Category: Microbiology SOP

1. SOP Title: Entry Exit Procedure in Microbiology Section
2. SOP Title: Cleaning of Used Glassware in Microbiology Section
3. SOP Title: Procedure for Media Preparation and Sterilisation for various Microbiological Testing
4. SOP Title: Decontamination of Media, Cultures and Microbial Waste
5. SOP Title: Sampling of Water for Microbiological Analysis
6. SOP Title: Environmental Monitoring
7. SOP Title: Procedure for Validation of Laminar Air Flow
8. SOP Title: Validation of Laboratory Autoclave located in Microbiology Laboratory
9. SOP Title: Maintenance of Various Cultures used in Microbiological Analysis
10. SOP Title: Determination of Minimum Inhibitory Concentration of Disinfectant
11. SOP Title: Validation of Disinfectant’s Effectiveness
12. SOP Title: Procedure for Fumigation in Microbiology Section
13. SOP Title: Area Cleaning and Sanitation of Microbiology-Section
14. SOP Title: Receipt and Storage of Dehydrated Culture Media/Bases and Reagents used for Microbiological Analysis
15. SOP Title: Serial Dilution Preparation of Microbial Cultures
16. SOP Title: Identification and Purity Verification of Master Microbial Cultures
17. SOP Title: Testing Growth Promotion, Inhibitory and Indicative Properties of Culture Media
18. SOP Title: Procedure for Surface Swab Analysis for Microbial Limits
19. SOP Title: Procedure for Efficiency test for UV Light
20. SOP Title: Operation and Cleaning of Autoclave
21. SOP Title: Procedure for Operation and Cleaning of Microscope
22. SOP Title:-Operation, Cleaning and Calibration of Colony Counter
23. SOP Title: Procedure for Operation, Cleaning and Calibration of Incubator
24. SOP Title: Operation and Cleaning of Laminar Air Flow Bench
25. SOP Title: Operation, Cleaning and Calibration of Refrigerator
26. SOP Title: Operation, Cleaning and Calibration of B.O.D. Incubator
27. SOP Title: Receiving, Storage and Qualification of Biological Indicator
28. SOP Title: Procedure to Determine Shelf Life of Reconstituted Media
29. SOP Title-Bio-Load Analysis of Compressed Air
30. SOP Title-Operation and Cleaning of Micropipette
31. SOP Title-Operation and Cleaning of Microscope
32. SOP Title-Operation and Cleaning of Pass Box
33. SOP Title-Procedure for Fumigation in Microbiology
34. SOP Title-Operation and Cleaning of Antibiotic Zone Reader

SOP Title-Operation and Cleaning of Microscope

 

• OBJECTIVE

To lay down the procedure for operation and cleaning of microscope.

• SCOPE

This SOP is applicable for operation and cleaning of microscope in Microbiology Lab.

 

• RESPONSIBILITY

3.1  QC Executive and above shall be responsible for implementation of this procedure.

3.2  QC Head / designee shall be responsible for compliance of this procedure.

 

• PROCEDURE :-
• Precautions :
• Check the cleaning status of microscope.
• Operation of Microscope:
• Switch ON the Mains Switch.
• Switch ON the microscope (Ensure that Intensity of light controller of the microscope is kept to minimum level every time before switching OFF or switching ON the microscope)
• Select the objective.
• Adjust the diaphragm & intensity controller to get sufficient light towards the stage micrometer.
• Place the slide to be examined, on the stage, underneath the lens.
• Set the magnification as per requirement.
• Focus the slide with the help of Coarse Adjustment Knob. (Coarse & Fine adjustments are coaxially mounted. The outer knob being for the coarse focusing and the smaller inner knob for fine focusing).
• Focus for clarity with the help of the fine adjustment knob and make observations.
• Adjust the ABBE condenser in upward or lower direction as per the chosen objective.
• For low magnification the condenser is moved to a lower position by provided knob and for higher magnification to upper position.

• Cleaning:
• After use, instrument shall be cleaned with lint free cloth/tissue paper by using 70% IPA.

 

• ABBREVIATION(s)

Abbreviation	Full Description
SOP	Standard Operating Procedure
IPA	Isopropyl Alcohol

 

 

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SOP Title-Operation and Cleaning of Micropipette

 

• OBJECTIVE

To lay down the procedure for operation and cleaning of micropipette.

• SCOPE

This SOP is applicable for operation and cleaning of micropipette in Microbiology Lab.

