Category: Microbiology SOP

SOP Title: Operation and Cleaning of Laminar Air Flow Bench

 

1. Objective:

To lay down the procedure for Operation and Cleaning of the Laminar Air Flow Bench.

 

2. Scope:

This SOP is applicable for Operation and Cleaning of Laminar Air Flow Bench located in the Microbiology Section.

3. Responsibility:

• Quality Control: To prepare and review the SOP. To follow the procedures laid down for Operation and Cleaning of Laminar Air Flow Bench 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 LAF is fixed in the socket properly.
5.1.2	Wear personal protective equipment’s such as protective hand gloves, nose masks and over gown during operation of equipment.
5.1.3	Never work under LAF when the UV lamp is ON.
5.1.4	HEPA filters must never be touched by personnel and no attempt should be made to clean or disinfect filters using chemical or mechanical methods.
5.1.5	Do not disturb the air flow or switch off the instrument if analyses are not completed.
5.1.6	Avoid direct exposure of UV lights on skin and body parts.
5.1.7	Dispose pre-filters if they are dirty in appearance or when the Magnehelic gauze pressure shows less than half of its initial reading.
5.2	Operation  Procedure:
5.2.1	Switch ‘ON’ the equipment
5.2.2	Put ‘ON’ the switch of air flow blower.
5.2.3	Check the pressure on magnehelic gauze on the top of the equipment should be within the specified limits (between 05-15 mm).
5.2.4	Record the reading in Annexure.
5.2.5	If the reading is outside the operating limits, the Microbiologist should inform Quality Control Head immediately.
5.2.6	After investigation and rectification, analysis to be carried out.
5.2.7	Put ‘ON’ the switch of UV light and close the cover of LAF unit and keep UV lamp ON for 30 minutes. Avoid direct view of burning of UV light.
5.2.8	After 30 minutes switch ‘OFF’ the UV Light and put ‘ON’ the switch of tube light.
5.2.9	Maintain the record for UV Lamp burning hour in Annexure.
5.2.10	Care to be taken the UV tube burning hours should not exceed 2000 hrs.
5.2.11	Clean the laminar air flow Bench and side arms with 70 % Isopropyl alcohol. Record the data on Annexure.
5.2.12	Switch ON LPG cylinder and rotate knob clock wise direction and light burner with lighter. Perform all the microbiological work/analysis under LAF.
5.2.13	After the analysis, switch OFF the burner, by rotating the knob in anticlock direction
5.2.14	Switch OFF the LAF Unit.
5.2.15	Clean up any spillage immediately and sanitize the work surface, panels and surrounding area with 70 % IPA. Switch off tube light.
5.2.16	Switch OFF the main LPG connection
5.2.17	Put ‘ON’ the switch of UV light and keep it ON for 30 minutes.
5.3	Cleaning Procedure:
	Frequency: Daily twice i.e. before starting and after the completion of analysis.
5.3.1	De-dust the external surface of LAF with lint free cloth.
5.3.2	Before switching the LAF Unit ‘ON’, clean the LAF bench, side glass and grills with lint free nylon cloth damped with 70 % v/v IPA daily.
5.3.3	Clean the pre-filters & UV light fortnightly carried out by Maintenance department.
5.4	Validation:
	Frequency: Once in a year or after carrying out any major maintenance work
5.4.1	For Validation of LAF refer SOP ‘Procedure for validation of LAF’.
5.5	Preventive Maintenance:
5.5.1	Perform the preventive maintenance as per Annexure
5.5.2	If preventive maintenance activity is outsourced, the observations are to be transcribed in the Annexure and external party certificate to be attached.

  

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
LAF	Laminar Air Flow
HEPA	High Efficiency Particulate air
UV	Ultra Violet
hrs	Hours
LPG	Liquid Petroleum Gas
No.	Number
%	Percent
v/v	Volume by volume
IPA	Iso Propyl Alcohol
PM	Preventive Maintenance

 

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

 

SOP Title: Procedure for Operation, Cleaning and Calibration of Incubator

1. Objective:

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

2. Scope:

This SOP is applicable for Operation, Cleaning and Calibration of Incubator (30-35°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 Incubator (30-35°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	Switch ON the mains.
5.2.3	Set the incubator to its desired temperature 32° (i.e. 300 C to 350 C) by rotating the temperature knob.
5.2.4	Green coloured indicator light will glow indicating the working of the thermostat is satisfactory.
5.2.5	Incubator temperature monitoring should be recorded twice in a day. Morning session and end of the shift and its record shall be maintained in Annexure ‘Temperature Monitoring Record’.
5.2.6	All petri plates, tubes, slants placed in the incubators should be labelled with sample name, batch number and date manually using a marker pen.
5.2.7	Update the “Instrument/Equipment Usage Log Book” for Incubator as per SOP.
5.2.8	After appropriate time of incubation, remove the samples to prevent overcrowding or contamination of incubator.
5.2.9	Do not switch OFF or disturb the incubators after setting the desired temperature parameter during performance of unit.
5.2.10	If any time incubator shows the reading out of range, follow the procedure as per deviation SOP.
5.2.11	The equipment service engineer must be contacted for appropriate 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 : 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	Parameters Set value Acceptance Criteria   Temperature 30°C 30°C ± 1°C 33°C 33°C ± 1°C 35°C 35°C ± 1°C
5.4.2	Generate the A. R. No. as per SOP and give requisition to QA department for calibration formats of equipment on the due date of calibration.

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
hrs	Hours
No.	Number
v/v	Volume by volume
°C	Degree Celsius
QC	Quality Control
A.    R. No.	Analytical Reference Number

 

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

 

SOP Title:-Operation, Cleaning and Calibration of Colony Counter

1. Objective:

To lay down the procedure for Operation, Cleaning and Calibration of Colony Counter.

