Antibacterial Screening By Kirby Bauer Method Biology Essay

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The quality control strain Staphylococcus aureus NCIM-5021 equivalent to S.aureus ATCC 29213 was procured from National Collection of Industrial Microorganism, Pune and all clinical isolates were obtained from Microbiology lab, Sri Ramakrishna Hospital and maintained at the Department of Pharmaceutical biotechnology, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore.

Maintenance of culture

The selected strains were confirmed for their purity and identity by Grams staining method and by their characteristic biochemical reaction. The selected strains were preserved by subculturing them periodically on nutrient agar slants and storing them under frozen condition. For the study fresh 24 hrs broth cultures were used after standardization.

Standardization of inoculum

All organisms were grown overnight (24 hr) at 370C on Nutrient agar (NA) and harvested during the stationary growth phase. For preparing fresh cultures a loopful of cells from the stock cultures were added to Mueller-Hinton broth and incubated for 24 hrs at 370C.Inoculum was standardized by matching the turbidity of the culture to 0.5 McFarland standards .This was produced by mixing 0.5 ml of

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0.048 M BaCl2 (1.175% W/V Barium Chloride dehydrates) with 99.5 ml of

0.36 N H2SO4.This produced an inoculating suspension of approximately 2.0x106(CFU/ml) for bacteria.

EXPERIMENTAL PROTOCOL:

Antibacterial screening by kirby-bauer (kb) method

Antibacterial activity of phytoconstituents

Antibacterial activity of antibacterial agents

Minimal inhibitory concentration (MIC) determination

E) Minimal bactericidal concentration determination(MBC)

F) Checkerboard broth dilution method

G) Study of possible synergism of phytoconstituents with antibacterial agents by double well diffusion method

H) Nanoemulsification of phytoconstituent (curcumin) and antibacterial (amikacin)

I) Antibacterial activity of nanoemulsified curcumin and amikacin

J) Study of possible synergism of nanoemulsified phytoconstituents with nanoemulsified antibacterial agent.

K) Minimum bactericidal concentration determination (MBC) of nanoemulsified phytoconstituents with nanoemulsified antibacterial agent.

METHOD

Antibacterial screening by Kirby-Bauer method:

Mueller Hinton agar plates were prepared aseptically to get a thickness of 5-6mm. The plates were allowed to solidify and inverted to prevent condensate falling on the agar surface. The plates were dried at 370C before inoculation. The organism was inoculated in the plate. It was allowed to dry at room temperature, with the lid closed. The sterile disc containing test drugs, standard and blank were placed on the previously inoculated surface of the Mueller Hinton agar plate and kept in the refrigerator for one hour to facilitate uniform diffusion of the drug. Plates were prepared in duplicate and they were then incubated for 18-24hrs. Observations were made for zone of inhibition around the drugs and compared with that of standard.

Antibacterial activity of phytoconstituents:

Agar well diffusion method was used. The test organism (Staphylococcus aureus) was spread on Mueller Hinton agar plates by means of sterile cotton swab. The wells were then punched into agar medium and filled with 60μl of the phytoconstituent dissolved in DMSO. The plates were incubated for 24 hrs at 370C. Antimicrobial activity was evaluated by measuring the zone of inhibition.

Antibacterial activity of the antibacterial agent:

Agar well diffusion method was used. The test organism (Staphylococcus aureus) was spread on Mueller Hinton agar plates by means of sterile cotton swab. The wells were then punched into agar medium and filled with 60μl of the antibacterial agent. dissolved in DMSO. The plates were incubated for 24 hrs at 370C. Antimicrobial activity was evaluated by measuring the zone of inhibition

Minimum inhibitory concentration determination (MIC)

Two fold serial dilution method:

Serial two fold dilutions of the antimicrobial agent were made in 1ml of MH broth series of 10-15 dilutions of the test antimicrobial agents were prepared.

Overnight cultures were grown at 370C and diluted in MH broth. This overnight culture was diluted to 10-2.[5Ã-105CFU/ml]

All tubes including a control tube without the drug (representing positive control) were inoculated with 50μl of the diluted inoculum and incubated for 18-24hrs at 37oC.

The tubes were examined for visible growth indicated by turbidity. The MIC was noted as the lowest drug concentration at which there was no growth. A negative control was also kept.

Fig. 5 MIC determination by two-fold serial dilution in liquid media

Minimum bactericidal concentration determination (MBC)

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The method was performed by sampling all the macroscopically clear tubes and the first turbid tube from the MIC determined test tubes series. Before being sampled, the tubes were gently mixed by flushing them and a loopful of broth was inoculated on sterile Mueller Hinton agar plate. Plates inoculated were then incubated at 370C for 24 hours. After incubation, the subculture plates are examined to determine the dilution or drug level at which 99.9% killing was achieved. The first plate with minimal dilution or drug level at which 99.9% killing achieved was taken as MBC.

