Standard Operating Procedure Requirements for BSL2 and/or ABSL2 Containment

The Principal Investigator (PI) has the responsibility to inform the laboratory personnel of the appropriate research procedures. When using hazardous or regulated biological agents the PI must prepare a written Standard Operating Procedure (SOP) outlining the necessary precautions to safely conduct research. An SOP is a set of specific guidelines designed to address the methods that will be used and the safe handling of biological agents. The SOP must be available in the laboratory.

The SOP is a valuable tool and worth the preparation time. A well-written SOP can be used to satisfy several compliance requirements. SOP should be written for all procedures that pose an identified potential risk to the health and safety of the laboratory personnel, although a separate SOP does not need to be written for each individual experiment, procedures with the same hazards can be combined into one SOP.

The process of writing SOPs requires an individual to think through all steps of a procedure and perform a risk assessment before work has begun. The best approach to writing an SOP is to do it, write it and test it. Be brief and succinct; the shorter the better. An SOP template is provided below.

ABSL2 and BSL2 requirements also include appropriate biohazard labeling. An example of appropriate signage for a door is attached at the end of the template for your use. Remember, other signs may also be appropriate, as long as they include the necessary information (Biohazard Symbol, Biocontainment Level, name of agent and any necessary requirements to take prior to entering or exiting the lab, and PI and lab contact information).

Sample Standard Operating Procedure Template for Safe Handling of

(List organisms and/or animals) at BSL2/ABSL2 [select appropriate environment(s)] Containment

Please edit and complete as necessary to address Biosafety Risks within your laboratory and/or animal housing area.

Title of Procedure: One safety SOP can be used for more than one experimental protocol if the material, equipment being used and potential hazards are the same.

Introduction and Purpose of Work: Provide a brief description of work.

If work will include animals, please submit the approved SOP to DLAR or the CCIF, as well as the OESO. If you are working with viral vectors and claiming replication deficiency, please provide details describing confirmation of competency testing. Otherwise, the same biosafety precautions and animal housing requirements must be followed as used for the wild type virus.

PI:	Lab Location: Animal Housing Location (must be approved by DLAR or CCIF):
Issue Date:	Revision Date:
Prepared by:	Approval Signature:

Applicable Regulatory Statutes / Guidelines: List only the appropriate.

[Examples: Export Control (http://www.bis.doc.gov/licensing/exportingbasics.htm), Select Agent Rules (http://www.cdc.gov/od/sap/), OSHA Bloodborne Pathogens Standard (http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051) , CDC/NIH’s Biosafety in the Microbiological and Biomedical Laboratories (http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm)

NIH rDNA Guidelines: http://oba.od.nih.gov/rdna/nih_guidelines_oba.html]

RISK ASSESSMENT:

Hazard Identification and Risk of Exposure to the Hazards: Describe the risk of the agents being handled in the laboratory. If applicable, describe the signs and symptoms of illness and/or disease. Determine if immunization is needed.

Routes of Transmission: Prior to assigning containment requirements, it is imperative to understand the routes of transmission.

Some issues to address:

1. What are the exposure routes/risks of most concern? (Examples: Sharps exposures, Splash exposures, Non-intact skin exposures, other exposures such as food, drink, inanimate objects).

2. If applicable, are there any off target effects (insertional mutagenesis, etc.) from exposure to the biohazardous and/or recombinant material??

3. What are the consequences of exposure to the biohazardous and/or recombinant material?

MEDICAL CONSIDERATIONS:

Medical Screening and Surveillance (if necessary): For example, all personnel that actively handle human cell cultures or human specimens in the lab must complete the following through Employee Occupational Health and Wellness (EOHW):

• EOHW health screening for laboratory worker
• Must be offered the hepatitis B vaccination

Personnel may also be offered vaccines or special counseling depending on the organism(s) handled in the lab and availability of vaccines or prophylaxis.

Accidental exposures, such as splash to the face or a sharps injury shall be reported immediately to EOHW by dialing 684-8115. The EOHW representative will help categorize the risk of developing occupationally-acquired infection and provide advice on an appropriate post-exposure treatment.

