Environmental Monitoring; Purpose

Environmental Monitoring; To describe the procedure for Environmental monitoring.

Scope

Environmental monitoring in  XX Pharmaceuticals Ltd

Definitions

Environmental monitoring:

Monitoring of viable and non-viable quality of a controlled environment

Particulate count:

[] Enumeration of non-viable particulate of specific size from a particular volume of air of a controlled
Environment.
[] Settle Plate: Exposure of petri-plates of nutrient media in a controlled environment to estimate viable
Microorganisms from the environment.
[] TSA: Tryptone Soya Agar
[] CFU: Colony Forming Unit

Responsibilities

The roles and responsibility is as follows:

Lab. Attendant

Room preparation for Test

Executive, Microbiology

Carry out the test and incubation & documentation

Sr. Executive, Microbiology

To ensure test, incubation, report checking, document preservation & application of sound technical information.

Head of Plant

Review of the SOP & the relevant technical information is applied.

Head of Quality Assurance

Take initiative to Approve of this SOP.

Procedure:

Instructions

• Don’t rub during contact plate sampling.
• Use the sterile filter holder.
• Place the filter paper on the filter holder carefully under laminar airflow.
• Try to avoid unwanted personal contamination during air sampling.
• Monitoring to be carried out before production hours. (Sterile products)

Non-viable Particle monitoring:

• Bring the laser particle counter into the specific monitoring area.
• Enter into clean room, wearing approved designated dress. When entering into the clean area, before taking reading make sure that all the doors remain closed.
• Operate the Particle counter following approved SOP.
• Take count of different several position for each room as indicated in sampling point. Count the number particles (5µ and 0.5 µ) form each sampling point. The average of the counted particle indicates the total particles of a room.

The minimum sampling time should be as per following formula (Following EN ISO 14644-1)

Vs=(20/Cn,m ) x 100

Where,

• Vs = is the minimum single sampling volume per sampling point, expressed in liter.
• Cn,m = is the class limit (number of particles per cubic meter) for the largest considered
  particle size, specified for the relevant class.
• 20 = is the defined number of particles that could be counted if the particle concentration
  were at the class limit.

Specification: Follow Annexure-III.

Write down the result in the format of Annexure-I.

Viable count:

Settle Plate

• Prepare, sterilize & dispense the media TSA into the petriplates and pre-incubate the plates.
• Check the pre-incubated plates for any evidence of microbiological contamination under the LAF bench.
• Discard the plates contains microbiological growth.
• Decontaminate the external surface of petriplates with sterile cloth /cotton soaked in a sanitizing agent (70% IPA).
• Place required number of petriplate in a sterilized /sanitized SS container; close the lid of SS container.
• Transfer SS box to the area to be monitored.
• Enter respective area as per the SOP of Entry and Exit procedure.

Mark Petri plates with the following details-

1. Name of the sampling point
2. Room No.
3. Date of exposure

• Place petriplates on corresponding designated plate exposure area and remove
  the upper lid of the petriplates. Note the beginning time of the exposure and write it on
  the plate.
• Place upper lid on edge of petriplates in slanting position.
• Expose media plates at sampling point for Maximum 4 hours.
• After completion of exposure, close petriplates with lid.
• Collect petriplates in the SS container and bring back exposed plates to microbiology Laboratory.
• Incubate all exposed petri plates in inverted position in laboratory Incubator.
• Incubate at (22.5 ± 2.5)°C for mold and Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5)°C. After incubation count the number of colony forming units (CFU) per plate per sampling point with colony counter.

• Incubate an unexposed media filled petriplates along with the exposed plates and mark it as negative control.
• Count the number of Colony Forming Units (CFU) per plate per location with colony counter. The average of colony indicates total CFU of a room.

Write down observation in the format attached as Annexure-II.

Air Sampling:

• Set instrument for desired sampling time & flow rate as per Approved SOP.
• Take air sampler to location where air is to be sampled, hold it with filter facing at required direction and take air sample following approved SOP.
• Collect filter under laminar airflow & place it inside on petriplates containing TSA[Tryptone Soya Agar ] media.
• Incubate all exposed petri plates in inverted position in the microbiology laboratory Incubator. Incubate at the (22.5 ± 2.5)°C for mold & Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5)°C. After incubation count the number of colony forming units (CFU) per plate per location with colony counter.
• Follow sampling location.
• Record observation in format attached as Annexure-II.
• Calculate number of organisms per cubic meter of air. Average colony indicates total cfu of a room.

