Category: Microbiology SOP

SOP Title: Receipt and Storage of Dehydrated Culture Media/Bases  and Reagents used for Microbiological Analysis

1. Objective:

To lay down the Procedure for Receipt and Storage of Dehydrated Culture Media/Bases and Reagents used for Microbiological Analysis.

        

2. Scope:

This Procedure is applicable for Receipt and Storage of Dehydrated Culture Media/Bases and Reagents used for Microbiological Analysis in microbiology Section of Quality Control Department.

 

3. Responsibility:

• Quality Control Department: To prepare and review the SOP. To follow the procedure for Receipt and Storage of Dehydrated Culture Media/Bases and Reagents used for Microbiological Analysis 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	The Media Shall be preserved away from direct light, Humidity and temperature.
5.1.2	In case of accidental spillage of any Media /Bases, collect the powder with duster on the paper and clean the area with water and finally swab with 70% v/v IPA solution.
5.1.3	Always use safety masks, goggles, gloves while handling these Media, Bases and Reagents.
5.2	Procedure for Receipt:
5.2.1	On receipt of intimation from security, the concerned Microbiologist or Officer should verify the list of items ordered and received against Purchase Order given to the respective supplier.
5.2.2	The Officer or Microbiologist should check the number of items, Name of the Media, Lot or batch No, item code no. and quantity received with date of Manufacturing and date of Expiry.
5.2.3	If material received is not as per the Purchase Order, cancel the entry of the respective item on all copies of challan and invoices (if attached) by drawing single line and duly signed with date and handover or return the material to the supplier.
5.2.4	Verify the received Material for any external damage.
5.2.5	If any discrepancy is observed then the Microbiologist or Officer shall inform the Head of the Quality Control Department and follow instructions given by the Head of the Quality Control.
5.2.6	If the materials confirm to the requirement then the Microbiologist or Officer shall accept the material and counter sign on the challan and submit to the security personnel for making entry in the Inward Register.
5.2.7	Challan and invoices shall be sent to Warehouse Department for preparing GRN.
5.2.8	Signed GRN copies shall be verified with invoice copies and submitted to the Accounts Department for further processing.
5.2.9	Enter the details of the media received in “Receipt of Media, Bases and Consumption Record” as per Annexure.
5.2.10	Individual index sheet shall be maintained for Media coming under one alphabet as Index A, Index  B and so on as per refer Annexure.
5.2.11	Under Index A, all Media which starts with ‘A’ alphabet shall be assigned code as A1, A2,.. and so on.
5.2.12	For example if MacConkey Agar is received then it will come under index M and the Media code shall be given as M1 and for MacConkey Broth it is M2.
5.2.13	This procedure shall be followed for all the individual index sheets.
5.2.14	Affix the receipt label as per define Annexure.
5.2.15	For each and every container of the media/bases or reagent received, are pasted with receipt label with necessary details.
5.2.16	Download the media Certificate of analysis by entering respective batch no. or lot no. mentioned on the containers or bottles from the Website or supplier.
5.2.17	Assign the media code and same shall be updated in the list.
5.2.18	Store the containers designated place away from light, humidity and temperature as per storage condition of respective Media / Bases/ Reagents.
5.2.19	If Media / Bases/ Reagents insist special storage conditions on the bottle label shall be stored as per specific requirement.
5.2.20	For routine analysis if bottle or containers are removed from stock for use, the date of opening and valid up to date shall be entered on the receipt label.
5.2.21	Make Entry into the Media Stock format after removing any Media or reagent bottle from the stock.
5.3	For Ordering the New Dehydrated Culture Media, Bases and Reagents
5.3.1	The Microbiologist is responsible for evaluation of the quantity in stock and preparing the required list.
5.3.2	Microbiologist shall prepare the indent for listed requirements and submit the requirement copy to Department Head for approval.
5.3.3	Quality control Head shall review and approve the Indent and Purchase Order raised against approved supplier and proceed to the Plant Head for Authorization.
5.3.4	After authorisation PO copies are sent to the supplier to supply the items.
5.4	Shelf life of Media and bases and reagents
5.4.1	In an unopened condition the expiry date of Medias, Bases and Reagents shall be as recommended by the Manufacturer. For Example. MacConkey  Agar: Mfg. date:  Mar-2024 Exp. date: Mar-2028
5.4.2	After Opening a container of Media, expiry shall be assigned as two years from date of opening or expiry given by manufacturer whichever comes earlier.
5.4.3	For e.g. If a container of MacConkey Agar  is opened on 10th of April 2024 then date of opening and valid up to date to be mentioned on the label as: Date of opening: 10/04/2024 Valid up to: 09/04/2026
5.4.4	Any leftover sample after the expiry date should be disposed as per Procedure for Destruction of Cultures and Microbial waste SOP.
5.4.5	If any culture Media, Bases or Reagent shows deterioration e.g. discoloration, change in physical appearance, sedimentation, precipitation, and crystallisation shall be discarded regardless of validity.
5.5	Storage of Media, Bases and Reagents:
5.5.1	Solid Media, Bases and Reagents shall be stored in the designated cupboards labelled as ‘Media, Bases /Reagents in stock’ in at room temperature or as specified storage conditions on containers or certificate.
5.5.2	Liquid chemicals shall be stored in the designated cupboards labelled as ‘Microbiological Liquid in Stock’ in at room or as specified storage conditions on containers or certificate.
5.5.3	All the cupboards containing Media, Bases should have the stock list.
5.5.4	Frequency of stock verification: Last week of every month.

 

6. Definitions / Abbreviations:

• Definitions:
•       Abbreviations:

Abbreviation	Expansion
°C	Degree Centigrade
%	percent
v/v	Volume by volume

ml	millilitre

 

 

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SOP Title: Area Cleaning and Sanitation of Microbiology–Section

1. Objective:

To lay down a procedure for Area Cleaning and Sanitation of Microbiology Section

 

2. Scope:

The scope of this SOP is applicable for Area Cleaning and Sanitation of Microbiology Section of Quality Control Department.

 

3. Responsibility:
   • Quality Control: To prepare and review the SOP. To follow the procedures laid down for the Procedure for Area Cleaning and Sanitation Section as per this SOP.
   • Quality Assurance Department: To review and approve the SOP and Annexure.
   • House Keeper: To follow the procedures laid down for the Procedure for Area Cleaning and Sanitation Section as per this SOP.
4. Accountability:

Head Quality Control Department, Head Quality Assurance Department.

