To detail the Standard Operating Procedure for Entry and Exit in Sterility Testing Area of XX Pharmaceuticals Ltd.
Entry and exit procedure; Scope
This SOP define the Entry & Exit Procedure of personnel who will get entry & exit in sterility testing area in Microbiology Laboratory of General Building at XX Pharmaceuticals Limited.
Definitions/Abbreviations
[] Not Applicable
Responsibilities
The roles and responsibility are as follows
Executive/Senior Executive, Microbiology
Follow the instructions of this procedure correctly.
Assistant Manager, Microbiology QC
To confirm that this mentioned procedure is kept up to date.
To confirm appropriate personnel from the section are trained on this procedure.
Head of Quality Assurance
Take initiative approval of SOP
To confirm the overall implementation of the SOP
Procedure
Instructions
Only certified, trained & validated employees can enter into the sterility testing area.
All employees are responsible to maintain their personnel hygiene & cleaning regularly.
To follow aseptic technique during change procedure
Always confirm sterility of the garments to be worn in the proper area.
Always confirm that sterile garments in the Laminar Air Flow dress cabinet.
The person infected by flue, cold, open lesions (i.e.: eczema) should not enter into sterility test area.
Confirm that AHU[Air Handling Unit] of sterile testing area is switched on at least one hour before starting testing in that mentioned area.
Step 01: Entry into Change room- 01 (Room no: GMB012, Grade: C)
Take away the whole garments (except undergarments), wrist watch, Jewelry, & any kind of ornaments after entering into the change room 01.
Personal/Street shoe must be put off into shelf of the step over bench. Cross step over bench & put on dedicated shoe kept into shelf of the other site.
Use 70% IPA from the IPA dispenser to rub Hand.
Step 02 : Entry into Change room 02 (Room Grade: C)
Put on a pair of sterile hand gloves very carefully.
Wear sterile primary gown having two parts (Trouser & shirts) & a hood to cover the hairs.
Rub the hands with 70% sterile IPA.
Step 03: Entry into Change Room 03 (Room Grade: B)
Take sterile garments from dress cabinet which was formerly kept into the dress cabinet in change room 03 of sterility testing area.
Put on sterile dress with sterile overall, head gear, face mask, Shoes, Sterile hand gloves & safety goggles and confirm aseptic clothing. The procedure of proper clothing is described below:
Putting on Gloves
Open the bag of sterile cleanroom latex free gloves & fold out to expose folded cuff area of the gloves.
Put on the glove by put in the fingers into latex free glove & pulling on the folded cuff area to cover the palm, confirming that only inside of the cuff is touched.
With gloved fingers, fold gloves over the arms, pulling them up over the wrists & forearms, confirming that gloves endure its sterility.
Putting on Hood
Open bag having sterile hood ensuring the contents remain sterile.
With care & only touching inside of hood, turn hood inside out & place on the head, confirming that all head is covered & no bouffant cap is unprotected.
Clasp hood around the neck by clipping self-possessed buttons under chin.
Being cautious not to touch outside surfaces, adjust hood to cover all of head.
Putting on Mask
[] Put on facemask & by touching as minimal area as possible tie on mask & making sure it fits comfortably.
Putting on Safety Eyewear
[] Put on sterile safety eyewear glasses remain on isolated portion of Laminar air Flow of dress cabinet.
Putting on Safety Overalls
Open bag having sterile overall, confirming innards remain sterile. Unfold left & right sides of overall.
Taking top folded edge of overall unfold it towards you until it is lying flat to reveal full length of zipper.
Take hold of upper edge of each leg inaugural & gather a handful of the garment in each hand, to catch up legs of overall.
Continue to gather garment in this way so that when overall is held up it does not touch floor.
Lifting overall, hold in front of you & step into one leg at a time, gradually releasing gathered fabric to allow each foot to push through.
Take care not to allow arms of overall to touch floor.
Pull overall up to waist height, taking care not to touch outer side of the overall.
Slip hands into sleeves, one at a time & then slip remainder of overall over shoulders.
Check that hood skirt is completely inside collar of overall & fasten overall with the zipper.
Step 04 : Entry into Sterility testing room 04 (Room Grade: B)
Spray gloved hands & wrists with sterile 70% IPA.
Flashing door with help of elbow, enter into sterility test room.
Step 05: Exit from testing room
After completion test remove used hand gloves put off into waste bin.
Rub hands with the help of 70% Iso Propyl Alcohol
Pull door to enter into change
Pull door to enter into second Change room then enter into first change room.
Take clean laboratory dress from cabinet put in the change room 01.
Send used garments to laundry for washing.
Enter information regarding entry exit procedures such as entry time, exit time, purpose,
The tenacity of this SOP is to define the operation, calibration & cleaning of Water Bath
Water Bath; Scope
This procedure is applicable for Water Bath installed in Microbiology section at XX Pharmaceuticals Limited.
Definitions/Abbreviations
LED: Light Emitting Diode
Responsibilities:
The roles and responsibility are as follows
Executive/Senior Executive, Microbiology
To confirm that the instructions of this procedure are properly followed.
To maintain the record correctly as per SOP.
To confirm cleaning of shaking water bath maintaining safety guidelines.
Assistant Manager, Microbiology
To confirm that this process is kept up to date.
To ensure that the SOP is technically sound & reflects required working practices.
To arrange training on the SOP to all concerned personnel & to ensure implementation of the SOP after training.
Schedule calibration of the instrument at the defined intervals.
Procedure
Instructions
Laboratory coat, hand gloves & safety glasses must be worn while handling instrument.
Never touch the liquid within the bath as it may be very hot.
Each time use the display or a thermometer to check the temperature.
Never block ventilation slots during operation.
Each time disconnect bath from electricity supply before cleaning.
Allow the liquid in bath to cool down to 40°C before draining.
Use only purified water, never operate water bath without water.
Fill the it prior to connection to power supply.
It should be covered with a lid or polypropylene spheres to achieve optimum performance.
Always use the stainless steel flat lid with concentric rings when the instrument is not in use to avoid contaminants landing in the bath liquid.
Don’t touch any electrical contacts or open any closure panel that may be risk of electrical shock when above 60˚C or below room temperature,
Operating Procedure
Check the calibration date of the instrument before each time use. If the calibration is out of date, calibrate it and then use.
Check water level, If require adjust the water bath to an appropriate level with purified water.
Switch on the power from the main.
Using the switch “I” ON, “O” OFF button located at the rear side of it, Switch on the Water bath When ON (I) the switch is illuminated & unit performs a self-test where all segments of the 3 digit LED display & indicators illuminated.
To display “SP1” to set temperature, press & hold down key for more than 1.5 seconds. “SP1” will show blinking.
To select required temperature, use up or down arrow keys.
Heater indicator will illuminate. Wait until the display flashes between “SP1” & set temperature and will revert to show actual liquid temperature.
When the bath temperature is either 4°C above or below set temperature, LED indicator will illuminate.
Open concentric ring & place beaker or suitable container with sample on sphere of it.
After completion of work, switch off bath by using “I” ON, “O” OFF switch, located at rear side of back of it.
Calibration Procedure
Calibrate Water bath once in a year.
Fill purified water in water bath to an suitable level.
Switch on the bath using “I” ON, “O” OFF switch, located at the rear side of back of the Water bath.
Set desired temperature against calibrated standard thermometer at 35⁰C, 45⁰C, 65⁰C.
Wait 30 minutes to equilibrate.
Verify temperature using a calibrated standard thermometer & record the temperature in the Calibration information sheet for Water bath (as per Annexure-I).
Cleaning Procedure
When water becomes dirty, clean instrument otherwise clean once in a week.
Switch ‘OFF’ the instrument.
Disconnect main power supply.
Open stainless steel flat lids with concentric rings of the water bath.
Use purified water to wash the stainless steel flat lids and concentric rings.
To reduce potential biological contamination, remove the water from water bath.
Wash it with soapy water & then purified water.
Mop with clean dry cotton cloth.
To remove any deposits, use 10% nitric acid on a cloth (wear suitable gloves).
Gently heat to 50°C for an hour after adding 1 liter of vinegar to water in the stainless steel tank, empty and brush the lime away for descaling then rinse thoroughly afterwards.
Fill with purified water up to desired level .
Close the stainless steel flat lids with concentric rings.
To operate, clean and calibrate Cooled Incubator in order to support the growth of Yeast and mould in Microbiological analysis.
Cooled incubator Scope
This SOP applies for operation, cleaning and calibration of Cooled Incubator, Model: ICP600 in Microbiology Section of at XX Pharmaceuticals Limited.
Definitions/Abbreviations
N/A
Responsibilities
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Operation, cleaning & calibration
Assistant Manager, Microbiology
Ensure Operation, cleaning, calibration and application of sound information.
Head Quality Assurance
Take initiate to approval of SOP
Procedure:
Instructions
Do not wipe with damped cloth at on position.
Use only 70% IPA or ethanol to wipe chamber
Do not overload chamber with tested items.
Do not keep items those may produce inflammation with air.
Keep all tested items to avoid touch inner surface of the chamber.
Do not move the Cooled Incubator at on position. Severe vibrations may cause serious damage of the temperature probes.
Operating Procedure
Press push/turn control key to put on main power switch in front of instrument. The Cooled Incubator will start in normal mode with display of timer, chamber temperature, alarm temperature (red color indication).
Hold down SET key & turn the push/turn control key at the clockwise or anti clockwise for setting date, local time, operating temperature at [23.5± 2.5]0C, alarm temperature at 300 After setting, SET key will be released the display briefly flashes the set point. The display then changes to the actual current temperature and starts to the setting temperature. The temperature will be automatically increased at setting temperature and display the setting temperature digitally.
Observe the display temperature until stable position.
Hold down the SET key (approximately 3 seconds) to select the operation mode, if require. The current operating mode will be flashed on the display. There are three operating mode in the Cooled Incubator:
Normal Operation
Weekly Programmer
Ramp time Programme Operation
Select required programme and set as per operation manual.
Select fan speed to set air changes. Selected position of fan speed will show in display.
Turn the push/turn control at clockwise until fan symbol flashing to move the air slider opens and closes the air valve to control the supply and discharge of air.
Check chamber temperature using by a calibrated digital thermometer, when the setting Temperature reaches.
Keep the required materials inside the chamber.
Cleaning & Disinfection :
Switch off Cooled Incubator and disconnect the power plug.
Remove all test flasks and others items from chamber.
Clean inside of chamber initially with dry cloth and wipe finally with 70% IPA wetted cloth.
Clean outside of chamber with dry cloth.
Disinfect all surfaces with 5% Savlon solution and dry the outer surface.
Reload all items into chamber when reach to dry the chamber surface.
At the end of cleaning, connect the power plug and switch on.
Clean the chamber once in a month.
Clean the outer surface every day.
Performance Check
Check chamber temperature daily once.
Record chamber temperature in Daily temperature Record
Temperature range ± 2.5C from the set temperature.
Calibration of Cooled Incubator
Insert standard thermometer or thermocouples into chamber.
Place at different location of chamber at least 6 position.
Press & hold down SET key until the normal mode light blinking.
Turn push/turn control key clock wise to reach SETUP light.
Select Calibration to rotate push/turn control key.
Use three calibration temperatures as below :
1 : 150C
2 : 220C
3 : 300C
Select required calibration temperature in SETUP key & set the corresponding calibration correction to 0.00
Measure deviation from selected calibration temperature under the steady conditions, using a reference instrument.
Set calibration correction in SETUP key. If the measured reference temperature is too low, the calibration correction setting is negative sign.
Adjust temperature if require from adjusting value of calibration temperature (CAL). The correction value should not more than ±10C.
Carry out check measurement using the reference thermometer.
Carry out others two calibration temperatures in the same manner.
Calibrate the incubator once in a year ± 15 days.
Record calibration in Calibration Record, as per Annexure-V of Engineering SOP
Maintenance
If Cooled Incubator shows any mechanical, electrical or any others problem, inform to supplier or Engineering Department for corrective action.
After corrective action, recalibrate Cooled Incubator.
To dry all properly cleaned glassware which is subject to use in chemical analysis & also to sterilize cleaned glassware which is subject to use in different microbiological analysis.
Oven Scope
This SOP applied for operation of Oven, which model is: UNE600 in QC Section, Microbiology Section & Product Development Section of at XX Pharmaceuticals Limited.
Definition/Abbreviation
N/A
Responsibilities
The roles and responsibility is as follows
Executive, Microbiology/ Executive, QC
Operation, cleaning & disinfection and performance checking of oven.
Assistant Manager, Microbiology
Ensure Operation, cleaning & disinfection and performance checking.
Ensure that oven is calibrated.
Checking that SOP is technically informative.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure
Instructions
Never enter open hands in the chamber of oven.
To keep or remove glassware, wear temperature resistance gloves.
Avoid to keep glassware into chamber with water or any other solution.
Do not dry plastic materials using oven.
Always keep door closed & avoid opening door for long period.
Operating Procedure
Sterilization of Glassware for Microbiological Analysis
To put on the main power switch, Press push/turn control key in front of the instrument. Oven will start in normal mode with the display of the chamber temperature, timer, alarm temperature.
Turn the push/turn control key by hold down the SET key & at the clockwise or anti clockwise for setting date, local time, operating temperature, alarm temperature. After setting, SET key will be released the display briefly flashes the set point.
To select the programme, Press & hold down SET key and rotate push/turn control key at clockwise and select “Ramp timer” mode.
Set the work days group using the push/turn control by holding down the SET key and Press push/turn control to select the ramp segment “t1” If the day is not required, it will be off position.
Set the time using push/turn control by hold down the set key &
Use the push/turn control, to Select ramp segment “t2”. Hold down SET key and using the push/turn control set the time.
Until the temperature display is flashing, Turn push/turn control clockwise. Set the required temperature setpoint using push/turn control
Select setpoint waiting time by hold down the SET key. Hold down SET key and set on using the push/turn control. After the SET key has been released the function for setpoint waiting time is stored.
Hold down SET key & set time using the push/turn control then Select hold time “t3”.
Hold down SET key & set time using push/turn control after selecting cooling time “t4”.
Select program repeats “loop”. Hold down the SET key and set 2 for repeats using the push/turn control.
To set the air changes, select the fan speed. Turn the push/turn control clockwise until the fan symbol is flashing. Set fan speed 50% suing the push/turn control holding down the SET key.
Turn the push/turn control clockwise until monitor temperature display is flashing. Set the alarm temperature using the push/turn control holding down the SET key & select alarm temperature.
Turn push/turn control at clockwise until the fan symbol flashing to move the air slider opens & closes the air valve to control the supply and discharge of air.
Turn the push/turn control clockwise until the stop symbol ▀ is flashing. Select start ► using the push/turn control holding down the SET key and On releasing the SET key the program starts to run
Glassware Drying
To put on main power switch. Press push/turn control key in front of the instrument. The oven will start in normal mode with display of chamber temperature, timer, alarm temperature.
Select operating mode “Normal operation” holding down SET key (approximately 3 seconds) the current operating is flashing.
Select temperature set point by hold down the SET key & use the push/turn control to select required temperature set point. After the SET key has been released the oven briefly flashes the temperature setpoint. Heating is indicated by the orange heater symbol.
Select fan speed. Turn push/turn control clockwise until fan symbol is flashing while the holding down the SET key. To set 50% fan speed use the push/turn control
Turn the push/turn control clockwise until the monitor temperature display is flashing. Select the alarm temperature. Use the push/turn control to set the alarm temperature by holding down the SET key.
The display then changes to the actual current temperature and starts to the setting temperature. The temperature will be automatically increased at setting temperature and display the setting temperature digitally.
Observe the display temperature until stable position.
Remove dried glassware from oven at the end of drying,
Cleaning of Oven
Clean the inner benches of Oven with a cleaned cloth.
Use 70% IPA or ethanol to disinfect the inner surfaces.
Clean the outer surfaces with dry cleaned cloth.
Disinfect the outer surfaces with 5% Dettol /Savlon solution.
Calibration of Oven
Insert standard thermocouples or thermometer into the chamber.
At least 6 position to be consider to place different location of chamber.
Press & hold down SET key until the normal mode light blinking.
To reach SETUP light, turn push/turn control key clock wise.
Select Calibration to rotate push/turn control key.
Use the following temperature for calibration:
1 : 1500C
2 : 2000C
3 : 2500C
Select required calibration temperature in SETUP key & set the corresponding calibration correction to 0.00
Under the steady conditions, using a reference instrument, measure the deviation from the selected calibration temperature.
If the measured reference temperature is too low, the calibration correction setting is negative sign, set the calibration correction in SETUP key.
The correction value should not more than ±10C.
Adjust the temperature if require from adjusting value of calibration temperature (CAL), carry out the check measurement using the reference thermometer.
Carry out others two calibration temperatures in the same manner.
Calibrate incubator once in a year.
Record calibration in Oven Calibration Record, Annexure-II.
Maintenance
Inform to supplier or Engineering Department for maintenance, If Oven shows any mechanical, electrical or any others problem, after maintenance, recalibrate the oven.
To establish the procedure for ideal operation and cleaning of Vortex Mixer, Model: Genius 3 KIA, Fisher Scientific, UK in microbiology test.
Vortex Mixer; Scope
This SOP applies for operation, cleaning of Vortex Mixer, Model: Genius 3 KIA, Fisher Scientific, UK in Microbiology Section at XX Pharmaceuticals Limited.
Definitions/Abbreviations
NA
Responsibilities
The roles and responsibility is as follows:
Executive/ Senior Executive, Microbiology
Operate & clean of Vortex Mixer.
Asst. Manager, Microbiology
Ensure Operation, cleaning & application of sound technical information.
Keep the up to date of SOP.
Review that SOP is technically informative.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure:
Instructions
Do not press hardly on platform.
To avoid movement during vortex, keep Vortex Mixer in a fixed place.
Do not vortex any materials for long time.
To avoid overflow during vortex, maintain the level of test solution into test tube/Vial/test flask.
Operation Procedure
Switch ON the power button. The red indicator light will be illuminated.
Press SET switch to select the automatic vortex or manual vortex.
Set time to press TIMER button as per requirement.
Adjust vortex speed by turning SPEED button at clockwise for increasing or anti clockwise for decreasing.
Place test tube/vial/test flask on the platform.
Attach tube/vial with the machine for more than 1 minute vortex.
Press START button if set manual vortex.
Touch test tube/vial on platform to start the vortex automatically.
Remove test tube at the end of vortex.
Switch OFF power button.
Disconnect machine.
Press MAINS switch off.
