # 1 2 3 4 5 6 7 Concentration (g/mL) 800 160 32 6.4 1.28 0.256 0.0512 MIC (growth +/-) + + + + MBC (growth +/-) + + + +

8 9 10 11 12

0.01024 2.048x10-3 4.096x10-4 8.192x10-5 1.6384x10-5

+ + + + +

+ + + + +

Computations and Solution:

(1/250)106 5 mL = 800 (800)1 5 mL = 160 (160)1 5 mL= 32 (32)1 5 mL= 6.4 (6.4)1 5 mL= 1.28 (1.28)1 5 mL = 0.256 (0.256)1 5 mL = 0.0512 (0.0512)1 5 mL = 0.01024 (0.01024)1 5 mL = 2.048x10-3 (2.048x10-3)1 5 mL = 4.096x10-4 (4.096x10-4)1 5 mL = 8.192x10-5. (8.192x10-5)1 5 mL = 1.639x10-5

Table 2: Results of Plate Dilution Method Plate # Antibiotic Concentration (g/mL) 1 2 3 4 5 190.476 9.070 0.4319 0.0209 0.000979 Growth (+/-) S. aureus + + + + E.coli + + +

Streptomycin is an antibiotic drug, the first of a class of drugs called aminoglycosides to be discovered, a bactericidal antibiotic, was tested against Escherichia coli and its Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) was obtained. The Minimum Inhibitory Concentration is the lowest concentration of the substance which causes apparently complete inhibition of the growth of organism. It confirms the resistance of microorganisms to an antimicrobial agent and monitors the activity of new antimicrobial agents. A better antimicrobial agent is produced if the MIC is lower. An MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism. The MIC was obtained using the Tube Dilution Technique and the Plate Dilution Technique. In Tube Dilution Technique, serial dilution performed in 12 tubes of Mueller-Hinton Broth. After 24-48 hours of incubation, results were already obtained. Tube 1-3 showed no growth, showing the bactericidal effect of the antibiotic while Tube 4-12 exhibited growth. The MIC is shown in Tube 3, which has a concentration of 32 g/ml, the values after that were inhibitory. Gram Positive Cell walls are thicker than gram negative cell walls, however, gram negative cell walls are more complicated than gram positive because it is high in lipid content and low in Peptidoglycan and there it also consists of two separate areas (Outer Membrane and Inner Membrane) with an additional membrane besides the cellular membrane which makes it hard for the antibiotics to penetrate. And also, Penicillin destroys the ability of the Peptidoglycan to bear the stress of osmotic pressure that acts on a bacterium. Therefore, a higher concentration of antibiotics is needed for it in order to penetrate through the walls of gram

negative bacteria. The results were compared with the other even numbered groups that also preformed the same experiment, all the results were almost identical and they only differ within the range of one variable. However, in Group 6 there was a growth on tube 2 and their MIC is on Tube 5. In group 8, the MIC is on tube 4, and on Group 10 in Tube 3, which proves that a higher concentration of antibiotic is needed in order for it to inhibit the microorganism. The Minimum Bactericidal Concentration (MBC) which is the lowest concentration that will kill the bacteria was also obtained. Tube 12 which has a concentration of 1.6384x10-5 g/ml exhibited the MBC. The MBC must be plated afterwards, because this will determine whether the antibiotic is bactericidal and bacteristatic, If there is growth on the plate, then the antibiotic is bacteristatic, the cells were just inhibited and when there is no growth it is bactericidal, meaning all the cells were killed. Plate Dilution Technique was also performed, Escherichia coli and Staphylococcus aureus were used this time. They were both subjected to Penicillin. The advantage of Plate Dilution from Tube Dilution is that in Plate Dilution many organisms can be tested unlike in Tube Dilution, however, the large doses of the antibiotic are applied to Tube Dilution while in Plate Dilution, it is administered in small doses only. Plate Dilution also differentiated the susceptibility of gram positive bacteria from gram negative bacteria. And based from the results obtained S. aureus is more susceptible to penicillin because in Plate 2, there was growth already unlike in E.coli in which there was still no growth. When compared to the results of other groups, Group 6 showed growth in all the plates, Group 8 were similar to our result and Group 10 showed equal susceptibility between E.coli and S.aureus. The results varied a little maybe because of the way the antibiotic was administered or maybe human error, but nevertheless, the result should be S.aureus being more susceptible to Penicillin than E coli since penicillin acts on Peptidoglycan by preventing its assembly, gram positive bacteria is more susceptible to penicillin because its cell wall is mostly composed of peptidoglycan.