2. Scope:

This SOP is applicable for Operation, Cleaning and Calibration of Colony Counter 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 Colony Counter 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 Colony Counter is fixed in the socket properly.
5.1.2	Ensure that the ON/OFF switch is working properly by switching “ON” the Colony Counter.
5.2	Operation  Procedure:
5.2.1	Check the validity of Calibration before starting any operations.
5.2.2	Switch ON the mains.
5.2.3	Switch ON the power button of instrument.
5.2.4	The internal light will be flashed ‘ON’ and the display will show ‘000’ figure.
5.2.5	Place the petri plate on the glass grid in inverted position and adjust the magnifying lens.
5.2.6	Now, remove the cap of marker pen and press firmly keeping the pen straight/vertical, on the petri plate where microbial colony is located.
5.2.7	The colony counter will register a count.
5.2.8	On pressing the tip of the marker a beep sound will be made and an ink dot will be marked on the petri plate.
5.2.9	Mark each and every colony one after the other by pressing the tip of the marker on the plate.
5.2.10	The number of colonies will be automatically displayed on the screen.
5.2.11	Note down the final reading from the counter.
5.2.12	When the counting is over, the screen shall be set to 000 by pressing the reset button.
5.2.13	Switch ‘OFF’ the instrument by pressing the power button again.
5.2.14	Update the “Instrument/Equipment Usage Log Book” for Colony Counter as per SOP.
5.2.15	If any time Colony Counter shows the reading out of range, follow the procedure as per SOP.
5.2.16	The equipment service engineer must be contacted for appropriate servicing/repair of the equipment followed by a recalibration that must be done before the reuse of the same.
5.3	Cleaning :
5.3.1	De-dust the external surface of colony counter and magnifier with lint free cloth.
5.3.2	Clean the external surface as well the magnifying lens of the colony counter with a wet cloth soaked in 70% v/v Iso Propyl Alcohol.
5.3.3	Frequency of cleaning is daily.
5.4	Calibration :
5.4.1	Frequency:   1)  Once in a year by external agency. 2) In-house calibration to be performed Quarterly or after carrying out any   major maintenance work.
5.4.2	For external calibration refer Annexure for calibration test to be performed and acceptance criteria.
5.4.3	Generate the A. R. No.as per SOP
5.4.4	Issue calibration report as per QA procedure.
5.4.5	Perform the In-house calibration of instrument as per Annexure and procedure mentioned below.
5.4.6	Ensure that the display shows ‘000’ figure.
5.4.7	Place the petri plate on the glass grid in inverted position and adjust the magnifying lens.
5.4.8	Now, remove the cap of marker pen and press the tip firmly for the given number of times as per define Annexure
5.4.9	The number of times marker pen has been pressed will be automatically displayed on the screen.
5.4.10	Note down the final reading from the counter.
5.4.11	Enter the details in calibration report as per Annexure. Ensure that the results obtained, comply with the acceptance criteria.     Parameters Set Count Observed Count Complies / Does Not Complies   Number Displayed on pressing of pen marker tip 01     20     40     60     80     100     200     300
5.4.12	Update the “Instrument/Equipment Usage Log Book” as per SOP
5.4.13	Affix the “Calibration Status Label” on the Instrument as per SOP.
5.4.14	If calibration activity is outsourced, the values are to be transcribed in the Annexure, and external party certificate to be attached.

6. Definitions / Abbreviations:

• Definitions:
• Breakdown: Any activity leading to operation of instrument other than the set parameters and/ or unusual sound or vibration observed in the instrument.
• Calibration: Tests performed to ensure that the instrument 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 instrument will function smoothly and to avoid breakdowns. These activities are performed as per predefined frequency.

• Abbreviations:

 

Abbreviation	Expansion
No.	Number
A.R. No.	Analytical Record Number
v/v	Volume by volume
QC	Quality Control
%	Percentage

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

 

SOP Title: Procedure for Operation and Cleaning of Microscope

  

1. Objective:

To lay down the procedure for Operation and Cleaning of Microscope.

 

2. Scope:

This SOP is applicable for Operation and Cleaning of Microscope located in the Microbiology Section.

 

3. Responsibility:

• Quality Control: To prepare and review the SOP. To follow the procedures laid down for Operation and Cleaning of Microscope 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:

 

Sr. No	Procedure
5.1	Safety Precautions/Instructions:
5.1.1	Wear personal protective equipments such as protective hand gloves during operation of instrument.
5.1.2	Be cautious when handling glass slides and cover-slips.
5.1.3	Cover the microscope with the polythene bag provided.
5.2	Operation  Procedure:
5.2.1	Place the microscope on a rigid, firm, non-shaking, flat surface.
5.2.2	Adjust the low power objective, by rotating the revolving nose piece in position.
5.2.3	Adjust the focus of light with the help of mirror and condenser.
5.2.4	Now position the required objective (low power, high power or oil immersion) by moving the resolving nose piece.
5.2.5	Place the slides on the stage, lower the objectives, while observing through the eye piece, till the field appears clearly.
5.2.6	If the oil immersion objective is in use, place one drop immersion oil on the slide before placing it on the stage and then lower the oil immersion Objective just to touch the oil drop.

 

5.2.7	Make the field clearer with the help of fine adjustment and then observe the slide.
5.2.8	Slide can be moved in left, right, upper and lower direction to observe the complete smear area with help of slide stage screw and coarse adjustment.
5.3	Cleaning Procedure:
5.3.1	Clean the microscope carefully with clean dry lint free napkin or a lens paper.
5.3.2	Clean the eye piece and nose piece with 70% v/v IPA applied on the lens paper.
5.3.3	After completion of observation, remove the slide, clean the objective lens and condenser with 70% v/v of IPA on the lens paper.
5.3.4	Cover the microscope with the polythene bag provided.

 

6. Definitions / Abbreviations:

• Definitions:
• Breakdown: Any activity leading to operation of instrument other than the set parameters or unusual sound or vibration observed in the instrument.
• Preventive Maintenance: Maintenance activity performed to ensure that the instrument will function smoothly and to avoid breakdowns. These activities are performed as per predefined frequency.
• Abbreviations:

 

	Abbreviation		Expansion
	No.		Number
v/v		Volume by volume
IPA		Iso Propyl Alcohol
QC		Quality Control
%		Percentage

  

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

 

 

SOP Title: Operation and Cleaning of Autoclave

1. Objective:

To lay down the procedure for Operation and Cleaning of Autoclave.