Surface monitoring (By swab sampling):

• Use sterilized swab or sterilize the swab in the autoclave.
• Place required number of sterilized swab sticks with tube containing 5 ml of sterile saline
  solution in a Sterilized SS container & tightly secure lid of the SS container.
• Transfer SS box to area to be monitored.
• Enter respective area as per SOP of entry and exit procedure.

Remove swab sticks with tubes from the SS box & take it to the location to be monitored and Mark tube with following details:

1. Location number or Name of the location
2. Swab No.
3. Date of sampling

• Hold swab stick from bottom & place tip on surface to be monitored. Gently wipe the swab bi-directionally to cover an area about 25 cm2.
• Perform swab sampling for each defined locations.
• Keep swab sample standing in the SS container & bring it to Laboratory.
• Gently vortexes swab sampling tube containing swab stick & pour contents in filter holder funnel and filter it. Uses the respective SOP.
• Rinse tube with 3 x 10 ml of sterile saline solution.
• Filter test sample under partial vacuum.
• Rinse Funnel with the portions of sterile Purified Water. This flushes residue from walls of funnel & helps to secure a uniform distribution of colonies on filter surface. Upon completion of rinse & filtration process, shut off vacuum.
• Transfer membrane filter with sterile smooth-tip forceps on to Microbial content test agar (TSA) plate.
• Place filter with a rolling motion to avoid entrapment of air.
• Keep a negative control by filtering 100 ml Purified Water(Sterile) before filtering actual test sample.
• Pass 20 to 30 ml of sterile Purified Water through funnel, between different test samples when the same funnel used for multiple sample.
• Incubate all exposed petriplates in inverted position in microbiology laboratory Incubator. Incubate at (22.5 ± 2.5)°C for mold and Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5°)C. After incubation count number of colony forming units (CFU) per plate per location with colony counter.
• Count number of colony forming units (CFU) per plate per location with colony counter.

Note down the observation in the format attached as Annexure-II

Surface monitoring (By contact plate):

• Fill Petridishes with TSA culture medium & pre-incubate.
• Take off the lid of plates.
• Invert & press agar surface for 10 seconds onto surface to be examined.
• Replace lid & mark plate with appropriate data.
• Clean sampling area on surface in order to remove any remaining of agar.
• Return plates to laboratory.
• Incubate all exposed petriplates in inverted position in microbiology laboratory Incubator. Incubate at (22.5 ± 2.5)°C for mold and Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5°)C. After incubation count number of colony forming units (CFU) per plate per location with colony counter.
• Take count of visible colonies within mean of plate
• Express results as CFU per contact plate.
• Perform appropriate identification of germs found by surface monitoring..Classified area should free of pathogenic microorganism.

Note down the observation in the format attached as Annexure-II.

Sampling Location:

Determine Number of sampling location by following formula.

N_L    = √A

Where,

N_L   = the minimum number of sampling locations, rounded up to a hole number.

A= Area of the clean room or clean air controlled space in m^2.

^{Environmental Monitoring Procedure as per Pharmacopeia}

Download All Annexure Here: Environmental Monitoring

• Annexure-I: Environmental Monitoring (Non-Viable Particle Count) Test Report
• Annexure-II: Environmental Monitoring (Viable Particle Count) Test Report
• Annexure-III: Environmental Monitoring specification
• Annexure-IV: Log book of environmental monitoring

Schedule: Environmental Monitoring

• Environmental monitoring Test Schedule

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

Microbiological Media disposal; Purpose

Microbiological Media disposal; To dispose of the used media properly in order to lessen Microbiology Laboratory Contamination as well as environmental pollution.

 Scope

This SOP is applicable for the disposal of used Media in the Microbiology Laboratory at XX Pharmaceuticals Ltd.

Definition

N/A

Responsibilities

The roles and responsibilities are as follows:

Laboratory Attendant

Clean & disinfect used media

Executive/ Sr. Executive, Microbiology

• Monitor disposal activity accordingly.
• Follow the instructions of this procedure appropriately.
• Follow the instructions of this procedure properly.

Asst. Manager, Microbiology

• Confirm proper disposal of used media.
• Confirm that this procedure is kept up to date.
• Confirm suitable personnel from the section are trained in this practice.
• Confirm that SOP is technically sound and reflects the required practices.