Cleaning Procedure
Switch off Vortex Mixer and disconnect the power plug.
Clean outer surface with dry cloth.
Disinfect outer surfaces of Microscope with 5% Dettol/Savlon solution.
Clean daily once.
Maintenance
Inform to supplier or Engineering Department for repairing, If Colony counter shows any mechanical, electrical or any others problem.
To establish the procedure for ideal operation and cleaning of Colony Counter, Model: SC6, Stuart UK, in microbiology test.
Colony counter; Scope
This SOP applies for operation and cleaning of Colony Counter, Model: SC6, Stuart, UK in Microbiology Section of General Building at XX Pharmaceuticals Limited.
Definitions/Abbreviations
N/A
Responsibilities
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Perform operation & cleaning.
Manager, Microbiology
Confirm that operation is carried out properly.
Keep the SOP up to date.
Review that SOP is technically informative & application of sound knowledge.
Head of Quality Assurance
Take initiative to Approval of this SOP
Procedure:
Instructions
Do not press hardly on counting platform.
Do not use any concentrate cleaning or disinfection solution to outer surface of machine.
Do not open petridish during colony counting because all micro-organisms are unscrupulous pathogen.
Cover with a pack of clear discs to protect receiver plate from dust and scratches.
Operation Procedure
Turn on unit pressing by <ON/OFF> switch located at back of the unit.
Select plate receiver background as per requirement either dark or white pressing by side panel.
Select appropriate adaptor for use of less than 90 mm petridish & place it on the receiver plate.
Adjust sensitivity turning the control knob at clockwise for increasing and at anticlockwise for decreasing.
Place covered Petridish on receiver plate, using centering adapter if required.
Press & hold the <correct/reset> key to ensure display is set to zero before counting.
Mark on each colony with a felt tip pen. Every time a colony is marked, apparatus will register count with a bleep sound and counter advance.
Press <correct/reset> key once for removal of each count If unwanted counts are made.
Place first petridish on receiver plate to use averaging facility.
At end of the count, press the <save> key to store the count in the memory. This will indicated by three dashes on the display. Replace petridish with next and press <save>key to resume the count. Repeat until all dishes have been counted.
At the end of the run press the <average> key to display the average count. When average facility is active a red LED spot at the top left hand corner of the display will be visible.
Press & hold the <correct/reset> key until the display returns to zero when count is completed. This will clear the memory of saved counts.
Switch unit OFF at mains after all the counting is completed.
Maintain Colony Counter Log Book, Annexure-I for each operation.
Cleaning & Disinfection Procedure
Remove plate from instrument for cleaning the receiver plate,
Make sure that instrument is switch off position.
Disconnect power plug.
Remove petridish from platform.
Clean outside of the equipment initially with dry cloth.
Disinfect the outer surfaces with 5% Dettol/Savlon solution.
Finally clean with Purified water.
Clean after each work.
Maintenance
Inform to supplier or Engineering Department for corrective action, If Colony counter shows any mechanical, electrical or any others problem.
To establish the ideal operation of Bio-safety Cabinet to confirm that the air are free from any microorganisms & any particle in order to favor microbiological test.
Biosafety Cabinet; Scope
This SOP applies for operation of Bio-safety Cabinet, Model: AC2-4E1 in Microbiology Section at XX Pharmaceuticals Limited.
Definitions
BSC : Bio-safety Cabinet
DOP : Dioctyl Phthalate
HEPA : High Efficiency Particulate Air.
IPA : Iso Propyl Alcohol.
PAO : Poly alpha olefin.
Responsibilities
The roles and responsibility is as follows
Executive, Microbiology
Operation, cleaning & disinfection and performance checking of Bio-safety Cabinet
Manager, Microbiology
Ensure Operation, cleaning & disinfection, performance checking and application of sound technical information.
Review that SOP is technically informative.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure:
Instructions
Do not wipe HEPA filter side of BSC.
Do not turn on UV light without full cover of slash.
Move always gently during operation of BSC to minimize air turbulence.
Care should be taken that water do not enter into HEPA filter through working station of down flow.
Operating Procedure
Press UV light button to turn on the UV light.
Wait for 30 minutes.
Press light button to turn on.
Pull up front slash at 50% open position. Open position (%) will be indicated on the display.
If show alarm, adjust position of slash.
Press FAN button to start up and wait for 3 minutes.
After 3 minutes, fan will be automatically started and
Use 70% ethanol/IPA to wipe all the surfaces of workstation, except in front of HEPA filter.
Use 70% ethanol to wipe all materials (Glassware’s, containers and other articles) before bringing them inside the Bio-safety Cabinet.
Start the work.
Use 70% ethanol to wipe again entire platform after finishing work.
Press light button to turn off.
Pull down to cover front slash at 0% open position. Open position (%) will be indicated on the display.
Press UV light button to turn on the UV light
Wait for 30 minutes to disinfect the whole cabinet.
After 30 minutes, press UV light button to turn off.
Disconnect the power plug of the cabinet.
Performance Check
Check the performance of HEPA filter by monitoring the Particle count and Microbial count once in a year and prepare the report in Annexure-I & Annexure-II
Check efficiency of HEPA filter by DOP/PAO test once in six months and prepare the report in Annexure-I & Annexure-II
Maintenance
Inform to the supplier or Engineering Department for maintenance, if Bio-safety Cabinet shows any mechanical, electrical or any others fault.
After maintenance, check HEPA filter efficiency & monitor Particle Count and microbial count.
To establish the procedure for ideal operation & cleaning of Microscope for use in microbiological test.
Microscope; Scope
This SOP applies for operation, cleaning and of Microscope, Model: CX21-LED SET1, Olympus, Japan in Microbiology Section at XX Pharmaceuticals Limited.
Definitions/Abbreviations
N/A
Responsibilities
The roles and responsibility is as follows
Executive/ Sr. Executive, Microbiology
Operation, cleaning and performance checking of Microscope
Manager, Microbiology
Ensure Operation, cleaning & disinfection and performance checking.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure:
Instructions
Use 2.5% Dettol/Savlon solution for cleaning of objectives lens, eyepieces and condenser.
Do not use any concentrate alcohol or any others solution.
Use only immersion oil on slide.
Wipe properly objective lens to remove immersion oil at the end of use.
Do not disassemble the microscope without approval of Head of Microbiology Section.
Operation Procedure
Wipe entire platform with clean cloth.
Prepare specimen (stain, if necessary) on a glass slide & place it on stage properly under objective lens with aid of adjustment knob.
Put on switch for light source.
Rotating light intensity adjustment knob for increase or decrease brightness clockwise or anticlockwise.
Adjust objective lens (10x, 40x, 100x) according to requirement.
Use both coarse focus and then Fine focus for better visualization.
Focus an area on the specimen, which is clearly defined or not over crowded.
Do not move specimen holder directly by hand this will damage rotator mechanisms of the knob.
First focus specimen with 10x then with 45x, and then with 100x objective lens (if necessary).
After work is done wipe again lens & platform with another clean cloth, separately.
Cover the microscope at end of the work.
Cleaning & Disinfection Procedure
Switch off Microscope & disconnect power plug.
Remove slide from stage.
Clean outside of chamber with dry cloth.
Use 2.5% Savlon/Dettol solution to Disinfect outer surfaces of Microscope
Clean Objective lens smoothly & Ocular lens with clean, soft, fiber free cloth before and after use.
Clean objective lens with cider wood oil if found any stain on lens.
Maintenance
If Microscope shows any mechanical, electrical or any others problem, inform to supplier or Engineering Department for corrective action.
To operate, clean & calibrate incubator in order to support the bacterial growth in microbiological test.
Incubator; Scope
This SOP applies for operation, cleaning and calibration of Incubator, Model : INE500 & INE600 in Microbiology Section At XX Pharmaceuticals Ltd.
Definitions/Abbreviation
[] CAL.1: Calibration Temperature 1
[] CAL.2: Calibration Temperature 2
[] CAL.3: Calibration Temperature 3
Responsibilities
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Follow the instructions of this procedure correctly.
Assistant Manager, Microbiology
Confirm that this procedure is kept up to date.
Confirm appropriate personnel from the section are trained on this procedure.
Confirm that SOP is technically sound and reflects the required working practices.
Head of Quality Assurance
Take initiative to Approval of this SOP
Procedure
Instructions
Do not wipe with damped cloth at on position.
Use only 70% IPA or Ethanol to wipe chamber with any disinfectants
Never overload the chamber with tested items.
Do not keep the items those may produce inflammation with air.
Keep all tested items to avoid the touch inner surface of the chamber.
Do not move the incubator at on position. Severe vibrations may cause serious damage of the temperature probes.
Operation Procedure
To the front of the instrument, Press push/turn control key to put on the main power switch. The incubator will start in normal mode with display of the timer, chamber temperature, alarm temperature (red color indication).
Turn the push/turn control key by holding down SET key, at the clockwise or anti clockwise for setting date, local time, operating temperature, and alarm temperature. After setting, SET key will be released the display briefly flashes the set point.
The display then changes to the actual current temperature & starts to the setting temperature. The temperature will be automatically augmented at setting temperature and display the setting temperature digitally.
Observe display temperature until its stable position.
Hold down the SET key (Approximately 3 Seconds) to select the operation mode, if require. The current operating mode will be flashed on the display. There are three operating mode in the incubator.
Normal Operation
Weekly Programmer
Ramp time Programme Operation
Select required programme & set as per operation manual.
Select fan speed to set air changes.
Turn the push/turn control at clockwise until the fan symbol flashing to move air slider opens & closes the air valve to control supply and discharge of air.
Check chamber temperature using by a calibrated digital thermometer, when setting Temperature reaches.
Keep required materials inside incubator.
Incubator will automatically control Temperature.
Instrument will automatically adjust the temperature. When the temperature exceed the setting temperature, “off” light will illuminate and if the temperature decrease the “on” light will illuminate.
Cleaning & Disinfection Procedure
Switch off incubator & disconnect power plug.
Remove all test flasks & others items from chamber of incubator.
Clean inside of chamber initially with dry cloth & wipe finally with 70% IPA wetted cloth.
Clean outside of chamber with dry cloth.
Disinfect all surfaces with 5% Savlon solution & dry outer surface of the incubator.
Reload all items into chamber when reach to dry chamber surface.
At the end of cleaning, connect the power plug and switch on the incubator.
Clean chamber once in a month.
Clean outer surface of the incubator every day.
Performance Check
Check chamber temperature daily once.
Record chamber temperature in Daily temperature Record, Annexure-I.
Temperature range ± 2.5° C from set temperature.
Calibration Procedure
Press and hold down SET key until normal mode light blinking.
Turn push/turn control key clock wise to reach SETUP light.
Select Calibration to rotate push/turn control key.
Use three calibration temperatures as below
1 : Temperature calibration at low temperature
2 : Temperature calibration at medium temperature
3 : Temperature calibration at high temperature
Select required calibration temperature in SETUP key & set corresponding calibration correction to 0.00
Measure the deviation from selected calibration temperature under the steady conditions, using a reference instrument.
Set calibration correction in SETUP key. If measured reference temperature is too low, calibration correction setting is negative sign.
Adjust temperature if require from adjusting value of calibration temperature (CAL). Correction value should not more than ±10C.
Carry out check measurement using reference thermometer.
Carry out others two calibration temperatures in the same manner.
Calibrate incubator once in a year ± 15 days.
Record calibration in Calibration Record, as per Annexure-V of Engineering SOP
Maintenance
If Incubator shows any mechanical, electrical or any others problem, inform to supplier or Engineering Department for corrective action.
To confirm that the air are free from any microorganisms and any particle in order to favor of microbiological test.
Laminar Air Flow Operating Procedure; Scope
This SOP applies for operation and cleaning of Laminar Air Flow, Model: AHC-4D1 in Microbiology Section at XX Pharmaceuticals Ltd.
Definitions/Abbreviations
[] DOP: Dioctyl Phthalate
[] HEPA: High Efficiency Particulate Air
[] LAFWS: Laminar Air Flow Work Station
[] LAF: Laminar Air Flow
[] PAO: Poly-alpha-olefin
Responsibilities:
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Operation, cleaning and disinfection and daily performance check.
Manager, Microbiology
Confirm Operation, cleaning and disinfection and performance checking.
Confirm that SOP is technically informative & application of sound knowledge.
Confirm that Laminar Air Flow is suitable for working.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure:
Instructions
Do not wipe HEPA filter side of LAFWS.
Do not move rapidly in a sweeping motion during operation of LAF to minimize air turbulence.
Do not use any disinfectant containing chlorine-based substances as this may be corrosive of the stainless steel.
Do not use Bunsen burner & aerosol generating instruments whenever possible as they interfere with airflow.
Do not turn on UV lamp without the front cover setting.
Operating Procedure:
Switch on the main power.
Set the front cover & make sure that the clean bench is fully closed & the interlock is working effectively.
Press UV/Exit button to turn on the UV lamp. UV can only be turned on when fan and light both are off.
Wait for around 60 minutes.
After 60 minutes, remove front cover from LAFWS.
Turn on Fan/Up button to start fan.
Press to turn on Light/Down to illuminate light.
To turn electrical socket of inner cabin, Press Socket/Set button
Use 70% Ethanol/IPA to wipe the work surface.
Before continuing the work, Leave LAF for 10 minutes
After 15 minutes, wipe all materials with 70% ethanol before bringing them inside the LAF Hood.
Start the work.
Work in cleaned bench in a slow & controlled manner.
Move hands in & out of the work zone opening slowly.
Use 70% ethanol to wipe again the entire platform after end of the work,
Press Fan/Up button to turn off the fan.
Press Light/Down Turns off the light.
Set front cover and press UV/Exit button to turn on the UV lamp to decontaminate the work bench.
Switch off the UV/Exit to turn off the UV lamp after 30 minutes.
Cleaning Procedure
Work surface and wall
Clean work surface & walls with appropriate 5% Dettol/ Savlon solution.
Exterior surface
Use a damp cloth to clean the exterior surface, predominantly on the front & top in order to remove dust that accumulated there.
Use fresh with Sterile Purified Water (which is sterilized at 1210C for 15 minutes or filtered by 0.2 µm) to eliminate any remaining of cleaning agent.
Use MEK (Methyl-Ethyl-Ketone) solution to eliminate stubborn stains or spots on the stainless steel surface. In such cases, wash the stainless steel immediately afterwards with clean water and liquid detergent. Then use polyurethane cloth or sponge for washing.
Microbiological Performance Check
Check performance of HEPA filter by monitoring Particle count & Microbial count before conducting sterility test.
For non-sterile preparation, check performance of HEPA filter by monitoring the particle count & microbial count in every six months.
HEPA Filter Integrity Test
Check efficiency of HEPA filter by DOP (Dioctyl Pthallate)/PAO(Poly Alpha Olefin) test once in a year.
Maintenance
If LAF exhibits any mechanical, electrical or any others difficulty, notify to the supplier or Engineering Department for repairs.
Working Guideline in Microbiology Laboratory; Purpose
To confirm that microbiological best laboratory practice and special precautions are maintained in Microbiology laboratory in order to prevent laboratory & personal contamination.
Working Guideline in Microbiology Laboratory; Scope
This SOP applies for maintaining microbiological best laboratory practices and special precautions in Microbiology Laboratory at XX Pharmaceuticals Limited.
Definitions/ Abbreviation
None
Responsibilities:
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Maintain microbiological best laboratory practices & special precautions.
Manager, Microbiology
Ensure microbiological best laboratory practices, special precautions and application of sound technical information.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure:
Media Preparation and Quality Control
Working Guideline in Microbiology Laboratory; Media Preparation
Choose correct media or components in making media based on the use of accepted sources or references for formula.
Check Certificate of Analysis describing expiry date, storage conditions etc.
Read instructions on the label carefully before media selection and preparation.
Follow if any special instruction such as heating, additives & pH adjustment etc.
Use always purified water for media preparation.
Record accurate weight of media & make up volume with purified water.
Dissolve media thoroughly into water prior to dispensing & sterilization.
Avoid overheating or less heating media.
Sterilize the media as per label of the container.
Media Storage
Label media properly with batch or lot numbers, preparation & expiry dates.
Store media according to manufacturer’s instructions.
Store prepared media under validated conditions.
Do not store agar at or below 00 Because freezing could damage the gel structure.
Protect stored media from exposure to light & excessive temperature.
Before prolonged storage, place into a sealed package or container to retard moisture loss.
Re-melt agar media in a hot water bath or by using free flowing steam only once.
Quality Control Testing of Media:
Check pH & growth promotion to confirm media efficacy.
Perform limited growth promotion test for each lot, if media is sterilized using a validated method.
Do not use media if any parameters do not comply.
Pre-incubate & 100% inspection prior to use the media.
Maintain double-wrapped condition for those media used in critical environmental monitoring.
Maintenance of Microbiological Cultures:
Handle with care microbiological cultures due to their pathogenicity and toxicity.
Use as standard culture from international recognized organization such as American Type Culture Collection (ATCC), National Collection of Type Culture (NCTC) or any other manufacturer.
Select Standard culture as freeze dried lyophilized condition in vial or ampoules form or ready to use condition with Certificate of Analysis.
Ensure identification of culture prior to its use.
Use only permitted method for culture maintenance.
Maintain storage and sub-culture as per standard operating procedure for standard culture maintenance.
Be care to prevent extreme sub-culturing working control cultures that increase the risk of contamination.
Preserve all typical culture at 2 to 80
Inoculate frozen stock monthly or weekly.
Discard any unused portion to minimize risk of loss of viability & contamination of the stock.
Maintenance of Laboratory Equipment
Perform standard validation practices such as Installation Qualification, Operational Qualification and Performance Qualification for equipment.
Calibrate equipment periodically.
Check performance of all equipment’s on a routine basis.
Maintain always approved protocol for IQ, OQ & PQ of equipment’s.
Clean & disinfect all equipment’s as per standard operating procedure for that equipment.
Laboratory Operation
Isolate items between sterility testing room & microbial limit test and bio-assay room.
Do not interchange item between those area without sterilization.
Sanitize carefully both hands with appropriate sanitizer.
Before entrance into the test room, wear garments, mask and gloves as appropriate.
Always use sterile garments, gloves and mask into sterility testing area.
Aseptically handle the items into microbial limit test room.
Disinfect all items with 70% IPA or any others suitable sanitizer, before transfer the items into testing room. Open growth plate only into bio-assay room or microbial testing room.
Aseptically collect the sample.
Identify contaminants at least genus level.
Use specific sample area to preserve all samples.
Isolate all contaminated samples to reduce the false-positive results.
