Procedure for Doc Padla

8/3/2019 Procedure for Doc Padla

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Concentration of Extracts

1. The crude extract with an initial concentration of 22 g/mL will be diluted with

distilled water to the following concentrations: (suggestion: 1000 g/mL, 500

g/mL and 100 g/mL) wala masyadong basis yan!:P ---scan journal articles

about banana peel w/ MIC for S. aureus. Paki-note ung MIC and STRAIN ng S.

aureus.. we can use it as basis on what concentrations we will use.

2. To prepare 1000 ug/mL, 220 mL dH2O will be a)dded to 1mL stock solution.

3. To prepare 500 ug/mL, 20 mL dH2O will be added to 10 mL of the 1000 ug/mL

solution.

4. To prepare 100 ug/mL, 20 mL dH2O will be added to 10 mL of the 1000 ug/mL

solution.

Initial Concentration: 100 g banana peel / 450 mL methanol= 0.22 g/mL = 220 000 g/mL

Final Concetrations needed: 1000 g/mL, 500 g/mL and 100 g/mL

(Initial Concentration) (Initial Volume) : (Final Concentration) (Final Volume)

1000 g/mL(220 000 g/mL) (1mL) = (1000 g) (x)

x = 220 mL

(1000 g/mL) (10mL) = (500 g/mL) (x)x = 20 mL

100 g/mL(1000 g/mL) (10mL) = (100 g/mL) (x)x = 100 mL

Preparation of bacterial suspension

1. Staphylococcus aureus maintained on nutrient agar slants at 4C will be

aseptically transferred using a sterilized needle into fresh sterile broth culture.

2. For standardization of bacterial density, the turbidity of the fresh 24 hour old

cultures will be adjusted to 0.5 MacFarland standard (1 108

cells/ml) by diluting

with the sterile broth. In preparation of 0.5 MacFarland standard the following

steps will be followed: not sure kung prepared na ung MacFarland or tayo pa

gagawa, pero feeling ko prepared na, nilagay ko lang din ung procedure anyway

:P

a. Add a 0.5-ml aliquot of a 0.048 mol/liter BaCl2 (1.175% wt/vol BaCl2 2H20)

to 99.5 ml of 0.18 mol/liter H2SO4 (1% vol/vol) with constant stirring to

maintain a suspension.

b. Verify the correct density of the turbidity standard by measuring absorbance

using a spectrophotometer with a 1-cm light path and matched cuvette. The

absorbance at 625 nm should be 0.08 to 0.13 for the 0.5 McFarland

standard.

c. Transfer the barium sulfate suspension in 4- to 6-ml aliquots into screw-cap

tubes of the same size as those used in standardizing the

- Graduated cylinder (100mL)

- Micropipette (1000 L)

- S. aureusculture

- Sterilized needle- Fresh Broth culture (200 mL


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bacterialinoculums.

d. Tightly seal the tubes and store in the dark at room temperature.

3. To adjust the turbidity of 24-hour old culture, both the standard and the inoculum

tube will be held side by side and no more than 1 inch from the face of the

Wickerham card (with adequate light present) then the appearance of the lines

through both suspensions will be compared. The tubes flush should not be held

against the card.

4. If the bacterial suspension appears lighter than the 0.5 McFarland standard, more

organisms should be added to the tube from the culture plate. If the suspension

appears more dense than the 0.5 McFarland standard, additional sterile broth

should be added to the inoculum tube in order to dilute the suspension to the

appropriate density. In some cases it may be easier to start over rather than to

continue to dilute a bacterial suspension that is too dense for use.

Paper disk diffusion assay

1. Mueller Hinton Agar plates will be used. Each plate will be divided into 6 equal

parts with a marking pencil on the bottom of the plates and will be labeled as

follows:

Musa sapientum Musa paradisiaca

Musa balbisiana

- Wickerham card

-

- 3 MH Agar plates

- Marking pen

CONC.

(A)

CONC.

(B)

CONC.

(C)

(+)

CONTROL

(-)

CONTROL

SOLVENT

ONLY

CONC.

(A)

CONC.

(B)

CONC.

(C)

(+)

CONTROL

(-)

CONTROL

SOLVENTONLY

CONC.

(A)

CONC.

(B)

CONC.

(C)

(+)

CONTROL

(-)

CONTROL

SOLVENTONLY


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2. A sterile cotton swab will be aseptically dipped into the S. aureus broth culture.

The cotton swab will be rotated against the inside wall of the tube to remove

excess broth, then the swab will be streaked evenly over the surface of the plate

and in three directions, leaving no gaps in between strokes.

3. The plate will be allowed to dry for 3-5 minutes with the lid in place. Filter paper

disks (6mm) will be impregnated with the respective treatments and controls by a

micropipette then will be allowed to dry for 10 minutes.

Research on the volume capacity of 6mm paper disk.. in microliters.

4. The individual disks will then be placed and pressed lightly on the surface of the

nutrient agar in the different sections marked on the bottom of the plates. The

plates will be incubated at 37C for 24 hours.

5. The zone of inhibition will be measured (in millimeters) after overnight incubation.

Zone of inhibition with a diameter of ? mm will be considered significant. Research on the what diameter (zone of inhibition) we will consider significant :/

Determination of MIC

1. The concentration of extract will the smallest zone of inhibition will be used in MIC.

2. Broth dilution test method will be used in determining the minimum inhibitory

concentration of the plant extracts.

3. In ten sterile tubes labeled 1 through 10, 0.5ml of sterile broth will be aseptically

added. Then, 0.5 ml of the peel extract will be added to tubes 1 and 2.

4. Serial dilution of the peel extracts will be carried out from tube 2 until tube 9. All the

tubes will then be inoculated with 0.5 mL of S. aureussuspension and incubated at

37C for 24 hours. Tube 10, containing only the sterile broth and S. aureus

suspension will serve as the control tube. Bacterial growth in each tube will be

examined by observing turbidity. The highest dilution without growth is the minimal

inhibitory concentration.

Determination of MBC

1. Aliquots from the tube with the least amount of drug that showed no growth and

the two tubes that immediately precede it will be collected and inoculated (streak

plating) in a Nutrient Agar medium. The plates will also be incubated at 37 C for

24 hours.

- Sterile cotton swab

- 18 Filter Paper Disk (6mm)

- Micropippette- Forceps

- Ruler / caliper

- 10 test tubes (? mL)

- Test tube rack

- Permanent Marker

- Tape for Labelling

- Alcohol lamp / Bunsen burne

- Pipette ( 1mL)

- Micropipette / pipette

- 3 Petri dish with Nutrient Aga

- Alcohol lamp / Bunsen burne

- Sterilized needle / wire loop


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2. The plates will be examined for bacterial growth by observing presence of S.

aureuscolonies. No growth indicates that the extract is bactericidal at that dilution

whereas growth indicates that the antibiotic is bacteriostatic but not bactericidal at

that dilution.

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Procedure for Doc Padla