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Exercise 9

The document provides instructions for performing an antibacterial assay to test plant extracts against bacteria. It involves the following steps: 1. Preparing bacterial cultures, Mueller Hinton agar plates, plant extracts, filter paper disks, and turbidity standards. 2. Making a bacterial suspension adjusted to the 0.5 McFarland standard for inoculum preparation. 3. Inoculating Mueller Hinton plates by swabbing the bacterial suspension onto the agar surface. 4. Applying prepared filter paper disks containing plant extracts or antibiotic controls to the inoculated plates and incubating them overnight.

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Nueva Ilona Cagampang
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8 views

Exercise 9

The document provides instructions for performing an antibacterial assay to test plant extracts against bacteria. It involves the following steps: 1. Preparing bacterial cultures, Mueller Hinton agar plates, plant extracts, filter paper disks, and turbidity standards. 2. Making a bacterial suspension adjusted to the 0.5 McFarland standard for inoculum preparation. 3. Inoculating Mueller Hinton plates by swabbing the bacterial suspension onto the agar surface. 4. Applying prepared filter paper disks containing plant extracts or antibiotic controls to the inoculated plates and incubating them overnight.

Uploaded by

Nueva Ilona Cagampang
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Pre Lab Procedures:

 Re– inoculate bacterial cultures into nutrient agar slants and incubate for 18-24 hours.
 Prepare Mueller Hinton Agar (MHA) plate, following manufacturer's instructions and 4 test tubes
with 5mL dH2O per group and sterilize.
 Grind prepared plant extracts using mortar and pestle. If plant material is less succulent, add a small
amount of water.
 Filter the extract with filter paper. Final volume must be at least 5 ml
 Store extract in an amber vial or test tube wrapped in aluminum foil. Do not sterilize the extracts.
 Prepare 6mm Whatman filter paper disks by using a paper puncher and place in clean dry beaker
and sterilize.
 Prepare 0.5 McFarland turbidity standards by mixing 0.5 aliquot of 0.048 mol/L BaCI 2 and 99.5 mL
of 0.18 mol/L H2SO4. Mix constantly.
 Store in a screw-cap tube.
 Sterilize 10 pcs cotton swabs in autoclavable cellophane.
 Prepare alcohol lamps and forceps.

Procedures:
I. Inoculum Preparation
 Pick 3-5 well isolated pure colonies from the agar slant culture.
 Use an inoculating loop and transfer to 5-mL sterile distilled water.
 Compare turbidity to that of the 0.5 McFarland turbidity standards by using a card with a white
background and contrasting black lines.
 Add more colonies if inoculum is less cloudy. Add sterile water if it’s too cloudy.
 Result should contain approximately 1-2 x 108 CFU/mL of E. coli.

II. Inoculation of Test Plates


 Dip a sterile cotton swab within 5 minutes of adjusting the turbidity of the inoculum suspension into
the adjusted suspension.
 Swirl swab repeatedly and press firmly on the inside of the wall of the tube above fluid level to re-
move excess.
 Swab the cotton into the dried surface of the Mueller-Hinton agar plate.
 Perform swabbing procedures 3 times, rotating the plate 60° after each swab to ensure even distri-
bution.
III. Application of Disks to inoculated Agar Plates
 Use sterile 6 mm in diameter filter paper disks.
 Use a pipette to extract 20mL of plant extract treatment and dispense into the disk. (III-2)
 Position the disks from the inoculated MHA plate in a sterile place.
 Place the positive control disk (antibiotic disk) on the center of the agar plate.
 Use antibiotic capsule amoxicillin diluted in dH2O (500 mg/20 mL dH2O) if antibiotic disks are not
available. Repeat procedure III– 2.
 Press the disks down to ensure full contact to the agar plate and ensure even distribution to avoid
overlapping zones of inhibitions.
 Distance of each disk should be 24 mm from each other.
 Incubate plates at 35 ± 2°C for 18 hours.

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