2. Scope:

This procedure is applicable Operation and Cleaning of Autoclave in the Microbiology Section.

3. Responsibility:
   • Quality Control: To prepare and review the SOP. To follow the procedures for Operation and Cleaning of Autoclave 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 the appropriate personal protective equipments are used during handling of Autoclave.
5.1.2	Do not open the Autoclave before the pressure attains the zero.
5.1.3	Check the water level before each cycle in the Autoclave
5.1.4	To achieve adequate sterilization, avoid overloading the autoclave
5.1.5	Ensure no items should touch the top or sides of the autoclave container.
5.1.6	Ensure containers do not touch each other, so that all surfaces are sterilized uniformly.
5.2	Procedure for Operation of Autoclave:
5.2.1	Ensure that the equipment is clean & free from dust and is disconnected from the mains prior to its use.
5.2.2	Open the lid of the autoclave.
5.2.3	Check the water level of the autoclave if necessary and fill the autoclave with Purified Water up to the red mark of the water level indicator.
5.2.4	Connect the plug to the mains supply.
5.2.5	Keep the pressure release valve in ON position.
5.2.6	Arrange loading in autoclave so that the maximum amount of surface can be exposed to the jets for better sterilization. Avoid crowding or stacking.
5.2.7	Ensure containers do not touch each other, so that all surfaces are sterilized uniformly.
5.2.8	Place empty flasks, test tubes or other non-porous containers on their sides with loose covers. This provides a horizontal pathway and prevents trapping of air pockets.
5.2.9	Load the glassware’s or media to be sterilized along with a strip of self adhesive chemical indicator tape.
5.2.10	Glassware’s are plugged with non-absorbant cotton.
5.2.11	Test tubes are sealed with autoclavable caps.
5.2.12	Petri plates and pipettes are sterilised in respective canisters.
5.2.13	Close the lid by sliding it and tighten all the nuts.
5.2.14	Switch ‘ON’ the mains and set the temperature at 121°C or desired temperature.
5.2.15	Allow the temperature to reach upto 100°C to remove out vapour along with trapped air and water droplets.
5.2.16	Close pressure release valve.
5.2.17	Slowly temperature will increase and reach set temperature.
5.2.18	Digital temperature display will show set temperature and the pressure gauze will display the pressure.
5.2.19	Set the desire pressure by adjusting pressure release valve.
5.2.20	Record the sterilisation cycle details in Annexure.
5.2.21	Refer SOP ‘Procedure for media preparation and sterilisation’ for media preparation.
5.2.22	For Glassware’s, sterilisation period is 30 minutes at 121°C at 15 psi. Media sterilisation period is 15 minutes at 121°C or as per media requirement.
5.2.23	If the autoclave cycle fails to be completed due to a power outage, the load must be discarded.
5.2.24	At the end of sterilization period, switch the mains off and wait for the pressure gauge of the autoclave to show zero pressure on gauge.
5.2.25	Open the lid of the autoclave.
5.2.26	Slowly open autoclave lid, Using heat resistant gloves carefully transfer the content to the tray.
5.2.27	Check the colour of chemical indicator tape used for sterilization cycle.
5.2.28	If colour change is not satisfactory, then discard the load and investigate the cause.
5.2.29	At the end of day’s work, open water drain valve and allow water to drain out.
5.2.30	For assigning the Autoclaving Load number for every cycle: This sequence is carried out for one calendar year.
5.3	Cleaning: Frequency is weekly
5.3.1	Switch OFF the autoclave.
5.3.2	Disconnect the plug from Mains supply.
5.3.3	Wipe the exterior surface of the autoclave with 2.5% savlon or dettol.
5.3.4	Scrub the chamber using 1% v/v (1 ml in 100 ml) of commercially available liquid soap solution.
5.3.5	Open the drain valve at the bottom and drain out the water into the sink.
5.3.6	Rinse the chamber thoroughly with water to remove all residual particles and detergent. Rinse finally with purified water.
5.3.7	Wipe the outside body of the autoclave with clean lint free cloth.
5.4	Preventive Maintenance:
5.4.1	Perform the preventive maintenance as per Define Annexure.
5.4.2	If preventive maintenance activity is outsourced, the values are to be transcribed in the Annexure, and external party certificate to be attached. Frequency of Preventive maintenance is Quarterly.

 

6. Definitions / Abbreviations:

• Definitions:
• Abbreviations:

 

Abbreviation

Expansion

 

°C	Degree Centigrade
ml	millilitre
psi	Per square inch
+	Plus
v/v	Volume by volume
%	percent

 

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

 

 

 

SOP Title: Procedure for Efficiency test for UV Light

1. Objective:

To lay down the procedure for determination of Efficiency test for UV Light.

2. Scope:

This procedure is applicable for determination of Efficiency test for UV Light in the Microbiology Section.

 

