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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.

Care of civilization

The selected strains were confirmed for their pureness and individuality by Grams staining method and by their characteristic biochemical reaction. The selected strains were preserved by subculturing them sporadically on alimentary agar angles and hive awaying them under frozen status. For the survey fresh 24 hour stock civilizations were used after standardisation.

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Standardization of inoculant

All beings were grown overnight ( 24 hour ) at 370C on Food agar ( NA ) and harvested during the stationary growing stage. For fixing fresh civilizations a loopful of cells from the stock civilizations were added to Mueller-Hinton stock and incubated for 24 hour at 370C.Inoculum was standardized by fiting the turbidness of the civilization to 0.5 McFarland criterions.This was produced by blending 0.5 milliliter of

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

0.36 N H2SO4.This produced an inoculating suspension of about 2.0×106 ( CFU/ml ) for bacterium.

EXPERIMENTAL PROTOCOL:

Antibacterial showing by kirby-bauer ( kilobit ) method

Antibacterial activity of phytoconstituents

Antibacterial activity of antibacterial agents

Minimal inhibitory concentration ( MIC ) finding

Tocopherol ) Minimal disinfectant concentration finding ( MBC )

F ) Checkerboard broth dilution method

G ) Study of possible synergy of phytoconstituents with antibacterial agents by double good diffusion method

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

I ) Antibacterial activity of nanoemulsified curcumin and amikacin

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

K ) Minimum disinfectant concentration finding ( MBC ) of nanoemulsified phytoconstituents with nanoemulsified antibacterial agent.

Method

Antibacterial showing by Kirby-Bauer method:

Mueller Hinton agar home bases were prepared aseptically to acquire a thickness of 5-6mm. The home bases were allowed to solidify and inverted to forestall condensate falling on the agar surface. The home bases were dried at 370C before vaccination. The being was inoculated in the home base. It was allowed to dry at room temperature, with the palpebra closed. The unfertile phonograph record incorporating trial drugs, criterion and space were placed on the antecedently inoculated surface of the Mueller Hinton agar home base and maintain in the icebox for one hr to ease unvarying diffusion of the drug. Home plates were prepared in extra and they were so incubated for 18-24hrs. Observations were made for zone of suppression around the drugs and compared with that of criterion.

Antibacterial activity of phytoconstituents:

Agar good diffusion method was used. The trial being ( Staphylococcus aureus ) was spread on Mueller Hinton agar home bases by agencies of unfertile cotton swab. The Wellss were so punched into agar medium and filled with 60I?l of the phytoconstituent dissolved in DMSO. The home bases were incubated for 24 hour at 370C. Antimicrobial activity was evaluated by mensurating the zone of suppression.

Antibacterial activity of the antibacterial agent:

Agar good diffusion method was used. The trial being ( Staphylococcus aureus ) was spread on Mueller Hinton agar home bases by agencies of unfertile cotton swab. The Wellss were so punched into agar medium and filled with 60I?l of the antibacterial agent. dissolved in DMSO. The home bases were incubated for 24 hour at 370C. Antimicrobial activity was evaluated by mensurating the zone of suppression

Minimum repressive concentration finding ( MIC )

Two fold consecutive dilution method:

Consecutive two fold dilutions of the antimicrobic agent were made in 1ml of MH broth series of 10-15 dilutions of the trial antimicrobic agents were prepared.

Nightlong civilizations were grown at 370C and diluted in MH stock. This nightlong civilization was diluted to 10-2. [ 5A-105CFU/ml ]

All tubings including a control tubing without the drug ( stand foring positive control ) were inoculated with 50I?l of the diluted inoculant and incubated for 18-24hrs at 37oC.

The tubings were examined for seeable growing indicated by turbidness. The MIC was noted as the lowest drug concentration at which there was no growing. A negative control was besides kept.

Fig. 5 MIC finding by double consecutive dilution in liquid media

Minimum disinfectant concentration finding ( MBC )

The method was performed by trying all the macroscopically clear tubings and the first turbid tubing from the MIC determined test tubes series. Before being sampled, the tubings were gently mixed by blushing them and a loopful of stock was inoculated on unfertile Mueller Hinton agar home base. Home plates inoculated were so incubated at 370C for 24 hours. After incubation, the subculture home bases are examined to find the dilution or drug degree at which 99.9 % violent death was achieved. The first home base with minimum dilution or drug degree 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 merely with Drug A

Dilutions above

MIC value

MIC

Dilutions below

MIC value

Column merely with drug B

A® Tube with no drug + 1 milliliter of stock incorporating trial being ( +ve control )

A® Tubes with drug B entirely in 0.5 milliliters + 0.5 milliliter of stock incorporating trial being