Follow SOP for disposal of used media to discard all contaminants.
Clean & disinfect all area according to SOP of cleaning and sanitizing Quality Assurance Department.
Lessen the movement into microbial testing room and bio-assay room.
After completion of sterility, exit from sterility testing room.
Wear another set of sterile dress, gloves, mask If require to re-enter into sterility test room.
Wash both hands with liquid soap or any skin cleanser solution, after completion of test and before exit from Laboratory, Finally disinfect both hands.
Documentation
Prepare documents in approved format.
Preserve documents into specific file up to defined period.
Review & update all standard operating procedure, specification & analytical Method within defined period.
Verify the data & calculation.
Maintenance of Laboratory Records
After completion of each work, prepare record in approved format
Follow approved SOP to reflect how the test is actually performed.
Keep calibration record after Perform calibration of any equipment’s
Preserve all records after approval.
Do not discard any approved record without permission and proper justification.
Forward test result of raw materials, in-process & finished products to the respected department.
Achieve and protect all laboratory record against catastrophic loss.
Spillage Management
Spills:
Report immediately to the reporting authority after spillages of culture.
Do not touch any spilled cultures & surrounding debris( e. g. glass , cotton or wool plugs) with exposed hands.
Wear disposable gloves & disinfect area by covering spill with several layers of paper towel/ cloth soaked in a suitable disinfectant.
Leave for it for 15-30 minutes.
Using paper towels, sweep spill debris into dustpan.
Transfer all disposable materials to a suitable container e.g. an roasting bag/autoclave for autoclaving and disposal.
Decontaminate dustpan either by autoclaving or by soaking (at least 24 hours) in hypochlorite solution.
Broken glass
Sweep carefully into a suitable container.
Dispose in a puncture proof container.
Splashes on clothing and skin
Soak in disinfectant contaminated cloth.
Treat to splash on skin as soon as possible.
Wash thoroughly with soap & finally with hot water.
To validate Moist Heat Sterilizer by Biological Indicator.
Validation of Moist Heat Sterilizer; Scope
This SOP applies for validation of Moist Heat Sterilizer in Sterile Processing area and Microbiology Laboratory at XX Pharmaceuticals Limited.
Definitions/Abbreviation
Validation
Validation is the process to establish documented evidence, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications & quality attributes.
Biological Indicator
Biological Indicator is a defined preparation of viable spores made from Bacillus stearothermophillus & Bacillus subtilis & has a particular spore count per indicator of not less than 104 and not more than 109 spores which is used to monitor the efficacy of sterilization process.
BI: Biological Indicator
ATCC: American Type Culture Collection
NCTC: National Type Culture Collection
Responsibilities
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Preservation of Biological Indicator, perform Moist Heat Sterilizer validation and report preparation.
Manager, Microbiology
Ensure Moist Heat Sterilizer Validation, documentation and application of sound technical information.
Head of Quality Assurance
Take initiative to Approval of this SOP
Procedure
Instructions
Biological Indicator is usually non-pathogenic but all microorganisms are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if required).
Discard all items after autoclave.
At the end of work, leave all used items at designated containers safely.
Make sure that all personal ornaments, cell phone are left to prevent unauthorized contamination, before entrance into the test room.
Handling of Biological Indicator
Check expiry date of Biological Indicator (Bacillus stearothermophilus, ATCC-7953).
Discard it after autoclaving if expiry date is exceeded
Observe Certificate of Analysis of Biological Indicator.
Ensure that the paper strip is fully intact.
Preserve always BI at 250C or as per manufacturer instructions.
Do not preserve it at freezer.
Validation Frequency
Perform the validation once in six months interval.
Exposure of Indicator:
Carry out validation as per test schedule.
Map the chamber with most critical area for exposure of Biological Indicator.
Define location & label on each Biological Indicator.
Place spore strip of Biological Indicator of Bacillus stearothermophilus (ATCC 7953) at 8 points as per chamber mapping, table-1.
Complete sterilization cycle along with product to be sterilized or empty sterilizer.
Analysis of Indicator
After sterilization, Cut cover paper aseptically & transfer paper strip into separate test tube containing 9 ml of sterile Tryptone Soya Broth including a positive control & negative control.
Incubation of Indicator:
Incubate all strips including positive & negative control at [50 to 55]0C for 7 days.
Interpretation of Result
Observe indicator strip after 7 days for growth. Turbidity of culture media indicates positive growth. Sterilizer is valid if the following criteria are met:
No growth found in exposed all indicator strip
No growth found in negative control indicator strip.
Growth found in positive control indicator strip.
Repeat validation if following result is found
Growth found in one or more than one exposed indicator strip
Growth found in negative control indicator strip.
No growth found in positive control strip.
The sterilizer is invalid if the following result are found
Growth found in one or more than one exposed indicator strip
No growth found in negative control indicator strip.
Growth found in positive control indicator strip.
Correction Action:
If growth found in anyone indicator strip in repeat test, inform test result to concerned department and Engineering Department for corrective action.
After corrective action, Engineering Department shall inform to Microbiology Lab. for the repeat test.
Microbiology Section shall perform the complete test.
Report Preparation
Report the validation in Moist Heat Sterilizer Validation Report, Annexure-I.
To validate Dry Heat Sterilizer by Biological Indicator & endotoxin Indicator.
Validation of Dry Heat Sterilizer; Scope
This SOP applies for validation of Dry Heat Sterilizer in Sterile Processing area and Microbiology Laboratory at XX Pharmaceuticals Limited.
Definitions/Abbreviation
Validation
Validation is the process to establish documented evidence, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications & quality attributes.
Biological Indicator
Biological Indicator is a defined preparation of viable spores made from Bacillus stearothermophillus & Bacillus subtilis & has a particular spore count per indicator of not less than 104 and not more than 109 spores which is used to monitor the efficacy of sterilization process.
Endotoxin Indicator
Endotoxin Indicator are designed for monitoring depyrogenation process or validation or which may be measured by comparing the levels of endotoxin before and after a depyrogenation cycle using LAL reagent . USP[United State Pharmacopoeia] suggests that a depyrogenation cycle should be reduce the endotoxin by at least 1000 fold ( 3- log reduction ) in endotoxic activity as measured by LAL method.
Endotoxin
It is the component of cell wall of certain bacteria which type of bacteria is known as gram negative bacteria. Endotoxin induce strong immune response and enhance release of cytokine.
[] ATCC: American Type Culture Collection
[] BI: Biological Indicator
[] CSE: Control Endotoxin Standard
[] EI: Endotoxin Indicator
[] λ: Lambda(Lysate Sensitivity)
[] LAL: Limulus Amebocyte Lysate
Responsibilities
The roles and responsibility is as follows
Executive/ Sr. Executive, Microbiology
Preservation of all Indicator, perform Dry Heat Sterilizer validation & report preparation.
Manager, Microbiology
Ensure Sterilizer Validation, documentation and application of sound technical information.
Head of Quality Assurance
Take initiative to approval of this SOP
Procedure:
Instructions
Biological Indicator is usually non-pathogenic but all microorganisms are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if required).
CSE[Control Standard Endotoxin] is pyrogenic in humans. Care should be exercised when handling to avoid ingesting it.
Use caution if handling hot vials. Wear gloves or wait until vials are cool.
Before transferring BI[Biological Indicator] from Microbiology Laboratory, disinfect whole surface of all apparatus with the help of 70% IPA or any others disinfectants.
Leave it at designated containers safely.
Ensure that waste container is tightly capped until autoclaving.
Handling of Biological Indicator & Endotoxin Indicator :
Use BI[Biological Indicator] for sterilization cycle validation & EI[Endotoxin Indicator] for Depyrogenation cycle validation.
Read insert package carefully for instruction of use.
Check expiry date of Biological Indicator (Bacillus subtilis, ATCC-9372) and Endotoxin Indicator.
Discard it after autoclaving if expiry date is exceeded.
Observe Certificate of Analysis of all indicators.
Ensure BI[Biological Indicator] paper strip & EI [Endotoxin Indicator] vial is intact.
Preserve always BI at 250C and at [2 to 8]0C for EI.
Do not preserve it at the freezer.
Culture Media Sterilization :
Prepare required amount of CSDM [Casein Soyabean Digest Medium] for the test & distribute [15 to 20] ml of medium in different test tubes.
Sterilize media at 1210C for 15 minutes.
Preserve those at [2 to 8]0C for use.
Validation Frequency
Perform the validation once in six months.
Exposure Condition:
Carry out validation as per test schedule.
Map chamber with most critical area for exposure of BI[Biological indicator] or EI[Endotoxin Indicator].
Define location & label each BI or EI.
Place EI at 4 points & BI at 12 points as per chamber mapping, mentioned on Table-1.
Complete sterilization cycle along with product or empty sterilizer.
After sterilization, transfer all indicators to Microbiology Laboratory.
BI[Biological Indicator] Assay
Perform whole analysis under Laminar Air Flow workstation.
Aseptically open envelopes of BI[Biological Indicator] test strips.
Place each test strips including negative control and positive control strips in individual tubes containing [15 to 20]ml of TSB.
Identify all tubes.
Incubation of Biological Indicator
Incubate the BI strips at [300 to 35]0C for 7 days.
Interpretation of Result
Observe BI[Biological Indicator] tube daily for growth. Growth should occur in positive control tube within 48 hours. The turbidity indicates positive growth of tubes. Sterilizer is valid if following criteria are met:
No growth found in exposed all tubes.
No growth found in negative control tube.
Growth found in positive control tube.
Repeat validation if following result is found :
Growth found in one or more than one exposed tube.
Growth found in negative control tube.
Sterilizer is invalid if following result are found
Growth found in one or more than one exposed tube.
No growth found in negative control tube.
Growth found in positive control tube.
EI Assay Procedure of LAL Test(Gel Clot Method) :
Use the LAL reagent sensitivity 0.125 EU/ml for this assay.
Reconstitute each EI vial with 1.0 ml LAL water including Positive control & vortex at least five minutes and then dilute 1:8 by using LAL water at least duplicate.
Make a control series (2 λ, λ, λ/2, λ/4) if a new lot of Endotoxin indicator or LAL reagent is used. Otherwise test at λ (Lysate sensitivity) only.
Carry out LAL test for exposure vials, positive control and negative control as per Standard Operating Procedure for Bacterial Endotoxin Test.
Incubation of Indicator
Incubate all LAL test tubes including positive & negative control at [37±1]0C for [60±1] minutes.
Interpretation of Result
A more exact calculation of endotoxin reduction is made by finding the end-point of the positive control, by ten-fold & then two fold dilution, & subtracting the logarithms of the exposed vial from the logarithms of the positive control.
Use the logarithm of lamda for the endotoxin concentration in exposed vials when there are negative LAL test results for the undiluted solutions.
The sterilizer is valid if the following criteria are met
No clot formed in all exposed tube
No clot formed in negative control tube
Clot formed in positive control tube
Repeat validation if the following result is found
Clot formed in one or more than one exposed tube
Clot formed in negative control tube
Not clot formed in positive control tube
The sterilizer is invalid if the following result is found
Clot formed in one or more than exposed tube
No clot formed in negative control tube
Clot formed in positive control tube
Corrective Action:
If endotoxin found in anyone exposed vial in the repeat test, inform the test result to concerned department & Engineering Department for corrective action.
After corrective action, Engineering Department or concerned department shall inform to Microbiology Lab. to perform the validation again.
Microbiology Section shall perform the complete test.
Report Preparation
Report the validation in Dry Heat Sterilizer Validation Report by Biological Indicator, Annexure-I and Dry Heat Sterilizer Validation Report by Endotoxin Indicator, Annexure-I
To initiate that the procedure is suitable for ideal operation, cleaning and calibration of Micropipette for use in microbiology test.
Scope
This SOP applies for Operation, Cleaning & Calibration of Micropipette, Model: 8-105-00-9 & 8-106-00-9 in Microbiology Section at XX Pharmaceuticals Limited.
Definitions/Abbreviations
N/A
Responsibilities
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Operation, Cleaning and Calibration of Micropipette.
Manager, Microbiology
Ensure Operation, Cleaning, Calibration and application of sound technical information.
Review that SOP is technically informative.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure:
Instructions
Keep Micropipette always at vertical position on the respective stand.
Never sink Micropipette into water.
Never reset or adjust the volume.
Care should be taken during pipetting that solutions do not enter into Micropipette.
Do not try to volume highly viscous solution by this device.
Do not use hot solution in this device.
Operation Procedure
Check is calibration status of Micropipette. If Micropipette is not calibrated, calibrate it before use.
Set desired volume by turning red colored setting screw button at clock wise for increasing and anticlockwise for decreasing the volume.
Set micropipette tips on bottom side in tight condition.
After setting desired volume, observe display digit.
Draw desired volume of solution by pressing red colored setting screw in single punch.
Rinse tips least three times with solution at first time.
Deliver total volume by pressing same button at double punch.
Draw total volume of the solution.
Press setting screw for delivery the solution.
After use, always keep it on the stand at vertical position.
Cleaning Procedure
Clean outer surface with dry cloth.
Use 70% IPA solution to disinfect outer surfaces except display of Micropipette.
Clean Micropipette before & after use.
Calibration Procedure
Set Micropipette reading as 100 µL for Model: 8-105-00-9 & 500 µL for Model: 8-106-00-9.
Set tips with Micropipette properly.
Take distilled water of temperature at 250C±10C into a beaker.
Weigh & tare another 100 ml beaker.
Draw 100 µL or 500 µL of distilled water by Micropipette & deliver it into tarred beaker.
Record weight of distilled water.
Repeat same procedure up to nine times.
Calculate the accuracy(% of error) & Precision(% CV) as below :
Accuracy (% error) = Mean Value – Reference value/Reference value x 100
Precision (% CV) = Standard deviation/mean x 100.
Record the result in Micropipette Calibration Record, Annexure-I.
Calibration Frequency: Perform the calibration once in a year.
Maintenance
If Micropipette shows any error or any mechanical fault, inform to the supplier or Engineering Department for maintenance, after repairing or maintenance, recalibrate it before use.
Antimicrobial preservative effectiveness test; To confirm that the concentration of Antimicrobial Preservatives used in different drug preparations are able to kill or destroy or inhibit or prevent or terminate the growth of microorganisms and thus make the product stable throughout its declared shelf life.
This SOP is applicable for Oral liquids tests at Microbiology Laboratory in XX Pharmaceuticals Ltd.
Definition/Abbreviation:
Preservative:
Antimicrobial preservatives are the substances added to different dosage forms or products to protect or defend them from microbial growth or from different microorganisms that are introduced unintentionally during or succeeding in the manufacturing process.
[] ATCC: American Type Culture Collection
[] SDA: Sabouraud Dextrose Agar
[] TSA: Tryptone Soya Agar
Responsibilities:
The roles and responsibilities are as follows:
Lab Attendant
Sample collection
Executive/ Sr. Executive, Microbiology
Sample collection, analysis of preservative test & respective test report preparation.
Manager, QC/Microbiology
Ensure sampling, analysis, documentation & application of sound technical information.
Review of this SOP and confirm that the whole procedure is technically sound.
Head of Quality Assurance
Take initiative to Approval of this SOP.
To ensure the overall implementation of this SOP
Procedure:
Instructions
Use Bio-safety Cabinet to Perform tests of Antimicrobial preservative effectiveness of products.
Before preparation of media and conduct antimicrobial preservative effectiveness test, Wear a mask, Hand gloves and headgear Bring the media to the boil to dissolve completely before autoclave.
Mix well before pouring the substances.
Cool the broth media at room temperature (250C) and the agar media at 500C & for use as per the requirement of the test.
Media & Biochemical Preparation
As per Media preparation for Media, preparation SOP prepare the same.
Inoculum Preparation:
Remove the vial of pellets from refrigerated storage & allow equilibrating to the room condition.
Warm hydrating & diluting fluids to [34 to 38]0C, before use.
To achieve a concentration of about 108 cfu per ml, transfer pellets to hydrating fluid.
Immediately place the microbial suspension into a [34 to 38]0C incubator for 30 minutes
To assure complete hydration, immediately following incubation, vortex the hydrated material to achieve a homogenous suspension.
Inoculation of Microbial Suspension to Product
To give inoculums of 105 to 106 microorganisms per ml of product and mix well, inoculate the product to be examined, each with a suspension of the test organisms.
The volume of the suspension of inoculums does not exceed 1 percent of the volume of the product.
Maintain the inoculated product at the temperature range from [20 to 25]0C and protected it from light.
According to the type of the product, Remove 1 ml sample from each container at suitable intervals & determine the number of viable microorganisms using the Pour plate method.
Suitability of counting method for non-sterile products
As per suitability of counting method for suitability of Microbial count, method SOP perform the same.
Test Sample Preparation
Aseptically accurately measured 1 ml sample to be transferred from each inoculated product container to a market sterile capped dilution tube containing 9 ml of sample diluent & mix well. The ratio of this dilution is 1:10.
Aseptically accurately measured 1 ml sample to be transferred from 1:10 dilution to a second dilution tube containing 9 ml of sterile diluent and mix well. This is the ratio of this dilution is 1:100.
Continue this dilution up to 10-5 or as essential levels.
Plating and incubation:
Take two sterile petri plates then Take 1 ml quantity sample from each dilution.
Maintain the temperature not more than 450C, then For Bacterial count pour 15 to 20 ml of sterile TSA medium into each plate being at and mix well.
Maintain the temperature not more than 450C, For Fungal count, pour 15 to 20 ml of sterile SDA medium into each plate being and mix well.
Pour two plates with TSA & SDA medium each with 1 ml diluents as the negative control.
After solidification incubate them at [20 to 25]0C for 5 to 7 days for the fungal count and [30 to 35]0C for 3 to 5 days for the bacterial count.
Acceptance Criteria
For Oral Liquid & Other than Antacids
For Bacteria (S. aureus, E.coli & P. aeruginosa)
Not less than 1.0 log reduction from the initial count at 14 days.
No increase from the 14 days counts at 28 days.
For Fungi (C. albicans, A. brasiliensis)
No escalation from the initial calculated count at 14 and 28 days.
For Oral Liquid Antacids Preparation
For Bacteria (E.coli, P. aeruginosa, S. aureus)
No upsurge from the initial calculated count at 14 and 28 days.
For Fungi (C. albicans, A. brasiliensis)
No increase from the initial calculated count at 14 and 28 days.
To validate Laminar Air Flow in order to support of processing area and Microbiology Test.