3. Responsibility:
   • Quality Control: To prepare and review the SOP. To follow the procedures for determination of Efficiency test for UV Light 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 mask and gloves while performing micro activities.
5.1.2	Use clean spatula, preferably wiped and dried with 70% v/v Isopropyl Alcohol for weighing of each media.
5.1.3	Switch OFF the UV light before collecting the plates.
5.2	UV Efficacy Study test of UV light
5.2.1	Frequency: Half Yearly
5.2.2	Applicable to Sampling booth RLAF and Pass box, Dispensing booth RLAF and pass box, Microbiology Section LAF and Pass boxes.
5.3	Procedure to Determine the UV light Efficiency test:
5.3.1	Transfer the required weighed quantity of SCDA media based on the number of UV light tubes and the positive and negative controls petriplates to a suitable flask and proceeds as per SOP for preparation of Culture media.
5.3.2	Aseptically pour 15-20 ml of sterile molten SCDA media cooled at approximately 45°C in required number of 90 mm Petri plates under LAF. Ensure that the plates are prepared in duplicates, one exposed and the other for unexposed test.
5.3.3	After solidification, incubate the plates at 30-35°C for 24 hours.
5.3.4	After pre-incubation, transfer not less than 100 cfu per 0.1 ml culture of Bacillus subtilis (as it is one of the most resistant bacteria) on to SCDA plates using a sterile pipette and spread the colony using sterile spreader. Prepare of serial dilution cultures to get 100 CFU per 0.1 ml cultures as per SOP.
5.3.5	Wrap one petriplate with Aluminium foil and second one keep as it is.
5.3.6	Under LAF, open the lid of unwrapped petri plate. Follow the same procedure for Sampling Booth, Dispensing booth and Pass boxes. Keep Wrapped plate as it is under UV light along with unwrapped plate.
5.3.7	Switch ON the UV light for 30 minutes.
5.3.8	After 30 minutes of exposure switch OFF the UV light and close the lid of unwrapped petriplate.
5.3.9	Simultaneously carry out positive control by streaking Bacillus subtilis and negative control without streaking onto SCDA plates.
5.3.10	Incubate all the test plates along with control plate at 30-350C for 24 hours.
5.3.11	After incubation observe the plates for total bacterial count and record the results in annexure.
5.4	Limit /Acceptance criteria:
5.4.1	1) There should be no growth in unwrapped petriplate and there should be growth in wrapped Aluminium foil petriplate. 2) There should be growth on positive control and no growth on negative control.
5.4.2	After recording of all results, decontaminate the plates and other used media by referring SOP, Decontamination of culture media and microbial waste.
5.4.3	If the UV efficacy test fails, investigate the cause and replace the tube if necessary. Perform efficacy test on new UV tube.
5.4.4	UV Burning hours for a UV tube should not exceed 2000 hours.

 

6. Definitions / Abbreviations:

 

• Definitions:

• Abbreviations:

Abbreviation	Expansion
LAF	Laminar Air Flow
SCDA	Soyabean Casein Digest Agar

 

UV	Ultra Violet
°C	Degree centigrade
v/v	Volume by volume
ml	milliliter
mm	millimeter
%	Percentage

                        

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

 

 

SOP Title: Procedure for Surface Swab Analysis for Microbial Limits

1. Objective:

To lay down the Procedure for Surface Swab Sample Analysis for Microbial Limits.

2. Scope:

This Procedure is applicable for Surface Swab Sample Analysis for Microbial Limits.

 

3. Responsibility:

• Quality Control Department: To prepare and review the SOP. To follow the Procedure for        Surface Swab Sample Analysis for Microbial Limits 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	Methods for Swab Testing:
5.3	Surface Monitoring Using Swab
5.3.1	On receipt of intimation, Microbiologist should do necessary arrangement for collecting swab sample.
5.3.2	Prepare swab, solutions and Media as per Media Preparation and Sterilisation SOP .
5.3.3	To the location, carry sterile test tubes with cotton swabs containing 10 ml of sterile normal saline or Buffered Sodium Chloride Peptone Solution pH 7.0 to collect the swab sample as per define swabbing procedure.
5.3.4	Mark the with location, sample name and date of sampling.
5.3.5	With the help of sterile forceps remove swab and hold the end of the swab stick and take a sample of 10 x 10 cm2 area and dipped the swab stick back in 10 ml of same sterile normal saline/BSCP. Immediately close the test tube.
5.3.6	Bring all the test tubes in microbiology section and do the analysis by pour plate method.
5.3.7	Mix the sample and pipette out 1 ml of sample in 4 sterile petriplates.
5.3.8	In two plates, add 15 to 20 ml of sterile SCDA media previously cooled at 45°C mix slowly by rotating the plates and allow to solidify.
5.3.9	Incubate these plates at 30-35°C for 5 days for bacterial count along with negative control. i.e. plate out 1 ml of diluent in a single petriplate, and positive control i.e. plate out 1 ml of any positive culture (Bacterial), preferably S.aureus in single.
