Group 4 Cadelinia, Anthony Fabian Candolita, Shiema Chris Carbonell, Mariah Kristianne Catahay, Jesus Alfonso

Exercise 3

Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)
I.

Objectives:
To allow the students to practice the microbiological assay of antibiotics by first using the Tube Dilution method and the Plate Dilution Method

II.

Materials:

50mL Mueller-Hinton Broth 200mL Mueller-Hinton Agar 1 vial of Benzylpenicillin in sterile 200mL phosphate buffer

III.

Methodology

A. Determination of MIC using the Broth Dilution Method

1mL

1mL

1mL

1mL

1mL 1mL 1mL 1mL

1mL 1mL

1mL

1mL

1 6oomg Benzylpenicillin in sterile 200 mL phosphate buffer = 3000g/mL Antibiotic sol.

10

11

12

4mL Mueller-Hinton Broth (each tube)

After the dilution, the tubes were inoculated with 2 drops of a 24-hr old broth culture of Enterococcus faecalis and were incubated at 30 deg. Celcius for 24 hours.

B. Determination of the Minimum Bactericidal Concentration

6oomg Benzylpenicillin in sterile 200 mL phosphate buffer = 3000g/mL Antibiotic solution

Tubes were observed after incubation and the top 3 tubes without growth (tube nos. 3, 4, 5) and lowest 3 tube with growth (tube nos. 6, 7, 8) were inoculated into 6 plates containing Mueller-Hinton Agar using multiple interrupted streaking. The plates were incubated in an incubator set at 36 deg. Celsius. The plates were observed after 24 hours. C. Determination of the MIC using the Plate dilution method 10mL 10mL 10mL 10mL 10mL

1
6oomg Benzylpenicillin in sterile 200 mL phosphate buffer = 3000g/mL Antibiotic solution

200mL Mueller-Hinton Agar (each flask)

Five organisms were inoculated in each plate, namely: S. aureus, E. faecalis, E. coli, Klebsiella & P. aeruginosa to test their susceptibilities in the different concentrations of antibiotic from each flask. The plates were incubated for 24 hrs and the organisms susceptibilities were recorded.

IV.

Results and Discussion

The tube Dilution Method Tube # 1 2 3 4 5 6 7 8 9 10 11 12 (broken) Conc (g/mL) 600 g/mL 120 g/mL 24 g/mL 4.8 g/mL 0.96 g/mL 0.192 g/mL 0.038 g/mL 7.68x10-3 g/mL 1.536x10-3 g/mL 3.072x10-4 g/mL 6.144x10-5 g/mL 1.229x10-5 g/mL MIC (Growth +/-) + + + + + + + MBC (Growth +/-)

+ (<25 colonies) + (<25 colonies) + (<25 colonies) + + +

The Plate Dilution Method Plate # Antibiotic concentration (g/mL) 142.86 g/mL 0.68 g/mL 3.24x10-3 g/mL 1.54x10-5 g/mL 7.35x10-8 g/mL Growth (+/-) S. aureus E. faecalis + + + + + +

E. coli + + + +

Klebsiella + + + + +

1 2 3 4 5

P. aeruginosa + + + + +

Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial solution that will inhibit the visible growth of a microorganism after overnight incubation, and Minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial solution that will prevent the growth of an organism after subculture on to antibiotic-free media. In the experiment, the MIC and MBC of Enterococcus faecalis was determined using Tube Dilution method and the Plate Dilution Method. In tube dilution method, serial dilutions of the

antibiotic (Benzylpenicillin) are made in a liquid growth medium (Mueller-Hinton Broth) which is inoculated with a standard number of organisms (2 drops of E. faecalis) and incubated in a period of time (24 hrs). After the incubation period, it is observable that the organism, E. faecalis is susceptible to concentrations of antibiotics ranging from 4.8 g/mL and 600 g/mL. This means that from 4.8 g/mL, the antibiotic can already kill the bacteria. However, as the antibiotic gets more diluted, its antimicrobial property gets weaker. It is observable also in the table that from 0.96 g/mL or lower, E. faecalis is resistant to the antibiotic. This means at this levels of concentrations of the antibiotic, the organism cannot be killed or terminated. Thus, 4.8 g/mL is considered to be the MIC. MBC plates were streaked using multiple interrupted streaking to observe the isolation of colines in the medium. However, there was an error in the experimenters results. The appearance of tubes 3, 4, and 5 after incubation was clear, light yellow liquid. So it is assumable that it indicated no growth of E. faecalis. But after the inoculation of the organism from those tubes into the plates, growth was observed by the presence of few colonies. The colonies were identical to the colonies present in tubes 6, 7, 8. The possible cause of these growths is that the organism (E. faecalis) might really grew in the medium, but the was very light that it wasnt observable at all in the test tubes. Hence, the MBS in this experiment is still uncertain due to the presence of colonies after a subculture from the test tube nos.3-8. The plate dilution method is slightly similar to tube dilution method. The differences are in plate dilution method, 5 organisms (S. aureus, E. faecalis, E. coli, Klebsiella & P. aeruginosa) are observed instead of one. Same principle is applied in interpreting the results of the suceptibility of each organism and it is seen that Klebsiella & P. aeruginosa remain resistant even in the highest concentration of the antibiotic present in the medium. This means that their MIC is still higher that the highest concentration of antibiotic used. V. References

https://www.boundless.com/microbiology/antimicrobial-drugs/measuring-drugsusceptibility/minimal-inhibitory-concentration-mic/ http://www.antimicrobialtestlaboratories.com/Minimum_Inhibitory_Concentration_Test_MIC. htm