 Head of Quality Assurance

Approval of this SOP

Procedure

Instructions

• Appropriately wear heat-resistant gloves, eye protection, and laboratory coat during handling autoclaved media.
• Avoid autoclaving the sealed containers or completely filled bottles with narrow necks as they may explode.
• Don’t expose any used media container or plate outside the Laminar Air Flow cabinet.
• Wash and disinfect both hands after handling used media.

Collection of Used Media

• Wear suitable garments, gloves, and mask.
• Wear safety goggles if hazardous media are subject to being discarded.
• Collect all the media to be disposed of in the designated vessel after use.
• Close the mouth of the vessel tightly.

 Sterilization

• Attach the Autoclave Tap with the vessel.
• Place the vessel into a sterilizer and sterilize at 121⁰ C & 15 lbs for 30 minutes.
• Check that the color of the autoclave tap is turned black.

Discard Method

• After autoclaving, collect the media in a specific container when the temperature comes down to 50⁰ to 55⁰ C and overlay the media with 40% formaldehyde solution.
• Keep the media for 30 minutes.
• Dispose of the media into the drain which is linked with ETP.
• Rinse the vessel properly with hot water.
• Wash the vessel with detergent.
• Disinfect the whole vessel with 70% Iso Propyl Alcohol and dry the vessel.

Record Maintain:

Maintain Register of Media Disposal Record in Annexure-I.

Download the Annexure: Media Disposal Record

[Microbiological Analysis of water; this article describes the basic procedure of Microbiological Analysis of water as per different guidelines]

Purpose

Microbiological Analysis of water; To confirm that different type of Water used for different drug processing, cleaning and drinking purpose meets the required Pharmacopoeia & In-house specifications.

Scope

This SOP applies for sampling and analysis of all types of water used in this plant.

Definition/Abbreviation

None

Responsibilities

The roles and responsibility is as follows

Lab Attendant

Sample collection of different types of water.

Executive / Sr. Executive, Microbiology

Verify, Monitoring of Sample collection, analysis of Water, and test preparation of report accordingly.

Asst. Manager/Manager, Microbiology

• Confirm sampling, analysis, documentation, and application of appropriate technical information.
• Review of this SOP that the whole procedure is technically informative and in execution condition.

Head of Quality Assurance

Take initiate to the approval of SOP

Procedure

Instructions

• Disinfect the outer surface of the sampling point with the help of 70% IPA.
• Wear sterilized latex-free gloves and an appropriate mask. Never forget to wear a beard mask where required.
• Never open the sample container before & after the collection of a specific sample.

Sample Collection

• Select the sampling point as per the schedule of the Water Test accordingly.
• Before sampling sterilized the sampling containers for microbiology test at 121⁰C for 15 minutes and label it accordingly.
• Wear appropriate Laboratory garments, gloves, and mask as required.
• Disinfect the outer surface of the sampling points with the help of 70% IPA.
• Discharge water for at least 2 minutes for the user points during the collection of sample and 1 minute for a storage tank in the water treatment plant.
• Collect water from each point at least 200 ml into the sterilized container for microbiology test.
• Collect the sample as soon as possible.
• Close the container after sampling and don’t expose the container for microbiology test.

Sample Preservation

• Samples shall be analyzed as soon as possible after being collected. If it is not possible to test the sample within about 3 hours of collection.
• The sample may be preserved at refrigerated temperatures (2-8⁰C) for maximum 12 hours to maintain the microbial attributes until analysis.

Test Schedule:

Perform the test as per the following schedule

Microbiological tests are as follows:

Potable/Pretreated Water

• Perform the test as per Analytical Method

Drinking water

• Perform the test as per QC Analytical Method

Purified Water

• Perform the test as per QC Analytical Method

Water for Injection

• Perform the test as per QC Analytical Method

Report preparation:

• Report of Potable/ Pretreated/ Drinking water Test Result in Annexure-I,
• Report of Purified Water Test Result in Annexure-II &
• Report of Water for Injection in Annexure-IV.

 Distribution of Water Test Result

• After completion of the analysis, inform the status of water test to a specific department.
• If any test result exceeds alert level or is out of specification, immediately inform to concerned department Head and engineering department also for corrective measurement.
• After taking corrective action, Engineering Department shall inform to Microbiology Section for further sample collection.
• Microbiology Section shall collect the sample from that area to carry out the analysis.
• After completion of the test, inform about the test result to concerned department after approval of Head Quality Assurance

Microbiological Analysis of water:

 Download All Annexure Here
Potable Water Test Report Annex I
Purified Water Test Report Annex II
Water Test Record (Log Book) Annex III
Water for Injection Test Report Annex IV

Microbial Examination of Non-Sterile Raw Materials and Products Purpose

Microbial Examination of Non-Sterile Raw Materials and Products, To confirm that the bacterial & fungal count in the non sterile products & raw materials are within the In-house / Pharmacopoeia specification & free from certain microorganisms indicated in Pharmacopeia.

