Microbial Examination of Empty Bottle, Cap & Stopper

Microbial Examination of Empty Bottle, Cap & Stopper; Purpose

To confirm that the bacterial & fungal count & specified microorganisms into empty bottle during filling are within In-house specification.

Scope

This SOP is applicable for microbiological test of empty bottle during filling in Microbiology Section at XX Pharmaceuticals Ltd.

Definitions/Abbreviation

[] CSDA: Casein Soyabean Digest Agar

[] CSDM: Casein Soyabean Digest Medium

[] SDA  :  Sabouraud Dextrose Agar

[] TAMC: Total Aerobic Microbial Count

[] TYMC: Total Yeast & Mould Count

Responsibilities

The roles and responsibility is as follows

Executive, Microbiology

Sample collection, analysis and documentation.

Manager, Microbiology/Quality Control

Ensure analysis of empty bottles, documentation and application of sound technical information.

Head of Quality Assurance

Take initiative to Approval of this SOP

Procedure

Instructions

• When enter into the test area, wear sterile latex free gloves, mask, laboratory coat & eye protection (if required).
• Make sure that all personal ornaments cell phone are left to prevent unauthorized contamination, before entrance into test area.
• Move always gently and never move vigorously into the test area.

General Requirements for the test

Glass Apparatus

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

Media and Reagents

• Casein Soyabean Digest Agar(CSDA)
• Casein Soyabean Digest Medium(CSDM)
• Mac-Conkey Broth
• MacConkey Agar
• Neutralized Peptone
• Sabouraud Dextrose Agar

Others Requirements

• Surgical Gloves
• Surgical Cotton
• 70% IPA or ethanol

Enumeration Method(TAMC & TYMC)

This test quantifies the enumeration of mesophillic bacteria & fungi that may grow under aerobic conditions.

Test Conditions

• Disinfectant both hands, bottles surface, Laminar Air Flow workstation with 70% IPA or 70% ethanol before starting test.
• Carry out the test under Laminar Air Flow to avoid contamination.

Culture Media Preparation

• Prepare different culture media as per requirement.
• Weigh accurate amount mentioned in the manufacturer label into appropriate flask.
• Bring to boil completely to dissolve media.
• Sterilize at 121⁰C for 15 minutes or as per manufacturer label.
• Store prepared culture media in air tight flask at 2 to 8⁰

Stock Buffer Solution

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

Glassware Cleaning & Sterilization

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

Sample Size

• Collect 5 empty sealed bottles from each batch during filling of the products at three stages of Starting, Middle and Ending of operation.

Test Method

• Carry out anyone from the following mentioned method

Pour plate method

• Transfer all bottles under Laminar Air Flow.
• Deseal the cap of all bottles.
• Add 10 ml sterile meat peptone or phosphate buffer pH 7.0 ± 0.2.
• Cap the bottles & shake well to mix properly.
• Mark all petridish as product name, batch number, plate name (CSDA/ SDA) and bottle number & test date.
• Take 1 ml of rinsing solution from each bottle & pour into two 90 mm sterilized petridish.
• Add 15-20 ml CSDA into one plate & add 1 ml SDA into another plate.
• Maintain same manner for the rest 4 bottles.
• Mix sample with the media by tilting & rotating the plate.
• Allow to solidify all plates & invert after solidification.
• Incubate CSDA at 30 to 35⁰C for 3 to 5 days & at 20 to 25⁰C for 5 to 7 days.
• After incubation, calculate number of cfu per bottle.

Negative Control

Use the diluents as sample in place of test preparations and follow the steps mentioned steps.

Membrane Filtration Method

• Prepare sample as per Pour Plate Method
• Filter whole rinsing solution of all bottles individually through 0.45 µm and transfer the filter paper to the surface of CSDA slant for bacterial count and SDA slant for yeast & mold count.
• Invert plates & incubate all CSDA plates at 30 to 350C for 3 to 5 days & SDA plates at 20 to 250C for 5 to 7 days.
• After incubation, count the colony of each plate.
• Calculate number of cfu per bottle.

Negative Control

Use the diluents as sample in place of test preparations and follow the steps mentioned steps.

Surface spread Method

• Spread not less than 0.1 ml of rinsing solution on surface of two CSDA & two SDA Plate.
• Dry all plates under Laminar Air Flow work station.
• Incubate CSDA at (30 to 35)⁰C for 3 to 5 days & at (20 to 25)⁰C for (5 to 7) days.
• After incubation, calculate number of cfu per bottle.

Negative Control

Use the diluents as sample in place of test preparations and follow the steps mentioned steps.

Interpretation of the results

• The bottles are passed if the observed count is less than specified count.
• The product is failed if the observed count is greater than specified count of that product.

Test Control

• Negative control must be negative growth. If found growth in negative control, the test is invalid.

Test for Specified Microorganisms

Suitability of Test Method

• Add each test strain separately not more than 100 cfu at the time of bottle test mixing with culture media as per standard operating procedure of Suitability of Microbial Count Method.
• The test is suitable if growth found the specific microorganisms. The test is not suitable if no growth found the specific microorganisms. In that case, add any neutralizer or increase dilution for removal any inhibition of product.

 Test for E. coli

• Add 10 ml of test sample to 90 ml of CSDM. Incubate at (30 to 35)⁰C for 18 to 24 hours.
• Shake the container and transfer 1 ml of CSDM to 100 ml of MacConkey Broth.
• Incubate at (42 to 44)⁰C for 24 hours.
• Sub culture on MacConkey Agar plate from MacConkey broth.
• Incubate at (30 to 35)⁰C for (18 to 72) hours.
• The product complies with the test for E. coli if no red colonies are present with precipitated zone & the biochemical tests are negative.

Test Report Preparation

Microbial Examination Report of  Cap & Stopper, Annexure-I.

Microbial Examination Report of Empty Bottle, Annexure-II.

Download All Annexure Here

 Microbial Examination Report of Cap & Stopper Annexure-I.
 Microbial Examination Report of Empty Bottle Annexure-II.