5.3.10	In other two plates add SCA/SDA media previously cooled at 45°C, mix slowly by rotating the plates and allow to solidify and then incubate the plates at 20-25°C for 5 days for fungal count along with negative control and positive control. Use Candida albicans as a positive control for fungi count.
5.3.11	Record the observations in Surface Swab Analysis record Annexure.
5.3.12	If the report are out of limits, intimate to Department Head and Quality Assurance Head, raise an incident and take further course of action.
5.3.13	If required, repeat the test one more time to confirm the result.
5.3.14	All equipments, petriplates and media are decontaminated as per SOP -Procedure for Destruction of Cultures and Microbial waste.
5.4	Procedure for Contact Plate:-
5.4.1	This Technique should be used for flat surfaces such a Working benches, walls and floors, Machine exterior surfaces etc.
5.4.2	Use Dey- Engley’s Agar Contact plate for carrying out this method. Refer SOP for media preparation.
5.4.3	Open the Pre prepared contact plate (from supplier) and gently press the convex surface on the area to be tested. Close the lid immediately. Label the plates appropriately. Single plate per area to be used for this test.
5.4.4	Immediately after sampling, clean the area of sampling thoroughly with 70% IPA or suitable disinfectant to prevent microbial growth on the surface.
5.4.5	After sampling, close each plate with the lid provided. Incubate at 20-250 C for first 3 days and 30-350 C for next two days for total bacterial and fungal count.
5.4.6	Record the count after completion of incubation period of 5 days.
5.4.7	Count the number of colonies on each plate and record the number of colonies as CFU/25 cm2 or CFU/Contact plate. Enter observations in Annexure.
5.5	Swab of Walls and Floors:-
5.5.1	Use Dey- Engley’s Agar Contact plate for carrying out this method. If  Pre prepared plates are not available prepare plates in the microbiology laboratory and use.
5.5.2	Open the Pre prepared contact plate (from supplier) and gently press the convex surface on the wall or floor to be tested. Close the lid immediately. Label the plates appropriately. Single plate per area to be used for this test.
5.5.3	Immediately after sampling, clean the area of sampling thoroughly with 70% IPA or suitable disinfectant to prevent microbial growth on the surface.
5.5.4	After sampling, close each plate with the lid provided. Incubate at 20-250 C for first 3 days and 30-350 C for next two days for total bacterial and fungal count.
5.5.5	Record the count after completion of incubation period of 5 days.
5.5.6	Count the number of colonies on each plate and record the number of colonies as CFU/25cm2 or CFU/Contact plate. Enter observations in Annexure.
5.5.7	If contact plates are not available, swab method can be used as an alternative method.
5.6	Hands Gloved Finger Dabs:-
5.6.1	Use Dey- Engley’s Agar Contact plate for carrying out this method.
5.6.2	Open the Pre prepared contact plate and gently press four gloved fingers one by one of one hand onto the surface of Dey Engley’s agar plates. Then take impression of thumb on same plate. Close the plate immediately. Repeat the same procedure for other hand. Use single plate for each hand.
5.6.3	The hand should not be disinfected prior to finger dab as to get a actual count during work.
5.6.4	After sampling, close each plate with the lid provided. Incubate at 20-250 C for first 3 days and 30-350 C for next two days for total bacterial and fungal count.
5.6.5	Record the count after completion of incubation period of 5 days.
5.6.6	Count the number of colonies on each plate and record the number of colonies as CFU/25cm2 or CFU/Contact plate. Enter observations in Annexure.
5.7	Monitoring of personnel gear:-
5.7.1	Before using any sterile overgown, check for any wear and tear, loose threads or any stains or other deformities.
5.7.2	Do not use such overgowns, replace them with a proper overgowns to avoid any contamination.
5.7.3	The Swab of personnel gear has to be taken at the end of the shift.
5.7.4	Use SCDA contact plates for carrying out this activity. Refer for media preparation SOP.
5.7.5	Open the Pre prepared contact plate and gently press against overgown.
5.7.6	Take swabs from from overgown i.e. Headgear, right forearm, left forearm, middle of the chest using 25cm2 contact plate.
5.7.7	After sampling, close each plate with the lid provided. Incubate at 20-250 C for first 3 days and 30-350 C for next two days for total bacterial and fungal counts.
5.7.8	Record the count after completion of incubation period of 5 days.
5.7.9	Count the number of colonies on each plate and record the number of colonies as CFU/25 cm2 or CFU/Contact plate. Record observations in Annexure.
5.7.10	Decontaminate all the plates and tubes after result recording by referring SOP, Decontamination of microbial culture media.
5.8	Testing Frequency:-
5.8.1	Surface Monitoring of equipments should be done as per the intimation received from other departments or for doing cleaning validation study.
5.8.2	Surface Monitoring of Walls and Floors should be done Monthly for Grade A areas and Microbiology Laboratory and Half Yearly for other areas.
5.8.3	Hand gloved Finger Dabs to be performed monthly and Monitoring of Over gown should be done Monthly.

