DETERMINATION OF MIC

 

Determination of Minimal Inhibitory Concentration

AIM: To identify the MIC of given antimicrobial compound or antibiotic stock solution.

PRINCIPLE: The agar dilution technique is used to measure qualitatively the in vitro activity of an antimicrobial agent against the test bacteria. In this method, graded amounts of antibiotics are incorporated in agar plates and inoculated in spots with the organisms under study. If the organism under study is susceptible to the incorporated antibiotic, no bacterial growth is expected in agar plates with higher amounts of the drugs. Bacterial growth is observed as the antibiotic concentration in the agar plate diminishes. Inhibition of growth at the minimum or lowest concentration of antibiotic is regarded as the end point.

MATERIALS REQUIRED: Culture media, solvents, antimicrobial agents, control strains and apparatus needed for the minimal inhibitory concentration (MIC) test.

PROCEDURE:

1.      Dissolve the antimicrobial agent powder in solvent to make a concentration of 1,000 µg/ml.

2.      Dispense the stock solution into sterile diluents using two-fold dilution technique.

3.      Prepare MHA and Keep in a water bath at 48-50°C until use.

4.      Label each empty sterile plate in order to identify the antimicrobial agent and their concentrations.

5.      Pipette 1 ml of appropriate dilutions of the test antimicrobial agent into the labeled plate. Two replicates must be made for each concentration.

6.      Pipette 9 ml of MHA (keep warm at 48- 50°C), add into the plate with appropriate dilution of the test antimicrobial agent and mix thoroughly.

7.      Allow the agar to solidify at room temperature and Control agar plates/Drug-free agar plates

8.      Do not add any antimicrobial agent. There should at least be 2 control plates.

9.      From a pure 18-24 hour bacterial culture get 4- 5 isolated colonies.

10.  Shake vigorously in a water bath at 30°C until it achieves or exceeds the turbidity of 0.5 MacFarland standard (prepared by adding 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.36 NH2 SO4 ; commercially available). The inoculum may also be standardized based on optical density [OD625 of 0.08-0.1 (1cm light path)] using a spectrophotometer. This is usually achieved after 18- 24 hours.

11.  Dilute the standardized inoculum 1:10 in sterile saline solution to obtain the desired concentration of 10⁶ cfu/ml.

12.  Pipette 0.1 ml of the 10⁶ cfu/ml inoculum and transfer to a well, sterile test tubes of the same size, may be used to hold the diluted standardized inoculum.

13.  Inoculate plates with 10 µl.

14.  When inoculating manually, it is only important to include a drug free or control plate at the beginning of the inoculation series.

15.  Inoculate the bacterial suspensions onto the surface of the agar plate.

INCUBATION

16.   Incubate the plates in an inverted position at 30°C for 18-24 hours.

17.  Read and record the MIC at the lowest concentration of antimicrobial agent that completely inhibits growth of the organism as detected by the naked eye.

18.  Report result as Resistant (R), Intermediate (I) or Susceptible (S).

Example: Antibiotic: Oxytetracycline MIC breakpoint: 0.2 µg/ml Interpretation: susceptible.

Stock Solution Preparation

a. At least 1,000 µg/ml or 10 times the highest concentration to be tested is to be prepared as an antimicrobial agent stock solution.

Some antimicrobial agents are of limited solubility. Therefore, lower concentration may be required.

 b. Some drugs must be dissolved in solvents other than water.

Prepare stock solutions using formula

1000  xVxC=W    P

 

  

P-potency (µg/mg), V-Volume required (ml), C-Final concentration of solution (mg/L), W-Wt of antibiotics dissolved in volume V (mL).

 

CONJUGATION

 

CONJUGATION

AIM:

To study the process of bacterial conjugation through transfer of genes coding for antibiotic resistance.