PRECAUTIONS:

All laboratory work shall fully comply with biosafety level 2 (BSL2) containment as described in the current edition of the CDC/NIH’s Biosafety in the Microbiological and Biomedical Laboratories: http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

Procedural Methods and Materials: Incorporate each category as it pertains to your work:

Door Signage & Equipment Labeling: (ex., doorway (Complete the attached sign(s) when applicable), refrigerators, incubators, cage signs)

1. Posting of signs is research staff’s responsibility!
2. Signs will be posted at all times when hazardous material is present.
3. Signs will be removed by research staff when hazardous material is no longer present.

Access to laboratory: (ex., describe restrictions, locks.)

Personal Protective Equipment (PPE): (ex., entry and exit procedures, PPE use during work)

Methods to minimize personal exposure: (work practices: ex., use of conveniently located sharps containers, safer needles and sharps, absorbent material on countertops)

Methods to prevent the release of infectious agents/protect workers from aerosols, splashes, splatters: (ex., equipment/engineering controls: ex., Class II Biological Safety Cabinets (BSC), covered centrifuge cups)

Specimen transport and removal of material(s) from the laboratory: (ex., leak proof transport containers)

Standard microbiological methods: (ex., handwashing after removal of gloves and before leaving the work area, no mouth pipetting, no food or drink in refrigerators where material is stored, no eating in work area)

Cleaning & Disinfection: Describe surface decontamination, cleaning procedures and type of disinfectant(s) used.

Waste Generation and Disposal Methods: Identify the types of waste generated (liquid waste, dry waste, sharps waste, animal carcasses) and procedures for handling/disposing of biological waste including contaminated, non-contaminated waste and use of sharps containers.

Spill and Accident Response Procedure: Describe all emergency procedures including spill clean-up. Describe disinfectant (dilutions/contact times) and environmental decontamination. (ex., Outside of a BSC: If spill is a respiratory hazard, evacuate 30 minutes to allow aerosols to settle. Place absorbent towels over the spill, apply freshly prepared 1:10 bleach solution to entire area of spill starting on the outer edges and working inward, contact time: 20-30 minutes, pick up sharp items with mechanical device (not hands), place all clean-up materials in a biohazard bag, autoclave all waste.)

Personnel Exposure to Biohazards

a. Report exposure immediately by calling the Employee Health Exposure Hotline (115 from a campus phone/684-8115 from a non-campus phone)

b. Complete the Report of Occupational Injury or Illness form found at http://www.hr.duke.edu/forms/injury.html

TRAINING:

Training Requirements: Workers conducting research under this procedure must comply with the following training requirements:

• Complete General Lab Safety Training provided by Duke’s Occupational and Environmental Safety Office (www.safety.duke.edu). This training is required annually and is documented by OESO.
• Complete the BSL2 Training provided at the same website.
• Those working with human cells or human specimens must complete OSHA Bloodborne Pathogens Training at the same website.
• All personnel shall read and fully adhere to this SOP.
P.I. will keep documentation of personnel reading and understanding this lab-specific SOP using a signature page (example attached).

For animal use only

PRECAUTIONS:

All animal work shall fully comply with animal biosafety level 2 (ABSL2) containment as described in the current edition of the CDC/NIH’s Biosafety in the Microbiological and Biomedical Laboratories: http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

Required Procedures for Work in ABSL2 Animal Facilities:

The researcher is responsible for:

1. Communicating the start date of the study and conveying the OESO-approved SOP for the animal work to the DLAR Senior Operations Manager, as well as the Operations Manager of the animal facility where the hazardous agent work will occur (for CCIF contact the Shared Resource Manager and the Staff Specialist).

- Contact information for these DLAR personnel can be found on the DLAR website (http://labanimal.duke.edu/) under "Contact Us" and then "Husbandry and Facilities."

- This communication must occur at least five (5) days prior to initiation of the work.