 

6. Definitions/Abbreviations:

• Definitions :
• Abbreviations:

Abbreviation	Expansion
°C	Degree Centigrade
%	percentage

 

ml	millilitre
No.	Number
v/v	Volume by volume
SCDA	Soyabean Casein Digest Agar
IPA	Isopropyl Alcohol
cm2	Centimetre square
BSCP	Buffered Sodium Chloride Peptone solution pH 7.0

  

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SOP Title: Testing Growth Promotion, Inhibitory and Indicative Properties of Culture Media

1. Objective:

To lay down the procedure for Testing Growth Promotion, Inhibitory and Indicative Properties of Culture Media.

2. Scope

This procedure is applicable for Testing Growth Promotion, Inhibitory and Indicative Properties of Culture Media in the Microbiology Section of Quality Control Department.

3. Responsibility:

• Quality Control Department: To prepare and review the SOP. To follow the procedures for Testing Growth Promotion, Inhibitory and Indicative Properties of Culture 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/Instructions:
5.1.1	Always wear protective nose masks and gloves while carrying out the microbiological activities.
5.1.2	Use spatula which is clean, preferably wiped with 70% v/v Isopropyl alcohol and dried, for weighing of each media.
5.2	Procedure for Testing:
5.2.1	Growth Promotion, Indicative and Inhibitory Properties of each lot of dehydrated media container shall be carried out before its usage and after every six months until its consumption or expiry, as applicable.
5.2.2	On receipt of Culture Media, make the entry in respective format of SOP, ‘Procedure for Receipt and Storage of Dehydrated Culture Media used for Microbiological Analysis’.
5.2.3	For testing, prepare the required quantity of Culture Media based on the number of organisms used for the test and sterilise as per Procedure for Media Preparation SOP. Refer annexure for Media name, media tests (Growth promotion, Inhibitory, Indicative tests) and test culture to be used with incubation temperature and time.
5.2.4	Allow the media to cool at 40-45°C after sterilization.
5.2.5	In case of Agar medium pour the media in plates and let them solidify.
5.2.6	Carry out the analysis under Laminar Air Flow unit. Refer SOP ‘Procedure for Operation of Laminar Air Flow bench’.
5.2.7	Remove the specified culture dilution for a given media by referring annexure of not more than 100 CFU from the refrigerator. Refer SOP ‘Procedure for Serial Dilution Preparation of Microbial Cultures.’
5.2.8	Allow the culture dilutions to attain room temperature.
5.3	Test for Growth Promoting Properties of Solid Media:
5.3.1	In case of solid media perform the surface spread method using spreader.
5.3.2	Pipette and spread on the surface of the agar plate with 0.1 ml of known number of microorganisms ( approximately NMT 100 CFU).
5.3.3	Incubate for not more than the specified shortest period of time and temperature till a clear visible growth is obtained. Record the details and observations in Annexure.
5.3.4	Media under test should be considered suitable if growth obtained is comparable to that obtained on the same medium, previously tested and approved.
5.3.5	Growth obtained on solid media must not differ from the calculated CFU of the standardized inoculum by a factor greater than 2 or should be comparable to that obtained on the same medium previously tested and approved. i.e. recovery should be within 50% to 200%. Refer Annexure.
5.4	Test for Growth Promoting Properties of Liquid Media:
5.4.1	Inoculate 10 ml of the medium with 0.1 ml of required culture using pipette.
5.4.2	Incubate for not more than the specified shortest period of time and temperature till a clear visible growth is obtained. Record the results in annexure.
5.4.3	The liquid media shall be observed for turbidity to confirm the growth of microorganism. If no turbidity is observed then it is considered as no growth.
5.5	Tests for Inhibitory Properties of Liquid/Solid Media
5.5.1	Inoculate each plate in case of solid media or tube in case of liquid media with 0.1 ml of required culture and incubate at the specified temperature for the time specified in the test. Refer Annexure.
5.5.2	There should not be any growth of the micro organisms on this medium.
5.6	Tests for Indicative Properties of Liquid/Solid Media:
5.6.1	Perform the surface spread method, in-case of solid media inoculating each  plate with 0.1 ml of required culture and spreading; in case of liquid media directly inoculate 0.1 ml of required culture in the media.
5.6.2	Enter the records in Annexure.
5.6.3	Incubate at the specified temperature for the time specified in the test Refer Annexure.
5.6.4	Colony morphology and Biochemical indication reaction should be similar to that obtained with the previously approved batch of the medium.
5.6.5	Perform a negative control for specified time and temperature as per the test, wherein no growth of microorganisms should occur.
5.6.6	Refer Annexure for Growth Promotion Record.
5.6.7	Growth of the micro organisms must be comparable to that obtained with previously tested and approved batch of medium.
5.6.8	If  the media passes the Growth Promotion Test (GPT), an approved label shall be affixed on the all the media containers as per Annexure and the same should be used for routine analysis. Note: – If Multiple containers of same batch numbers are received at one time, then the Growth promotion test of every container not necessary to perform. If same batch number container are received on two different days then perform GPT separately.
5.6.9	If the media fails for the Growth Promotion Test, discard the media and prepare fresh media again to repeat the test. Incident report should be filled in case on non-compliance of the test.
5.6.10	After completion of growth promotion test, all the used media and glasswares should be decontaminated as per SOP “Destruction of Cultures and Microbial waste.

 

6. Definitions / Abbreviation:

• Definitions:

 

• Abbreviation:

Abbreviation	Expansion
°C	Degree Centigrade
CFU	Colony Forming Unit
ml	millilitre
LAF	Laminar Air Flow
%	percent

 

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SOP Title: Identification and Purity Verification of  Master Microbial Cultures

1. Objective:

To lay down the procedure for Identification and Purity Verification of Master Microbial Cultures.      

2. Scope:

This procedure is applicable for Identification and Purity Verification of Master Microbial Cultures    in Microbiology Section of Quality Control.

 