5. Procedure:

5.1	Preparation of disinfectant solution:
5.1.1	Dettol – 2.5% v/v solution in purified water. i.e. 2.5 part dettol diluted to 97.5 parts of  purified water
5.1.2	Savlon – 2.5% v/v solution in purified water. i.e. 2.5 part savlon diluted to 97.5 parts of purified water.
5.1.3	IPA – 70% v/v solution in purified water. i.e. 70 parts IPA diluted to 30 parts of purified water.
5.2	Procedure for Cleaning and Sanitisation of Floor:
5.2.1	The mopping operation is to be carried out before start of activity and after completion of analysis or at the end of the shift.
5.2.2	Use a clean dry lint free mop/cloth and remove any dust.
5.2.3	Use a lint free mop and dedicated buckets/containers for different disinfectants.
5.2.4	Clean the area with purified water, allow to dry and then mop with disinfectant solution.
5.2.5	Use freshly prepared disinfectant solution for mopping the floors.
5.2.6	Measure the required amount of disinfectant and add purified water to make up final desired volume.
5.2.7	Moping should be carried out as per the sequence:
5.2.8	LAF room to be cleaned first with purified water, then the third and second air lock followed by the incubation room and first airlock. Finally mop the media preparation room.
5.2.9	Follow the same pattern for disinfectant solution.
5.2.10	Rotate the disinfectants every week to avoid the development of resistance by microorganisms.
5.2.11	For the first and third week use dettol and second and fourth week use savlon.
5.3	Procedure for Area Cleaning:
5.3.1	Perform the area cleaning and sanitation when there is no activity.
5.3.2	De-dust the surfaces of the ceilings, walls, pass boxes and exposed parts of the equipments with the aid of dry clean cloth twice a week before mopping. Preferably on Monday and Thursday (if Monday/Thursday is a holiday than next working day).
5.3.3	Record the activity in Annexure ‘Area cleaning and sanitation record’.
5.3.4	For cleaning glasses, use Colin as cleaning solution every week.
5.3.5	Tables, pass boxes and other surfaces shall be cleaned with a lint free cloth using 70%IPA solution.
5.4	Cleaning  Frequency:
5.4.1	Floor – twice a day. Walls, Glass, Ceilings – twice in a week

 

6. Definitions / Abbreviations:

• Definitions:

 

• Abbreviations:

Abbreviation	Expansion
SOP	Standard Operating Procedure
QC	Quality Control
QA	Quality Assurance
%	Percent
v/v	volume by volume
IPA	Isopropyl Alcohol
No.	Number

 

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SOP Title: Procedure for Fumigation in Microbiology Section

1. Objective:

To lay down the Procedure for Fumigation in Microbiology Section of Quality Control Department.

 

2. Scope:

This procedure is applicable for the Procedure of Fumigation in Microbiology Section of Quality Control Department.

 

3. Responsibility:
   • Quality Control Department: To prepare and review the SOP. To follow the procedures for

Fumigation in Microbiology Section 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/Instructions:
5.1.1	AHU and air conditioners should be switched OFF before proceeding with the Fumigation Activity.
5.1.2	The person, handling the fumigants, shall be thoroughly familiar with application, procedures, safety equipment, first-aid treatment, and disposal procedures.
5.1.3	Person performing Fumigation Activity should leave the area immediately.
5.1.4	No one should enter the fumigated area for at least 8 to 10 hrs after Fumigation or until the completion of the Fumigation Activity.
5.2	Fumigation Procedure:
5.2.1	Switch OFF the AHU.
5.2.2	If the LAF is ON, put it OFF before starting the Activity.
5.2.3	All the Materials, Equipments shall be removed or covered.
5.2.4	Wear safety hand gloves and nose mask.
5.2.5	Weigh and transfer about 5gm of Potassium Permanganate in a container or an evaporating dish.
5.2.6	Measure about 50 ml volume of Formaldehyde solution 37% w/v in the measuring cylinder and add to the weighed quantity of Potassium Permanganate powder.
5.2.7	Personnel who perform Fumigation Activity should leave the area being fumigated as soon as possible.
5.2.8	Entrance of the Fumigated area must be labelled as “FUMIGATION IN PROGRESS – DO NOT ENTER” as per Annexure.
5.2.9	At the end of the Fumigation period or after minimum 8-10 hrs of Fumigation, remove Formaldehyde pans or containers.
5.2.10	Switch ON the Air conditioner or AHU and run it for 30 minutes, prior to entering this area.
5.2.11	Dissolve the left over residue from the evaporating dish or container in about 500 ml of raw water & transfer the contents in the container labelled as ‘Chemical Waste’ and send it for disposal.
5.2.12	The area and equipments should be cleaned and disinfected as per SOP ‘Procedure for Area Cleaning and Sanitation of Microbiological section’.
5.2.13	Update the Fumigation record as per Annexure.
5.2.14	Frequency – Weekly once, preferably on Saturday or prior to a holiday, at the end of the shift.

6. Definitions / Abbreviation:

• Definitions:
• Abbreviation:

Abbreviation

Expansion

 

AHU	Air Handling Unit
LAF	Laminar Air Flow
%	percentage
ml	millilitre
hrs	hours
g	gram
w/v	Weight by volume

  

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SOP Title: Validation of Disinfectant’s Effectiveness 

1. Objective:

To lay down the Procedure for Validation of Disinfectant’s Effectiveness. 

2. Scope:

This procedure is applicable for Validation of Disinfectant’s Effectiveness in Microbiology Section of Quality Control Department.