V. Conclusion This study showed some important findings like higher MIC values of antibiotic tested. The study informs the doctors communities the important information about the the MIC and MBC of condition of the antibiotics. The distribution of streptomycin MICs in E. coli can be greatly influenced by the genes encoding resistance to streptomycin. The low-level streptomycin resistance represents an obstacle in classifying E. coli as susceptible or resistant to streptomycin. Furthermore, the determination of an

epidemiological cut-off value for surveillance purposes is also complicated by dissemination. Therefore, we conclude that, overall, based on in vitro evaluation of inhibitory and bactericidal activities against E.coli, streptomycin have about equivalent potentials and are clearly promising effective. VI. Guide Questions:

1. Give the advantages and disadvantages of MIC determination using the Tube and Plate Dilution Methods. MIC determination using tube dilution is the standard method for determining levels of resistance to an antibiotic. Its advantages are: it provides better information on relative susceptibility, provides greater accuracy and repeatability and provides information that can be used to modify dose regimes, while its disadvantages are: it is more expensive, extra" information leads to confusion which means only one organism can be tested. While in MIC determination using Plate Dilution, not only one test organism can be tested however, only a small growth can be obtained because the organism can only be added in minimal doses unlike in Tube Dilution. 2. Give the reason for the streaking process required by MBC determination. To know whether it is bacteriostatic or bactericidal, because if it is bactericidal it would not grow on the plate anymore because the organism has already been killed by the antibiotic however in bacteriostatic, the organism was just inhibited but not killed and it would be able to grow on the plate. 3. How will one report the MIC value for the Tube Dilution method if all tubes showed growth and if all tubes did not show growth. If all the tubes exhibited growth the Minimum Inhibitory Concentration (MIC) will not be determined because in order for the MIC to obtained, there must be negative and positive growth that would serve as a basis for the least concentration that will show negative growth. And this same principle also applies for tube dilution showing no growth at all. 4. Give the MIC value for your test microorganism using the Tube Dilution Method. The Minimum Inhibitory Concentration in our experiment using the Tube Dilution Method was seen on Tube 3 with a concentration of 32 g/ml after that the succeeding tubes all exhibited growth.

5. What is meant by a 2-fold, 3-fold, 4-fold and a 10-fold series using the Tube Dilution Method? What is the advantage of a lower fold dilution series compared to a higher fold dilution series? The 2-fold, 3-fold, 4-fold and 10-fold series using the Tube Dilution simply means that the higher the dilution the lesser the amount of the antibiotic would be present in the tubes. Lower dilutions are more advantageous than the higher ones because the lesser the dilution, the higher the amount of the antibiotic is present, which enables the antibiotic to display its full potential. 6. Which is more susceptible to penicillin, Gram-positive or Gram-negative bacteria? Explain the basis of the MIC result. Since penicillin act on the Peptidoglycan by preventing its assembly, gram positive bacteria is more susceptible to penicillin because its cell wall is mostly composed of Peptidoglycan. 7. Are penicillin and streptomycin bacteriostatic and bacteriocidal antibiotics? Streptomycin and Penicillin are bactericidal because they act on the Peptidoglycan layer of Gram-positive and Gram-negative bacteria. By preventing the assembly of the Peptidoglycan, penicillin antibiotics destroy the ability of the Peptidoglycan to bear the stress of osmotic pressure that acts on a bacterium. VII. References Dedeles, G.R., Hutalle, K..M., Quinto, E. 2010. Exercises in Analytical Microbiology Laboratory. Retrieved from: http://cpharm.vetmed.vt.edu/vm8784/ANTIMICROBIALS/principles.htm Retrieved from: http://filebox.vt.edu/users/chagedor/biol_4684/Methods/cellwalls.html