Scope

This SOP is applicable for microbiological test of Non-sterile Products such such as Oral Liquid, Semi-solid, Solid preparations and Raw Materials in Microbiology Section.

Definitions

Microbial Examination: Microbial examination is designed to determine the microbial contamination in non-sterile   products intended for Oral liquid, Topical Preparations or other non-sterile applications & Raw Materials.

• CSDA: Casein Soyabean Digest Agar
• CSDM: Casein Soyabean Digest Medium
• SDA  : Sabouraud Dextrose Agar
• SDB  : Sabouraud Dextrose Broth
• TAMC : Total Aerobic Microbial Count
• TYMC: Total Yeast & Mould Count

Responsibilities

The roles and responsibility is as follows:

Laboratory Attendant

Preparation Room for Microbiological Test

Microbiologist

Perform the test and incubation and in time proper documentation

Asst. Manager/Manager, Microbiology

Confirm test, incubation, report checking, document preservation and application of precise technical information.

Head of Quality Assurance

Take initiative regarding approval of this SOP

Procedure

Personal Precautions

• During enter into the test area, wear sterile gloves, Lab coat and eye protection (if necessary).
• To prevent unauthorized contamination, make sure that all personal ornaments, cell phone are left before entrance into the test room. The use of all type of Cell phone in the test area is strictly prohibited.
• Don’t move forcefully into the test area. Move always gently.

General Requirements for the test

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)
• Cetrimide Agar
• Mac-Conkey Broth
• MacConkey Agar
• Mannitol Salt Agar
• Meat peptone
• Neutralized Peptone
• Rapport Vasiliadis Salmonella Broth
• Sabouraud Dextrose Agar
• Xylose Lysine Deoxycholate(XLD) Agar

All of these media can be purchased from commercial available manufacturer.

Others Requirements

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

Types of Test for Microbiological Examination

Enumeration Method (TAMC &TYMC)

This test quantify enumeration of mesophilic bacteria & fungi which may grow under aerobic 

Condition.

Test Conditions

• Wear latex free gloves, Head gear, mask and beard cover [if required], before enter into Test Room
• Use 70% IPA or ethanol to disinfectant the hands, the outer surface of test sample, LAF workstation with before start test.
• Perform the test under LAF to avoid contamination.
• Monitor the test area microbiologically with the help of Microbial Air Sampler at each working day.

Culture Media Preparation

• Prepare the different culture media as per specific requirements.
• Weigh the exact amount stated in the manufacturer label into right flask.
• Bring to boil completely to dissolve the media properly.
• Sterilize at 121⁰C for 15 minutes or as directed by the Manufacturer label.
• Store the prepared culture media in air tight flask properly at controlled environment.
• Store the prepared agar media at 2-8⁰C.
• Preserve the dehydrated culture media up to expiry date.
• Never use the expired culture media.
• Use the agar media when the temperature reduce near at 45⁰C & cool in case of the broth media.

Stock Buffer Solution

• Place 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 the prepared buffer at 2-8⁰C for a validated period.

Glassware Cleaning & Sterilization

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

Testing of Products

Sample Size

• Collect 10 g or 10 ml of the products to be taken. 10 containers of the products from a batch.
• Collect the amount is not less than the amount present in 10 dosage units or 10 g or 10 ml of the respective product,  if amount per dosage unit is less than or equal to 1 mg.
• Take 1% of the batch size when batch size is less than 1000 ml or 1000 gm.
• Take 2 units or 1 units if the batch size is less than 100.

Types of Method

• Membrane Filtration
• Most Probable Number Method
• Pour Plate Method
• Surface spread Method

Membrane Filtration Method

• Prepare sample as per Method Suitability.
• Filter the sample through 0.45 µm & transfer the filter to the surface of CSDA for bacterial count and SDA[Sabouraud Dextrose Agar] for fungal count.
• Incubate CSDA[Casein Soyabean Digest Agar] at [30-35]⁰C for 3-5 days & at [20-25]⁰C for 5-7 days.
• After incubation, calculate the number of the cfu per gm or ml of the product.

Negative Control

Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.