3. Responsibility:

3.1 Quality Control: To prepare and review the SOP. To follow the Procedure for Validation of Disinfectant’s Effectiveness 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 Iso Propyl Alcohol before carrying out operation.
5.2	Procedure of preparation of swab sample:
5.2.1	1.   Refer SOP of Media Preparation and Sterilisation for preparation of media and solutions.
5.2.2	Prepare a swab test sample by measure 10 ml of Buffered Sodium Chloride peptone pH 7.0 solution in a test tube.
5.2.3	Cotton swabs are inserted in test tubes containing 10 ml buffered Sodium Chloride pH 7.0 and plugged with non absorbent cotton.
5.2.4	Autoclave these test tubes at 1210C for 15 minutes.
5.3	Swabbing Procedure:
5.3.1	Before mopping, swab should be taken from an area which is difficult to clean and when in operation. Refer SOP for swabbing procedure.
5.3.2	With the help of a Swab template, swab the 10 x 10 cm2 area of the floor by moving the swab in an zig zag motion within the template in around 10 strokes with sterile swab before mopping and dip it in the sterile buffer sodium chloride peptone solution contained in the test tube. Immediately close the mouth of the tube with cotton plug or autoclavable cap. Mix for 1 minute. Refer Annexure for Swab procedure diagram.
5.4	Testing Procedure:
5.4.1	Bring the test tubes in microbiology section. Carry out the Microbiology analysis for Total microbiology count & detection of pathogens.
5.5	For Total Microbiology Count:
5.5.1	Pipette out 1 ml of the buffered solution into empty Sterile petriplates in duplicates. Mark all details on the plates.
5.5.2	Pour about 15 to 20 ml of Sterile Soyabean  Casein Digest Agar (40- 450C) in two plate & 15 to 20 ml of sterile Sabouraud Chloramphenicol or Dextrose Agar (40- 45°C) in the remaining two plates.
5.5.3	Mix gently and allow it to solidify. Incubate Soyabean Casein Digest Agar plate at 30-35° C for 3 to 5 days.
5.5.4	Incubate Sabouraud Chloramphenicol or Dextrose Agar plate at 20-25 0C for 5 to 7 days.
5.5.5	On completion of the incubation period, note down the colonies on the petriplate.
5.5.6	For pathogen: Swab solution is streaked on selective Agar plates. Use MacConkeys Agar For E.coli, Cetrimide Agar for P. aeruginosa, Mannitol Salt Agar for S.aureus and XLDA agar for Salmonella for detection of pathogens and incubate at 30-35°C for 48 hours. Refer SOP for culture identification.
5.5.7	Positive Controls: streaked a loopful of each of Escherichia coli, Salmonella, Pseudomonas aeruginosa & Staphylococcus aureus on selective Agar plates (as mentioned above), for detection of pathogens and incubate at 30-350C for 48 hours.
5.5.8	Observe the colonies obtained. Test is valid only if positive control shows the Characteristic colonies.
5.5.9	Negative Controls: Maintain a Negative control plates for each selective Agar plates i.e. MacConkey Agar, Xylose lysine deoxycholate agar, Cetrimide Agar & Mannitol Salt Agar.
5.6	After Mopping:
5.6.1	Mop the area and repeat the same procedure at the same area one hour after mopping with fixed concentrated disinfectant, later six hours and eight hours after mopping with selected MIC concentration solution of Dettol and Savlon, IPA sheet. Refer SOP, MIC of Disinfectants.
5.6.2	Repeat the procedure with one disinfectant at least for 3 days for interval of 1 hour, 6 hours and 8 hours. Collect and record the Validation of Disinfectant Data in Annexure.
5.7	Interpretation:
5.7.1	A Reduction, not less than 85 % in the viable counts after one hour of mopping from the initial count and absence of pathogens.
5.7.2	Not less than 70% reduction in the viable counts after Six hours and eight hours of mopping and absence of pathogens after each interval of mopping indicates that mopping with the respective disinfectant is effective. Refer Annexure.
5.7.3	Test is valid only if positive control shows the Characteristic colonies.
5.7.4	If the result is not within the accepted limit, then increase the concentration of respective disinfectant and carry out the Validation for increased concentration. Compile the Data in Annexure.
5.7.5	Decontaminate all the plates and Media after completion of analysis. Refer SOP  Decontamination of Microbial waste.

6. Definitions / Abbreviations:

• Definitions
• Abbreviations:

Abbreviation	Expansion
°C	Degree Centigrade
%	percent
v/v	Volume by volume
MIC	Minimum Inhibitory Concentration
IPA	Isopropyl alcohol

 

 

  

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SOP Title: Determination of Minimum Inhibitory Concentration of Disinfectant

1. Objective:

To lay down the procedure for Determination of Minimum Inhibitory Concentration of Disinfectant.

2. Scope

This procedure is applicable for Determination of Minimum Inhibitory Concentration of Disinfectant in all sections.

3. Responsibility:
   • Quality Control: To prepare and review the SOP. To follow the procedures laid down for Determination of Minimum Inhibitory Concentration of Disinfectant 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	Ensure that all glassware’s to be used are properly cleaned and dried.
5.1.2	All the following operations shall be carried out under laminar Air Flow in quality control Lab using aseptic techniques.
5.1.3	Ensure that sterilized pipettes, tubes, media are kept under Laminar air Flow.
5.2	Procedure to Determine the MIC of Disinfectant:
5.2.1	For dilution of cultures, refer SOP: Procedure for Serial Dilution Preparation of Microbial Cultures.
5.2.2	Streak bacterial cultures, Escherichia coli (E. coli) ATCC 8739, Salmonella abony (S. abony) NCTC 6017, Pseudomonas aeruginosa (P. aeruginosa) ATCC 9027 and Staphylococcus aureus (S. aureus) ATCC 6538 SCDA slants from working culture slants. Refer SOP ‘’Maintenance of cultures.
5.2.3	Fungal cultures, Candida albicans (C. albicans) ATCC 10231 and Aspergillus brasiliensis (A. Brasiliensis) ATCC 16404 shall be streaked on SDA slants from working culture slants.
5.2.4	Incubate the bacterial culture slants  at 30°C – 35°C for 24 hours and fungal culture slants at 20 °C – 25 °C for 48 hours in case of C. albicans and 72-120 hours for A. Brasiliensis.
5.2.5	After completion of incubation period observe the slants. If the growth is not satisfactory then again streak the slants. scrape the growth from the slants using a sterile nichrome loop in 10 ml sterile Buffered Sodium Chloride Peptone Solution which is considered as culture stock solution. Thoroughly mix for about 1 minute.
5.2.6	Prepare the dilutions of known disinfectants such as Dettol and Savlon  from 0.5 % to 5 %  i.e. 0.5, 1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0. Report in Annexure No. 02.
5.2.7	Eg. For Preparing 0.5%  Dettol solution in 100 ml of water 0.5 ml Dettol + 99.5 ml Sterile Purified Water = 100 ml of 0.5% Dettol solution. Refer Annexure No. 04 for dilution preparations.
5.2.8	For IPA from 50 % to 100% i.e. 50, 60, 70, 80, 90, 100. Label the test tubes as per above dilutions. Using sterile purified water make up to volume to get required concentration. Report in Annexure .
5.2.9	Eg. For Preparing 70%  IPA solution in 100 ml of water 70 ml IPA + 30 ml Sterile Purified Water = 100 ml of 70% IPA solution. Refer Annexure No. 04 for dilution preparations.
5.2.10	Prepared dilution concentrations should be mixed well (Dettol, Savlon and IPA). Dispense these 100 ml disinfectant solutions in 10 tubes of 10 ml each and add to this, 0.1 ml of individual culture stock solution (E. coli, S. abony, P. aeuruginosa, S. aureus, C. albicans and A. brasiliensis.  Shake and keep the tubes standing for 5 minutes. Consider this as Dilution 1.
5.2.11	Mark sets of tubes containing 9 ml of sterile Nutrient broth or SCDM and SDB, according to the dilutions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% and 5.0%) for Dettol and Savlon.
5.2.12	Prepare 50%, 60%, 70%, 80%, 90% and 100% dilution solutions for IPA.
5.2.13	Add 1 ml of each prepared dilution(Dilution 1) of disinfectant containing bacterial culture to the 9 ml of sterile nutrient broth or SCDM and fungal cultures in SDB. Consider this as Dilution 2.
5.2.14	For positive control inoculate 0.1 ml of bacterial culture stock solution of each organism directly in each separate 9.9 ml of nutrient broth or SCDM and fungal cultures in SDB.
5.2.15	Keep one test tube of nutrient broth or SCDM and SDB without inoculation as negative control.
5.2.16	Shake all the tubes and then incubate at 30 to 350 C  for bacteria for 24 hours and at 20 to 25C0 C for fungi 48 hours (C. albicans) and upto 120 hours (A. brasilliensis).
5.2.17	Minimum Inhibitory Concentration of a disinfectant shall be determined depending on the turbidity or clearity of the media in test tubes. If the media is found turbid then it should be considered as bacterial or fungal growth. If found clear then it shall be considered as inhibition of growth.
5.2.18	Observe the inhibition of the growth in tube and make entries of results in Report of Minimum Inhibitory Concentration of Disinfectant Annexure depending on type of disinfectant used.
5.2.19	For Confirmation, prepare SCDA and SDA plates as per SOP. Pipette out 0.1 ml from tubes that showed no visible growth and spread on the SCDA and SDA plates and incubated the plates for 24 hours at 30 – 35° C for bacteria(SCDA) and 48 hours to 120 hours at 20-25° C for fungi (SDA). There should not be any growth on the plates, which confirms Inhibition of specified organisms. Report in Annexure. If growth is observed, discard the plates and perform new study.
5.2.20	Select the concentration of disinfectant for validation of disinfectants which shows growth inhibition at minimum disinfectant concentration for the above microorganisms.
5.2.21	Frequency: Initially (One time) for routine disinfectants or immediately before the introduction of a new disinfectant.