PRINCIPLE:

Conjugation is the mode of gene transfer in many species of bacteria. In 1950 William Hayes, Francis Jacob and Elie L. Wollman established that conjugating bacteria are of two mating types. Certain “male” types (designated as F+) donate their DNA and other “female types” (designated as F-) receive the DNA. F- cells become F+ when they acquire a small amount of DNA. Hence the F factor is called as the Fertility factor. In contemporary microbiology, the donor’s F factors are known to be plasmids which are the extrachromosomal elements. The factors (plasmids) contain about 20-30 genes, most of which are associated with conjugation. These genes encode enzymes that replicate DNA during conjugation and structural proteins needed to synthesize special pili at the cell surface. Known as F pili or sex pili, these hair like fibres contact the recipient bacteria, and then retract so that the surfaces of donor and recipient are very close or touching one another. At the area of contact, a channel or conjugation bridge is formed. Once contact via sex pili has been made, the F factor (plasmid) begins replicating by the rolling circle mechanism. A single strand of the factor then passes over through the channel to the recipient. When it arrives, enzymes synthesize a complementary strand, and a double helix is formed. The double helix bends to a loop and reforms an F factor (plasmid), thereby completing the conversion of recipient from F- cell to F+ cell. Meanwhile, back in the donor cell a new strand of DNA forms, to complement the leftover strand of the F plasmid. The transfer of F factors involves no activity of the bacterial chromosome; therefore the recipient does not acquire new genes other than those on the F factor.

MATERIALS REQUIRED:

Glass wares: Conical flask, Measuring cylinder, Sterile test tubes, Petri plates

Reagents: Distilled water

Other requirements: Incubator, Shaker, Spectrophotometer, Micropipettes, Tips, Sterile loops and spreaders

PROCEDURE:

MATERIALS

1X PBS (Phosphate-buffered saline )

Lysogeny agar

Lysogeny

STEP MATERIALS

1X PBS (Phosphate-buffered saline )

1X PBS (Phosphate-buffered saline )

PROCEDURE:

Day 1:

1.      Open the vials containing Donor and Recipient cultures and resuspend the cells with 0.25 ml of LB broth respectively.

2.      Pick up a loopful of Donor culture and streak onto LB plates with Tetracycline (30 μg/ml).

3.      Pick up a loopful of Recipient culture and streak onto LB plates with Streptomycin (100 μg/ml). 4. Incubate overnight at 370 C.

Day 2:

1.      Pick up a single colony from Donor and Recipient Strain grown overnight on LB plates and inoculate a single colony in 6 ml of LB broth having respective antibiotics.

2.      Incubate the test tubes overnight at 37o C.

Day 3:

1.      Take 25 ml of LB broth and add 25 μl of tetracycline into it and inoculate 1 ml of overnight grown culture into it. Incubate at 37oC in a shaker.

2.        Take 25 ml of LB broth with streptomycin at a concentration of 100 μg/ml and inoculate 3 ml of overnight grown culture in it. Incubate at 37oC in a shaker.

3.      Grow the cultures till O.D of the donor culture reaches 0.8-0.9 at A600.

4.      Add 0.2 ml of each donor and recipient cultures in a sterile test tube labeled as conjugated sample. Mix by gentle pipetting and incubate at 37oC for 1-1.5 hours.

5.      Take 2 sterile test tubes and label them as donor and recipient. Add 0.2 ml of respective cultures to the test tubes and incubate at 37oC for 1-1.5 hours.

6.      Add 2 ml of LB broth into each tube after incubation. Incubate the tubes at 37oC for 1.5 hours.

7.      Plate 0.1 ml of each culture on the antibiotic plates as indicated in Table .

8.      Incubate the plates overnight at 37oC overnight.

Observation and Result:

 

	LB + (Streptomycin)+X gal	LB + (Tetracycline)+IPTG	LB + (Streptomycin, Tetracycline)+X gal+ IPTG
Donor Strain A
Recipient Strain B
Conjugated Sample

NOTE: Keep uninoculated LB plate as control.

Interpretation:

On observing colonies on different plates the following interpretation can be made:

1.        Donor strains will grow only on tetracycline plates, similarly recipient strains will grow only on streptomycin plates.

2.        Donor strain is sensitive to streptomycin and recipient strain is sensitive to tetracycline, hence no growth will be seen in these plates.

3.        The conjugated sample will grow on tetracycline and streptomycin plate. The reason being, transfer of gene has occurred by means of conjugation.

4.        The donor and recipient strain will not grow on tetracycline + streptomycin plate since each of the strain is sensitive to one antibiotic in the plate.