Pour Plate Method

• Prepare the sample as per the Method Suitability
• Pour 1 ml of prepared sample into the four 90 mm petridish, Add 15-20 ml CSDA[Casein Soyabean Digest Agar] into two plate & SDA into the others two plate.
• Allow to solidify & invert all plates.
• Incubate CSDA at [30-35]⁰C for 3-5 days and at [20-25]⁰C for 5-7 days.
• After incubation, calculate the number of the cfu per gm or ml of the product.

Negative Control

Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.

Surface spread Method

• Prepare the sample as per Method Suitability.
• Spread not less than 0.1 ml of sample on the surface of two CSDA[Casein Soyabean Digest Agar]  and two SDA[Sabouraud Dextrose Agar] Plate.
• Dry all plates at Laminar Air Flow.
• Incubate the CSDA at [20-25]⁰C for 5-7 days and at [30-35]⁰C for 3-5 days.
• After incubation, calculate the number of the cfu per gm or ml of the product.

Negative Control

Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.

Interpretation of the results

Count as Total Yeast/ Mould Count (TYMC) on SDA plate and Total Aerobic Microbial Count(TAMC) in CSDA plate and The acceptable criterion for microbiological quality is prescribed as :

• 101 cfu : maximum acceptable count =20
• 102 cfu : maximum acceptable count =200
• 103 cfu : maximum acceptable count =2000

Declaration

• The Material/product is passed when the observed count is less than specified count of that Material/product.
• The Material/product is failed if the observed count is greater than specified count of that Material/product.
• In that case, repeat the test, if the count is greater than specified count, the product is failed.

Test for Specified Microorganisms

Suitability of Test Method

Cary out the test in presence of the product. Add each test strain distinctly not more than 100 cfu at the time of product mixing with the culture media.The test will be suitable if found growth of the specific microorganism. The test will not suitable if no growth found the specific microorganism. In thatcase, add any neutralizer or increase the dilution for removal any inhibition of product.

Testing of Products

Test for E. coli

• Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]⁰C for 18-24 hours.
• Shake the container then transfer 1 ml of CSDM to the 100 ml of MacConkey Broth. Incubate at [42-44]⁰C for 24 hours.
• Sub-culture on MacConkey Agar plate from MacConkey broth. Incubate at [30-35]⁰C for 18-72 hours.
• The product complies with the test for E. coli if no red colonies are present with precipitated zone and the biochemical tests found negative[-ve].

Test for Salmonella

• Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]⁰C for 18-24 hours.
• Shake the container; transfer 0.1 ml of CSDM to 10 ml of RVS [Rappaport Vassiliadis Salmonella] Broth. Incubate at [30-35]⁰C hours for 18-24 hours.
• Sub-culture on XLD [Xylose Lysine Deoxycholate] Agar plate from RVS [Rappaport Vassiliadis Salmonella] Broth . Incubate at [30-35]⁰C for 18-48 hours.
• The product complies with the test for Salmonella if no red colonies are present with or without black centres and the biochemical tests are negative[-ve].

Test for Pseudomonas aeruginosa

• Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]⁰C for 18-24 hours.
• Sub-culture on Cetrimide Agar plate from CSDM [Casein Soyabean Digest Medium]. Incubate at [30-35]⁰C for 18-72 hours.
• The product complies with the test for Ps. aeruginosa if no bluish green colonies are present and the biochemical tests are negative[-ve].

Test for C. albicans

• Add 10 g or 10 ml of test sample to 90 ml of SDB [Soubaurad Dextrose Broth]. Incubate at [30-35]⁰C for 3-5 days.
• Sub-culture on SDA[Soubaurad Dextrose Agar] plate from [Soubaurad Dextrose Broth]. Incubate at 30-35⁰C for 24-48 hours.
• The product complies with the test for C. albicans if no white colonies are present and the   biochemical tests are negative[-ve].

Test Report Preparation

• Report the result in Microbial Count Report of Non-sterile RM, Annexure-I.
• Report the result in Microbial Count Report of Non-sterile Products, Annexure-II.

This is all about the Microbial Examination of Non-Sterile Raw Materials and Products and based on this information you can generate a SOP for Microbial Examination of Non-Sterile Raw Materials and Products.

Download all Annexure

Annexure I Microbial Count Report of Non-Sterile Raw Materials

Annexure II Microbial Count Report of Non-Sterile Products

Annexure III Non Sterile Raw Materials Log book

Annexure IV Non Sterile Products Log book

Culture Media Purpose

Culture Media, To make the culture media for the development of microorganisms in the microbiological test of raw materials, In-process sample & finished products.