Validation of Laminar Air Flow; Scope
This SOP applies for validation of Laminar Air Flow used in Processing area and Microbiology Section XX Pharmaceuticals Ltd.
Definitions/Abbreviation :
Validation: Validation is the established documented evidence, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications & quality attributes.
DOP Test: Dioctyl Pthalate is a combustible non-toxic colorless oily liquid with slight odor. This chemical is used in HEPA filter integrity test at vaporized condition by DOP Test Meter.
[] HEPA : High Efficiency Particulate Air
[] CSDA : Casein Soyabean Digest Agar
[] SDA : Sabouraud Dextrose Agar
[] LAF : Laminar Air Flow
Responsibilities
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Perform Microbiology Air monitoring of Laminar Air Flow & report preparation.
Executive/ Senior Executive, Engineering
Perform Air Velocity Test, DOP test and report preparation.
Microbiology/ Assistant Manager, Engineering
Ensure Laminar Air Flow Validation, documentation and application of sound technical information.
Head of Quality Assurance
Take initiative to Approval of SOP
Procedure:
Instructions
Wear protective items such as sterile latex free gloves, laboratory coat and eye protection (if required).
Disinfect whole surface of all apparatus with the help of 70% IPA or any others disinfectants before transferring into Laminar Air Flow.
Make sure that all personal ornaments, cell phone are left to prevent unauthorized contamination, before working under Laminar Air Flow.
Microbiological Monitoring
Start Laminar Air flow at least before 30 minutes of validation.
Select sampling point as mentioned on Table-1.
Expose sterile CSDA and SDA plate for 30 minutes under LAFWS at different location as per sampling points.
After sampling, cover all plates with the glass lids.
Incubate all CSDA plate at (30 to 35)0C for 72 hours and SDA plate at (22 to 25)0C for 5 days including positive control & negative control plate.
After incubation, count the CFU per plate.
Report in Laminar Air Flow Validation Report, Annexure-I.
Perform this test yearly.
Detection of Air Velocity
Disinfect outer surface of Anemometer with the help of 70% IPA before transferring into LAF.
Measure distance 6” from HEPA.
Take air flow reading of HEPA of LAF by moving smoker from left to right & top to down.
Check that any deviation of reading found for air velocity. If found mark that point.
Report Air Velocity & Filter Integrity Test Report of Laminar Air Flow, Annexure-II.
Filter Integrity Test
Set DOP Test Meter with DOP test port of LAF.
Run machine as per SOP of DOP Test Meter.
Take air by smoker of DOP Test & record the reading.
Check any deviation of reading.
Report Air Velocity & Filter Integrity Test Report of Laminar Air Flow, Annexure-II.
Validation Frequency
Perform LAF Validation once in a year.
Corrective Action:
[] If growth obvers in anyone plate, inform the test result to concerned department & Engineering Department for corrective action.
[] If the test result found out of specification in Air Velocity Test or Filter Integrity Test, Engineering department shall take necessary action.
[] Microbiology Section and Engineering Department shall perform the complete test.
Microbial Examination of Empty Bottle, Cap & Stopper; Purpose
To confirm that the bacterial & fungal count & specified microorganisms into empty bottle during filling are within In-house specification.
Scope
This SOP is applicable for microbiological test of empty bottle during filling in Microbiology Section at XX Pharmaceuticals Ltd.
Definitions/Abbreviation
[] CSDA: Casein Soyabean Digest Agar
[] CSDM: Casein Soyabean Digest Medium
[] SDA : Sabouraud Dextrose Agar
[] TAMC: Total Aerobic Microbial Count
[] TYMC: Total Yeast & Mould Count
Responsibilities
The roles and responsibility is as follows
Executive, Microbiology
Sample collection, analysis and documentation.
Manager, Microbiology/Quality Control
Ensure analysis of empty bottles, documentation and application of sound technical information.
Head of Quality Assurance
Take initiative to Approval of this SOP
Procedure
Instructions
When enter into the test area, wear sterile latex free gloves, mask, laboratory coat & eye protection (if required).
Make sure that all personal ornaments cell phone are left to prevent unauthorized contamination, before entrance into test area.
Move always gently and never move vigorously into the test area.
General Requirements for the test
Glass Apparatus
Sterilized 90 mm Glass Petridish
Screw capped Conical Flask 100 ml
Screw Capped Test Tube
Volumetric Flask 1000 ml
Volumetric Flask 500 ml
Pipette 2 ml, 10 ml
Media and Reagents
Casein Soyabean Digest Agar(CSDA)
Casein Soyabean Digest Medium(CSDM)
Mac-Conkey Broth
MacConkey Agar
Neutralized Peptone
Sabouraud Dextrose Agar
Others Requirements
Surgical Gloves
Surgical Cotton
70% IPA or ethanol
Enumeration Method(TAMC & TYMC)
This test quantifies the enumeration of mesophillic bacteria & fungi that may grow under aerobic conditions.
Test Conditions
Disinfectant both hands, bottles surface, Laminar Air Flow workstation with 70% IPA or 70% ethanol before starting test.
Carry out the test under Laminar Air Flow to avoid contamination.
Culture Media Preparation
Prepare different culture media as per requirement.
Weigh accurate amount mentioned in the manufacturer label into appropriate flask.
Bring to boil completely to dissolve media.
Sterilize at 1210C for 15 minutes or as per manufacturer label.
Store prepared culture media in air tight flask at 2 to 80
Stock Buffer Solution
Take 34 g of Potassium Dihydrogen Phosphate[KH2PO4] in a 1000 ml volumetric flask.
Dissolve in 500 ml of Purified Water, adjust to pH 7.2 ± 0.2 & dilute to 1000ml with Purified Water.
Dispense 90 ml into each screw capped flask.
Sterilize at 1210C for 15 minutes.
Store prepared buffer at 2 to 80C for a validated period.
Glassware Cleaning & Sterilization
Clean all glassware by 1% detergent initially & then rinse with sufficient tap water.
Rinse finally with sufficient Purified Water to remove residual content of detergent.
Sterilize all glassware at 2000C for 1 hour.
Sample Size
Collect 5 empty sealed bottles from each batch during filling of the products at three stages of Starting, Middle and Ending of operation.
Test Method
Carry out anyone from the following mentioned method
Pour plate method
Transfer all bottles under Laminar Air Flow.
Deseal the cap of all bottles.
Add 10 ml sterile meat peptone or phosphate buffer pH 7.0 ± 0.2.
Cap the bottles & shake well to mix properly.
Mark all petridish as product name, batch number, plate name (CSDA/ SDA) and bottle number & test date.
Take 1 ml of rinsing solution from each bottle & pour into two 90 mm sterilized petridish.
Add 15-20 ml CSDA into one plate & add 1 ml SDA into another plate.
Maintain same manner for the rest 4 bottles.
Mix sample with the media by tilting & rotating the plate.
Allow to solidify all plates & invert after solidification.
Incubate CSDA at 30 to 350C for 3 to 5 days & at 20 to 250C for 5 to 7 days.
After incubation, calculate number of cfu per bottle.
Negative Control
Use the diluents as sample in place of test preparations and follow the steps mentioned steps.
Membrane Filtration Method
Prepare sample as per Pour Plate Method
Filter whole rinsing solution of all bottles individually through 0.45 µm and transfer the filter paper to the surface of CSDA slant for bacterial count and SDA slant for yeast & mold count.
Invert plates & incubate all CSDA plates at 30 to 350C for 3 to 5 days & SDA plates at 20 to 250C for 5 to 7 days.
After incubation, count the colony of each plate.
Calculate number of cfu per bottle.
Negative Control
Use the diluents as sample in place of test preparations and follow the steps mentioned steps.
Surface spread Method
Spread not less than 0.1 ml of rinsing solution on surface of two CSDA & two SDA Plate.
Dry all plates under Laminar Air Flow work station.
Incubate CSDA at (30 to 35)0C for 3 to 5 days & at (20 to 25)0C for (5 to 7) days.
After incubation, calculate number of cfu per bottle.
Negative Control
Use the diluents as sample in place of test preparations and follow the steps mentioned steps.
Interpretation of the results
The bottles are passed if the observed count is less than specified count.
The product is failed if the observed count is greater than specified count of that product.
Test Control
Negative control must be negative growth. If found growth in negative control, the test is invalid.
Test for Specified Microorganisms
Suitability of Test Method
Add each test strain separately not more than 100 cfu at the time of bottle test mixing with culture media as per standard operating procedure of Suitability of Microbial Count Method.
The test is suitable if growth found the specific microorganisms. The test is not suitable if no growth found the specific microorganisms. In that case, add any neutralizer or increase dilution for removal any inhibition of product.
Test for E. coli
Add 10 ml of test sample to 90 ml of CSDM. Incubate at (30 to 35)0C for 18 to 24 hours.
Shake the container and transfer 1 ml of CSDM to 100 ml of MacConkey Broth.
Incubate at (42 to 44)0C for 24 hours.
Sub culture on MacConkey Agar plate from MacConkey broth.
Incubate at (30 to 35)0C for (18 to 72) hours.
The product complies with the test for E. coli if no red colonies are present with precipitated zone & the biochemical tests are negative.
Test Report Preparation
Microbial Examination Report of Cap & Stopper, Annexure-I.
Microbial Examination Report of Empty Bottle, Annexure-II.
To confirm that the processing area & processed equipment’s are free from any intolerable microorganisms & total aerobic count are within specification.
Microbiology Analysis of Surface Swab; Scope:
This SOP applies to detect or count of bacteria and Yeast/Mold in Non-sterile Processing, Sterile Processing Area & Testing area of Microbiology laboratory at XX Pharmaceuticals Ltd.
Definition:
Swab test & Finger Printing are to check with a view to take timely corrective measures for maintaining a favorable manufacturing environment, minimizing the risk of contamination of the products.
Responsibilities:
The roles and responsibility is as follows
Executive/ Senior Executive, Microbiology
Sample collection, analysis of swab and document preparation
Manager, Microbiology
Confirm sampling, analysis, documentation & application of updated technical information.
Head of Quality Assurance
Take initiative to Approval of this SOP
Procedure:
Instructions
Disinfect with the help of 70% IPA/Ethanol the whole outer surface of machine & all apparatus.
Wear gloves, mask & sterilized garments before entrance into aseptic area.
Minimize the movement into aseptic area or others sampling area.
Swab Test of Garments/Floor/Wall/Equipment
Sterilization of Swab Kits and Culture Media
Prepare swab kits with cotton bud as per requirement.
Roll tightly a small amount of cotton of surgical grade at one end of the glass rod.
Place cotton bud in a narrow test tube.
Plug end of test tube with the help of non-absorbent cotton.
Prepare CSDA [Casein Soybean Digest Agar], CSDB [Casein Soybean Digest Broth] according to the requirement of the test. Distribute 50ml of CSDB in each conical flask.
Sterilize media & test tubes containing cotton bud & Template steel plate at 1210C & 15lbs pressure for 15 minutes in an autoclave.
Swab Collection
Select a steel template of 5 x 5 cm. size.
Sterilize steel template by alcohol flaming before the test
Wipe cotton bud slowly & firmly in interior direction of steel template on the surface, selected for test.
Rotate cotton bud against direction of the overall wiping movement.
Repeat process for three times.
Collected Sample Preservation
Perform test within 30 minutes after collection of sample.
Preserve sample at 2 to 80C at not more than 12 hours if test is not performed within 30 minutes.
Test Method
Place swab immediately in a bottle containing 50ml of CSDB.
Pull cotton free in the medium.
Shake bottle containing swab for sometime in a shaker.
Pour 1ml of diluent into sterile petridishes with aid of a sterile pipette.
Add 20 to 25ml of Tryptone Soy Agar at about 45 to 500C to the plate.
Mix medium with sample by rotating petridish.
Allow medium to solidify & then keep plate for incubation at 370C for 48 hours.
After incubation period count number of colonies either by colony counter or visual inspection.
Carry out the test in every week.
Interpretation of Test Result
Report swab test if test result found in Swab Test Report, Annexure-I.
If test result found out of specification, repeat the test.
Recollect swab sample from same area or same person.
Carry whole test as previously performed
If test result found within specification as per table-1, report the test result in Annexure-I.
Personnel Finger Printing :
Culture Media Preparation and Sterilization
Prepare CSDA [Casein Soybean Digest Agar] for the test according to the requirement of the test following the instructions of the respective manufacturer.
Autoclave medium at 1210C & 15lbs pressure for 15 minutes.
Pour approximately (20 to 25) ml of medium in each plate after autoclaving.
Allow medium to solidify & then keep in refrigerator until use.
Dry surface of agar media.
Do not use prepared plate after 72 hours.
Take medium aseptically to respective department.
Collection of Finger Print
Take finger print of both the hands, of each person working in the aseptic area in the separate plate.
Mark each plate with person’s name, area /room no., hand (right/left) and date.
Aseptically bring plates back to microbiology laboratory
Incubate plates at (30 to 35)0C for 48 hours.
After incubation count number of colony forming unit formed on each plate either by visual examination or by colony counter.
Carry out test every week for sterile process operator & once in a month for Non-sterile process operator.
Interpretation of Test Result
Report swab test if test result found in Swab Test Report, Annexure-I.
If test result found out of specification, repeat the test.
Recollect swab sample from same area or same person.
Carry whole test as previously performed
If test result found within specification as per table-1, report the test result in Annexure-I.
Out of Specification
If test result found out of specification, send test report to concerned Sectional Head for corrective action.
After corrective action, repeat the test for specific area or person.
Maintain and preserve of standard culture; Purpose
To maintain & preserve the standard culture in order to use in specific microbiology test.
Maintain and preserve of standard culture;Scope
This SOP applies to preserve & maintain stock standard culture in Microbiology Laboratory of at XX Pharmaceuticals Ltd.
Definitions
Standard Culture
Standard culture is a specific microorganism of a specific strain that is recognized by BP/USP/Eur. Ph. and certified by ATCC/NCTC or any other standard culture bank. That culture is used in the different microbiological test such as Growth Promotion Test, Biological Assay of Antibiotics, Sterilizer Validation, Antimicrobial Preservative Effectiveness Test, Microbial Count suitability Test & Sterility Test Validation.
ATCC: American Type Culture Collection
NCTC: National Type Culture Collection
Responsibilities
Executive/ Senior Executive, Microbiology
Preparation of Culture Media, sub-culture, preservation and record keeping.
Follow the instructions of this procedure correctly.
Ensure appropriate personnel from the section are trained on this procedure.
Confirm that SOP is technically sound & reflects the required working practices.
Head of Quality Assurance
Take initiative to approval of this SOP
Procedure:
Instructions
Standard microorganisms are usually non-pathogenic but those are unscrupulous pathogen. So before handling it, wear protective items such as sterile latex free gloves, laboratory coat and eye protection (if necessary).
Move always gently. Don’t move vigorously into the test area.
Disinfect the whole surface with the help of 70% IPA or any others suitable disinfectants. After completion of sub-culture or transfer of pellets.
Take away all used items those are directly contacted with standard micro-organisms and Leave it at designated containers safely.
Confirm the waste container is tightly capped until autoclaving.
Never touch the apparatus directly in open hands those are used.
Make sure that all personal ornaments, cell phone are left before entrance into the test room, to prevent unwanted contamination.
Sterilization of Apparatus & Glassware:
Sterilize all glassware plus Latin Square Plate and others heat stable apparatus at 2000C for 60 minutes in hot air oven using a validated process.
Use the glassware when the temperature reduce to 400
Preparation of Culture Media:
Select media & diluents as per instruction of BP or USP.
Prepare required amount of Culture media of CSDA [Casein Soybean Digest Agar] & CSDB [Casein Soybean Digest Broth] and SDA [Sabouraud Dextrose Agar] as per indication by manufacturer instructions.
Bring to boil to dissolve it completely.
Distribute 10 ml of the media into each screw capped test tube.
Sterilize at 1210C for 15 minutes.
Cool media approximately to (45 to 50)0
Allow to solidify agar media to prepare slant.
Transfer broth media flasks into the test room.
Dry surface of agar slant keeping into Laminar Air flow or Bio-safety Cabinet.
Incubate sterilized CSDA [Casein Soyabean Digest Agar]/broth at (30 to 35)0C for 48 hours and SDA [Sabauroud Dextrose Agar] for 5 days at (22 to 25)0C for checking sterility of the media.
After incubation observe each test tube for growth. If no growth found, the media is suitable for use.
Store media at (2 to 8)0C into refrigerator until it use.
Preparation of Standard Microorganism:
Test Conditions
[] Disinfectant the surface of all equipments including LAF or BSC. [] Rub hands with the help of 70% IPA. [] Always maintain aseptic condition during handling of standard microorganisms.
Inoculation Technique
Opening of Standard Culture vial/ampoule
Read carefully label’s instructions of standard culture vial/ampoule.
Aseptically break the ampoule/vial under Laminar Air Flow.
Remove pellets from vial/ampoule aseptically as per instruction of the label.
Tight the container for the next time use after removing pellets.
Inoculation of Standard Microorganisms:
Label on the each tube with the name of standard culture.
Reconstitute standard culture pellets as per instructions of manufacturer.
Aseptically Transfer 1 or 2 pellets of dehydrated culture of bacteria and fungus in the test tubes containing CSB.
Incubate test tubes containing bacterial culture at (30 to 35)0C for 3 days and the tubes containing fungi culture at (20 to 25)0C for 5 days. This will serve as mother culture.
After incubation, observe the growth of standard culture that should be turbid or settle growth.
Transfer & streak from the broth culture on surface of agar slant.
Incubate test tubes containing CSA at (30 to 35)0C for 3 days & the SDA tubes containing fungi culture at 20 to 250C for 5 days.
Observe good growth, then preserve it at (2 to 8)0C in refrigerator with proper labeling.
Discard previous culture by autoclaving at 1210C for 30 minutes.
Subculture microorganism to freshly prepared culture media once in a month.
Preparation of spore suspension:
Transfer sterilized media under Laminar Air Flow or Bio-safety Cabinet.
Keep plate for incubation at 350C for 7 days.
After incubation period take out culture with aid of sterilized glass beads & pre-sterilized diluents (0.001 g/L containing Manganese sulfate) by rotating plate.
Pour culture suspension along with few of those glass beads in a 100ml flask containing 50ml of sterilized diluents.
Heat culture suspension at 700C for 30 minutes or 800C for 10 minutes in a water bath.
Cool suspension & then keep inside a refrigerator not exceeding at 40C
Don’t use spore suspension more than 60 days.
Preparation of standard culture suspension (Vegetative form):
Prepare culture suspension by regular subculture on Nutrient Agar slant or Sabouraud Dextrose Agar.