2. Initiating the work only after obtaining confirmation that your notification has been received.

3. Placing the proper signs on the animal room door and cages prior to the initiation of the study.

4. Removing the signs when the study is complete.

· Cage Cards and Door Signs: As soon as the animals have been dosed with the biohazardous agent, cages must be marked with the biohazard cards and the attached sign must be posted on the outside of the animal room door by research staff. This sign will be removed by research staff once the infected animals and biohazardous agents are no longer in the animal room.

Researcher’s Procedures in the ABSL2 Animal Facility: Describe procedures done within the animal facility and engineering controls used such as a biological safety cabinet.

• EXAMPLE: Animals will be dosed intranasally with the biohazardous agent. All work with the biohazardous agent will be done within a Class II biological safety cabinet.

Animal Cage-Change Procedures:

NOTE: at ABSL2, cage changes will be confined to a ventilated cage changing station or biological safety cabinet and unless otherwise instructed by the researcher, DLAR/CCIF staff will change cages. In addition, the researchers will train DLAR/CCIF staff regarding any agent specific hazards and any additional special precautions needed.

Standard ABLS2 cage change procedures:

a. Cages will be changed by DLAR/CCIF staff no sooner than 48 hours after the animals are exposed to the biohazardous agent.

b. Cages (with bedding) will be double bagged in two orange autoclave bags and taped shut.

c. The bags are then sprayed down with a DLAR/CCIF approved disinfectant that is effective against the agent in use. (Examples: Virkon-S, Trifectant or other approved disinfectant).

d. DLAR/CCIF staff will transport bagged cages to autoclave.

e. Only after cages have been autoclaved will they be dumped (by DLAR/CCIF staff) as normal waste.

Exceptions to Standard DLAR ABLS2 cage change procedures:

Describe necessary changes to the standard DLAR ABSL2 cage changing procedures

· EXAMPLE: Research staff will be responsible for performing all cage changing procedures described above.

· EXAMPLE: Bleach is the only disinfectant recommended when working with this agent.

For Lab use

Example of appropriate signage for BSL2 laboratory doorway: PLEASE COMPLETE

AUTHORIZED PERSONNEL ONLY!

BIOHAZARD

2

BIOSAFETY LEVEL

Principal Investigator: ________________________

Agent (s): ___________________________________

Bldg: ___________ Room: _____________

Special Instructions/ Requirements Prior to Entry or Exit (i.e. personal protective equipment, vaccination):

EMERGENCY CONTACT/ ADVICE	CONTACT	WORK PHONE	HOME PHONE or PAGER
PRIMARY
SECONDARY
BIOSAFETY OFFICE	Debra L. Hunt	684-8822	970-6012

For animal use

Example of appropriate signage for ABSL2 laboratory doorway: PLEASE COMPLETE

AUTHORIZED PERSONNEL ONLY!

BIOHAZARD

2

ANIMAL BIOSAFETY LEVEL

Principal Investigator: ________________________

Agent (s): ___________________________________

Bldg: ___________ Room: _____________ (must be space approved by DLAR for ABSL2)

Special Instructions/ Requirements Prior to Entry or Exit (i.e. personal protective equipment, vaccination):

EMERGENCY CONTACT/ ADVICE	CONTACT	WORK PHONE	HOME PHONE or PAGER
PRIMARY
SECONDARY
BIOSAFETY OFFICE	Debra L. Hunt	684-8822	970-6012

Example of Signature Page:

Standard Operating Procedure for Handling

[List organism(s)] at BSL2/ABSL2 [circle appropriate environment(s)] Containment

“I have read and understand this SOP. I agree to fully adhere to its requirements.”