• RESPONSIBILITY

3.1 QC Executive and above shall be responsible for Implementation of this procedure.

3.2 QC Head / designee shall be responsible for compliance of this procedure.

 

• PROCEDURE :
• Precautions;
• Check the cleaning status of micropipette.
• Check the calibration status of micropipette.
• Operation of micropipette:
• Adjust the required volume of micropipette by rotating the knobs provided..
• Attach tips to Micropipette by pressing at the base.
• Press the top central knob up to first step lock.
• Dip the Micro-pipette with tips in the solution in such way that only half of the tip will get dip.
• Then release the top central knob, it will suck the solution.
• Then remove the Micro-pipette from the dipped solution.
• Dispense sucked solution in required tube/container by pressing full top central knob up to second step lock & release the knob.
• To remove the tip attached, press top side knob.
• Use separate and sterilized tips for each product/materials.
• Cleaning:
• Clean the micropipette with the help of tissue paper/dry lint free cloth.
• After handling of culture, micropipette shall be cleaned with 70% IPA.

 

• ABBREVIATION(s)

Abbreviation	Full Description
SOP	Standard Operating Procedure
IPA	Isopropyl Alcohol

 

 

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SOP Title-Bio-Load Analysis of Compressed Air

 

• OBJECTIVE

To lay down the procedure for bio-load analysis of compressed air.

• SCOPE

This SOP is applicable for bio-load analysis of compressed air in Microbiology Lab.

 

• RESPONSIBILITY

3.1  QC Executive and above shall be responsible for implementation of this procedure.

3.2 QC Head / designee shall be responsible for compliance of this procedure.

• PROCEDURE
• Sampling schedule
• Sampling plan for compressed air analysis shall be prepared as per SOP.
• All points shall be covered for analysis on monthly basis.
• Sampling procedure
• Sterilize 100 ml of purified water or buffered peptone water in a 250 ml Bottle/conical flask along with cap for 15 minutes at 121^oC & 15 Psi pressure.
• Allow the sterile purified water or Buffered peptone water to attain room temperature.
• Carry the Bottle/conical flask of sterile purified water or buffered peptone water to the required sampling point.
• Connect the sampling point in the sampling bottle and bubble the compressed air for 10-15 minutes into sampling bottle.
• Carry the sample to the microbiology lab for analysis.
• After sampling make the entries in the Sample Entry Log Book.

 

• Analysis procedure
• Transfer 10 ml of the sample on to 90 ml soyabean casein digest medium and perform the bio-load test under the laminar air flow.
• Transfer 1 ml above prepared sample into sterile petriplate in duplicate.
• After inoculation of 1 ml sample into plates, pour the 20-25 ml SCDA media cooled to 45-50°C in the petriplates.
• Pour the 20-25 ml SCDA media cooled to 45-50°C in the petriplates into two sterile petriplate.
• Leave the plates for solidification.
• After solidification, expose one above prepared media plates in the environment and this plate shall be consider as positive control while one unexposed plate shall be consider as negative control.
• Incubate all the plates (Sample plates, negative control and positive control plate) in inverted position for a period of 72 hours at 20 to 25°C & further incubate all the plates in inverted position for a period of 48 hours at 30 to 35°C.
• After completion of incubation period, count the number of CFU per plate.
• Record shall be maintained as per annexure.
• Acceptance Criteria:

Standard Limit              :  NMT 100 CFU/Plate.

Alert Limit        :  NMT 50 CFU/Plate

Action Limit     :  NMT 75 CFU/Plate

Fungus              :  Should be Nil

ABBREVIATION(s)

Abbreviation	Full Description
SOP	Standard Operating Procedure
˚C	Degree centigrade
Psi	Pascal square inch

 

 

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SOP Title: Procedure to Determine Shelf Life of Reconstituted Media

1. Objective:

To lay down the Procedure to Determine Shelf Life of Reconstituted Media.

 

2. Scope:

This procedure is applicable to Determine Shelf Life of Reconstituted Media in the Microbiology Section of Quality Control Department.

 

3. Responsibility:

• Quality Control: To prepare and review the SOP. To follow the procedures to Determine Shelf Life of Reconstituted Media as per this SOP.
  • Quality Assurance Department: To review and approve the SOP and Annexure.

4. Accountability:

Head Quality Control Department, Head Quality Assurance Department.