6. Definitions / Abbreviation:

• Definitions:
• Abbreviation:

Abbreviation	Expansion
°C	Degree Centigrade
0.1 N	0.1 Normal
0.1 N	0.1 Normal
0.1 M	0.1 Molar
ml	millilitre
psi	Per square inch
+	Plus
IPA	Isopropyl Alcohol.
SCDA	Soyabean Casein Digest Agar
SDA	Saborauds Dextrose Agar
SDB	Saborauds Dextrose Broth
SCDM	Soyabean Casein Digest Medium

 

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SOP Title: Maintenance of Various Cultures used in Microbiological Analysis

1. Objective:

To lay down the procedure for Maintenance of Various Cultures used in Microbiological Analysis in Microbiology section of Quality Control Department.

2. Scope:

This procedure is applicable for Maintenance of Various Cultures used in Microbiological Analysis section of Quality Control.

3. Responsibility:

3.1 Quality Control Department: To prepare and review the SOP. To follow the procedures for Maintenance of Various Cultures used in Microbiological Analysis 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	Restrict the access to handle all live Microbial Cultures only to qualified and authorized persons.
5.1.2	All prepared Culture media and their components should be stored away from light and exposure to direct sunlight should be avoided at all times.
5.1.3	If any culture and surrounding debris is spilled (e.g. glass, cotton wool plugs) must not be touched with unprotected hands.
5.2	Procedure for Maintenance of culture:
5.2.1	Microorganisms used for Microbiological analysis should be procured from National or International Institute.
5.2.2	These Cultures are generally supplied in vacuum sealed glass ampoules or vials in freeze-dried condition or Sub Cultured on slants.
5.2.3	On receipt of the Culture, check the name of the organism and strain number as per the requirement. Check for physical damage if any.
5.2.4	Record the receipt details of Mother Culture in Annexure.
5.2.5	Use hand gloves, Nose mask and Overgown while handling live Cultures.
5.2.6	Report any incidence of spillage of live Microbial Culture or Microbial contamination to the Quality Control Head who will review the situation and take necessary measures.
5.2.7	Transfer the Cultures to Microbiological area after following Entry and Exit Procedure of Microbiological Section.
5.2.8	Carry out tests for identification and purity check of Culture slant or vials  as per SOP.
5.2.9	If the purity check fails, inform the Department Head and file an incident. Repeat the purity check.
5.2.10	If purity check fails again, inform the Department Head and discard the Culture as per Procedure for Destruction of Cultures and Microbial waste-SOP. Procure new Mother cultures.
5.2.11	If the purity test passes then proceed for the Maintenance of Cultures such that each Culture should never be used for more than five passages from its Original Mother Culture. The mother culture which is received from the supplying agency should be considered as Second passage.
5.2.12	Prepare specific growth medium as per Procedure for Media Preparation and Sterilisation- SOP.
5.2.13	Disinfect hands by using 70% v/v Iso propyl alcohol.
5.2.14	Disinfect the outer parts of Culture slant, ampoules, tubes or vials with 70% v/v isopropyl alcohol.
5.2.15	Break the Seal and remove the cotton plug from the neck of tube, vial or ampoule. Flame the mouth of tube by using a burner to sterilize the mouth of the tube to prevent contamination.
5.2.16	Transfer with a Nichrome wire a loopful of each culture in separate 10 ml tube containing 0.9% v/v sterile saline solution or Buffered Sodium Chloride Peptone pH 7.0. Refer SOP for preparation of reagents.
5.2.17	Mix the inoculated tube well to disperse the microorganisms and incubate at required temperature for 18-24 hours. Bacterial cultures at 30-35°C and fungal cultures at 20-25°C.Consider this as Culture suspension.
5.2.18	Before Sub Culturing ensure that fresh slants should attain room temperature if stored in refrigerator as microorganisms may not grow properly on cold temperatures. (For slant preparation refer SOP). Streak a loopful of Culture from 24 hours old Culture suspension on the respective media slants and incubate at 20-250 C for fungi and 30-350C. Streak 2 Slants each.
5.2.19	Incubate Bacterial cultures at 30-35°C for 48 hours and fungal cultures at 20-25°C for 72 hours Aspergillus brasilliensis can be incubated upto 120 hours for luxuriant growth. Use SCDA slants for bacteria and SDA slants for Fungal cultures.
5.2.20	First slant will be Mother Culture slant, second will be Working Culture slant, incubate at specified temperature as above.These slants along with initial inoculated Mother Culture suspension (point no.5.2.16) should be used only for culture identification and Purity check.
5.2.21	Proceed for the Identification and Purity Check of cultures as per SOP.
5.2.22	Label the slants and preserve the culture suspension in the refrigerator at 2° C to 8° C.
5.2.23	Streak each of the cultures on separate Nutrient Agar Plates from the culture suspension (Point No. 5.2.16) or the above slants (refer point No. 5.2.20) for obtaining pure isolated colonies which should be used for Identification tests. Incubate Bacterial cultures at 30-35°C for 48 hours and candida albicans at 20-25°C for 48-72 hours. Aspergillus brasilliensis should be incubated at 20-25°C for upto 120 hours for luxuriant growth.
5.2.24	Perform the Identification and Purity Check as per SOP.
5.2.25	If all the cultures are complying with Identification and Purity tests then proceed further to prepare  Master Culture Slants from culture suspension (Refer point No. 5.2.16)
	Preparation Of Master Slants
5.2.26	Prepare Six Master Culture Slants from the 10 ml of Culture suspension(Refer point No. 5.2.16). Master slants should be labelled as M1, M2, M3, M4, M5 and M6. Store at 2 – 80C.
5.2.27	Streak a loopful of the mother Culture suspension on the surface of 6 agar slants of Soyabean Casein Digest Agar for each Bacterial Culture and Sabourauds Dextrose Agar with chloramphenicol (SCA) or without antibiotic (SDA) for each fungal culture(Third passage). Incubate as per point No. 5.2.23 and observe the growth. If growth is not proper then streak again. These six Master slants shall be used for preparation of working and reference slants for next 12 months. Each Master slant is valid for 2 months.
5.2.28	Label the Agar Slants as Master Slants as follows. Eg. E. coli. For example MS/EC/01/03. MS -Master Slant EC – code number of organism 01 – serial No. of slant and 03 – passage No.
5.2.29	Incubate at 30 to 35°C for 48 hrs (Bacterial Cultures) and 20 to 25°C for 72-120 hours (Fungal Cultures).
5.2.30	Check for growth, if satisfactory growth is not observed then inform the Department Head and Subculture again on fresh agar slants.
	Preparation Of Working and Reference Slants
5.2.31	Prepare 2 SCDA slants for bacteria and SDA agar slants for fungi for each Culture from Master Slant. Prepare Media as per SOP.
5.2.32	Incubate the slants at 30 to 35°C for 48 hrs (Bacterial Cultures) and 20 to 25°C for 72-120 hours (Fungal Cultures). Consider this as Passage 4.
5.2.33	Keep one slant as Working Slant and another as Reference Slant, Working Slant should be use for routine analysis for 1 month from date of preparation.
5.2.34	Before due period, from Reference Slant further Sub Culture on two SCDA and SDA slants, use one for routine analysis as Working Slant  and another one as Reference Slant (Passage 5).
5.2.35	Working Slants should be used for a period 1 Month. If there is any accidental contamination with Working Slant, then prepare new working slant by using reference slant and use as a working slant for routine analysis.
5.2.36	Allocate the Reference Number to each Working Slant and Reference Slant for example WS or RS /EC/01/04 or 05. WS – Working Slant RS – Reference Slant EC- code no. of Organism. 01 – serial No. of Master Slant and 04 or 05 – passage No.
5.3.37	Record the sub Culturing details in Annexure and label the slants with as per annexure. Store the Working and Reference Slants at 2° C to 8° C.
5.2.38	Discard the expired Working Slant and Reference Slant as per procedure for Destruction of Cultures and Microbial Waste-SOP. The Master slant shall be discarded only when the incubation period of working and reference slant is completed and proper growth is observed.
5.2.39	If there is delay in new Culture, use 5th passage Reference Slant as a Working slant for another one month. Refer Point No. 5.2.34.
5.2.40	Procure a new Mother Culture before the 5th passage validity gets over.