Before using a culture maintained in a slant, subculture the organism in another slant containing a specified medium.
Keep the slant for incubation at (30 to 35)0C for bacteria for (24 to 30) hours and at (20 to 25)0C for fungi for (3 to 5) days.
Store slant in the refrigerator not exceeding at 40C if not used immediately.
Don’t use this suspension at more than 7 days.
Stock Maintenance of Standard Microorganisms:
Check expiry date before use of standard microorganisms.
Don’t use if the expiry date is exceeded & discard it after autoclaving.
Raise requisition for standard culture before exceed of expiry date.
Report Preparation
Maintain Register of standard culture maintenance Record, Annexure-I.
This SOP applies for identification of Microorganisms in Microbiology Section at XX Pharmaceuticals Ltd.
Definitions
GN-ID System
The GN-ID system employs 12(GNA) or 24(GN A+B) standardized biochemical substrates in microwells to identify the family of Enterobacteriaceae & other non-fastidious Gram negative bacilli (Oxidase negative and positive). The kit is planned for professional laboratory use only.
Identification of Microorganisms;Responsibilities:
Executive/ Sr. Executive, Microbiology
Selection of culture and identification of Microorganisms.
Assistant Manager, Microbiology
Ensure identification and application of sound technical information.
Head of Quality Assurance
Take initiative to Approval of SOP.
Identification of Microorganisms; Procedure:
Instructions
Before handling of micro-organisms, wear sterile latex free gloves, mask, laboratory coat & eye protection (if required).
Make sure that all personal ornaments, cell phone are left to prevent unauthorized contamination, before entrance into the test room. The use of Cell phone in the test area is firmly prohibited.
Do not touch any identification materials directly because all are hazardous materials.
Keep all used materials into specific designated container.
The reagents kits are for in vitro use only.
Discard all used items by immersion in an appropriate disinfectant e.g. 3% of sodium hypochlorite for 30minutes. Liquid waste containing acid must be neutralized before treatment.
Care should be taken when handling additional reagents as they may contain corrosive or irritant materials.
Read carefully the leaflet of all reagents before use.
General Requirements for the test
Glass Apparatus :
Sterile Screw Capped Test Tube
Sterile Pipette 1ml/2 ml/10 ml
Glass slide
Media and Reagents :
Casein Soyabean Digest Agar(CSDA)
Casein Soyabean Digest Broth(CSDB)
Cetrimide Agar
Hydrogen Peroxide
Kovac’s reagent
Mac-Conkey Broth
MacConkey Agar
Mannitol Salt Agar
Meat peptone
Mineral Oil
Neutralized Peptone
Nitrate Reagent
Oxidase strips
PYR Reagent
Rapport Vasiliadis Salmonella Broth
Sabouraud Dextrose Agar
Sterile 0.85% Saline
TDA reagent
VP I and VP II reagents
Xylose Lysine Deoxycholate(XLD) Agar
These media and reagent can be purchased from commercially available manufacturer.
Others Requirements
70% IPA or ethanol
Forceps
Micropipette
Micropipette sterile Tips
Surgical Gloves
Surgical Cotton
Scissors
Identification of E. coli/ Salmonella/ Others Enterobacteriaceae/ Pseudomonas aeruginosa :
Preparation of Specimens :
Isolate bacterial culture by streaking on the slant initially on MacConkey Agar for coli or Xylose Lysine Deoxycholate (XLD) Agar for Salmonella species & Cetrimide Agar for Pseudomonas aeruginosa.
Identification of Staphylococcus aureus:
Preparation of Specimens
Isolate the bacterial culture by streaking on the slant initially on Mannitol Salt Agar.
Sub-culture on the slant of Casein Soyabean Digest Agar.
Perform Staining as per SOP for Staining of Micro-organisms as per approved sop
Use always 18 to 24 hours pure culture for identification.
Ensure that the isolate bacteria is catalase positive and Gram Positive cocci in clausters in Gram staining test.
Inoculation and Incubation :
Confirm that the bacteria is the genus of Staphylococcus in slide Coagulate test (CAT)
Emulsify a single colony from an 18-24 hors culture in the suspending medium supplied in the kit.
Mix thoroughly.
Carefully peel back the adhesive strip sealing the microwell strip.
Do not discard sealing strip as they will be required later.
Using a sterile pastuer pipette, add 100 µL of the bacterial suspension to each well of the strip.
As a purity check, transfer 1 drop of the bacterial suspension on to the purity plate of Mannitol Salt Agar.
Incubate the purity plate aerobically at 35-370C for 18-24 hours.
After inoculation overlay wells 10 and 11 with 100 µL of mineral oil. This well is highlighted with a black circle around the well to assist in adding oil to the correct wells.
Seal the top of the microwell strip with the adhesive strip removed earlier and incubate at 35-370C for 18-24 hours.
Reading and Addition of Reagents
Remove adhesive strip & record positive reactions the aid of the color chart.
Record results on provided forms.
Add 1 drop of PYR reagent to well 12. Read & record the results after 10 minutes.
Perform nitrate reduction test on well 9 after reading & recording the ONPG result.
Add 1 drop of Nitrate A reagent &1 drop of Nitrate B reagent to well and read after 60 seconds.
Add small amount of zinc powder, If well 7 remains yellow or colorless after addition of nitrate reagents. After addition of zinc, colorless/yellow indicates positive & red color indicates negative.
Record these additional results on the form provided.
Identification
Report in the form the substrates have been organized into triples (set of 3 reactions) with each substrate assigned a numerical value (1, 2 & 4). The sum of the positive reactions for each triplet forms a single digit of the profile number that is used to determine the identity.
Enter the profile number into identification software which generates a report of the five likely organisms in the selected database.
The software provides identification based on probability in % up to species level.
Sub-culture on the slant of Casein Soyabean Digest Agar.
Perform Staining as per SOP for Staining of Micro-organisms
Use always 18-24 hours pure culture for identification.
Confirm that the isolated bacteria is Gram Negative bacilli.
Inoculation and Incubation :
Carry out an Oxidase test on the isolate. Oxidase positive organisms can only be identified by inoculating both GNA and GN B microwell strips.
Emulsify a single colony from an 18-24 hour culture in 3 ml sterile 0.85% saline for the GN A microwell strip. If both GN A and GN B strips are to be inoculated, the colony should be emulsified in 3-5 ml sterile 0.85% sterile.
Mix methodically.
Carefully peel back the adhesive strip sealing the microwell strip.
Do not discard sealing strip as they will be required later.
Using a sterile pastuer pipette, add 100 µL of the bacterial suspension to each well of the strip.
As a purity check, transfer 1 drop of the bacterial suspension on to the purity plate of MacConkey Agar/ Xylose Lysine Deoxycholate (XLD) Agar.
Incubate the purity plate aerobically at (35 to 37)0C for 18 to 24 hours.
After inoculation overlay wells 1,2 and 3 (GN A strip counting from the tabbed end) and well 20 and 24(GN B strip-well 13 is at eht tabbed end) with 100 µL drops of mineral oil.
Do not overlay well 20 if isolate bacteria is Oxidase positive. These wells are highlighted with a black circle around the well to assist in adding oil to the correct wells.
Seal the top of the microwell strip with the adhesive strip are over wells 7, 11 and 12 in the GN A strip and over well in the GN B strip.
GN A and GN B microwell strips are read after 18 to 24 hours incubation for Enterobacteriaceae and after 48 hours for Oxidase positive bacteria.
Reading and Addition of Reagents :
GN A Strip :
Remove adhesive strip & record positive reactions the aid of the colour chart.
Record results on the forms provided.
Add 2 drops of Kovac’s reagent to well 8. Read & record the results after 60 seconds.
Add 1 drop of VP I reagent and 1 drop of VP II reagent to well 10 and read after 15-30 minutes.
Add 1 drop of TDA reagent to well 12 and read after 60 seconds.
Perform the nitrate reduction test on well 7 after reading and recording the ONPG result.
Add 1 drop of Nitrate A reagent and 1 drop of Nitrate B reagent to the well and read after 60 seconds.
Add small amount of zinc powder, If well 7 remains yellow or colorless after addition of nitrate reagents, After addition of zinc, colorless/yellow indicates positive and red color indicates negative.
Record these additional results on the form provided.
GN B Strip
[] Remove the adhesive strip and record all positive reactions with the aid of the color chart.
Record the result
[] The gelatin well 13 must be read after 18-24 hours for Enterobacteriaceae and after 48 hours for Oxidase positive isolates. A positive gelatin liquefaction result is indicated by black particles visible throughout the well. [] Arginine well is interpreted differentially after 24 hours an 48 hours incubations as below :
[] Report form GN A+B, the substrates have been organized into triples (set of 3 reactions) with each substrate assigned a numerical value (1, 2 & 4). The sum of the positive reactions for each triplet forms a single digit of the profile number, that is used to determined the identity. [] Enter the profile number into identification software which generates a report of the five likely organisms in the selected database. [] The software provides identification based on probability in % up to species level.
To stain the microorganism in order to identify the bacteria or fungi.
Scope
This SOP is applicable to stain bacteria and Yeast/mould in Microbiology Section of General Building at XX Pharmaceuticals Limited.
Definitions
Staining
This the auxiliary technique which is mainly used to enhance contrast in microscopy on a microscopic image. The main application of stain and dyes in medicine and biology sector to highlight the structure of biological tissue.
Gram Staining
Gram stain is the most useful staining technique engaged in bacteriology, is a differential stain. By using this technique, it is possible to divide bacteria into two different groups- Gram Positive & Gram Negative.
Endospore Staining
Spore stain is a most useful staining technique engaged in bacteriology is a structural stains. By this technique, it is possible to detect the presence & position of endospore in the bacteria.
Responsibilities:
Executive/ Sr. Executive, Microbiology
Initiative of subculture of Microorganisms, perform staining & report preparation.
Standard microorganisms are usually non-pathogenic but those are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if necessary).
Don’t move vigorously into test area. Move always softly.
After completion of subculture or transfer of pellets, disinfect outer surface of the vial or test or plate with 70% IPA.
Remove all used items those are directly contacted with standard micro-organisms. Leave it at designated containers safely.
Ensure that the waste container is tightly capped until perform autoclaving.
Don’t touch the apparatus directly in open hands those are used.
Make sure that all personal ornaments, cell phone are left to prevent unauthorized contamination, before entrance into the test room.
Simple Staining
Staining Reagents
Methylene blue Stock Solution :
Methylene Blue 2.g
Distilled Water 100 ml
Methylene blue Staining Solution :
0.2 % of Methylene Blue Solution 12.5 ml
Distilled Water 87.5 ml
Crystal Violet Solution
Crystal Violet 2.0 g
Ethyl alcohol 95% 20 ml
NH4 Oxalate 0.8 g
Distilled Water 100 ml
Carbol Fuchsin(Zeihl-Neelsen) Solution
Fuschin 1 g
Ethanol 10 ml
Phenol 5 g
DistilledWater 200ml
Dissolve Fuschin in alcohol and dissolve phenol in water, then mix the two solutions.
Apparatus
Glass slide 3 x 1 inch
Pipettes
Gas Burner or spirit Lamp
Staining Technique:
Clean & dry microscope slides thoroughly.
Flame the surface in which the smear is to be spread.
Flame the inoculating loop properly.
Transfer a loop full of tap Water to the flamed slide surface.
Reflame the loop making sure that the entire length of the wire that will enter the tube has been heated to redness.
Remove the tube cap with the fingers of the hand holding the loop.
Flame the tube mouth.
Touch inoculating loop to the inside of the tube to make sure it is not so hot that it will distort the bacterial cells; then pick up a pinhead size sample of the bacterial growth without digging into the agar.
Reflame tube mouth, replace can, and put tube back in the holder.
Disperse bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear.
Reflame the inoculating loop to redness including the entire length that entered the tube.
Allow the smear to dry thoroughly.
Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.
Test the temperature of slide after each pass against back of the hand. It has been heated adequately when it feels hot but can still be held against the skin for several seconds. Excessive heat will distort the cells.
Stain smear by flooding it with one of the staining solutions & allowing it to remain covered with stain for time designated below.
Methylene blue: 1 minute
Crystal violet: 30 seconds
Carbol Fuchsin: 20 seconds
During staining the slide may be placed on the rack or held in the fingers.
At the end of the designated time rinse off the excess stain with gently running tap water. Rinse thoroughly.
Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.
Place the stained smear on the microscope stage smear side up and focus the smear using the 10x objective.
Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied.
Apply oil directly to the smear, and focus the smear under oil with the 100X objective.
Draw the cells observed.
Gram Staining Method
Stain and Reagents
Crystal Violet Solution
Crystal Violet 2.0 g
Ethyl alcohol 95% 20 ml
NH4 Oxalate 0.8 g
Distilled Water 100 ml
Gram’s Iodine
Iodine Crystals 1.0 g
Potassium Iodide 2.0 g
Distilled Water 100 ml
Docolorizer :
Acetone 50 ml
Ethanol 96% 50 ml
Counterstain :
Safranin 2.5 g
Ethanol 95% 100 ml
Distilled Water 100 ml
Apparatus :
Glass slide 3 x 1 inch
Pipettes
Gas Burner or spirit Lamp
Staining Technique
Use always young culture of (18 to 24) hours that the differentiation in cell wall structures is retained.
Do not use old cultures due to lose Gram Positiveness.
Make smear, and dry in air and fix by flaming.
Stain with crystal violet for about 30 seconds.
Rinse with the water.
Cover smear with Gram’s iodine for about 30 seconds.
Rinse with the water.
Decolorize with 95% ethanol. For a thin smear, 10-20 seconds is long enough; after the proper time interval, alcohol
Drippings from the slide are no longer colored.
Rinse with the water.
Counterstain with safranin solution for 20-30 seconds.
Rinse with water and blot dry.
Examine under the oil-immersion objectives.
Interpretation of Result :
Gram positive bacteria retain the crystal violet dye after de-colorization and appear deep blue or purple colour.
Gram negative bacteria are not capable of retaining the crystal violet dye after de-colorization and are counterstained red or pink by the safranin dye.
Endospore staining :
Stain and reagents :
Malachine Green solution
Malachite green 5 g
Distilled Water 100 ml
Counterstain Solution
Safranin 2.5 g
Ethanol 95% 100 ml
Distilled water 10 ml
Apparatus
Glass slide 3 x 1 inch
Pipettes
Gas burner
Slide Cleaning :
Clean the slide by immersion in concentrated Sulphuric Acid saturated with Potassium dichromate for several days for removal of grease from slide.
Place drop water on the surface of the slide, if water is spread over its surface indicates the slide is cleaned properly.
Staining Technique
Prepare smear, dry in air and fix by flaming.
Place the slides on a staining rack.
Cover the smear and keep saturated with malachite green 5% aqueous solution and continue heating for 5 minutes.
Wash gently with the water.
Counterstain with safranin for 30 seconds.
Wash with water and blot dry.
Examine under the oil-immersion objectives.
Interpretation of result :
The endospore stains green and the remainder of the cell (or a cell without an endospore) stains light red.
The phase microscope is effective in observing the endospore without staining where it appears as a dense white structure in the cell. If a phase microscope is available, observe the unstained endospore in cultures of Bacillus and Clostridium species.
Bioassay; To determine the potency of antibiotics of raw materials & products which are specified in different Pharmacopeia.
Bioassay; Scope
This SOP is applicable for Biological Assay of Antibiotics in Microbiology Section at XX Pharmaceuticals Limited.
Definitions
Microbial Assay
In Microbial assay the potency or concentration of a chemical substance (especially antibiotics) may be determined by its effect on the growth of a defined microorganism.
Responsibilities
Executive/Senior Executive, Microbiology
Assay plate preparation, assay dilution, inoculation, zone reading and report preparation.
Manager, Microbiology
Ensure that all activities of Biological assay, document preservation & application of sound technical information.
Checking SOP that the relevant technical information is applied.
Bioassay Procedure
Instructions
Standard microorganisms are usually non-pathogenic but those are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if required).
Advised to move softly at Test area not vigorously.
After completion of sub-culture or transfer of pellets, disinfect the outer surface of vial or test or plate with 70% IPA.
Remove all used items those are directly contacted with standard micro-organisms. Leave it at designated containers safely.
Ensure waste container is properly capped until autoclaving.
Don’t touch apparatus directly in open hands those are used.
To lessen the contamination, make sure that all personal ornaments, cell phone are left before enter into the test room/area’.
Sterilization of Apparatus& Glassware:
Sterilize all glassware including Latin Square Plate and others heat stable apparatus at 2000C for 60 minutes in hot air oven using a validated process.
Use the glassware when the temperature reduce below 400′
Preparation of Culture Media:
Select the media & diluents as per instruction of BP or USP.
Prepare 300 ml of particular media as per indication by manufacturer instructions.
Bring to boil for dissolve completely.
Sterilize at 1210C for at least 15 minutes.
Cool media approximately to 450C to 500C
Allow to solidify the agar media to prepare the slope.
Preparation of Test solution
Prepare different concentrations of Test Solution to determine of the lowest dose for detectable zone of inhibitions.
Select & prepare two concentrations of the Test solution as “High Dose” and “Low Dose”.
Preparation of Standard solution
Prepare different concentrations of Standard Solution to determine of the lowest dose for detectable zone of inhibitions.
Select & prepare two concentrations of Standard solution as “High Dose” & “Low Dose”.
Preparation of Microorganisms Suspension:
For Spore suspension preparation
Prepare 140ml of Nutrient agar medium.
Aseptically Pour the medium on a petri plate (190mm).
Allow the medium to solidify.
Keep plate in refrigerator for 30 minutes.
After 30 minutes take out plate & streak whole plate with the desired organism aseptically.
Keep plate for incubation at 350C for 7 days.
After incubation period take out the culture with the aid of sterilized glass beads & pre-sterilized saline water by rotating plate.
Pour culture suspension along with few of those glass beads in a 100ml flask containing 50ml of sterilized saline.
Heat culture suspension at 700C for 30 minutes in a water bath for spore formation.
Cool suspension & then keep inside a refrigerator between (2 to 8)0
Don’t use the spore suspension more than 60 days.
For Maintenance of Sub-culture of Vegetative bacteria
Before using a culture maintained in a slant, subculture the organism in another slant containing a specified medium.
Keep slant for incubation at (30 to 35)0C for 24 to 30 hours.
Store the slant in the refrigerator at not more than 7 days.
Method: Plate Diffusion:
Select Latin Square Plate of 12” X 12” size.
Place plate on a leveled surface after sterilization.
