Suitability of Microbial Count Method & its SOP

Suitability of Microbial Count Method; Purpose

Suitability of Microbial Count Method; To confirm the ability & the suitability of the test to detect microorganisms in the presence of a product or Raw Materials as per the In-house or Pharmacopoeia specifications.

 Scope

This SOP is applicable for Microbial Count Method Validation in Microbiology Section of XX Pharmaceuticals Limited.

 Definitions

Microbial count Suitability: Microbial Count suitability is to confirm that the test dilution is free from any type of interfering substances or antimicrobial properties that will recover by dilution or the addition of a neutralizer to detect microorganisms in the presence of the product.

• CSDA: Casein Soybean Digest Agar
• CSDB: Casein Soyabean Digest Broth
• LAF: Laminar Air Flow
• SDA: Sabouraud Dextrose Agar
• SDB: Sabouraud Dextrose broth

Responsibilities

The roles and responsibility is defined as follows:

Executive/ Sr. Executive, Microbiology

Preparation & inoculation of standard culture, carry out test & arrange appropriate document preparation.

Asst. Manager/Manager, Microbiology

Ensure of method suitability, documentation and application of sound technical information.

Head of Quality Assurance

Taking Initiative to Approval of this SOP

Procedure

Instructions

Safety Precautions

• When enter into the test area, wear sterile latex free gloves, Laboratory coat and eye protection (if required).
• To prevent unauthorized contamination, make sure that all personal ornaments, cell phone are left before enter into the test room. The use of Cellular phone in the test room is strictly prohibited.
• Don’t move vigorously into the test area. Move always gently.

General Requirements

Glass Apparatus

• Pipette 2 ml, 10 ml
• Sterilized 90 mm Glass Petridish
• Screw capped Conical Flask 100 ml
• Screw Capped Test Tube
• Volumetric Flask 500 ml
• Volumetric Flask 1000 ml

Media and Reagents

• Casein Soyabean Digest Agar(CSDA)
• Casein Soyabean Digest Broth(CSDB)
• Meat peptone
• Neutralized Peptone
• Sabouraud Dextrose Agar
• Sabouraud Dextrose Broth

These media can be purchased from commercially available manufacturers.

 Others Requirements

• 45 µm Membrane Filter
• 70% IPA or ethanol
• Filtration Unit( sterilized filter disk and filtering funnel)
• Forceps
• Glass spreader
• Scissor
• Surgical Cotton
• Surgical Gloves

General Procedures

Test Conditions

• Wear gloves, mask/beard mask Headgear before entrance into Testing Room.
• Disinfectant hands, the outer surface of test sample, LAF workstation with the help of 70% IPA or ethanol before starting the test.
• Carry out the test under LAF to avoid any type of contamination.
• Monitor the test area microbiologically using Microbial Air Sampler at each working day.

Culture Media Preparation

• Prepare the different culture media as per the requirement.
• Weigh the mentioned amount as per manufacturer label into appropriate flask.
• Bring to boil completely to dissolve the media.
• Sterilize at 121⁰C for 15 minutes or as per Manufacturer label.
• Store the prepared culture media in air tight flask at controlled environment.
• Store prepared agar media at (2-8)⁰C
• Preserve dehydrated culture media up to its expiry date.
• Never use expired culture media.
• Use the agar media when the temperature reduce to 45⁰C and cools in case of the broth media.

Stock Buffer Solution

• Take 34 g of Potassium Dihydrogen Phosphate[KH₂PO₄] in a 1000 ml volumetric flask.
• Dissolve in 500 ml Purified Water, adjust to pH [7.2 ± 0.2] then dilute to 1000 ml with Purified Water.
• Dispense 90 ml into each screw capped flask[Approx. 11 containers].
• Sterilize at 121⁰C for 15 minutes.
• Store the prepared buffer at 2-8⁰C for a validated period.

Glassware Cleaning & Sterilization

• Clean glassware by 1% detergent initially then rinse with sufficient tap water.
• Rinse finally with sufficient Purified Water to remove the residual content of detergent.
• Sterilize glassware at 200⁰C for 1 hour.