3. Responsibility:

3.1   Quality Control Department: To prepare and review the SOP. To follow the procedure for Identification and Purity Verification of Master Microbial Cultures 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	Do not overheat the smear.
5.1.3	Staining solution to be handled carefully. It should not fall on the floor.
5.1.4	For oil immersion technique, use only one drop of immersion oil. Do not over flood the slide with immersion oil.
5.2	Procedure for Identification and Purity:
5.2.1	Media preparation for all the tests has to be performed by referring SOP, Media Preparation and Sterilization.
5.2.2	Perform the Purity Check on receipt of Mother Culture by staining method by isolating the bacteria.
5.2.3	The staining procedure differentiate bacteria into gram positive and gram negative based on their ability to retain the primary dye (Crystal violet ) or loose the primary dye and accept the colour of the counter stain (safranin).
5.2.4	The gram positive bacteria produces blue/purple colour with this staining procedure while gram negative ones are pink or red in colour.
5.2.5	The staining reagent are: 1) Crystal violet: A primary dye. 2) Grams iodine: A complexing agent. 3) Alcohol: A decoloriser. 4) Safranin: A Counter strain.
5.2.6	Prepare a thin smear of the culture to be examined on a glass slide.
5.2.7	Air dry or heat fix the smear and flood the slide with Crystal Violet staining solution for 1 minute. Allow the slide to cool do not burn the slide.
5.2.8	Wash the smear with 70% v/v Ethanol /Grams decolouriser till all the extra Crystal Violet stain is washed off.
5.2.9	Wash with water and flood with Grams Iodine solution for 1 minute.
5.2.10	Flood the smear with safranin solution for 1 to 2 minutes. Wash with water and Air dry the slide.
5.2.11	Place a drop of cedarwood oil and examine under oil immersion lens 100X of the microscope.
5.2.12	Gram positive organisms should observed blue in colour and Gram negative organisms should observe pinkish red colour. Record the results.
5.3	Fungal  Staining Procedure:
5.3.1	Place a Lactophenol cotton blue reagent on a clean and dry slide and place a        small loopful Aspergillus fungal culture into Lactophenol cotton blue reagent and tease the Aspergillus culture into thin preparation.
5.3.2	Remove the excess stain using tissue paper.
5.3.3	Wait for 15 minutes and observe under microscope.
5.3.4	Place a coverslip on the slide before observing under the microscope. Avoid air bubbles.
5.3.5	Observe Initially under low power for screening in low intensity and then under high power intensity.
5.3.6	Aspergillus brasilliensis produce white or yellow growth colonies that are covered by Septate dark asexual fungal spores. Record the results.
5.4	Procedure for Motility testing:
5.4.1	Prepare suspension of the Bacterial culture in normal saline, with the help of sterile nichrome loop place a drop of suspension of Bacterial culture on the cover slip.
5.4.2	Grease the four corners of the cover slip.
5.4.3	Place a cavity slide on the top of the cover slip taking care that drop is not disturbed.
5.4.4	Invert the slide so that the cover slip is on the top of the slide.
5.4.5	Observe under the microscope, first under low power for screening in low intensity and then under high power.
5.4.6	For motile organisms eg. S boydii, S.aureus, motility can be seen towards the edge of the drop and for non motile organisms eg. B. Subtilis, E. coli, S. abony. C albicans, P. aeruginosa, the cells will be evenly distributed.
5.4.7	After confirmation of Purity Check of Culture from colony characteristic, staining and Motility, Carry out the Biochemical test for the Cultures and compare the results obtained, as per Annexure.
5.4.8	Confirm the growth of cultures on selective media as per SOP – Procedure for Testing Growth Promotion, Inhibitory and Indicative Properties of Culture Media.
5.4.9	The commonly used Biochemical tests are mentioned below. (a) Catalase test, (b) Coagulase test ,(c) Oxidase test,(d) Sugar fermentation test ,(e) Indole test ,(f) Citrate test and  (g) Urease test.
5.5	Biochemical tests for Escherichia coli:
5.5.1	Procedure for Acid and Gas Test:
5.5.2	Transfer a loopful of culture in 5 ml of sterile MacConkey Broth containing durhams tube. Incubate the test tube at 42 to 44°C for 24 hrs.
5.5.3	Observation: Change in colour of MacConkey Broth from purple to yellow indicates the production of acid and formation of bubble in Durham’s tube indicates the production of gas.
	Procedure for Indole Test:
5.5.4	Transfer a loopful of culture in 5 ml of sterile Peptone water. Incubate the test tube at 42 to 44°C for 24 hours.
5.5.5	Add a few drops of Kovacs reagent in the test tube and mix the content and allow the test tube to stand for about 2 minutes.
	Observation: Formation of red ring in the reagent layer indicates the production of Indole.
5.6	Biochemical Tests for Salmonella:
5.6.1	Procedure for acidic butt and alkaline slant on Triple Sugar Iron (TSI) Agar.
5.6.2	Streak a loopful of culture on the slant with sterile nichrome loop and stab the butt with straight sterile nichrome loop.
5.6.3	Incubate at 30 to 35°C for 24 hours.
5.6.4	Observation: Acidic butt should show Yellow colour and alkaline slant should show pink colour on TSI without production of H2S gas.
5.6.5	Transfer a loopful of culture in 10 ml of RVSEB. Incubate at 30 to 35°C for 24 hrs.
	Observation: Change in colour of the medium from blue to colourless.
5.7	Biochemical test for Pseudomonas aeruoginosa
5.7.1	Procedure for Oxidase Test:
5.7.2	Place 2 or 3 drops of freshly prepared 1% w/v solution of N,N,N’,N’, Tetramethyl-4-phenylene-diamine dihydrochloride on a filter paper and smear with P. aeuroginosa colonies. For solution Preparation, refer SOP, Preparation and Handling of Laboratory reagents.
5.7.3	Observation: Development of pink colour should change to purple colour.
5.8	Biochemical test for Staphyloccocus aureus:
5.8.1	Procedure for Coagulase test:
5.8.2	Transfer a loopful of culture to individual tube containing 0.5 ml of coagulase rabbit or horse plasma. Incubate in water bath at 37°C,  examine the tubes after 3 hours and subsequently at a 3 hour interval up to 24 hours.
5.8.3	Observation: Formation of Clot.
5.9	Biochemical Tests for Shigella boydii:
5.9.1	Procedure for acidic butt and alkaline slant on Triple Sugar Iron (TSI) Agar.
5.9.2	Streak a loopful of culture on the slant with sterile nichrome loop and stab the butt with straight sterile nichrome loop.
5.9.3	Incubate at 30 to 35°C for 24 hours.
5.9.4	Observation: Acidic butt should show Yellow colour and alkaline slant should show pink colour on TSI without production of H2S gas.
5.9.5	Transfer a loopful of culture in 10 ml of GN Broth. Incubate at 30 to 35°C for 24 hrs.
5.9.6	Observation: Change in colour from colourless to turbid.
5.10	KOH String test or Gram Staining Alternative test
5.10.1	Place a drop of 3% potassium hydroxide (KOH) on a glass slide. For solution Preparation, refer SOP, Preparation and Handling of Laboratory reagents.
5.10.2	By using a nichrome loop, place a visible loopful of cells from a single, well isolated colony is mixed into the drop.
5.10.3	Mix with Nichrome loop for about 60 seconds.
5.10.4	Observation: If the mixture becomes viscous and a string is formed on lifting the nichrome loop (KOH positive) than the colony is considerd as gram negative.
5.10.5	Note: This test is simple and can be done effectively on old cultures. This method can be done as a backup to gram staining.
	If Purity Check and Identification of any culture is not satisfactory, repeat the test. If Fails after rechecking, procure a new culture.
5.10.6	After Completion of the activity, decontaminate all the cultures and media by referring to SOP for Decontamination of microbial Waste.

6. Definitions / Abbreviations:

• Definitions:
• Abbreviations:

Abbreviation	Expansion
°C	Degree Centigrade
%	percent
v/v	Volume by volume
No.	Number
TSI	Triple Sugar Iron
IPA	Isopropyl Alcohol
RVSEB	Rappaport Vasiliadis Salmonella Enrichment Broth

 

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SOP Title: Serial Dilution Preparation of  Microbial  Cultures

1. Objective:

To lay down the Procedure for Serial Microbial Cultures Dilution.

 

2. Scope

This procedure is applicable for Serial Microbial Cultures Dilution in the Microbiology Section of Quality Control Department.