 

 

6. Definitions / Abbreviations:

• Definitions :
• Abbreviations :

Abbreviation	Expansion
v/v	Volume by volume
No.	Number
°C	Degree Centigrade
SCA	Sabouraud Chloramphenicol Agar
SDA	Sabouraud dextrose agar
SCDA	Soyabean casein digest agar
MS	Master Slant
RS	Reference Slant
Ec	code no. of organism

*Note – Ready to use SOP available in “DOWNLOAD” Section.

 

 

 

SOP Title: Validation of Laboratory Autoclave located in Microbiology Laboratory

1. Objective:

To lay down the procedure for Validation of Laboratory Autoclave in Microbiology section of Quality Control Department.

2. Scope

This procedure is applicable for Validation of Laboratory Autoclave section of Quality Control Department.

3. Responsibility:

3.1 Quality Control: To prepare and review the SOP. To follow the procedures for Validation of Laboratory Autoclave in Microbiology section 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:

Sr. No.	Procedure
5.1	Safety Precautions:
5.1.1	Ensure that the appropriate protective equipments are used during handling of Autoclave.
5.1.2	Check the water level before each cycle in the Autoclave.
5.1.3	Ensure that the instrument is cleaned and free from dust and is disconnected from the mains after use.
5.2	Procedure to carry out validation of Autoclave :
5.2.1	Protocol for Autoclave validation should be prepared.
5.2.2	Validation of Autoclave is carried out for 1)    Heat distribution/penetration study with temperature Mapping. 2)    Sterilization study of Biological Indicators.
5.3	Heat distribution study with temperature Mapping:
	Acceptance criteria: The load penetration study carried out in Autoclave should comply with the acceptance criteria of respective heat distribution study for Empty load, half load and full load.

 