Clean Room Classification with respect to particle caounts and Microbial load count

Clean room Classification with respect to particle caounts

Clean Aira ----Iso ------> 0.5 um Particles --Microbiological ------Microbiological*

Clasification--Designation--Per Cubic------Ative Air Action leve- Stteling plate *

100------ ----5----------3520------------1-----------------1

1000----- ----6---------35200-----------7-----------------3

10000--- -----7---------352000---------10----------------5

100000---- --8----------3520000-------100--------------50

Action Lvel CFU /4 HRS*

Procedure for monitoring the microbial quality of compressed air used in pharmaceuticals manufacturing

MATERIAL:-

1 Sterile Soybean casein digest medium

2 Sterile Soybean casein digest agar

3 Sterile 500 ml 01% peptone water

PROCEDURE:-

1 Pass about 1000 liters of compressed air from a predetermined points to the 500 ml of 01% peptone
water
2 Aseptically transfer the 250 ml of peptone water to each of two membrane filter funnel and immediately pass the peptone through the membrane of 045u with the aid of vacuum

3 Keep the membrane filter covered with liquid throughout the filtration for maximum efficiency of filter

4 Aseptically remove both the membrane from the filter holder and transfer one of the membrane filter
in sterile petri plate, intended for the enumeration of viable bacteria and other membrane filter to the
soybean casein digest medium for detection of pathogens

5 Pour 20 ml of sterile molten cooled soybean casein digest agar on plate Cool the plate

6 Carry out positive and negative control along with the test sample

7 Then incubates the plates and tubes at 35 - 370c for 72 hours for enumeration of viable bacteria

and 24 - 48 hours for detection of pathogens respectively

8 At the end of the incubation period count the colony forming units ( cfu ) on the plate and maintain
the records

9 If the tubes shows growth in terms of turbidity, carry out identification test for specified pathogens

Identification of microorganisms characteristics by gram staining method

PROCEDURES :

1 Pick up the microorganism to be identified and smear it on the clean slide with a
drop of sterile saline

2 Heat fix the smear on the slide by passing it through the flame 3 -4 times

3 Flood the slide with Grams crystal violet solution and keep it for i minutes

4 Wash with water and flood with Grams iodine and keep it for 1 minute

5 Wash with water and decolorise with Acetone alcohol solution till no further violet
colour discharges out

6 Wash with water and counter stain with 05 % saffaranin solution and keep it
for about 30 seconds

7 Wash the slide with water and air dry it

8 Observe the slide under oil immersion objective and observe for gram positive
and gram negative organisms Gram positive organisms will be violet in colour
and gram negative organism will be in pink colour

GENERAL METHOD FOR CHECKING OF BACTERIOLOGICAL QUALITY OF WATER FOR PHARMACEUTICALS

OBJECTIVE :- To sample and test the factory water supplies at various stages of processing,

storage and distribution in order to establish the quality of the water used directly or indirectly

in product manufacture

PRECAUTIONS :-

1 Use gloves when hot water is to be sampled

2 While sampling take care to avoid contamination

PRECEDURE :-

A) MONITORING -----------LOCATION

a) Main water source------- Tap point facility inlet

b) Storage tank ---------------- water from the storage tank

c) Demineralised Water (DM )---- Washing area of ointment section

d) Distilled water (DW)----------Distilled water still in QA Department

e) Tap ----------------------------Drinking water point


B) SAMPLING :-

a) Tap water main water suplly :- Unplug the sterile 500 ml capacity sample bottle containing

1 ml of sterile 3 % Sodium thiosulphate solution Fill the bottle with 3/4 th its capacity

Plug the bottle immediately with cotton plug

b) D/M & D/W point :- Unplug the sterile 500 ml capacity sample bottle Fill the

bottle with 3/4 th its capacity Plug the bottle immediately with the cotton

c) Storage tank :- Let some water run through out let of storage tank , and then collect from all pints in storage tank Unplug the sterile 500 ml capacity sample
bottle Fill the bottle with 3/4th its capacity Plug the bottle again with the cotton

C) NOTE :-
a) Hot samples should be cooled to ambient temperature before filteration
b) Samples should be tested within 1 hour of sampling or if cannot be tested within
1 hour

D) METHOD :-

1 Method used is membrane filteration method
2 For D/W 10 ml of sample is filetered and in case of raw water , storage and tap water
100 ml of sample is filtered

3 Remove the cap of the filter cone and transfer the above mentioned volumes for

mentioned samples and filter it through 045 u filter paper Following filteration

transfer the membrane filter using sterile forceps to the sterile petridish Pour

sterile molten and cooled to not more than 44 deg C Soybean Casien Digest

Agar medium Rotate the medium to cover the entire membrane filter , allow it
to set