To the medium( 45 to 50)0C add the organism mentioned in above chart for a particular antibiotic in required amount.
Shake flask gently to distribute organism throughout the medium.
Pour medium on plate & allow it to stand for 30 minutes before placing lid in position.
Transfer plate into refrigerator.
When required for use, cut cups in agar by means of a sterile cork borer of 8mm diameter.
Remove each disc of agar with a “spear” so that the surrounding is not lifted.
Application of solution to Assay Plate :
Enter details of the sample numbers, weight & dilutions on the assay report form, after diluting the standard and test solutions, There is an assay report form for each plate.
Concentrated solutions are coded with H (High dose) and the lower concentrated solutions are coded with L (Low dose).
Apply using a standard (100 ±1) µL the solutions to the assay plate in the order of the design.
Starting at top left hand corner & working from left to right across the rows down to the bottom right hand corner.
Once started, plating out should be continuous, as it is important that solutions are placed in cups at regular interval.
Keep the plate about one hour for proper diffusion.
After diffusion lid the plate with glass lid.
Incubation of Assay Plate
Incubate assay plate for (16 to 20) hours at 370C for antibiotic or 250C for antifungal.
Measurement of the Zone Diameters
Place the assay plate on a photographic light box.
Measure zone of inhibition using Varnier calipers.
Start at top left hand corner and measure the diameter of the zone accurately.
Continue measuring zones from left to right on row 1, then right to left on row 2.
Repeat the procedure until all 64 zones have been properly measured.
Bioassay; Calculation of Potencies:
Sum high & low doses for each standard ( S1&S2)
Sum high & low doses for each test sample (T1& T2)
Substrate test treatment totals from standard treatment total. This will give a plus or a
Minus figure = D, D = (T1 +T2)-(S1 +S2).
B = (Sum of high doses of test, T1 – Sum of low doses of test solution, T2) + (Sum of high doses of
Standard, S1– Sum of low doses of standard, S2)
Calculate the “Dilution factor of high-test sample (F)” by dividing “Weight of sample” taken by “Total
Volume of dilution” up to high doses.
Log ratio of dilution (I) = Log (High dose concentration ÷ Low dose concentration)
Actual weight
Calculate “Potency of High Standard (H) = ———————– X. High dose concentration Theoretical weight
Potency (P) = Antilog (D/B x I) x F x H.
Report the result in Biological Assay Report, Annexure-I.
Environmental Monitoring; To describe the procedure for Environmental monitoring.
Scope
Environmental monitoring in XX Pharmaceuticals Ltd
Definitions
Environmental monitoring:
Monitoring of viable and non-viable quality of a controlled environment
Particulate count:
[] Enumeration of non-viable particulate of specific size from a particular volume of air of a controlled
Environment. [] Settle Plate: Exposure of petri-plates of nutrient media in a controlled environment to estimate viable
Microorganisms from the environment. [] TSA: Tryptone Soya Agar [] CFU: Colony Forming Unit
Responsibilities
The roles and responsibility is as follows:
Lab. Attendant
Room preparation for Test
Executive, Microbiology
Carry out the test and incubation & documentation
Sr. Executive, Microbiology
To ensure test, incubation, report checking, document preservation & application of sound technical information.
Head of Plant
Review of the SOP & the relevant technical information is applied.
Head of Quality Assurance
Take initiative to Approve of this SOP.
Procedure:
Instructions
Don’t rub during contact plate sampling.
Use the sterile filter holder.
Place the filter paper on the filter holder carefully under laminar airflow.
Try to avoid unwanted personal contamination during air sampling.
Monitoring to be carried out before production hours. (Sterile products)
Non-viable Particle monitoring:
Bring the laser particle counter into the specific monitoring area.
Enter into clean room, wearing approved designated dress. When entering into the clean area, before taking reading make sure that all the doors remain closed.
Operate the Particle counter following approved SOP.
Take count of different several position for each room as indicated in sampling point. Count the number particles (5µ and 0.5 µ) form each sampling point. The average of the counted particle indicates the total particles of a room.
The minimum sampling time should be as per following formula (Following EN ISO 14644-1)
Vs=(20/Cn,m ) x 100
Where,
Vs = is the minimum single sampling volume per sampling point, expressed in liter.
Cn,m = is the class limit (number of particles per cubic meter) for the largest considered
particle size, specified for the relevant class.
20 = is the defined number of particles that could be counted if the particle concentration
were at the class limit.
Specification: Follow Annexure-III.
Write down the result in the format of Annexure-I.
Viable count:
Settle Plate
Prepare, sterilize & dispense the media TSA into the petriplates and pre-incubate the plates.
Check the pre-incubated plates for any evidence of microbiological contamination under the LAF bench.
Discard the plates contains microbiological growth.
Decontaminate the external surface of petriplates with sterile cloth /cotton soaked in a sanitizing agent (70% IPA).
Place required number of petriplate in a sterilized /sanitized SS container; close the lid of SS container.
Transfer SS box to the area to be monitored.
Enter respective area as per the SOP of Entry and Exit procedure.
Mark Petri plates with the following details-
Name of the sampling point
Room No.
Date of exposure
Place petriplates on corresponding designated plate exposure area and remove
the upper lid of the petriplates. Note the beginning time of the exposure and write it on
the plate.
Place upper lid on edge of petriplates in slanting position.
Expose media plates at sampling point for Maximum 4 hours.
After completion of exposure, close petriplates with lid.
Collect petriplates in the SS container and bring back exposed plates to microbiology Laboratory.
Incubate all exposed petri plates in inverted position in laboratory Incubator.
Incubate at (22.5 ± 2.5)°C for mold and Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5)°C. After incubation count the number of colony forming units (CFU) per plate per sampling point with colony counter.
Incubate an unexposed media filled petriplates along with the exposed plates and mark it as negative control.
Count the number of Colony Forming Units (CFU) per plate per location with colony counter. The average of colony indicates total CFU of a room.
Write down observation in the format attached as Annexure-II.
Air Sampling:
Set instrument for desired sampling time & flow rate as per Approved SOP.
Take air sampler to location where air is to be sampled, hold it with filter facing at required direction and take air sample following approved SOP.
Collect filter under laminar airflow & place it inside on petriplates containing TSA[Tryptone Soya Agar ] media.
Incubate all exposed petri plates in inverted position in the microbiology laboratory Incubator. Incubate at the (22.5 ± 2.5)°C for mold & Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5)°C. After incubation count the number of colony forming units (CFU) per plate per location with colony counter.
Follow sampling location.
Record observation in format attached as Annexure-II.
Calculate number of organisms per cubic meter of air. Average colony indicates total cfu of a room.
Surface monitoring (By swab sampling):
Use sterilized swab or sterilize the swab in the autoclave.
Place required number of sterilized swab sticks with tube containing 5 ml of sterile saline
solution in a Sterilized SS container & tightly secure lid of the SS container.
Transfer SS box to area to be monitored.
Enter respective area as per SOP of entry and exit procedure.
Remove swab sticks with tubes from the SS box & take it to the location to be monitored and Mark tube with following details:
Location number or Name of the location
Swab No.
Date of sampling
Hold swab stick from bottom & place tip on surface to be monitored. Gently wipe the swab bi-directionally to cover an area about 25 cm2.
Perform swab sampling for each defined locations.
Keep swab sample standing in the SS container & bring it to Laboratory.
Gently vortexes swab sampling tube containing swab stick & pour contents in filter holder funnel and filter it. Uses the respective SOP.
Rinse tube with 3 x 10 ml of sterile saline solution.
Filter test sample under partial vacuum.
Rinse Funnel with the portions of sterile Purified Water. This flushes residue from walls of funnel & helps to secure a uniform distribution of colonies on filter surface. Upon completion of rinse & filtration process, shut off vacuum.
Transfer membrane filter with sterile smooth-tip forceps on to Microbial content test agar (TSA) plate.
Place filter with a rolling motion to avoid entrapment of air.
Keep a negative control by filtering 100 ml Purified Water(Sterile) before filtering actual test sample.
Pass 20 to 30 ml of sterile Purified Water through funnel, between different test samples when the same funnel used for multiple sample.
Incubate all exposed petriplates in inverted position in microbiology laboratory Incubator. Incubate at (22.5 ± 2.5)°C for mold and Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5°)C. After incubation count number of colony forming units (CFU) per plate per location with colony counter.
Count number of colony forming units (CFU) per plate per location with colony counter.
Note down the observation in the format attached as Annexure-II
Surface monitoring (By contact plate):
Fill Petridishes with TSA culture medium & pre-incubate.
Take off the lid of plates.
Invert & press agar surface for 10 seconds onto surface to be examined.
Replace lid & mark plate with appropriate data.
Clean sampling area on surface in order to remove any remaining of agar.
Return plates to laboratory.
Incubate all exposed petriplates in inverted position in microbiology laboratory Incubator. Incubate at (22.5 ± 2.5)°C for mold and Yeast for 72 hours followed by another 48 hours for bacteria at (32.5 ± 2.5°)C. After incubation count number of colony forming units (CFU) per plate per location with colony counter.
Take count of visible colonies within mean of plate
Express results as CFU per contact plate.
Perform appropriate identification of germs found by surface monitoring..Classified area should free of pathogenic microorganism.
Note down the observation in the format attached as Annexure-II.
Sampling Location:
Determine Number of sampling location by following formula.
NL = √A
Where,
NL = the minimum number of sampling locations, rounded up to a hole number.
A= Area of the clean room or clean air controlled space in m2.
Suitability of Microbial Count Method; To confirm the ability & the suitability of the test to detect microorganisms in the presence of a product or Raw Materials as per the In-house or Pharmacopoeia specifications.
Scope
This SOP is applicable for Microbial Count Method Validation in Microbiology Section of XX Pharmaceuticals Limited.
Definitions
Microbial count Suitability: Microbial Count suitability is to confirm that the test dilution is free from any type of interfering substances or antimicrobial properties that will recover by dilution or the addition of a neutralizer to detect microorganisms in the presence of the product.
CSDA: Casein Soybean Digest Agar
CSDB: Casein Soyabean Digest Broth
LAF: Laminar Air Flow
SDA: Sabouraud Dextrose Agar
SDB: Sabouraud Dextrose broth
Responsibilities
The roles and responsibility is defined as follows:
Executive/ Sr. Executive, Microbiology
Preparation & inoculation of standard culture, carry out test & arrange appropriate document preparation.
Asst. Manager/Manager, Microbiology
Ensure of method suitability, documentation and application of sound technical information.
Head of Quality Assurance
Taking Initiative to Approval of this SOP
Procedure
Instructions
Safety Precautions
When enter into the test area, wear sterile latex free gloves, Laboratory coat and eye protection (if required).
To prevent unauthorized contamination, make sure that all personal ornaments, cell phone are left before enter into the test room. The use of Cellular phone in the test room is strictly prohibited.
Don’t move vigorously into the test area. Move always gently.
General Requirements
Glass Apparatus
Pipette 2 ml, 10 ml
Sterilized 90 mm Glass Petridish
Screw capped Conical Flask 100 ml
Screw Capped Test Tube
Volumetric Flask 500 ml
Volumetric Flask 1000 ml
Media and Reagents
Casein Soyabean Digest Agar(CSDA)
Casein Soyabean Digest Broth(CSDB)
Meat peptone
Neutralized Peptone
Sabouraud Dextrose Agar
Sabouraud Dextrose Broth
These media can be purchased from commercially available manufacturers.
Others Requirements
45 µm Membrane Filter
70% IPA or ethanol
Filtration Unit( sterilized filter disk and filtering funnel)
Forceps
Glass spreader
Scissor
Surgical Cotton
Surgical Gloves
General Procedures
Test Conditions
Wear gloves, mask/beard mask Headgear before entrance into Testing Room.
Disinfectant hands, the outer surface of test sample, LAF workstation with the help of 70% IPA or ethanol before starting the test.
Carry out the test under LAF to avoid any type of contamination.
Monitor the test area microbiologically using Microbial Air Sampler at each working day.
Culture Media Preparation
Prepare the different culture media as per the requirement.
Weigh the mentioned amount as per manufacturer label into appropriate flask.
Bring to boil completely to dissolve the media.
Sterilize at 1210C for 15 minutes or as per Manufacturer label.
Store the prepared culture media in air tight flask at controlled environment.
Store prepared agar media at (2-8)0C
Preserve dehydrated culture media up to its expiry date.
Never use expired culture media.
Use the agar media when the temperature reduce to 450C and cools in case of the broth media.
Stock Buffer Solution
Take 34 g of Potassium Dihydrogen Phosphate[KH2PO4] in a 1000 ml volumetric flask.
Dissolve in 500 ml Purified Water, adjust to pH [7.2 ± 0.2] then dilute to 1000 ml with Purified Water.
Dispense 90 ml into each screw capped flask[Approx. 11 containers].
Sterilize at 1210C for 15 minutes.
Store the prepared buffer at 2-80C for a validated period.
Glassware Cleaning & Sterilization
Clean glassware by 1% detergent initially then rinse with sufficient tap water.
Rinse finally with sufficient Purified Water to remove the residual content of detergent.
Sterilize glassware at 2000C for 1 hour.
Sample Preparation
Water-soluble samples
Take 1 gm or 1 ml into the 9 ml CSDB [Casein Soybean Digest Broth] or phosphate buffer or Sodium chloride peptone solutions.
Water-insoluble samples
Take 1 g or 1 ml into the 9 ml CSDB [Casein Soybean Digest Broth] or phosphate buffer or Sodium chloride peptone solutions and add 0.1% Polysorbate 80.
Fatty Products
Dissolve with Isopropyl Myristate sterilized by filtration with low amount of Polysorbate 80 or anther non-inhibitory sterile surface active agent or necessary heat not more than 450C.
Inoculation and Dilution:
To maintain of not more than100 cfu, add adequate volume of suspension of inoculums to the sample.
Add the inoculums suspension not more than 1% of diluted product.
Prepare lowest possible dilution for acceptable microbial recovery of sample.
Add neutralizer for removal of Interfering factor when [If] the sample contains any antimicrobial properties.
Follow Table-1 for the inactivators of Antimicrobial agents.
If growth is inhibited, then increase use of diluents or membrane filtration or combination of all above.
Suitability of Counting Method:
[] Membrane Filtration Method
[] Most Probable Number Method
[] Pour Plate Method
[] Surface-spread plate Method
Pour Plate Method :
Add 1 ml prepared sample to the 90 mm diameter Petridish
Pour (20~25) ml of Sabouraud Dextrose Agar(SDA) for fungi and Casein Soybean Digest Agar(CSDA) for Bacterial count both media being not more than 450
If larger Petridish [more than 90 mm diameter] is used amount of media to be increased accordingly.
Perform plate count methods at least in duplicate for each medium and use the mean count of the result.
Inoculate the microorganisms not more than 100 cfu as indicated Table 2.
Mix properly & incubate CSDA plate at (30~35)0C for 3 days, SDA plate at (20~25)0C for 5 days.
After incubation, count the colony on each plate.
Take arithmetic mean of the count per medium.
Surface-Spread Plate Method
Add (20~25) ml of SCDA and SDA to 90 mm diameter petridish at least duplicate.
Allow to solidify and dry the plate under Laminar Air Flow cabinet or incubator.
Spread not less than 0.1 ml (equal to or less than 100 cfu) of each microorganisms as indicated in Table 1 on the surface of medium
Incubate at CSDA plate at (30~35)0C for 3 days, SDA plate at (20-25)0C for 5 days.
After incubation, count the colony of each plate.
Take arithmetic mean of the count per medium.
Membrane Filtration Method
Use the membrane filter which nominal pore size is not more than 0.45µm.
Filter prepared sample through membrane.
Rinse membrane filter with sterile 0.1% meat peptone solution or any others suitable diluents for neutralization of the sample.
Add inoculums to filter as indicated in Table 1 & rinse again.
Transfer filter on the surface of CSDA plate & SDA plate.
Incubate CSDA plate at (30 to 35)0C for 3 days, SDA plate at (20 to 25)0C for 5 days.
After incubation, ten count the colony of each plate.
Take arithmetic mean of the count per medium.
Test Control
Negative Control: Use diluents in place of test preparations. There must be no growth found in the negative control. If found any growth in the negative control, then the test is invalid & repeat the test.
Result and Interpretation
Mean count of any of test microorganisms not differing by a factor greater than 2 from value of the control defined in absence of product must be obtained.
Repeat test by increasing neutralizer or dilution or any treatment for overcome of antimicrobial properties of products. If the above criteria cannot be met for one or more microorganisms.
Test Report Preparation
Prepare Report the result in Suitability Report of Microbial Count Method, Annexure-I.
Microbiological Media disposal; To dispose of the used media properly in order to lessen Microbiology Laboratory Contamination as well as environmental pollution.
Scope
This SOP is applicable for the disposal of used Media in the Microbiology Laboratory at XX Pharmaceuticals Ltd.
Definition
N/A
Responsibilities
The roles and responsibilities are as follows:
Laboratory Attendant
Clean & disinfect used media
Executive/ Sr. Executive, Microbiology
Monitor disposal activity accordingly.
Follow the instructions of this procedure appropriately.
Follow the instructions of this procedure properly.
Asst. Manager, Microbiology
Confirm proper disposal of used media.
Confirm that this procedure is kept up to date.
Confirm suitable personnel from the section are trained in this practice.
Confirm that SOP is technically sound and reflects the required practices.
Head of Quality Assurance
Approval of this SOP
Procedure
Instructions
Appropriately wear heat-resistant gloves, eye protection, and laboratory coat during handling autoclaved media.
Avoid autoclaving the sealed containers or completely filled bottles with narrow necks as they may explode.
Don’t expose any used media container or plate outside the Laminar Air Flow cabinet.
Wash and disinfect both hands after handling used media.
Collection of Used Media
Wear suitable garments, gloves, and mask.
Wear safety goggles if hazardous media are subject to being discarded.
Collect all the media to be disposed of in the designated vessel after use.
Close the mouth of the vessel tightly.
Sterilization
Attach the Autoclave Tap with the vessel.
Place the vessel into a sterilizer and sterilize at 1210 C & 15 lbs for 30 minutes.
Check that the color of the autoclave tap is turned black.
Discard Method
After autoclaving, collect the media in a specific container when the temperature comes down to 500 to 550 C and overlay the media with 40% formaldehyde solution.
Keep the media for 30 minutes.
Dispose of the media into the drain which is linked with ETP.
Rinse the vessel properly with hot water.
Wash the vessel with detergent.
Disinfect the whole vessel with 70% Iso Propyl Alcohol and dry the vessel.