	Frequency:  Initial once after installation and then once in a Year.
5.3.1	To ensure that the autoclave is capable to attain the temperature of 121°C during the sterilization hold at a specified hold period with the steam pressure on the pressure gauge is 15 psi.
5.3.2	Ensure that the autoclave temperature and pressure gauges are calibrated. Check the calibration status of Data logger and sensors used for calibration.
5.3.3	Protocol for validation and revalidation of autoclave to be prepared as per SOP.
A.	Heat Distribution study at 121°C / 15 psi  in Empty chamber
5.3.3	Empty cycle load heat distribution study shall be performed.
5.3.4	Fill purified water in autoclave upto the mark before starting.
5.3.5	Set the parameters for the sterilisation cycle set to be operated.
5.3.6	Insert minimum 8 to 12 nos. of sensors or suitable numbers depending upon the volume of autoclave into the chamber through the port provided for Autoclave.
5.3.7	Place one sensor near the autoclave controller probe, one sensor near the drain and one sensor in the middle position, distribute the remaining probes uniformly in the chamber. Place a chemical indicator (Autoclave sterilization indicator) tape/strip and a biological indicator near each of the first three sensors mentioned above. Define Annexure for sensor locations.
5.3.8	Seal the logger port with silicon sealant so that steam leakage does not take place.
5.3.9	Connect the sensors to a suitable calibrated data logger which can scan and print the actual temperature with time.
5.3.10	Set the Autoclave to attain the desired temperature of 121°C at 15 psi during the complete sterilisation hold period.
5.3.11	Start the autoclave cycle after ensuring all the necessary precautions are taken. In Empty load cycle the chamber must be totally empty except for data logger sensors and chemical and Biological indicators.
5.3.12	Start the data logger recording once the cycle is started; record the actual temperature at an interval of 1 minute during sterilization cycle of 15 minutes or 30 minutes by using a data logger. After completion of sterilization, record the data in datalogger until temperature drops down to 100 °C.
5.3.13	The temperature spread should be within the range of 121°C to 124°C during sterilisation for set temperature of 121°C at 15 psi pressure for 15 minutes and 30 minutes.
5.3.14	Review the temperature data for all sensors printed on datalogger print, check and compare the temperature observed at different location.
5.3.15	For Sterilization study of Biological Indicators refer point No. 5.4.
5.3.16	For Heat Distribution study details record in Annexure.
5.3.17	Define Annexure for the list of cycles and for sterilization parameters.
B.	Heat Distribution/Penetration study at 121°C / 15 psi for Media(Half Load/Full Load)
5.3.18	Media Sterilization cycle is validated for 121°C at 15 psi pressure for 15 minutes.
5.3.19	Place one sensor near the autoclave controller sensor, one sensor near the drain and one sensor in the middle position, distribute the remaining sensors uniformly in the chamber. Place a chemical indicator (Autoclave sterilization indicator) tape/strip and a biological indicator near each of the first three sensors mentioned above. Define sensor locations.
5.3.20	Use same numbers of sensors as used in the Empty load distribution cycle.
5.3.21	Carry out the autoclave cycles as per Empty load method. Refer Point No.5.3.4 to 5.3.17.
5.3.22	Prepare Solid and Liquid media for carrying out the media cycles as per SOP. Prepare preferably SCDA and R2A agar medium as solid media. SCDM and MacConkeys broth shall be prepared as liquid media. If these media are not available, any other media can also be used. Enter Media prepared in Annexure.
5.3.23	Half load refers to the the media load which occupies half or minimum space in the Autoclave during sterilization cycle. Record in Annexure.
5.3.24	Full load refers to the Media load which occupies the maximum loadable space in autoclave during sterilization cycle. Record contents of cycle in Annexure.
5.3.25	Place sensors in between the media glasswares and in the media at different locations to map the temperature distribution and penetration of steam.
5.3.26	For Steam sterilization study of Biological Indicators refer point No. 5.4
5.3.27	For Heat Distribution study record in Annexure.
C.	Heat Distribution/Penetration study at 121°C / 15 psi for Garments/ Glasswares/Accessories.(Half Load/Full Load)
5.3.28	Garments/ Glassware/Accessories Sterilization cycle is validated for 121°C at 15 psi for 30 minutes as to sterilize the garments and other items for maximum duration.
5.3.29	Place one probe near the autoclave controller probe, one probe near the drain and one probe in the middle position, distribute the remaining sensors uniformly in the chamber. Place a chemical indicator (Autoclave sterilization indicator) tape/strip and a biological indicator near each of the first three sensors mentioned above.
5.3.28	Use same numbers of sensors as used in the Empty load distribution cycle.
5.3.30	Carry out the autoclave cycles as per Empty load method. Refer point No. 5.3.4 to 5.3.17.
5.3.31	Half load refers to the the Garment/Glassware/ Accessories load which occupies half or minimum space in the Autoclave.
5.3.32	Full load refers to the Glasswares/ Garments/ Accessories which occupies the maximum loadable space in autoclave. Record in Annexure for loading pattern and contents of cycle.
5.3.33	Place sensors in between the Garments and glasswares at different locations to map the temperature distribution and penetration of steam.
5.3.34	For Analysis of Biological Indicators refer point No. 5.4
5.3.35	For Heat Distribution study record in Annexure.
D.	Heat Distribution/Penetration study at 115°C / 10 psi for Media.(Full Load)
5.3.36	Media Sterilization cycle for some media like Rappaport is to be validated for 115°C / 10 psi pressure for 15 minutes.
5.3.37	Follow the same procedure as media cycle point No. 5.3.19 to 5.3.25
E.	Heat Distribution study at 121°C at 15 psi for Media Discard autoclave.(Full Load)
5.3.38	Media Decontamination/Sterilization cycle is validated for 121°C at 15 psi for 30 minutes.
5.3.39	Prepare Solid and Liquid media (Refer point No. 5.3.22) for carrying out the media decontamination cycles. Glassware should be included for this type of cycle. Record media prepared in Annexure.
5.3.40	Perform the validation as per Empty cycle method point No. No.5.3.4 to 5.3.17.
5.4	B) Steam Sterilization study of Biological Indicators
	Frequency:  Once a year during validation, every six months as to check sterilization efficiency or as per validation requirements.
5.4.1	Place 3 spore strips/ampoules of Biological indicator Geobacillus stearothermophilus (Bacillus Stearothermophilus) ATCC 7953 in the most difficult to sterilize positions in the chamber.
5.4.2	Sterilize in cycles as per procedure for individual cycle.
5.4.3	After sterilisation, incubate each spore strip in a individual 10 ml Soyabean Casein Digest Medium test tube for 7 days at 55°C to 60°C. For media preparations refer SOP QC 034. Keep positive and Negative control with the test strips. Positive control (Unexposed) should be incubated in SCDM along with test strips for 7 days at 55°C to 60°C. Negative control (Unexposed) Indicators to be incubated at 30°C to 35oC for 7 days for checking the spore growth if any in normal conditions. Check the Biological Indicator tubes daily for 7 consecutive days and record in Annexure.
5.4.3	The unexposed spore strip (positive control) shows growth (turbidity), indicates that spores are viable.
5.4.4	Negative control shows no growth (turbidity) means spores are not activated.
5.4.5	If  the  positive  control  shows  turbidity  and  all three  test  samples  are  not showing  turbidity after 7 days of   incubation  period,  then  the Autoclave  is  working  satisfactory  as  per  the  requirements.
5.4.6	In case of ampoules, incubate the ampoules after sterilization at 55°C to 60°C for 48 hours. Keep positive and Negative control with the test ampoules. Incubate positive control (Unexposed) along with test ampoules. Incubate the negative control (Unexposed) at 30°C -35°C for 48 hours.
5.4.7	Exposed ampoules should not show any colour changes.
5.4.8	The unexposed ampoule (positive control) shows colour change, indicates that spores are viable.
5.4.9	Negative control should show no colour change.
5.4.10	Record the data on Annexure
5.4.11	If  validation fails then do not use the Autoclave. Inform the Head of Quality control and the Head of Quality assurance,  raise an incident and investigate  the  reasons  &  take  immediate  action  to  rectify  the  same  and  perform  Revalidation.
5.4.12	Decontaminate the spore strips in autoclave before disposing off. Refer SOP for Decontamination of culture media and Microbial waste.

 

6. Definitions / Abbreviations:

 

• Definitions
• Abbreviations :

Abbreviation	Expansion
psi	Pressure square inch

 

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SOP Title: Procedure for Validation of Laminar Air Flow

1. Objective:

To lay down the procedure for Validation of Laminar Air Flow / Reverse Laminar Air Flow.