Culture Media Scope

This designated SOP is applicable for the preparation of Culture Media in Microbiology Laboratory at XX Pharmaceuticals Limited.

Definitions/Abbreviation: 

Culture Media: The Culture media is a liquid or gel designed to support growth of microorganisms or cells  or small plants.  There are different type of media for growing different type of cells. 

There are two major types of growth media: those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or yeast.

Responsibilities:

The roles and responsibilities are as follows:

Officer/Sr. Officer, Microbiology

To follow the instructions of the described procedure accordingly.

Asst. Manager/Manager, Microbiology

• Ensure that the procedure is kept up to date.
• Ensure right personnel from the section are trained on this specific procedure.
• Ensure the media preparation, sterilization, requisition, maintain accurate storage & proper documentation.
• To Confirm that this SOP is technically sound and reflects the required working practices as current practices.

Head of Quality Assurance

• Approval of SOP
• To ensure the overall implementation of the SOP

Procedure

Instructions

Dehydrated media are hygroscopic & are sensitive to light, heat and moisture. They are adversely affected by extreme changes in temperature e.g. hot/ cold cycling temperatures which may occur between day and night laboratory temperatures in winter season.

Condition of Media Preparation:

• Take clean & dry flask as per required volume.
• Wear appropriate Laboratory garments, gloves and mask.
• Wear safety goggles during selection of hazardous media.

Storage Condition of Dehydrated Media:

• Mention receipt date on the label when enter into laboratory.
• Store as per directions on the label; typically below 25⁰C in a dry area, away from direct sunlight,  autoclaves, drying ovens or other heat sources, Where indicated store at (2-8)⁰C.
• Check expiry date on the specific label, some media have suggestively shorter shelf life than others.
• Use/maintain stock in lot/batch number order. Maintain FIFO [First In First Out], FEFO [First Expiry First Out].
• Do not open a new bottle until the previous bottle has been emptied. Ensure date with label on the supplied container when it first opened.
• After intended use, ensure the container is tightly closed and store it into the designated storage area.
• Collect/procure/order the medium in an appropriate size of container and in a quantity which harmonies to normal use requirements.

• A medium in a large container which has been opened many times will deteriorate on storage. Discard the medium if the powder is not free flowing, if the colour has changed or if it   appears abnormal in any way.

• Temperature of media storage room/facility/area/location should be monitored through min./max. thermometer and limit shall be followed as per the media storage requirements.

• List of media having designated storage condition and specific pH limit shall be prepared as per Annexure-II and shall be displayed near media storage facilities.

pH Check:

Check pH of the specific medium before sterilization with calibrated pH meter. If required adjust the pH with the help of 1N or 0.1N NaOH and 1N or 0.1N HCl solutions.

Culture Media Preparation:

• Select the media as per specific requirements.
• Read the instructions on the label very carefully before preparation of the specific media.
• Weigh the media according to the instructions of the manufacturer of the supplied media.
• Close the media container tightly just after weighing in order to avoid the moisture acquisition.
• Reconstitute the media with purified water and boil it appropriately until entirely dissolve.
• Distribute the reconstituted media into clean and dry flask as per requirements.
• Cap the flask using cotton plug or screw cap appropriately.
• Transfer the media flask into the detest room for use if instructed on the label as “DO NOT AUTOCLAVE” the media.

Sterilization:

• Place all prepared media into the autoclave.
• Sterilize the media at 121⁰C for 15 minutes.
• Wait until completion of cycle, and collect sterilized media from the autoclave when the chamber temperature reduce at 60⁰C.

Storage of Sterilized culture Media:

• After completion of autoclaving activities, transfer all flasks containing the broth media to the test room for use.
• Store the agar media at the warming condition (50⁰C) into autoclave until use.
• Do not keep the prepared media for more than two weeks [14 days].
• Keep the prepared agar plate at (2-8)⁰C into the refrigerator for not more than two weeks.

Handling of Sterilized culture Media:

• Ensure the aseptic condition during handle of sterilized media.
• Do not de-cap or expose the sterilized media outside Laminar Air Flow or Bio-Safety Cabinet.

Record Keeping:

Keep in practice to Maintain Register for Media Preparation Record, Annexure-I and Annexure-II to keep record for list of media with storage condition and pH limit.

List of Annexure: Download Here

Annexure I Register of Media Preparation

Annexure II List of Media with storage condition and pH Limit