Name: IAN REY M.

DE LOS SANTOS Course: BSFT-2A

TOTAL YEAST AND MOLD COUNT

MATERIALS:
APPARATUS & EQUIPMENT
o Balance Machine
o Stomacher
o Autoclave o Stomacher bag
o Micro pipette o Water bath
o Biological Safety Cabinet o Petri Dish
o Pipette tips o Bunsen Burner
o Incubator o Glass spreader

MEDIA & CHEMICAL

➢ DRBC Agar (Dichloran Rose Bengal Chloramphenicol Agar)

➢ Peptone

STEP-1: DILUENT & MEDIA PREPARATION

Prepare 0.1% Peptone Water Solution

Weigh 0.5g Peptone

Dissolve the 0.5g peptone on 500 mL distilled water

Swirl the flask to mix the content

Label the bottle with tag

Distribute the mix content on the falcon tube. (9ml each 6-falcon tube) and attach the
cap loosely.

Autoclave the diluent at 121 °C and 15 lbs. for 15 mins.

 CULTURE MEDIA PREPARATION

DRBC (Dichloran Rose Bengal Chloramphenicol Agar) Agar Media

Weigh 6.32g DRBC agar dehydrated medium

Take 200 mL distilled into Duran bottle and transfer the DRBC Agar Medium

Boil the content in water bath at 100˚C for 5 mins

Shake the bottle thoroughly

Autoclave the media and diluent at 121 °C and 15 lbs. for 15 mins.

After autoclaving, distribute 20 mL DRBC agar medium to each petri plate.

(Cooldown for 20 minutes to solidify)

STEP-2: SAMPLE PREPARATION

Use sterilized spoon to transfer the sample

(Spray 70% isopropyl alcohol if they have any chance of contamination)

Weigh 25 g of homogeneous sample into a sterile stomacher bag.

Measure 225 mL of 0.1% Peptone water solution aseptically and transfer the peptone solution
into stomacher bag with the homogeneous sample.
8
8 sample and peptone water solution.
Using stomacher mix and homogenize the
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 STEP-3: SERIAL DILUTION OF SAMPLE SOLUTION

Dilute sample 10 times by mixing 25g sample in 225 mL diluent.

(Label the first sample solution 10 -1)

(Shake the diluted sample properly before pipetting)

-1
Transfer 1 mL from 10 solution into 9 mL sterile diluent to make 100 times dilution of the
sample.
(Label the 2nd dilution 10 -2)

-2
Transfer 1 mL from 10 dilution into another 9 mL sterile diluent to make 1000 times dilution of
the sample.
(Label the 3rd dilution 10 -3)

-3
Transfer 1 mL from 10 dilution into another 9 mL sterile diluent to make 10000 times dilution of
the sample.
(Label the 4th dilution 10 -4)

-4
Transfer 1 mL from 10 dilution into another 9 mL sterile diluent to make 100000 times dilution of
the sample.
(Label the 5th dilution 10 -5)

-5
Transfer 1 mL from 10 dilution into another 9 mL sterile diluent to make 1000000 times dilution
of the sample.
(Label the 6th dilution 10 -6)

And one of the falcon tube contain only an autoclave diluent, which can be to determine if the diluent is
contaminated.
(CONTROL)
 STEP- 4: INOCULATION OF SAMPLE

SPREAD PLATE METHOD

(Each dilution must have two petri plates for Trial-1 and for Trial-2)

Label each, DRBC Agar plate

10 -2 10 -3 10 -4 10 -5 10 -6

Trial 1 Trial 1 Trial 1 Trial 1 Trial 1

10 -2 10 -3 10 -4 10 -5 10 -6

Trial 2 Trial 2 Trial 2 Trial 2 Trial 2

Diluent Control Plate

Media Control Plate

Contains only media which is for contamination check of the prepared media
 Biosafety Cabinet Control (BSC)

Note: Keep the BSC Control plate open to check Biosafety Cabinet contamination during all the time
working on the cabinet.

SPREADER PREPARATION

Keep the spreader soaked into 70% Isopropyl Alcohol.

Light the Bunsen burner or alcohol lamp.

Burn all of the spreader after making wet with 70% Isopropyl Alcohol
(Cooldown the spreader inside the BSC to avoid cross-contamination)

Vortex the 10 -2 dilution tube

Transfer 100 µ𝐿 from the 2nd dilution into Trial-1 plate and transfer another 100 µ𝐿 into Trial-2
plate
(Discard the pipette tip)

Spread the sample solution on DRBC agar surface using sterile glass rod.
(Use different glass rod for spreading the sample solution)

Repeat the process of transferring 100 µ𝐿 from the dilution 10 -3 up to dilution 10 -6.

Spread the sample solution on DRBC agar surface using sterile glass rod.
(Use different glass rod for spreading the sample solution)

Transfer 100 µ𝐿 diluent into the Diluent Control plate.

Spread the sample solution on DRBC agar surface using sterile glass rod.
 Close the BSC control plate and keep it with other plates.

Incubate the plates at 25-30 °C for 5 days.

STEP-5: COLONY COUNTING

Take the plate into Biological Safety Cabinet

Take a close look.

Count the colony formed into the plate

Notes all of the colony counts

Check to Biological Safety Cabinet control plate, Diluent control plate and Media Control Plate.

Notes all of the colony counts.

Determine the Acceptability Range of Fungal Count (per plate) = Below 150 CFU

Calculate the Total Yeast and Mold colony for the tested sample. Using the formula
∑𝐶
𝑁=
(𝑛1 + 0.1 𝑛2) 𝑑

Record the Total CFU/g or CFU/ml.

STEP-6: RESULT INTERPRETATION

Write a conclusion of the experiment.
(State or discuss the principal findings and analyze the strengths and weaknesses of the
experiments)