2. Scope

This procedure is applicable for Validation of LAF in the Microbiology, Sampling Booth RLAF and Dispensing Booth RLAF.

3. Responsibility:

3.1   Quality Control Department: To prepare and review the SOP. To follow the procedures for  Validation of LAF 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.

Procedure:

5.1	Safety Precautions/Instructions:
5.1.1	Ensure that Laminar Air Flow Unit is properly installed in a clean environment.
5.1.2	Ensure that pre filters of Laminar Air Flow Unit are clean.
5.1.3	Before Starting the activity ensure that LAF is properly Cleaned.
5.2	Procedure for Validation:
5.2.1	Validation of LAF is carried out by performing following tests. 1. HEPA Filter Integrity by PAO Test. 2. Air flow Velocity: Unidirectional Airflow. 3. Non-Viable Particle Count Test.
5.2.2	Validation study of LAF is applicable to LAF in Microbiology, Sampling  Booth RLAF and Dispensing Booth RLAF.
5.3	1. HEPA Filter Integrity by PAO Test:
5.3.1	LAF consists of two filters and are named as F1(Left side) and F2(Right side).
5.3.1	Arrange single phase power supply as well as air connection with 20psi (1.5kg) pressure.
5.3.2	Switch ON Aerosol Generator System, before starting the measurements and Connect the Spike Guard to Photometer.
5.3.3	Start the air to generate the aerosol particles of PAO at 20 psi pressure by using Aerosol Generator System.
5.3.4	Direct the generator face to the return air point or fresh air intake of the LAF.
5.3.5	Connect one end of the tube to filter face upstream port and another end to Photometer in Downstream port.
5.3.6	Switch ON the photometer and set it to zero.
5.3.7	Put the photometer to upstream mode and visually note the reading on display, the reading should be in between 20 mg/Ltr to 80 mg/Ltr.
5.3.8	If it is between the above range then set the value to 100%, wait until the photometer displays 100% upstream concentration.
5.3.9	Switch ‘ON’ the photometer, enter the clear mode and enter zero. Wait until photometer display shows ‘0’ (zero).
5.3.10	Change photometer mode to downstream mode and scan the entire filter by holding the probe approximately one inch away from the filter face.
5.3.11	Scan the corners of HEPA filter for checking any leakages.
5.3.12	If the reading on the photometer is more than 0.01%, rectify the leakages by tightening the locking bolts.
5.3.13	Repaired patches on filters should not exceed maximum of 5% of the total filter face area and the maximum width x length of each patch should not be more than 1.5 inches.
5.3.14	Total number of patches should not exceed 5 numbers / filters.
5.3.15	Note down the final readings after rectification.
5.3.16	If the reading on the Photometer is more than 0.01% or total number of patches exceed 5 numbers per filter, then HEPA Filter Integrity Test fails.
5.3.17	Replace with new HEPA Filter and carry out PAO Test again.
5.3.18	Acceptance Criteria: During scanning, percentage of the PAO penetration shown by photometer should be less than 0.01% throughout the filter.
5.4	2. Air Flow Velocity: Unidirectional Airflow
5.4.1	Switch ‘ON’ Anemometer for measuring Air Velocity before starting the measurement. Use Vane type anemometer.
5.4.2	1        Filter face Air Velocity should be measured at distance of about 150 mm.
5.4.3	Switch ‘ON’ the LAF air flow,  place the Anemometer in front of the filter surface at a distance of about 150 mm. Check the air velocity readings at the four corners and centre. Note readings as 1, 2, 3, 4, and 5 on annexure no. 01. Refer below figure.        1                                                                         4                                    5 2                                                                                     3
5.4.4	Measuring time at each position should be at least 10 seconds. Record the average, maximum and minimum values. Note down the readings in annexure.
5.4.5	Acceptance Criteria: The measured average velocity for each filter shall be within 0.36m/s to 0.54 m/s as per ISO-14644.
5.5	3. Non- Viable Particle Count test.:
5.5.1	The test method specifies the measurement of air borne particle concentration with size distributions between 0.5µm and 5.0µm.
5.5.2	Measurements can be made in any of three states as-built, at-rest and operational.
5.5.3	LAF Comes under ISO CLASS 5 Classifications. At rest the particles should not exceed 3520 (0.5µm) size and 29 (5 µm) size for compliance of LAF for Non- Viable Particle Count test.
5.5.4	Switch ‘ON’ the LAF for 2 hours before starting the measurements.
5.5.5	Connect the Spike Guard to particle counter and set the Particle counter at center position or as per the the working level of LAF.
5.5.6	Position the sampling probe vertically upward.
5.5.7	Switch ‘ON’ the Particle Counter and adjust the Particle Counter for ‘0’ display.
5.5. 8	Start taking readings of LAF and take print of the reading. Take at least 3 readings from same position in LAF. Refer ISO-14644 for the test.
5.5.9	Thermal paper print should be pasted on a blank page and should be Photocopied immediately.
5.5.10	Record validation data on Annexure.
5.5.11	Acceptance Acceptance Criteria: The LAF should meet the criteria as per ISO Class 5 classifications i.e. NMT 3520 particles/m3, for particles size equal to or greater than 0.5 µm and NMT 29 particles/m3 for particles size equal to or greater than 5 µm.
	Frequency – Yearly by External agency
5.5.12	After the test is completed, the LAF and the surrounding areas shall be cleaned properly.
5.5.13	If the validation study fails, file an deviation, rectify the problem and again validate the LAF.

 

6. Definitions / Abbreviations :

• Definitions :

 

• Abbreviations :

Abbreviation	Expansion
ISO	International Standard Operating
RLAF	Reverse Laminar Air Flow
LAF	Laminar Air Flow
AHU	Air Handling Unit
psi	per square inch
HEPA	High efficiency particulate air
PAO	Poly Alfa  Olefin
mg	milligram
%	percent
lts	litres
IPA	Iso Propyl Alcohol
ft	feet
avg	average
μm	micrometer

 

*Note – Ready to use SOP available in “DOWNLOAD” Section.

 

SOP Title: Environmental Monitoring

1. Objective:

To lay down the procedure for Environmental Monitoring of Microbiology Lab and Production Area.

2. Scope:

This procedure is applicable for Environmental Monitoring of Microbiology Lab and Production Area.