 

5. Procedure:

5.1	Safety Precautions:
5.1.1	Always wear protective masks and gloves while performing micro activities.
5.1.2	Use clean Spatula, cleaned and dried with 70% v/v Isopropyl Alcohol for weighing of each media.
5.2	Procedure of Shelf Life  Determination :
5.2.1	Refer Procedure for Media Preparation and Sterilisation SOP for preparation of reconstituted media.
5.2.2	For the List of Media to be prepared and individual procedure refer respective SOP for preparation of reconstituted media.
5.2.3	Enter all dehydrated media weighed and update the stock in annexure.
5.2.4	Enter the Details for the weighing, dispensing, pH and autoclaving of Media in annexure.
5.2.5	Prepared liquid media shall be distributed aseptically in a suitable capacity previously sterilized conical flasks/ bottles/Test tubes.
5.2.6	Affix label containing name of the media, batch number, date of preparation and signature.
5.2.7	Sufficient Media Should be prepared considering the amount required to perform all the tests for all the days of the study. Number of organisms for the Growth promotion test has to be calculated before Media preparation.
5.2.8	Eg. If SCDM is to be prepared for study then consider the number of Organisms for GPT i.e. 7 organisms + Media control= 8 x 10 ml + pH= 1 x 10 ml + Other test together (discolouration, sedimentation, precipitation and contamination) = 1 x 10 ml: Total = 100 ml per day of study. This step has to be repeated for 8 days of the study, accordingly minimum of 800 ml SCDM media has to be prepared for the study.
5.2.9	Store the media in an incubator at 20-25°C or in the refrigerator at 2-8° C. Ensure that the media should be at room temperature after removing from the refrigerator and before performing the study.
5.3	Different Time Intervals Check for Growth Promotion study and physical observation.
5.3.1	The growth promotion capability to be determine at following intervals. Initial, 1st day, 3rd day, 7th day, 14th day, 21st day, 28th day and 31th day.
5.3.2	While ascertaining the shelf life of media, following observations are to be recorded in the Annexure.
5.3.3	At each stage, prepared Media are checked for physical observation such as discolouration, sedimentation, precipitation and contamination. Record the parameters in Annexure. and observe its validity at each stage.
5.3.4	At any given time within the study days, pH should not vary from given range for individual reconstituted media.
5.3.5	Growth promotion test should comply when tested initially and during the particular storage period. Refer SOP “Procedure for Growth promotion, Inhibitory and Indicative Properties of Culture Media.”
5.3.6	For Culture Medium, incubation parameter and No. of organisms to be tested for growth promotion, Refer SOP “Procedure for Growth promotion, Inhibitory and Indicative Properties of Culture Media.”.
5.3.7	Enter results of each day in the Annexure for comparing and noting down the Results to determine the Shelf life.
5.3.8	If reconstituted media fails for any one of following tests:      Physical appearance or pH or Growth promotion test, previous stage results which are complying will be considered as shelf life of that media.
5.3.9	Enter the Details in Equipment usage log books like Incubator, pH Meter, Autoclave, LAF etc. and other Documents like Media Consumption to be updated online to prevent any discrepancy in recording.
5.3.10	All the Used media and glasswares used for study, after completion of incubation and recording of results has to be Decontaminated in autoclave before disposal as per SOP -Destruction of Microbial waste.
5.4	Acceptance Criteria:
5.4.1	The shelf life of each individual reconstituted media will be determined based on no major change in physical appearance, pH and growth promotion test values from the initial values upto the maximum duration of study.
5.4.2	Above steps are performed Initially once to validate shelf life of each media.

 

6. Definitions / Abbreviation:

• Definitions:

 

• Abbreviation:

 

Abbreviation

Expansion

 

°C	Degree Centigrade
%	percent
v/v	Volume by volume
ml	millilitre
GPT	Growth Promotion Test
SCDM	Soyabean Casein Digest Medium
pH	-ve log of hydrogen ion concentration

  

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SOP Title: Receiving, Storage and Qualification of Biological Indicator

1. Objective:

To lay down the procedure for Receiving, storage and qualification of biological indicator.

 

2. Scope

This procedure is applicable for procedure for receiving, storage and qualification of biological indicator in the Microbiology Section.

3. Responsibility:

3.1      Quality Control: To prepare and review the SOP. To follow the procedures for Receiving, storage and qualification of biological Indicators as per this SOP.

•     Quality Assurance Department: To review and approve the SOP and Annexure.

