METHOD OF ANALYSIS

Lactobacillus spp.

1.0 OBJECTIVE:
To determine the total viable count for Lactobacillus spp. in given sample.

2.0 Requirements:
➢ Autoclave
➢ Stomacher and sterile stomacher Bag
➢ Incubator
➢ Weighing balance
➢ Laminar air flow cabinet
➢ Calibrated micropipettes and sterile tips
➢ Vortex mixer
➢ Sterile Petri plates (90 mm diameter)
➢ Sterile tubes or autoclaved tubes
➢ Peptone Bacteriological powder
➢ Anaerobic Jar
➢ Gas Pak Container system with indicator
➢ MRS agar
➢ MRS broth
➢ Cysteine HCL powder

3.0 PROCEDURE:
3.1 Preparation of 0.1% Peptone diluent:
➢ Take 1.0gm. of peptone and 1000 ml distilled water.
➢ Dissolve 1.0gm peptone in 1000 ml distilled water.
➢ The pH of diluent is 7.0.
➢ Transfer 400 ml of the Peptone diluent to media bottles and cap bottle loosely.
➢ Autoclave for 20 min. at 121°C and 15 lbs Pressure.
3.2 Preparation of MRS Agar (Himedia):
➢ Preparation for MRS agar medium as per manufacturer instruction.
➢ Autoclave for 20 min. at 121°C and 15 lbs Pressure.

3.3 Preparation of 5% Cysteine HCL:

➢ Weight 5gm. Cysteine powder dissolve in 100 ml purified water or distilled water and filter
sterilize and protect from light. (Sterile 0.22µ syringe filter).

3.4 Preparation of MRS broth(Himedia):

➢ Preparation for MRS broth as per manufacturer instruction.
➢ Autoclave for 20 min. at 121°C and 15 lbs Pressure.

3.5 Sample Preparation:

➢ Aseptically weigh 10 grams of the sample into a sterile stomacher or homogenizer bag.
➢ Aseptically add 90 mL of sterile, room temperature, MRS broth to the 10 grams of sample
in the sterile stomacher or homogenizer bag. This called 10-1 dilution.
➢ Turn stomacher blender on and allow to blend for two minutes.
➢ Hold the sample at room temperature for 30 minutes to sample.
➢ Return the sample to the stomacher blender and blend for an additional two minutes.
➢ Make serial dilutions in 99 mL 0.1% peptone dilution blanks by adding 1 mL of the primary
10-1 dilution (from the stomacher bag) to 99 mL of diluent with a 1 mL pipette so as to
obtain a 10-3 dilution. Rinse the pipette three times. Repeat this operation until the desired
dilution series is obtained.
➢ Proceeding in plates, transfer 1 mL of each appropriate dilution to labelled, sterile Petri
plates with sterile 1 mL pipettes. At least minimum make two plates of each diluent.
➢ Take a bottle of sterile MRS agar that has been melted (100 C for 30 minutes) and tempered
to 45 C in a 45 C water bath and sanitize the bottle by dipping it into a 200 PPM chlorine
solution (made fresh daily), or by flaming the lip of the bottle.
➢ Under a laminar hood, aseptically add 1 mL of sterile 5% cysteine-HCl solution to each
100 mL of the MRS agar to achieve a final cysteine- HCl concentration of 0.05% in the
MRS agar.
➢ Pour approximately 15 ml-20 ml of the MRS/0.05% cysteine-HCl agar into each plate.
Swirl the plates to mix and let solidify at room temperature on a cool level surface.
 ➢ Prepare one blank plate that contain only MRS/0.05cysteine HCL media.
➢ Incubate the plates at 38° C (±1°C) under anaerobic conditions (Gas Pak Container Systems
with indicator in an anaerobic jar) for 72 hours.

3.6 Calculation of CFU:

➢ After 72 h of incubation, count the colonies on the prepared plates.
➢ Plates containing between 25 and 250 colonies are considered ideal for counting.
➢ Calculate the average number of colonies per plate, then multiply the average number of
colonies counted by the reciprocal of the dilution factor to obtain the Lactobacillus spp.
CFU/g of the sample.
➢ Blank plates should be entirely free of any type of colonies.
➢ In the case of blank plates that contain colonies, the entire procedure must be
repeated, potentially including the preparation of the Diluent and the MRS/0.05% cysteine-
HCl agar, depending on which plate(s) contain colonies.