3. Responsibility:

3.1 Quality Control: To prepare and review the SOP. To follow the procedures for Environmental Monitoring of Microbiology Lab and Production Area 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	Microbiologist should wear hand gloves, nose mask and disinfect the hand gloves with 70% v/v IPA before carrying out operation.
5.2	Procedure for plate exposure:
	Frequency: Every 15 days ± 3 days.
5.2.1	Refer Procedure for Media Preparation and Sterilisation – for preparation of Soyabean Casein Digest Agar plates.
5.2.2	Pre Incubate these plates at 30-35°C for 24 hours in the incubator.
5.2.3	Carry these pre-incubate SCDA plates through canister to Production Areas. For Plate exposure locations, refer Annexure for location of plate exposure.
5.2.4	Expose pre-incubated sterile SCDA plates in classified areas at locations as per Annexure. Expose plates on a SS plate exposure stand, or any clean surface like instruments minimum above one feet from ground.
5.2.5	Mark the plates with location number and date of exposure.
5.2.6	Open the lid of plate and keep adjacent to plate on the exposing surface. Never expose the plates on floor.
5.2.7	Expose these plates for two hours.
5.2.8	At the end of the exposure time close the lid of plate and carry the plates in Microbiology section through canister.
5.2.9	Incubates the plates at 20-25°C for 72 hours and continue the incubation at 30 to 35°C for 48 hours along with an unexposed plate as Negative control.
5.2.10	After completion of specified incubation period, count the number of colonies on each plate.
5.2.11	Ensure that the negative control does not show any growth otherwise the results are invalid in this case raise an incident report.
5.2.12	Report the Total bacterial and fungal count observed in Environment Monitoring, refer Annexure.
5.2.13	If the observed microbial count is exceed than the alert limit, intimate the concerned Department by forwarding the note through Quality Assurance Department.
5.2.14	Stop the operation from the affected areas till further intimation by Quality Assurance.
5.2.15	After necessary action taken by related Department, Microbiologist should repeat the Environment Monitoring activity for verification.
5.2.16	Dispose off the Media plates by following the procedure given in SOP (Decontamination of Media and Cultures.)

6. Definitions and Abbreviations :

• Definitions

 

• Abbreviations

Abbreviation	Expansion
SCDA	Soyabean Casein Digest Agar
v/v	Volume by volume
%	Percentage
°C	Degree centigrade
±	Plus or minus
IPA	Isopropyl  Alcohol

 

*Note – Ready to use SOP available in “DOWNLOAD” Section.

 

SOP Title: Sampling of Water for Microbiological Analysis

 

1. Objective:

To lay down the procedure for Sampling of Water for Microbiological Analysis.

2. Scope:

This procedure is applicable for Sampling of Water for Microbiological Analysis in the

Microbiology Section of Quality Control Department.

3. Responsibility:

• Quality Control: To prepare and review the SOP. To follow the procedures laid down for Sampling of Water for Microbiological Analysis as per this SOP.
• Quality Assurance Department: To review and approve the SOP and Annexure.

4. Accountability:

Head – Quality Control / Head – Quality Assurance

5. Procedure:

5.1	Safety Precautions/Instructions:
5.1.1	Before carrying out sampling operation microbiologist should wear personal protective equipments such as protective hand gloves, nose masks and disinfect the hand gloves with 70% v/v IPA.
5.1.2	Immediately cap the bottle After collecting water sample.
5.2	Operation Procedure for Sampling :
5.2.1	For water sampling, carry the transparent screw cap bottle or stoppered test tubes or stoppered flask of 100 ml capacity which is previously sterilized one in a tray or suitable container.
5.2.2	Prior to taking samples from the user points, sanitise the sampling point by spraying with 70% IPA and drain OFF about 2 – 5 litres of water.
5.2.3	Collect the sample aseptically into the sterile container, and secure the lid tightly leaving a minimum headspace in the bottle.
5.2.4	After sampling, sanitise the port of the sampling points with 70% IPA using a sprayer or mop the sample port.
5.2.5	Affix label or mark with marker pen on the sample bottle with sampling point number, Date and time of sampling.
5.2.6	Bring all the samples to microbiology section, make entry in water sample Inward register and assign the A.R.No. Refer Annexure .
5.3	Assigning the A. R. Number :
5.3.1	Code number for Raw Water as RW, for Potable Water as PO, for Purified Water as PW and for Drinking water as DW.
5.3.2	Collection points for Raw Water are–IDC water (IW) and borewell water (BW).
5.3.6	A.R.Number for raw water (RW) for IW & BW collection point, Potable water (PO) and for Purified water (PW) is as follows. 1 2 / 3 4 / 5 6 7
5.3.7	First two characters (1 and 2) stand for code number of sample. Third, fourth characters (3 and 4 ) Stands for last two digit of year 2018 Fifth, sixth and seventh characters (5, 6, 7) stand for serial number.
5.3.8	For Ex.  : PW/24/001 PW : Code number stand for Purified water 24  : Stands for last two digit of year 001  : First serial number of the series
5.3.9	Sampling points IW & BW-(near solvent store room) comes under Raw water and are allotted one A.R .No.
5.3.10	Sampling points coming under Potable water- SP3, SP4, SP5, SP6 (in RO unit) are allotted one A.R.No. and SP7, UP1, UP2, UP3 and UP4 (in Production area) and UP5(Quality Control)are allotted one A.R.No.
5.3.11	Single sampling point for drinking water. i.e. canteen
5.3.12	The serial number will continue till the end of the current academic year and should not be   repeated.
5.3.13	Analysis to be carried out within 2 hrs of sampling. If not then refrigerate the samples at 2°C to 8 °C up to 8 hours.
5.3.14	Due to some reason if not possible to carry out analysis within 8 hrs of sampling  than collect a fresh sample for analysis and this information should be transferred to water inward register with justification.
5.3.15	For the location of sampling points, refer the list of sampling point, Annexure.
5.3.16	Sampling should be carried out as per the sampling plan attached as Annexure.
5.3.17	Drinking water from the canteen should be analysed twice in a month for TAVC and pathogen tests.
5.3.18	The Raw water points should be checked for pathogen and TAVC test every working day and other user points(UP2 to UP5) once a week for TAVC and Pathogens.
5.3.19	Main User point UP1 should be analysed daily for TAVC test and once in a week for pathogen test as per Annexure .
5.3.20	If due to holiday or any other reason, it is not possible to analyse the sample on the scheduled day of the week, perform sampling and analysis of those points on next working day.
5.4	Seasonal validation of water system plan
5.4.1	Seasonal validation should be carried out in three seasons as follows. Winter   –  between December to January Summer – between April to May Monsoon – between July to August
5.4.2	Samples should be drawn daily on fifteen consecutive working days.
5.4.3	Sampling should be carried out as per the sampling plan attached to Annexure.
5.4.4	For microbiological testing should be done every user points as per specifications.

 

6. Definitions / Abbreviations :

• Definitions :
• Abbreviations :

Abbreviation	Expansion
TAVC	Total Aerobic Viable Count
UP	Utility Point
SP	Sampling point
A. R. No	Analytical Reference Number
etc	etcetera
No.	Number
%	Percentage
v/v	Volume by volume
IPA	Isopropyl Alcohol

 

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