4 Filer the second set of sample and filter the sample with volumes mentioned above
of the mentioned samples and filter it through 045 u filter paper Folowing
filteration transfer the membrane filter using sterile forceps into the 100 ml of sterile
Soybean Casien Digest Medium
5 Incubate the plates at 35 - 37 deg C for 72 hours and the media tubes for minimum
period of 24 hours

6 Observe the plates every 24 hours till the completion of 72 hours and the tubes for
48 hours

7 In case of tubes if there is a evidence of growth after 48 hours of incubation proceeed
as tabulated for detection of pathogens viz Escherechia coli , Salmonella spp
Staphylococcus aereus, Pseudomonas spp resp In case there is no evidence of
growth after 24 hours of incubation it indicates the absence of pathogens

8 After 72 hours of incubation examine all the membrane filter Count the total colony
forming units per membrane and calculate the results in cfu/ml and record

9 After proceeding for the pathogen detection test record the observations

10 Using autoclaved water samples, prepare negetive controls for membrane filteration
for each days of testing Transfer the membrane filters into the sterile pertidish and
pour 20 ml of sterile soybean Casien digest agar medium, cool it and incubate it
along with the test samples Similarly the autoclaved water sample is filtered and
the membrane filter transferred using sterile forceps into the 100 ml of Sterile
Soybean casien digest medium Incubate them at 35- 37 deg c for 72 hours and
48 hours resp


ACTION TO BE TAKEN IN CASE OF NON COMPLIANCE /DEVIATION :-

a) Check for proper technique of sampling and test method to rule out external

or personal contamination

b) Report to the Production Head to take necessary steps to disinfect the source where
the pathogen has been detection

c) Resample the source and check again for pathogens so as to assure that the
pathogens are completely removed

Types of water-------Location----------------------------Frequency

a)Main water source ---Tap point ------------------------ As per requirement

b) Storage tank----Water from the storage tank---As per requirement

c) Demineralised Water (DM )--Washing area-----As per requirement

d) Distilled water (DW)---Distilled water ----------- As per requirement

e) Tap water -----Drinking water point in Canteen---- As per requirement

ENVIRONMENTAL MICROBIAL MONITORING OF ASEPTIC AREA IN PHARMACEUTICAL MANUFACTURING BY FINGER DAB TESTING

To check the microbial load on the gloved hands as they come in direct
contact with the product and the machine As the operators frequently disinfect
their gloved hands with 70 % isopropyl alcohol, this method checks the
microbial quality of the gloved hands.

IMPORTANT POINTS/NOTES/PRECAUTIONS:-

1 After the Finger dab testing the gloved fingers are cleaned thoroughly with suitable
disinfactant such as 70 % isopropyl alcohol or the gloves are discarded as they may
contaminate the product or the machine

2 The media used should be asigned a batch no

MATERIAL:-

1 Sterie Soyabean casein digest agar plates


PROCEDURE:-

1 In the aseptic area instruct the persons to place their fingers on the sterile
gently taking care that the media will not break due to pressure

2 Close the plates immediately Put the name of the person on the plate along
with the date

3 Disinfect the gloved hands immediately with 70 % isopropyl alcohol solution

4 Incubate the plates, after solidifying of the medium in bacteriological incubator for 72
hours at 35 deg C to 37 deg C

5 Count the total number of colony forming units (cfu) Calculate the number of cfu/ml
and express in number of cfu/ person

6 If any organisms observed on the plate is recognisable as potential pathogen, investigatate
furthur

7 Positive and negative control is kept along with the test sample

8 Record the observations in the attachment 1



OTHER RELEVANT DETAILS, IF ANY:-

1 Limits:- NMT 3 cfu / person

FREQUENCY OF OPERATION :- As per your requirement

ENVIRONMENTAL MICROBIAL MONITORING OF ASEPTIC AREA AIR SAMPLING METHOD

OBJECTIVE :- The purpose of this procedure is to describe the method for sampling a measured quantity of air so that viable particles ( cfu ) are impinged upon an agar surface
to allow subsequent culture and quantitification