Record Maintain:
Maintain Register of Media Disposal Record in Annexure-I.
[Microbiological Analysis of water; this article describes the basic procedure of Microbiological Analysis of water as per different guidelines]
Purpose
Microbiological Analysis of water; To confirm that different type of Water used for different drug processing, cleaning and drinking purpose meets the required Pharmacopoeia & In-house specifications.
Scope
This SOP applies for sampling and analysis of all types of water used in this plant.
Definition/Abbreviation
None
Responsibilities
The roles and responsibility is as follows
Lab Attendant
Sample collection of different types of water.
Executive / Sr. Executive, Microbiology
Verify, Monitoring of Sample collection, analysis of Water, and test preparation of report accordingly.
Asst. Manager/Manager, Microbiology
Confirm sampling, analysis, documentation, and application of appropriate technical information.
Review of this SOP that the whole procedure is technically informative and in execution condition.
Head of Quality Assurance
Take initiate to the approval of SOP
Procedure
Instructions
Disinfect the outer surface of the sampling point with the help of 70% IPA.
Wear sterilized latex-free gloves and an appropriate mask. Never forget to wear a beard mask where required.
Never open the sample container before & after the collection of a specific sample.
Sample Collection
Select the sampling point as per the schedule of the Water Test accordingly.
Before sampling sterilized the sampling containers for microbiology test at 1210C for 15 minutes and label it accordingly.
Wear appropriate Laboratory garments, gloves, and mask as required.
Disinfect the outer surface of the sampling points with the help of 70% IPA.
Discharge water for at least 2 minutes for the user points during the collection of sample and 1 minute for a storage tank in the water treatment plant.
Collect water from each point at least 200 ml into the sterilized container for microbiology test.
Collect the sample as soon as possible.
Close the container after sampling and don’t expose the container for microbiology test.
Sample Preservation
Samples shall be analyzed as soon as possible after being collected. If it is not possible to test the sample within about 3 hours of collection.
The sample may be preserved at refrigerated temperatures (2-80C) for maximum 12 hours to maintain the microbial attributes until analysis.
Test Schedule:
Perform the test as per the following schedule
Microbiological tests are as follows:
Potable/Pretreated Water
Perform the test as per Analytical Method
Drinking water
Perform the test as per QC Analytical Method
Purified Water
Perform the test as per QC Analytical Method
Water for Injection
Perform the test as per QC Analytical Method
Report preparation:
Report of Potable/ Pretreated/ Drinking water Test Result in Annexure-I,
Report of Purified Water Test Result in Annexure-II &
Report of Water for Injection in Annexure-IV.
Distribution of Water Test Result
After completion of the analysis, inform the status of water test to a specific department.
If any test result exceeds alert level or is out of specification, immediately inform to concerned department Head and engineering department also for corrective measurement.
After taking corrective action, Engineering Department shall inform to Microbiology Section for further sample collection.
Microbiology Section shall collect the sample from that area to carry out the analysis.
After completion of the test, inform about the test result to concerned department after approval of Head Quality Assurance
Microbial Examination of Non-Sterile Raw Materials and Products Purpose
Microbial Examination of Non-Sterile Raw Materials and Products, To confirm that the bacterial & fungal count in the non sterile products & raw materials are within the In-house / Pharmacopoeia specification & free from certain microorganisms indicated in Pharmacopeia.
Scope
This SOP is applicable for microbiological test of Non-sterile Products such such as Oral Liquid, Semi-solid, Solid preparations and Raw Materials in Microbiology Section.
Definitions
Microbial Examination: Microbial examination is designed to determine the microbial contamination in non-sterile products intended for Oral liquid, Topical Preparations or other non-sterile applications & Raw Materials.
CSDA: Casein Soyabean Digest Agar
CSDM: Casein Soyabean Digest Medium
SDA : Sabouraud Dextrose Agar
SDB : Sabouraud Dextrose Broth
TAMC : Total Aerobic Microbial Count
TYMC: Total Yeast & Mould Count
Responsibilities
The roles and responsibility is as follows:
Laboratory Attendant
Preparation Room for Microbiological Test
Microbiologist
Perform the test and incubation and in time proper documentation
Asst. Manager/Manager, Microbiology
Confirm test, incubation, report checking, document preservation and application of precise technical information.
Head of Quality Assurance
Take initiative regarding approval of this SOP
Procedure
Personal Precautions
During enter into the test area, wear sterile gloves, Lab coat and eye protection (if necessary).
To prevent unauthorized contamination, make sure that all personal ornaments, cell phone are left before entrance into the test room. The use of all type of Cell phone in the test area is strictly prohibited.
Don’t move forcefully into the test area. Move always gently.
General Requirements for the test
Glass Apparatus:
Pipette 2 ml, 10 ml
Sterilized 90 mm Glass Petridish
Screw capped Conical Flask 100 ml
Screw Capped Test Tube
Volumetric Flask 500 ml
Volumetric Flask 1000 ml
Media and Reagents:
Casein Soyabean Digest Agar(CSDA)
Casein Soyabean Digest Broth(CSDB)
Cetrimide Agar
Mac-Conkey Broth
MacConkey Agar
Mannitol Salt Agar
Meat peptone
Neutralized Peptone
Rapport Vasiliadis Salmonella Broth
Sabouraud Dextrose Agar
Xylose Lysine Deoxycholate(XLD) Agar
All of these media can be purchased from commercial available manufacturer.
Others Requirements
70% IPA or ethanol
0.45 µm Membrane Filter
Filtration Unit(sterilized filter disk and filtering funnel)
Forceps
Glass spreader
Scissors
Surgical Gloves
Surgical Cotton
Types of Test for Microbiological Examination
Enumeration Method (TAMC &TYMC)
This test quantify enumeration of mesophilic bacteria & fungi which may grow under aerobic
Condition.
Test Conditions
Wear latex free gloves, Head gear, mask and beard cover [if required], before enter into Test Room
Use 70% IPA or ethanol to disinfectant the hands, the outer surface of test sample, LAF workstation with before start test.
Perform the test under LAF to avoid contamination.
Monitor the test area microbiologically with the help of Microbial Air Sampler at each working day.
Culture Media Preparation
Prepare the different culture media as per specific requirements.
Weigh the exact amount stated in the manufacturer label into right flask.
Bring to boil completely to dissolve the media properly.
Sterilize at 1210C for 15 minutes or as directed by the Manufacturer label.
Store the prepared culture media in air tight flask properly at controlled environment.
Store the prepared agar media at 2-80C.
Preserve the dehydrated culture media up to expiry date.
Never use the expired culture media.
Use the agar media when the temperature reduce near at 450C & cool in case of the broth media.
Stock Buffer Solution
Place 34 g of Potassium Dihydrogen Phosphate[KH2PO4] in a 1000 ml volumetric flask
Dissolve in 500 ml of purified water, adjust to pH [7.2 ± 0.2] & dilute to 1000ml with purified water.
Dispense 90 ml into each screw capped flask
Sterilize at 1210C for 15 minutes.
Store the prepared buffer at 2-80C for a validated period.
Glassware Cleaning & Sterilization
Initially clean all glassware by 1% detergent & then rinse with sufficient tap water.
Finally Rinse with sufficient Purified Water to remove the residual content of detergent.
Sterilize glassware at 2000C for 1 hour.
Testing of Products
Sample Size
Collect 10 g or 10 ml of the products to be taken. 10 containers of the products from a batch.
Collect the amount is not less than the amount present in 10 dosage units or 10 g or 10 ml of the respective product, if amount per dosage unit is less than or equal to 1 mg.
Take 1% of the batch size when batch size is less than 1000 ml or 1000 gm.
Take 2 units or 1 units if the batch size is less than 100.
Types of Method
Membrane Filtration
Most Probable Number Method
Pour Plate Method
Surface spread Method
Membrane Filtration Method
Prepare sample as per Method Suitability.
Filter the sample through 0.45 µm & transfer the filter to the surface of CSDA for bacterial count and SDA[Sabouraud Dextrose Agar] for fungal count.
Incubate CSDA[Casein Soyabean Digest Agar] at [30-35]0C for 3-5 days & at [20-25]0C for 5-7 days.
After incubation, calculate the number of the cfu per gm or ml of the product.
Negative Control
Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.
Pour Plate Method
Prepare the sample as per the Method Suitability
Pour 1 ml of prepared sample into the four 90 mm petridish, Add 15-20 ml CSDA[Casein Soyabean Digest Agar] into two plate & SDA into the others two plate.
Allow to solidify & invert all plates.
Incubate CSDA at [30-35]0C for 3-5 days and at [20-25]0C for 5-7 days.
After incubation, calculate the number of the cfu per gm or ml of the product.
Negative Control
Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.
Surface spread Method
Prepare the sample as per Method Suitability.
Spread not less than 0.1 ml of sample on the surface of two CSDA[Casein Soyabean Digest Agar] and two SDA[Sabouraud Dextrose Agar] Plate.
Dry all plates at Laminar Air Flow.
Incubate the CSDA at [20-25]0C for 5-7 days and at [30-35]0C for 3-5 days.
After incubation, calculate the number of the cfu per gm or ml of the product.
Negative Control
Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.
Interpretation of the results
Count as Total Yeast/ Mould Count (TYMC) on SDA plate and Total Aerobic Microbial Count(TAMC) in CSDA plate and The acceptable criterion for microbiological quality is prescribed as :
101 cfu : maximum acceptable count =20
102 cfu : maximum acceptable count =200
103 cfu : maximum acceptable count =2000
Declaration
The Material/product is passed when the observed count is less than specified count of that Material/product.
The Material/product is failed if the observed count is greater than specified count of that Material/product.
In that case, repeat the test, if the count is greater than specified count, the product is failed.
Test for Specified Microorganisms
Suitability of Test Method
Cary out the test in presence of the product. Add each test strain distinctly not more than 100 cfu at the time of product mixing with the culture media.The test will be suitable if found growth of the specific microorganism. The test will not suitable if no growth found the specific microorganism. In thatcase, add any neutralizer or increase the dilution for removal any inhibition of product.
Testing of Products
Test for E. coli
Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]0C for 18-24 hours.
Shake the container then transfer 1 ml of CSDM to the 100 ml of MacConkey Broth. Incubate at [42-44]0C for 24 hours.
Sub-culture on MacConkey Agar plate from MacConkey broth. Incubate at [30-35]0C for 18-72 hours.
The product complies with the test for E. coli if no red colonies are present with precipitated zone and the biochemical tests found negative[-ve].
Test for Salmonella
Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]0C for 18-24 hours.
Shake the container; transfer 0.1 ml of CSDM to 10 ml of RVS [Rappaport Vassiliadis Salmonella] Broth. Incubate at [30-35]0C hours for 18-24 hours.
Sub-culture on XLD [Xylose Lysine Deoxycholate] Agar plate from RVS [Rappaport Vassiliadis Salmonella] Broth . Incubate at [30-35]0C for 18-48 hours.
The product complies with the test for Salmonella if no red colonies are present with or without black centres and the biochemical tests are negative[-ve].
Test for Pseudomonas aeruginosa
Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]0C for 18-24 hours.
Sub-culture on Cetrimide Agar plate from CSDM [Casein Soyabean Digest Medium]. Incubate at [30-35]0C for 18-72 hours.
The product complies with the test for Ps. aeruginosa if no bluish green colonies are present and the biochemical tests are negative[-ve].
Test for C. albicans
Add 10 g or 10 ml of test sample to 90 ml of SDB [Soubaurad Dextrose Broth]. Incubate at [30-35]0C for 3-5 days.
Sub-culture on SDA[Soubaurad Dextrose Agar] plate from [Soubaurad Dextrose Broth]. Incubate at 30-350C for 24-48 hours.
The product complies with the test for C. albicans if no white colonies are present and the biochemical tests are negative[-ve].
Test Report Preparation
Report the result in Microbial Count Report of Non-sterile RM, Annexure-I.
Report the result in Microbial Count Report of Non-sterile Products, Annexure-II.
This is all about the Microbial Examination of Non-Sterile Raw Materials and Products and based on this information you can generate a SOP for Microbial Examination of Non-Sterile Raw Materials and Products.
Culture Media, To make the culture media for the development of microorganisms in the microbiological test of raw materials, In-process sample & finished products.
Culture Media Scope
This designated SOP is applicable for the preparation of Culture Media in Microbiology Laboratory at XX Pharmaceuticals Limited.
Definitions/Abbreviation:
Culture Media: The Culture media is a liquid or gel designed to support growth of microorganisms or cells or small plants. There are different type of media for growing different type of cells.
There are two major types of growth media: those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or yeast.
Responsibilities:
The roles and responsibilities are as follows:
Officer/Sr. Officer, Microbiology
To follow the instructions of the described procedure accordingly.
Asst. Manager/Manager, Microbiology
Ensure that the procedure is kept up to date.
Ensure right personnel from the section are trained on this specific procedure.
Ensure the media preparation, sterilization, requisition, maintain accurate storage & proper documentation.
To Confirm that this SOP is technically sound and reflects the required working practices as current practices.
Head of Quality Assurance
Approval of SOP
To ensure the overall implementation of the SOP
Procedure
Instructions
Dehydrated media are hygroscopic & are sensitive to light, heat and moisture. They are adversely affected by extreme changes in temperature e.g. hot/ cold cycling temperatures which may occur between day and night laboratory temperatures in winter season.
Condition of Media Preparation:
Take clean & dry flask as per required volume.
Wear appropriate Laboratory garments, gloves and mask.
Wear safety goggles during selection of hazardous media.
Storage Condition of Dehydrated Media:
Mention receipt date on the label when enter into laboratory.
Store as per directions on the label; typically below 250C in a dry area, away from direct sunlight, autoclaves, drying ovens or other heat sources, Where indicated store at (2-8)0C.
Check expiry date on the specific label, some media have suggestively shorter shelf life than others.
Use/maintain stock in lot/batch number order. Maintain FIFO [First In First Out], FEFO [First Expiry First Out].
Do not open a new bottle until the previous bottle has been emptied. Ensure date with label on the supplied container when it first opened.
After intended use, ensure the container is tightly closed and store it into the designated storage area.
Collect/procure/order the medium in an appropriate size of container and in a quantity which harmonies to normal use requirements.
A medium in a large container which has been opened many times will deteriorate on storage. Discard the medium if the powder is not free flowing, if the colour has changed or if it appears abnormal in any way.
Temperature of media storage room/facility/area/location should be monitored through min./max. thermometer and limit shall be followed as per the media storage requirements.
List of media having designated storage condition and specific pH limit shall be prepared as per Annexure-II and shall be displayed near media storage facilities.
pH Check:
Check pH of the specific medium before sterilization with calibrated pH meter. If required adjust the pH with the help of 1N or 0.1N NaOH and 1N or 0.1N HCl solutions.
Culture Media Preparation:
Select the media as per specific requirements.
Read the instructions on the label very carefully before preparation of the specific media.
Weigh the media according to the instructions of the manufacturer of the supplied media.
Close the media container tightly just after weighing in order to avoid the moisture acquisition.
Reconstitute the media with purified water and boil it appropriately until entirely dissolve.
Distribute the reconstituted media into clean and dry flask as per requirements.
Cap the flask using cotton plug or screw cap appropriately.
Transfer the media flask into the detest room for use if instructed on the label as “DO NOT AUTOCLAVE” the media.
Sterilization:
Place all prepared media into the autoclave.
Sterilize the media at 1210C for 15 minutes.
Wait until completion of cycle, and collect sterilized media from the autoclave when the chamber temperature reduce at 600C.
Storage of Sterilized culture Media:
After completion of autoclaving activities, transfer all flasks containing the broth media to the test room for use.
Store the agar media at the warming condition (500C) into autoclave until use.
Do not keep the prepared media for more than two weeks [14 days].
Keep the prepared agar plate at (2-8)0C into the refrigerator for not more than two weeks.
Handling of Sterilized culture Media:
Ensure the aseptic condition during handle of sterilized media.
Do not de-cap or expose the sterilized media outside Laminar Air Flow or Bio-Safety Cabinet.
Record Keeping:
Keep in practice to Maintain Register for Media Preparation Record, Annexure-I and Annexure-II to keep record for list of media with storage condition and pH limit.
Bacterial Endotoxins Test (BET) is use to determine the quantity of bacterial endotoxins which present in the cell wall of the gram negative bacteria. Medical devices which are subject to contact[directly/indirectly] with the lymphatic system, cardiovascular system or cerebrospinal fluid must be undergo Bacterial Endotoxins Test (BET) as part of the lot release testing.
Bacterial Endotoxins Test (BET) is mandatory for the Injectable pharmaceutical products. The validated water system which is subject to routine monitoring and the starting/incoming materials must be ensure that the final product doesn’t effect by its endotoxins. The BET is also known as LAL[Limulus Amebocyte Lysate] Test and also known as Pyrogen test due to bacterial endotoxins can cause a fever in mammals, including humans. Don’t messed up with rabbit pyrogen test which is mentioned in USP chapter <151>.
Also read the following guidance for better understanding- for Industry Pyrogen and Endotoxins
ANSI/AAMI ST72:2011
FDA Guidance
EP 2.6.14
JP 4.01
USP Chapter <85>
USP Chapter <161>
In pharmaceutical industry and the industry which are manufacturing sterile products/products label claim itself sterile to its intended use must be pyrogen free and BET test must be done before release of the batch/lot to the market. Sterile pharmaceutical products and Water for injections are undergo BET in a pharmaceutical firm using gel clot method.
BET is an in-vitro test which is used to seek the endotoxins presence in the specific test sample/products. Endotoxins are frequently known as Pyrogen which are mainly produced by gram-negative bacteria. The main principle of BET makes it the utmost sensitive test that one can use to detect and quantify endotoxins, toxins which are notably known for causing fever in human.
During purification, production or packaging stages of Pharmaceutical products, it can be contaminated and BET must be perform before using its as sterile parenteral solutions. To identify the presence of endotoxins, add the sample to the Lysate, an enzyme found from horse shoe crab which hemolymph cells is mainly responsible to produce Lysate. The main principle of the BET is physiological reaction between the amoebocytes and endotoxins. The amoebocytes are fond on the blood of horse shoe crabs.
Amoebocytes found in the horse shoe crabs provide protection mechanism against pathogens. The Amoebocytes contains granules possess clotting factor which is generally released when encountered by the endotoxin causing coagulation. So this is very basic that the main mechanism of BET is physiologic effect between coagulating factor and endotoxins.