Checkerboard broth dilution method

(Victor Lorian 3rd edition)

Dilutions above MIC value

MIC

Dilutions below MIC value

Row only with Drug A

Dilutions above

MIC value

MIC

Dilutions below

MIC value

Column only with drug B

® Tube with no drug + 1 ml of broth containing test organism (+ve control)

® Tubes with drug B alone in 0.5 ml + 0.5 ml of broth containing test organism

® Tubes with drug A alone in 0.5 ml + 0.5 ml of broth containing test organism

® Tubes with drug A and B in 0.5 ml + 0.5 ml of broth containing test organism

The broth dilution method was used to determine the activity of drug A in combination with drug B for double combination. Four concentrations below the MIC and the two concentrations above the MIC value where taken in two fold serial dilution, for each drug .Drug C was used as fixed single concentration per plate/set in triple combination regimen. Sterilized glass tubes were taken in rows and columns representing x-axis (Drug A) and y-axis (Drug B) respectively. The drug A was serially diluted along the abscissa, while drug B was diluted along the ordinate .An inoculum equal to 0.5 McFarland turbidity standard was prepared . Each test tube was inoculated with 50 µl of a bacterial inoculum of 5x105 CFU/ml.The test tubes were incubated (16-24hrs) at 35-370C.

The fractional inhibitory concentration index (FIC index) was calculated by summing the separate FIC's for each of the drug present in that tube (or well). For double drug combination,

(A) (B)

--------- + ------------- =FICA+FICB= FIC index

(MICA) (MICB)

(A)& (B) are concentrations of drug A and B in a tube that is the lowest inhibitory concentration in its row or column respectively.

MICA and MICB are the MICs of the organism to drug A and drug B alone respectively;

FICA and FICB are the fractional inhibitory concentrations of drug A and drug B respectively.

Synergy (FIC index≤0.5) is defined as a four fold greater in MIC of both drugs in combination compared with the drug tested individually. Partial synergism (FIC index>0.5 but<1) is defined as four fold decrease in MIC with one drug and 2-fold decrease in other drug. Additive (FIC index=1) is defined as a two-fold drop in MIC with both drugs. Indifference (FIC index>1 but<4) is noted when there was no change in MIC whether the drugs were tested alone or in combination. Antagonism is defined as a four fold increase in MIC of both drugs in combination with the drug tested individually

Study of possible synergism of phytoconstituents with different antibacterial agents

Agar well diffusion method was used to study the synergistic interaction. The wells were punched at a predetermined distance after inoculation of the test organism so that their inhibitory circles touch each other without influencing each other. The wells were loaded with 60μl (500mg/ml) of phytoconstituent and antibiotic. The interaction of the combination was studied separately. The combination exhibited enhanced inhibition zone as compared to individual zones of inhibition clearly signifying synergism against the tested strain of S. aureus

Process for nanoemulsification of phyto constituent (Curcumin):

12 g of curcumin 98% was taken in a glass beaker and added 212 g of polysorbate 80 and 224 g polyethylene glycol 400 and sonicated the mixture for 30 min at 25 KHz, until completely dissolved.

Preparation of nanoparticles of antibacterial agent (Amikacin):

160 mg of amikacin powder was dissolved in deionided water containing 1% w/w tween 80.Then 314 mg of cholesterol as lipid phase was dissolved in 24 ml 0f the mixture of ethanol/acetone with the ratio of 3:1(v/v)(equal to 18 ml ethanol and 6 ml acetone) by heating to 700C and stirring. Then hot oily phase was added to aqueous phase in 250C and sonicated.

Antibacterial activity of nanoemulsified curcumin and amikacin

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Agar well diffusion method was used. The test organism (Staphylococcus aureus) was spread on Mueller Hinton agar plates by means of sterile cotton swab. The wells were then punched into agar medium and filled with 60μl of the nanoemulsified drugs. The plates were incubated for 24 hrs at 370C. Antimicrobial activity was evaluated by measuring the zone of inhibition

Study of possible synergism of nanoemulsified curcumin with nanoemulsified amikacin.

Agar well diffusion method was used to study the synergistic interaction. The wells were punched at a predetermined distance after inoculation of the test organism so that their inhibitory circles touch each other without influencing each other. The wells were loaded with 60μl of nanoemulsified phytoconstituent and antibiotic. The interaction of the combination was studied separately. The combination exhibited enhanced inhibition zone as compared to individual zones of inhibition clearly signifying synergism against the tested strain of S. aureus

Minimum bactericidal concentration determination (MBC) of nanoemulsified curcumin with nanoemulsified amikacin.

The method was performed by sampling all the macroscopically clear tubes and the first turbid tube from the MIC determined test tubes series. Before being sampled, the tubes were gently mixed by flushing them and a loopful of broth was inoculated on sterile Mueller Hinton agar plate. Plates inoculated were then incubated at 370C for 24 hours. After incubation, the subculture plates are examined to determine the dilution or drug level at which 99.9% killing was achieved. The first plate with minimal dilution or drug level at which 99.9% killing achieved was taken as MBC.