A® Tubes with drug A entirely in 0.5 milliliters + 0.5 milliliter of stock incorporating trial being

A® Tubes with drug A and B in 0.5 milliliters + 0.5 milliliter of stock incorporating trial being

The broth dilution method was used to find the activity of drug A in combination with drug B for dual combination. Four concentrations below the MIC and the two concentrations above the MIC value where taken in two fold consecutive dilution, for each drug.Drug C was used as fixed individual concentration per plate/set in ternary combination regimen. Sterilized glass tubings were taken in rows and columns stand foring x-axis ( Drug A ) and y-axis ( Drug B ) severally. The drug A was serially diluted along the abscissa, while drug B was diluted along the ordinate.An inoculant equal to 0.5 McFarland turbidness criterion was prepared. Each trial tubing was inoculated with 50 Aµl of a bacterial inoculant of 5×105 CFU/ml.The trial tubings 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 nowadays in that tubing ( or good ) . For dual drug combination,

( A ) ( B )

— — — — – + — — — — — — – =FICA+FICB= FIC index

( MICA ) ( MICB )

( A ) & A ; ( B ) are concentrations of drug A and B in a tubing that is the lowest repressive concentration in its row or column severally.

MICA and MICB are the MICs of the being to drug A and drug B entirely severally ;

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

Synergy ( FIC indexa‰¤0.5 ) is defined as a four crease greater in MIC of both drugs in combination compared with the drug tested separately. Partial synergy ( FIC index & gt ; 0.5 but & lt ; 1 ) is defined as four fold lessening in MIC with one drug and 2-fold lessening in other drug. Additive ( FIC index=1 ) is defined as a double bead in MIC with both drugs. Indifference ( FIC index & gt ; 1 but & lt ; 4 ) is noted when there was no alteration in MIC whether the drugs were tested entirely or in combination. Antagonism is defined as a four fold addition in MIC of both drugs in combination with the drug tested separately

Study of possible synergy of phytoconstituents with different antibacterial agents

Agar good diffusion method was used to analyze the interactive interaction. The Wellss were punched at a preset distance after vaccination of the trial being so that their inhibitory circles touch each other without act uponing each other. The Wellss were loaded with 60I?l ( 500mg/ml ) of phytoconstituent and antibiotic. The interaction of the combination was studied individually. The combination exhibited enhanced suppression zone as compared to single zones of suppression clearly meaning synergy against the tried strain of S. aureus

Procedure for nanoemulsification of phyto component ( 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 wholly dissolved.

Preparation of nanoparticles of antibacterial agent ( Amikacin ) :

160 milligram of amikacin pulverization was dissolved in deionided H2O incorporating 1 % w/w tween 80.Then 314 milligram of cholesterin as lipid stage was dissolved in 24 milliliter 0f the mixture of ethanol/acetone with the ratio of 3:1 ( v/v ) ( equal to 18 milliliters ethanol and 6 milliliters acetone ) by heating to 700C and stirring. Then hot oily stage was added to aqueous stage in 250C and sonicated.

Antibacterial activity of nanoemulsified curcumin and amikacin

Agar good diffusion method was used. The trial being ( Staphylococcus aureus ) was spread on Mueller Hinton agar home bases by agencies of unfertile cotton swab. The Wellss were so punched into agar medium and filled with 60I?l of the nanoemulsified drugs. The home bases were incubated for 24 hour at 370C. Antimicrobial activity was evaluated by mensurating the zone of suppression

Study of possible synergy of nanoemulsified curcumin with nanoemulsified amikacin.

Agar good diffusion method was used to analyze the interactive interaction. The Wellss were punched at a preset distance after vaccination of the trial being so that their inhibitory circles touch each other without act uponing each other. The Wellss were loaded with 60I?l of nanoemulsified phytoconstituent and antibiotic. The interaction of the combination was studied individually. The combination exhibited enhanced suppression zone as compared to single zones of suppression clearly meaning synergy against the tried strain of S. aureus

Minimum disinfectant concentration finding ( MBC ) of nanoemulsified curcumin with nanoemulsified amikacin.

The method was performed by trying all the macroscopically clear tubings and the first turbid tubing from the MIC determined test tubes series. Before being sampled, the tubings were gently mixed by blushing them and a loopful of stock was inoculated on unfertile Mueller Hinton agar home base. Home plates inoculated were so incubated at 370C for 24 hours. After incubation, the subculture home bases are examined to find the dilution or drug degree at which 99.9 % violent death was achieved. The first home base with minimum dilution or drug degree at which 99.9 % killing achieved was taken as MBC.

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