At the time of BET, the combination of endotoxins and calcium, a preclotting enzyme is typically activated which play a major role to catalyze the transformation of procoagulogen into a unit generally made of polypeptide. It is marked as coagulogen, its link up by a disulfide bond form gel-clot. With the help of a spectrophotometry, the formed precipitate subject to measure to identify if there are endotoxins in a test sample.
Types of Bacterial Endotoxins Test (BET)
Gel clot technique
Turbidimetric method
Chromogenic method
There are three endotoxin detection methods are available but among these methods, gel clot technique is widely used to detect the Bacterial Endotoxins which form gel that can be easily identified. Another method is known as turbidimetric method where the amount of endotoxins are measured based on the turbidity.
The selected sample introduce into a specific solution contains endogenous substrate, cleaved upon introduction of the endotoxin containing sample and generate turbidity. Intensity of the turbidity indicate the presence of endotoxin otherwise absence. The third and last method is chromogenic method which produce color. The selected sample is introduced into a solution contains synthetic complex made of peptide-chromo-gen. If the solution develop color then it indicate the presence of endotoxin to the suspected solution.
This method is very simple and time saving method. Generally 1 hour is required to determine the test result of the selected solution and more efficient compare to another method and very useful in pharmaceutical industry avoid using of animal for the similar purpose.
Procedure for Bacterial Endotoxin Test
Precautions:
Avoid touch contamination of closures.
Dehydrated endotoxin, LAL reagent and LAL water stored in a refrigerator in the temperature of (2-8)0C.
Use Endotoxin free apparatus during testing.
Preparation of Lysate:
Lysate must be reconstituted just before use by addition of the designated amount of LAL Reagent Water with the help of pipetting it directly into the vial after removing the stopper. Accumulate Lyophilized LAL powder into the bottom portion of the vial applying slight tapping on the hard surface.
Wear safety goggles if hazardous media is selected. Elude touch contamination of closures. Swirl gently but thoroughly for at 30 seconds until dissolve. Avoid any type of shaking/vibration.
Preparation of standard Endotoxin:
CSE [Control Standard Endotoxin] is an endotoxin preparation that has been standardized against the USP RSE [Reference Standard Endotoxin].Constitute the entire contents of 1 vial of the CSE with 5 ml of LAL water, mix spasmodically of 5 minutes, with the help of vortex mixture, and use this concentration to make appropriate serial dilutions.
Preserve the concentration in a refrigerator for making subsequent dilutions for 14 days only. Mix robustly with the help of vortex mixture, only for 3 minutes before use. Mix each dilution for 30 seconds before proceeding to make the next/another dilutions. Never/ever store the dilutions.
Determination of Maximum Valid Dilution (MVD):
Maximum Valid Dilution is maximum allowable dilution of a specimen/sample at which the Endotoxin Limit can be determined.
MVD applies to injection or to solution for parenteral administration in the form constituted or dilute for administration or wherever applicable, to the extent of drug by weight if the volume of dosage forms for administration could be varied.
General equation for the determination of MVD is defined as:
MVD = Endotoxin Limit × Concentration of sample solution and/ Sensitivity of reagent
When the sample under test comply with the test at a dilution less than Maximum Valid Dilution, repeat the test using greater dilution but not exceeding the MVD. The use of more sensitive Lysate permits a greater dilution of sample to be inspected.
Confirmation of labeled LAL reagent sensitivity:
Labeled sensitivity of LAL Reagent to be confirm using at least 1 vial of LAL Reagent lot. Prepare a series of two fold dilutions of the control standard endotoxin in LAL reagent water to provide concentration at 2λ, λ, 0.5 λ, 0.25 λ. Accomplish the test on four standard concentrations in quadruplicate and include the negative control.
Lysate sensitivity Test confirmation of to be carried out when a new batch of LAL Reagent is used. Mix the volume of LAL Reagent with an equal volume (such as 0.1 ml aliquots) of one of the standard solution in each test tube. Incubate reaction mixture for a constant period according to directions provided by the LAL manufacturer (usually 37±1ºC for 60±2 minutes), avoiding vibration, into incubator.
To verify/identify/test the integrity of gel, take each tube in turn directly from the incubator & invert it through about 180º in one smooth motion. If a firm/fixed gel formed which remains in place upon inversion, record the result as a positive. Mark result is negative if an intact gel is not formed/found. The test will not declare valid unless the lowest concentration of the standard solutions shows negative results in all replica test.
Solution
Endotoxin concentration/solution to which Endotoxin is added
Number of Replicates
A
None / Sample solution
2
B
2λ / Sample solution
2
C
2λ / Water of BET
2
D
None / LAL Reagent Water
2
Solution A: A sample solution of the preparation under test that is free of detectable Endotoxin
Solution B: Test for interference.
Solution C: Controlled for labeled LAL reagent Sensitivity.
Solution D: Negative control of LAL Reagent water.
Gel Clot Limit Testing:
Depyrogenate all related glassware & heat-stable materials in a hot-air oven using validated process at the time and temperature setting are 30 minutes at 250ºC respectively. Plastic apparatus like micro-plates & pipette tips for automatic pipette use only that which has been shown to be free of detectable Endotoxin and do not to interfere with test result. Perform the inhibition test on the sample dilution at dilution.
pH of the test mixture of the specimen & the LAL Reagent is in the range 6.0 to 8.0 The pH may be adjusted by the addition of sterile, Endotoxin free Sodium Hydroxide[NaOH] or Hydrochloride Acid[HCl] or Suitable Buffers to the Specimen before testing. Mix a volume of the LAL reagent with the equal volume [such as 0.1 ml aliquots] of one standard solution in each of (10 X 75) mm test tube.
Incubate reaction mixture for a constant period according to directions provided by the LAL manufacturer [usually 37±1ºC for 60±2 minutes, avoiding vibration, into incubator. To test/identify/verify the integrity of the gel, take each tube in turn directly from the incubator and invert it through about 180º in one smooth motion.
If a firm gel has formed that remains in place upon inversion, record the result as a positive. A result is negative if an intact gel is not formed. Calculate the detected Endotoxin, through used dilution factor and used LAL reagent [Lysate Sensitivity]. Generate Report the test result.
Sterility test is the basic requirements for the products claim it is sterile for its intended use. This is the core requirements to ensure the sterile status of the products which never contain the viable microorganism at the time of use to the patients or must confirm before released for sales.
Sterility testing need to be as precise as possible due to its critical point of use such as pharmaceutical products, tissue materials, blood products, serum preparations, vaccine preparation, Insulin preparations, powder for injections etc. and the other products which are claim to be sterile or free from viable microorganisms.
Various types of firm, food factory, pharmaceutical industry, beverage manufacturers, and medical device manufacturer’s etc. company use Sterility testing procedures which company deal with the sterile products and this is mandatory for them. Generally, a microbiologist or a group of microbiologists are involve to perform the sterility test based on company work flow.
A Consistent sterility testing is the core to the develop or validate a specific product or procedure. Continuous robust test method is mandatory get the accurate test result repeatedly. A robust quality infrastructure is required to support the biopharmaceutical, pharmaceutical, and medical device industries.
Direct Inoculation and Membrane Filtration Methods
Sterility testing is required to ensure viable contaminating microorganisms are not evident in a product. This testing is conducted by direct inoculation or membrane filtration methods and can be performed in an isolator or cleanroom environment.
Sterility Testing Techniques
There are several sterility Testing are available-
Recommended Sterility Test: Two Types
Direct inoculation
Membrane filtration
Additional Test: Two Types
Bacteriostasis/fungistasis testing–b/f testing
Vaporized Hydrogen Peroxide (VHP) ingress testing
Direct Inoculation
Here two types of media are used to directly inoculate the test article for the determination of the both aerobic and anaerobic microorganisms. The both media are for 14 day from the start of the test day and sporadic observations as well as final/end day observations are done to check the any type of evidence of microbial contamination.
A suitable volume of growth media is used to inoculate small volume of sample which is directly collect from sample container by applying aseptic technique then it incubate for 14 days. Direct Inoculation Sterility Testing has some significant limitations. The sensitivity is low for the test as small volume of a full container is inoculate to the respective culture media. At the starting of inoculation, if the sample appears cloudy or turbid then this very challenging to detect the turbidity and the end of the test period for the microbial growth.
Membrane Filtration Sterility Testing
Here the simultaneous filtration of test sample and standard preparation perform through two membrane filters and the samples are subsequently incubated for 14 days from the start of the test day, and finally check/determine the visibility of the microorganisms both aerobic and anaerobic.
The filterable pharmaceuticals product are subject to Membrane Filtration Sterility Testing which have been described in EU Pharmacopoeia < 2.6.1>, USP <71> & JP Pharmacopoeia <4.06>. To perform the test 0.45 µm membrane filter is used to pass the sample, then culture medium is added for incubation. The sensitivity of the test is more precise as the whole or composite sample is passed through the filter. Another best opportunity of the Membrane Filtration Sterility Testing is, its rinse away components present in the sample which may cause the turbidity or inhibit growth as for example preservatives or antibiotics.
Bacteriostasis/Fungistasis Testing–B/F Testing
Bacteriostasis/fungistasis testing is perform in conjunction with the sterility test evaluate whether or not the test article is inhibitory to the growth of the different microorganisms. To evaluate the sterility result Bacteriostasis/fungistasis test is essential to ensure that test article don’t contain any antimicrobial properties and it don’t inhibit the detection of microorganism at sterility test.
Vaporized Hydrogen Peroxide (VHP) Ingress Testing
An isolator required to perform the Vaporized Hydrogen Peroxide (VHP) Ingress Testing which undergo undergoes VHP decontamination. This assay assesses if VHP enter to the test article is apparent that may affect the validity of the result.
What is Sterility Test USP <71>?
Sterility test USP <71> is the chapter of USP[United States Pharmacopeia] which represents how the sterility test to be perform, and the detail description, methodology and how the product to be tested based on the fill volume and sample size.
Media use in sterility testing
The sterility testing of the all product subject to sterile require two types of media which to be cultured in separate two media. In sterility testing, two types of culture media are used to promote the growth of residual anaerobes, as well as aerobes and fungi.
FTM[Fluid Thioglycolate Medium] and SCDM[Soybean Casein Digest Medium], these two type of media are used use to culture anaerobic and some aerobic bacteria and fungi.
Generally FTM is use for culture of anaerobic and some aerobic bacteria on the other hand, SCDM is use for fungi and aerobic bacteria. Before examination the samples are incubated for 14 days at 32.5°C and 22.5°C. Media must be turbidity free. Presence of turbidity in the respective culture media subject to growth of microorganism and it must be investigate.
Sterility testing methods for medical devices
For medical devices testing, direct transfer sterility testing is recommended. The respective devices are tested is in direct contact with the designated test media during the incubation period where microorganism is growing on or in the device to be check. Transfusion and infusion assemblies related products which contain fluid pathway declared sterile then product flush sterility testing is preferred for this type of product. Here a rinsing fluid is used to flush the product lumen then the elute is passed through the membrane filter and after that it place on the suitable media for incubation for 14 days.
Sterility Testing Procedure
Precautions:
Be assured of strict devotion with aseptic technique and no occurrence of secondary contamination in every step of the test. Traffic in the LF workbench should be reduce and well-ordered. Use cellulose acetate membrane filters when strongly alcoholic solutions are subject to filter. If the solution being tested has antimicrobial properties, rinse the membrane at least three times with sterile dilution fluid.
Avoid piercing/ splitting of HEPA filter with liquid and spraying of solutions on the workbench. Use single syringe for single batch or test and avoid touch contamination of the needle and plunger of the syringes. All rubber stoppers of vials and neck of ampoules should be out of hand touch as hands are clean but not sterile. Check all the media for clarity as well as sterility before use. Perform positive control/growth promotion test in another separate area from sterility test area under Bio-safety Cabinet.
Media and Diluents:
Tryptone Soya Broth (TSB)
Fluid Thioglycollate Medium (FTM). [When medium is stored, store at a temperature between 2ºC and 25 ºC in a sterile, air tight container. If more than upper one third of the medium has acquired a pink color, then to remove the pink color, the medium may be restored once by heating the containers in a water bath until the pink color vanishes and by cooling quickly.] USP Diluting Fluid A/rinse solution (1g/L peptone water/Sterile water for injection)
USP Diluting Fluid D (To each Liter of Fluid A/rinse solution add 1 mL of polysorbate 80, adjust to a pH of 7.1± 0.2). For Cephalosporin/Penicillin, add a quantity of sterile β lactamase, adequate to deactivate any residual antibiotic activity on the membranes after the solution of the test specimen has been filtered. Filtered 70% IPA (Isopropyl Alcohol).
Prepare these dehydrated mediums and ensure effectiveness of the media before, or in parallel, with the sterility test on the product subject to be examined. Growth promotion test of medium should be justified.
Method of Testing:
Membrane filtration
Direct inoculation
Criteria of membrane filter:
Filter I: Cellulose Nitrate Filter[CNF] for aqueous, oily and weakly alcoholic solutions and Filter II: Cellulose Acetate Filter[CAF] for strongly alcoholic solutions.
Diameter of membrane filter: 47 mm
Pore size of the membrane filter: Not greater than 0.45 µm.
Test sample:
Sterility test is a destructive test [You can’t return the test sample] and it is impossible to test every single item for sterility. Sample for testing should be representative of the batch, which ensures that the results of the tests are substantial. Arbitrary samples are optimally selected every Lth unit, where L = the total units in the batch per number of sample required.
The following number of samples should be used in sterility test described in Table – 1 and Table -2.When the test samples are turbid and is impossible to filter the samples follow the direct transfer method unless in other case follow membrane filtration method.
Sample preparation:
Excessive care must be exercised when opening an article so that the sample to be tested for sterility is not contaminated by Microorganisms present on the exterior part of the container. The exterior surfaces of ampoules and closures of vials must be cleansed with 70% IPA or Hydrogen peroxide. Allow for 10/15 minutes and assemble the sampling unit’s previously sterile tray at inverted position.
Transfer the samples through pass box into the sterility testing area. FTM [Fluid Thioglycollate Medium] and SCDM[Soybean-Casein Digest Medium] or TSB[Tryptone Soya Broth] is used for the sterility test. Prepare these dehydrated mediums.
Before use, each batch of medium should justify for sterility by incubating portions of the medium for not less than 7 days. Growth promotion test [Nutritive properties] should be justified.
Test for Sterility of the product/material [Membrane filtration method]:
Prepare required amount of FTM, TSB, and USP Diluting Fluid–A and sterilized it. After completion of sterilization transfer all testing materials and accessories into sterility testing area by opening sterile side door of the autoclave.
Enter into the sterility testing area through change room. Clean & sanitize the working place. Accumulate the Sterilized filtration unit cautiously, using not more than 0.45µm cellulose nitrate filters (47mm dia.). Convey samples and other equipment into testing area from pass box after cleaning the surface with 70% IPA.
For Raw Materials transfer aseptically 5 -10 gm of tested sample into 500 ml screw caped conical flask containing 200 ml of USP diluting fluid-A and shake gently when it completely dissolve. For Finished Product collect required amount of test sample (see step Table) & transfer aseptically 300 mg of solids into a 500 ml screw caped conical flask containing 200 ml of USP Diluting Fluid-A and mix or constitute, as directed in the labeling, the containers and transfer a quantity of liquid equivalent to about 300 mg into a 500 ml screw caped conical flask containing 200 ml of USP diluting fluid-A.
Transfer the whole content to the filter cup assembly under strict aseptic condition. Filter the whole content with aid of vacuum pump [negative pressure] from the filter cup. If the tested sample under test has inherent Bacteriostatic & Fungistatic properties or contains preservative, rinse the filter paper using USP Diluting Fluid A.
After completing the previous step, cut the filter paper into two equal sections with sterile scissor and aseptically transfer into 100 ml bottle containing TSB & FTM media separately. Mark 1×100 mL of sterile FTM and 1×100 mL of sterile TSB medium without any sample and inoculum, as negative control.
After finishing of the test transfer out all the materials through pass box, clean the working place thoroughly and sanitize with approved disinfecting solution before leaving the area.
Direct Transfer Method:
Prepare required amount of FTM, TSB, (distribute it in screw cap 100 ml bottle) and USP diluting Fluid-A and sterilized it. After completion of sterilization transfer all testing materials and accessories into sterility testing by opening sterile side door of the autoclave.
Enter into the sterility testing area through change room. Clean & sanitize the working place. Prepare required amount of test sample (as per mention Table) and aseptically transfer it into 500 ml screw caped conical flask containing 200 ml of USP Diluting Fluid-A, and shake it gently.
Agitate the flask and aseptically withdraw 5 ml of test specimen into both of sterile TSB & FTM medium. Mix each test specimen with the appropriate medium, but do not aerate excessively. Incubate the test mixture and both negative controls as directed in the Incubation condition. Examine the media visually for growth.
Where the material being tested renders the medium turbid, so that the presence or absence of microbial growth cannot be determined by visual examination, transfer suitable portions of the medium to fresh containers of the same medium at least once during the period from the third to the seventh day after the test is started.
Continue incubation of the original and of the transfer containers for a total of not less than 14 days from the original inoculation.
Sterility Test of Syringes ( 5 mL, 10 mL & 20 mL)
Take the required quantity of the material followed by previously mentioned table. Aseptically disassemble the syringes into the components like barrel, plungers, needle and needle shield.
Transfer half of the syringe components into one media bottle having sufficient quantity of the FTM and another media bottle having TSB so that components can submerge completely within media and if require pour additional media.
Select the bottle for sterility test on the basis of the size of the components of syringes so that upon addition of media sufficient air space will be available.
Incubation Conditions:
All the test containers, incubated for not less than 14 days at 32.5 ± 2.50C for the Fluid Thioglycollate Medium and at 22.5 ± 2.50C for the Soybean-Casein Digest Medium or Tryptone Soya Broth Medium regardless of the method used for sterility testing.
Observe the media bottle on a periodic basis over Digest Medium regardless of the method used for sterility testing. Observe the tested as well as negative control incubated media bottles on each working day for any kind of macroscopic evidence of microbial growth and record the results in the report sheet with sign and date of the observer.
Interpretations of the Results:
At intervals during the incubation period and the completion of the prescribed incubation time, examine the media for any visible growth (Turbidity). If confusion arises, make subculture on TSA medium and/or justify evidence of growth.
If no evidence of growth is found, the preparation being examined, pass the sterility test and issue report form (Annexure I). If evidence of growth is found, isolate and identify the organism and make a full case investigation.
If the cause of microbial growth failed to reveal, perform repeat test with same number of test sample. Take double number the sample in case of high risk product.