3. Responsibility:

3.1     Quality Control: To prepare and review the SOP. To follow the procedures for Serial Dilution Preparation of Microbial Cultures 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 Serial Dilution:
5.2.1		Remove the slants of the Working Cultures from the refrigerator and allow to stand for at least 2-3 hrs to attain room temperature.
5.2.2		Refer Procedure for Media Preparation and Sterilisation as per SOP for Media preparation.
5.2.3		Inoculate a loopful of the bacterial cultures from working culture slants in test tubes containing 10 ml of Soyabean Casein Digest Medium and fungal cultures in Saborauds Dextrose Broth. Both SCDM and SDB are considered as stock cultures for dilution (100). Refer Annexure for List of cultures. Refer SOP for maintainance of cultures.
5.2.4		Incubate the bacterial cultures at 30-35°C for 18-24 hours, fungal cultures at 20-25°C, Candida albicans for 48hrs and Aspergillus brasilliensis for 72 to 120 hours or until good sporulation.
5.2.5		Use Buffered Sodium Chloride Peptone Solution to make the culture dilutions.
5.2.6		0.05% polysorbate 80 may be added to the buffered solution to suspend spores of Aspergillus brasilliensis
5.2.7		Switch ‘ON’ the LAF and proceed for operation.
5.3		A)  Small Volume Serial Dilutions:
5.3.1		Label four screw-capped tubes as 1:100, 1:10,000, 1:1,00,000 and 1:1,000,000.
5.3.2		In scientific notation this would be 10–2, 10–4, 10–5 and 10–6. For details, refer Annexure.
5.3.3		Using a sterile 10 ml pipette, add 9.9 ml of sterile BSCP aliquot into first, second and fourth tubes and add 9.0 ml in third tube.
5.3.4		Using a 100μL micropipette and sterile tip, transfer 0.1 ml of broth culture into the 10-2 tube and Cap the tube. Glass pipette of 0.1 ml can also be used.
5.3.5		Mix it for a few seconds vigorously flick the tube to adequately disperse the bacteria evenly throughout the tube and break up bacterial clumps.  Note: Do not shake the test tube.
5.3.6		With new sterile tip, transfer 0.1ml from first tube (1:100 or 10-2) and add it to the tube labeled as 1:10,000 or 10-4.  Repeat the tube mixing procedure.
5.3.7		With new sterile tip, transfer 1.0 ml broth culture from the second tube (1:10,000 or 10-4) and add it to third tube labeled as 1:100,000 or 10-5 Repeat the tube mixing procedure.
5.3.8		With new sterile tip, transfer 0.1 ml from the second tube (1:10,000 or 10-4) and add it to fourth tube labeled 1:1000,000 or 10-6. Repeat the tube mixing procedure.
5.3.9		Plate out 0.1 ml and 1.0 ml of each dilution on sterile Petri plates in duplicate. Also plate out stock cultures (100) in duplicates.
5.3.10		Pour about 20 ml of   Soyabean Casein Digest Agar cooled to 45°C in to petri plates for bacterial culture and Saborauds Dextrose Agar with or without antibiotic for fungal cultures.
5.3.11		Rotate clockwise and anticlockwise for uniform mixing. Repeat this procedure for all the cultures.
5.3.12		Incubate the plates at 30-35°C for 48 hrs except plate containing dilutions of Candida albicans which is incubated at 20-25°C for 48 hours and Aspergillus Brasilliensis for 72 to 120 hours.
5.3.13		After completion of incubation period observe the plates and note down the colonies and select the dilutions which show colonies NMT 100 CFU(approximately 10–100 CFU). If the dilutions are showing more than 100 CFU then further dilution to be done.
5.3.14		Record the results of Serial Culture Dilution in Annexure.
5.4		B) Large Volume Serial Dilutions:
5.4.1		Select four sterile bottles and labeled as 1:100, 1:10,000,100,000 and 1:1,000,000.
5.4.2		In scientific notation this would be 10-2, 10-4, 10-5 and 10-6. For details refer Annexure No. 01.
5.4.3		Add measure volume of sterile buffer solution in to the bottle as follows. Labeled Bottle as. 1:100(10-2), 1:10,000 (10-4) and 1:1,000,000.(10-6)  add 99 mL and for 100,000 (10-5) add 49 ml sterile buffer solution respectively.
5.4.4		Using a sterile 1mL pipette, transfer 1mL of broth culture into the 10-2 bottle Repeat the tube mixing procedure as per 5.3.5.
5.4.5		Using a new sterile 1ml pipette, transfer 1ml from the (1:100 or 10-2) and add it to the bottle labeled 1:10,000 (10-4). Repeat the bottle shaking procedure.
5.4.6		Using a new sterile 1 ml pipette, transfer 1 ml from the 1:10,000 (10-4) bottle and add it to the 1:1,00,000 (10-5) bottle. Repeat the shaking procedure.
5.4.7		Using a new sterile 1 ml pipette, transfer 0.1 ml from the 1:10,000 (10-4) bottle and add it to the 1:1,000,000 (10-6) bottle. Repeat the shaking procedure.
5.4.8		Follow the procedure as per 5.3.9 to 5.3.15.
5.4.9		After completion of incubation and result recording, the petriplates and test tubes shall be decontaminated as per define SOP.
5.4.10		Store the test tubes or bottles containing 100 CFU culture at 2-8°C in refrigerator for its further use. Discard the rest of the dilution tubes.
5.4.11		If the Serial dilution results are not proper, than the microbiologist should investigate the cause. The Serial Dilution has to be performed again to get count within range.
5.5		Serial Dilutions Preparations
5.5.1		Frequency : Monthly
5.5.2		Serial dilutions to be performed for all micro organisms.

6. Definitions / Abbreviations:

• Definitions:
• Abbreviations:

Abbreviation	Expansion
LAF	Laminar Air Flow
°C	Degree centigrade
ml	mililiter
µl	microlitre
%	percentage
v/v	Volume by volume
SCDA	Soyabean Casein Digest Agar
SDA	Saborauds Dextrose Agar
SDB	Saborauds Dextrose Broth
BSCP	Buffered Sodium Chloride Peptone Solution
SCDM	Soyabean Casein Digest Medium

 

 

List of Cultures for Serial Dilutions

Culture Name	Abbreviation
Bacillus subtilis	B. subtilis
Candida albicans	C. albicans
Escherichia coli	E. coli
Pseudomonas aeruginosa	P. aeruginosa
Salmonella abony	S. abony
Staphylococcus aureus	S. aureus
Shigella boydii	S. boydii
Aspergillus brassiliensis	A. brasilliensis

 

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