ANALYTICAL METHOD

1355

Subject: Total Cell Counts For Freeze Dried Products Containing Page 1 of 3
Lactobacillus acidophilus LA-14 Rev. No.: 3
Date of issue: 8/30/2012
Effective date: 8/30/2012

Field of application: This procedure is used to enumerate freeze dried pellet intermediates and
freeze dried finished goods products which contain Lactobacillus
acidophilus LA-14.

Definition: The total cell count is used to determine the number of bacteria per gram.
The results of this test are used to determine if a sample has the required
number of bacteria to qualify for an intermediate or finished goods.

Principle: Freeze dried products are rehydrated in MRS broth, serially diluted and
plated on MRS agar with cysteine. These plates are placed in an anaerobic
environment and incubated for 72 hours at 38o C. Those plates having
between 25-250 colonies per plate are recorded as total cell count per
gram.

Estimated time of analysis: Set up 45 minutes

Incubation 72 hours
Plate reading 15 minutes per set

Apparatus: Sterile Petri dish

Pipette apparatus
Sterile Pipette(s)
Sterile pipette tip(s)
Permanent Marker
Sterile Plastic bags
Certified Autoclave
Stomacher® or equivalent
Balance-500 gram capacity
Quebec Colony Counter or equivalent
Certified Incubator set at 38oC
Media Water bath at 45oC
Biological Safety Cabinet (BSC)
Anaerobic Jars
BD GasPak™ EZ Anaerobe System or equivalent
BBL Dry Anaerobic Indicator Strips or equivalent

Reagents: Difco™ Lactobacilli MRS broth or equivalent

Bacto™ Agar or equivalent
200 ppm Chlorine or equivalent
 ANALYTICAL METHOD
1355

Subject: Total Cell Counts For Freeze Dried Products Containing Page 2 of 3
Lactobacillus acidophilus LA-14 Rev. No.: 3
Date of issue: 8/30/2012
Effective date: 8/30/2012

Sterile 99 ml 0.1% Peptone Water -International BioProducts or equivalent

5% Cysteine HCl solution

Control sample: A control sample is plated using approximately 15 ml of MRS + cysteine for
one plate. After solidifying, this plate is incubated under the same
conditions as the test sample.

Procedure: Remarks:
Obtain sample and verify batch numbers from Daily QC Melt media at 100o C for 30 minutes then
culture lab schedule. temper for 30 minutes at 45o +1o C before
using.

Label Petri plates with sample name, batch number and

dilution(s).

Prepare a 10-1 dilution of the sample(s) by aseptically Dilution Scheme: Based on product
weighing 11 g + 0.1 g of the powder into a sterile plastic specification and theoretical number of
bag. Add 101 g + 2.0 g of room temperature MRS broth to organisms/ unit of measure:
the bag and blend in Stomacher® or equivalent for 2 minutes
@ 230 rpm.
•
<100 billion organisms/gram dilute 10-
8
Allow sample to rehydrate for 30 minutes then blend for an and 10-9 for plating
additional 2 minutes in the Stomacher® or equivalent.
• >100 billion organisms/gram dilute
Continue with serial dilutions in 99 ml peptone water until 10-9.25 for plating
proper dilution level has been obtained.
•
<300 billion organisms/gram dilute 10-
Shake the dilution blanks as directed in the current edition of 9
and 10-10 for plating
Standard Methods for the Examination of Dairy Products
(SMEDP).

Inoculate labeled plates with 1.0 ml of the appropriate

dilution.

Clean a bottle of MRS agar by dipping into a 200 ppm

chlorine solution.

In a BSC, add 1 ml of 5% Cysteine-HCl solution for each

 ANALYTICAL METHOD
1355

Subject: Total Cell Counts For Freeze Dried Products Containing Page 3 of 3
Lactobacillus acidophilus LA-14 Rev. No.: 3
Date of issue: 8/30/2012
Effective date: 8/30/2012

100 ml of MRS agar to be used and swirl to mix.

Prepare 200 ppm chlorine solution by adding
Pour approximately 15 ml of MRS/0.05% Cysteine-HCl agar 1ml of 12.5% Sodium Hypochlorite solution to
into each plate, swirl to mix and allow agar to solidify on a 1 liter of distilled H2O.
level surface.

After the plates have solidified, invert the plates and

incubate at 38o +1o C for 72 hours in an anaerobic
environment.

Anaerobic environment is created using BD

GasPak™ EZ Anaerobe Container System
sachets or equivalent and a BBL Dry
Anaerobic Indicator Strip or equivalent in an
Anaerobic Jar.

Calculation: After incubation, count colonies on the plates and record as total cell count
per gram, taking into account the dilution factor of the plates. Only plates
having between 25 and 250 colonies should be used in calculations.
Repeatability
and accuracy: In accordance with method limits.

Literature: The above listed procedure uses the following methods as references:

Standard Methods For The Examination of Dairy Products, 17th

edition, 2004

ISO 7889: 2003