4. Accountability:

Head Quality Control Department, Head Quality Assurance Department

 

5. Procedure:

5.1	Safety Precautions:
5.1.1	Wear personal protective equipments such as protective hand gloves, nose masks and over gown during operation.
5.1.2	Microbiologist should disinfect the hand gloves with 70% v/v IPA before carrying out operation.
5.2	Procedure for Receiving the Biological Indicators (BI)
5.2.1	Check the physical condition of biological indicator strip/ampoules at the time of receiving.
5.2.2	Record shall be maintained as per Annexure.
5.3	Storage :
5.3.1	Biological indicators shall be stored in refrigerator at 2-8 0C after receiving the BI or as per suggested by the Supplier.
5.3.2	Opened biological indicators shall be stored in refrigerator wrapped with parafilm/Aluminium foil.
5.3.3	Record shall be maintained as per Annexure.
5.4	Spore count (Label Claim) determination of Biological indicator:
5.4.1	Take randomly three paper strips or ampoules of Biological Indicator Geobacillus stearothermophilus ATCC 7953 (BI) which is used to confirm the efficiency of Steam Sterilization at 121 0C.
5.4.2	Tear off the outer covering of BI if it is a paper carrier and transfer it into a single glass tube/bottle containing 100 ml sterile purified water with help of a sterile forceps. In case of Glass ampoules, break the neck under LAF using a SS rod/glass rod which is sterile and transfer the contents of ampoules into a single glass tube/bottle containing 100 ml sterile purified water.
5.4.3	Gently vortex the tube about 15 minutes so as to achieve a homogenous suspension with 100 ml sterile purified water.
5.4.4	Heat the tube/bottle containing the suspension with the help of water bath at 95–1000 C for about 15 minutes. This shall be considered as Stock suspension.
5.4.5	Prepare 0.9% sterile saline solution and dispense in 10 tubes with 9 ml in each tube, Refer SOP No. 012 for reagents preparation.
5.4.6	Take 1 ml from stock suspension tube and inoculate into the tube containing 9 ml sterile 0.9% sterile normal saline solution under LAF. This constitute dilution is considered as 10 -1. Shake the tube well.
5.4.7	From tube 10 –1, transfer 1 ml into tube 10-2 using a pipette and shake well. Serially transfer 1 ml from tube 10-2 to tube marked as 10-3 and prepare further serial dilutions as 10-3 to 10-4, 10-4 to 10-5 upto 10-7. Record in Annexure No. 01. Refer Annexure No. 03 for dilutions.
5.4.8	Prepare Soyabean casein digest agar as per SOP No. 034
5.4.9	Inoculate 1.0 ml suspension from each dilution into plates in duplicate by pour plate method under LAF.
5.4.10	After inoculation of 1 ml of suspension into plates, pour 15-20 ml soyabean casein digest agar-medium cooled to 45-50°C in each petriplate.
5.4.11	Each Plate has to be marked with dilution number, SCDA Media lot number, Date and Sign.
5.4.12	Swirl gently to mix and allow it to solidify.
5.4.13	Incubate the plates at 55-60 ºC for 24-48 hrs. Check count at 24 hours and final count after 48 hours.
5.4.14	Note: Adjust the Incubator set at 20-25ºC to 55-60ºC for the above incubation as it is validated for 55-60ºC. Monitor the temperature of incubator twice in a day.
5.4.15	Examine the plates after 24 hours and 48 hours and record the number of CFU in each plate in Annexure. Take observations of 48 hours for final calculation.
5.4.16	Select the dilution for calculation which gives 10-100 CFU count per ml on petriplates.
5.4.17	Calculate average number of spores per specimen by dividing the CFU with dilution factor. Example.: Calculation for BI spore count:- Results of 3 strips of BI = Average spore count Dilution Factor Suppose, Average spore count  =  67 CFU for serial dilution 10-3 for 3 strips of BI   Then, Results of  spore count for  =   67   =  67 x 103 3 strips of BI                     10-3       Therefore, Results per BI  =  67 x 103 3 = 22.33 x 103
5.4.18	Divide the results obtained by 3 to get the spore count (Population) for single strip as the analysis is performed using three strips.
5.4.19	After completion of analysis and result recording, all the petriplates and glasswares used for the test to be Decontaminated as per procedure given in SOP for Decontamination of culture media and microbial waste.
5.4.20	Acceptance Criteria: The log of average spore count obtained should not be less than the theoretically calculated value by substracting 0.3 from the log of suppliers label claim value and should not be more than theoretically calculated value by adding 0.48 to the log of suppliers label claim value. For e.g:- 1.Suppliers label claim spore count = 3.8 x 104 2.Log of supplier label claim spore count = log (3.8x 104) = 4.5798 3. Lower limit of label claim                    = 4.5798 – 0.3 = 4.2798 3. Higher limit of label claim                    = 4.5798 + 0.48 = 5.0598 4. Acceptance criteria                               = 4.2798   to  5.0598
5.4.21	If the Biological Indicators does not show proper count within the labelled claim, then inform the Department head and discard the Biological Indicators and procure a new lot.