 Sample Preparation

Water-soluble samples

Take 1 gm or 1 ml into the 9 ml CSDB [Casein Soybean Digest Broth] or phosphate buffer or Sodium chloride peptone solutions.

Water-insoluble samples

Take 1 g or 1 ml into the 9 ml CSDB [Casein Soybean Digest Broth] or phosphate buffer or Sodium chloride peptone solutions and add 0.1% Polysorbate 80.

Fatty Products

Dissolve with Isopropyl Myristate sterilized by filtration with low amount of Polysorbate 80 or anther non-inhibitory sterile surface active agent  or necessary heat not more than 45⁰C.

Inoculation and Dilution:

• To maintain of not more than100 cfu, add adequate volume of suspension of inoculums to the sample.
• Add the inoculums suspension not more than 1% of diluted product.
• Prepare lowest possible dilution for acceptable microbial recovery of sample.
• Add neutralizer for removal of Interfering factor when [If] the sample contains any antimicrobial properties.

Follow Table-1 for the inactivators of  Antimicrobial agents.

If growth is inhibited, then increase use of diluents or membrane filtration or combination of all above.

 

 

Suitability of Counting Method:

[] Membrane Filtration Method

[] Most Probable Number Method

[] Pour Plate Method

[] Surface-spread plate Method

Pour Plate Method :

• Add 1 ml prepared sample to the 90 mm diameter Petridish
• Pour (20~25) ml of Sabouraud Dextrose Agar(SDA) for fungi and Casein Soybean Digest Agar(CSDA) for Bacterial count both media being not more than 45⁰
• If larger Petridish [more than 90 mm diameter] is used amount of media to be increased accordingly.
• Perform plate count methods at least in duplicate for each medium and use the mean count of the result.
• Inoculate the microorganisms not more than 100 cfu as indicated Table 2.
• Mix properly & incubate CSDA plate at (30~35)⁰C for 3 days, SDA plate at (20~25)⁰C for 5 days.
• After incubation, count the colony on each plate.
• Take arithmetic mean of the count per medium.

Surface-Spread Plate Method

• Add (20~25) ml of SCDA and SDA to 90 mm diameter petridish at least duplicate.
• Allow to solidify and dry the plate under Laminar Air Flow cabinet or incubator.
• Spread not less than 0.1 ml (equal to or less than 100 cfu) of each microorganisms as indicated in Table 1 on the surface of medium
• Incubate at CSDA plate at (30~35)⁰C for 3 days, SDA plate at (20-25)⁰C for 5 days.
• After incubation, count the colony of each plate.
• Take arithmetic mean of the count per medium.

Membrane Filtration Method

• Use the membrane filter which nominal pore size is not more than 0.45µm.
• Filter prepared sample through membrane.
• Rinse membrane filter with sterile 0.1% meat peptone solution or any others suitable diluents for neutralization of the sample.
• Add inoculums to filter as indicated in Table 1 & rinse again.
• Transfer filter on the surface of CSDA plate & SDA plate.
• Incubate CSDA plate at (30 to 35)⁰C for 3 days, SDA plate at (20 to 25)⁰C for 5 days.
• After incubation, ten count the colony of each plate.
• Take arithmetic mean of the count per medium.

Test Control

Negative Control: Use diluents in place of test preparations. There must be no growth found in the negative control. If found any growth in the negative control, then the test is invalid & repeat the test.

 

Result and Interpretation

Mean count of any of test microorganisms not differing by a factor greater than 2 from value of the control defined in absence of product must be obtained.

Repeat test by increasing neutralizer or dilution or any treatment for overcome of antimicrobial properties of products. If the above criteria cannot be met for one or more microorganisms.

Test Report Preparation

Prepare Report the result in Suitability Report of Microbial Count Method, Annexure-I.

Annexure

Annexure-I: Suitability of Microbial Count:
Suitability Test Report of Microbial Count Method