 

 

6. Definitions / Abbreviations:

• Definitions:
• Quality Control Associate: Executive or Officer Quality Control Department or any personnel trained to perform duties as per this SOP.
• Quality Assurance Associate: Executive of Quality Assurance Department or any personnel trained to perform duties as per this SOP.
• Abbreviations:

Abbreviation	Expansion
LAF	Laminar Air Flow
°C	Degree centigrade
ml	mililiter
CFU	Colony Forming Unit
%	Percentage
BI	Biological Indicator
IPA	Isopropyl Alcohol
ATCC	American Type Culture Collection.

 

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SOP Title: Operation, Cleaning and Calibration of B.O.D. Incubator

1. Objective:

To lay down the procedure for Operation, Cleaning and Calibration of B.O.D. Incubator.

2. Scope:

This SOP is applicable for Operation, Cleaning and Calibration of B.O.D. Incubator (20-25°C) located in the Microbiology Section.

3. Responsibility:

• Quality Control: To prepare and review the SOP. To follow the procedures laid down for operation, cleaning and calibration of B.O.D.  Incubator (20-25°C) as per this SOP.
• Engineering Department: To carry out preventive maintenance as per schedule and procedure defined.
• Quality Assurance Department: To review and approve the SOP and Annexures.

 

4. Accountability:

Head Quality Control Department, Head Quality Assurance Department.

5. Procedure:

5.1	Safety Precautions/Instructions:
5.1.1	Ensure all the electrical connections are properly earthed. Ensure the power plug of the Incubator is fixed in the socket properly.
5.1.2	Wear personal protective equipments such as protective hand gloves, nose masks during operation of equipment.
5.1.3	Ensure that the ON/OFF switch is working properly by switching “ON” the Incubator.
5.2	Operation  Procedure:
5.2.1	Check the validity of Calibration before starting any operations.
5.2.2	Connect the incubator to the mains and switch it ‘ON’.
5.2.3	Set the desired temperature with the help of Digital Temperature controller panel.
5.2.4	set the desired temperature at 22.5°C (20 to 25°C)
5.2.5	The set value 22.5°C remains in the memory of the digital controller.
5.2.6	Incubator temperature monitoring should be recorded twice in a day. Morning session and evening session. The record shall be maintained in Annexure
5.2.7	All petri plates, tubes, slants placed in the incubators shall be labelled with sample name, Batch or A.R. number and date manually using a marker pen.
5.2.8	Update the “Instrument/Equipment Usage Log Book” for Incubator as per SOP.
5.2.9	After appropriate time of incubation, remove the samples to prevent overcrowding or contamination of incubator.
5.2.10	Do not switch OFF or disturb the incubators after setting the desired temperature parameter during performance of unit.
5.2.11	If any time incubator shows the reading out of range, follow the procedure as per SOP.
5.2.12	The equipment service engineer must be contacted for servicing/repair of the equipment followed by a recalibration that must be done before the reuse of the same.
5.3	Cleaning Procedure:
5.3.1	De-dust the external surface of Incubator with lint free cloth.
5.3.2	Clean the internal surface of Incubator with wet cloth soaked in 70% v/v Iso Propyl Alcohol.
5.3.3	Frequency of cleaning is every fifteen days from inside and daily from outside.
5.4	Calibration Procedure:
	Frequency:  Once in a year or after carrying out any major maintenance work.
5.4.1	Generate the A. R. No. as per SOP.
5.4.2	Parameters Set value Acceptance Criteria   Temperature 20°C 20°C ± 1°C 23°C 23°C ± 1°C 25°C 25°C ± 1°C

 

6. Definitions / Abbreviations:

• Definitions
• Breakdown: Any activity leading to operation of equipment other than the set parameters and/ or unusual sound or vibration observed in the equipment.
• Calibration: Tests performed to ensure that the equipment is functioning as per set parameters. These tests are performed as per predefined frequency and also after any major repair or replacement of parts.
• Preventive Maintenance: Maintenance activity performed to ensure that the equipment will function smoothly and to avoid breakdowns. These activities are performed as per predefined frequency.

 

• Abbreviations:

 

Abbreviation

Expansion

 

B.O.D.	Biological Oxygen Demand
SP	Set Parameter
hrs	Hours
No.	Number
v/v	Volume by volume
°C	Degree Celsius
QC	Quality Control
ENT	Enter

 

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