UNIT OF RESPONSIBILITY : QA Executive and trwained QA Assistants

MATERIAL :-

1 Air Sampler

2 Sterile soyabean casien digest agar strips


Note :- The media preparation batch no is given to prepared media



IMPORTANT POINTS/NOTE/PRECAUTIONS:-

1 It is not advisable to expose the Agar strips beyond 7 minutes as the medium gets
desiccated

2 The coated surface of the Agar strips should face down during incubation or while
storing in order to avoid water of condensation


3 The instrument can be operated either on battery (6 dry cell batteries of 105 Volts)
or with battery eliminator of 9 volts & 1500 m amp

4 Ensure that the air flow rate is calibrated and certified



PROCEDURE:-

1 Prepare the Soyabean casien digest agar media as per media preparation SOP

2 Autoclave the media at 121 deg C for 20 minutes

3 After completion of autoclaving transfer the media to the microbiology room

4 Prepare the strips by pouring the sterilized Soyabean casien digest agar medium

aseptically taking care to avoid contamination from the person preparing them. Cool the strips

5 The prepared strips are labelled and incubated for 24 hours at 35 to 37 degree C

6 Sterilize the drum, impeller blade, timer box, cylinder the use of 70 % IPA and fumigation

respectively

7 Tear open the plastic tubing covering Agar strip

8 Slide back the sliding cover filted on the outer socket Remove the agar strip with the

medium filled side facing downwards & insert into the slot in the side of the drum of

the Air Sampler

9 Care must be taken that the medium does not touch the impeller and the end strip

produces enough for it to be pulled out of the instrument again after the sampling



10. To avoid contamination do not touch the layer of culture medium in the agar strips

before and after measuring process

11 Bring the Air Sampler to the required position Set up the running time and

switch on


12 The sampling time may be set as per the following table

Switch

1 ON 1 Min = 40 lts

2 ON 2 Min = 80 lts

4 ON 4 Min = 160 lts



All Switches On 7 minutes = 280 lts

13 Remove and incubate the agar strip for 72 hours at 35 to 37 deg C

15 After incubation count the cfu on the agar strip , and identify the growth observed on the

agar strip their gram character by Gram staining refer sop



14 For immediate second sampling using same drum & impeller blade, 70 % IPA may be

used for sterilization

15 Record the observation in the attachment 1



OTHER RELEVANT DETAILS:-

a) EVALUATIONS :-

Total no of colonies observed on the strip be counted with the help of colony counter or

directly by holding the strip against light The total microbial load may be arrived by using

the following formula -

Total no. of colonies on agar strip
c.f.u./litre of air = -----------------------------------------------------------
Volume of air sucked in X time of exposure

Total no. of colonies
c.f.u/ m /min = ------------------------------------------ X 25
Time of exposure in minutes

Limits

1) Manufacturing area : : NMT 10 cfu
2) Storage Area : : NMT 10 cfu

3) Filling area : : NMT 05 cfu

ENVIRONMENTAL MICROBIAL MONITORING OF ASEPTIC AREA IN PHARMACEUTICALS MANUFACTURING BY SURFACE SWAB METHOD

Surface swab testing of critical area in the manufacturing and filling
area by assesing the microbial contamination levels of any surface
against the predetermined action levels and efficiency of the operating
procedures in the aseptic areas during the manufacturing the product

UNIT OF RESPONSIBILITY:- QA Executive and trained QA Assistants


PRECAUTIONS:-

a) During the Swab testing the test portions are cleaned thoroughly with suitable

disinfactant to avoId contamination from the solutions used for the test

MATERIAL:-

1 Pre sterilized cotton buds/swabs

2 01 % w/v peptone water which has been previously sterilised

3 Sterilised molten soyabean casien digest agar maintained at a temperature

not below 45 degree C

Note :- The media preparation batch no is given to prepared media


PROCEDURE:-

1 Streak the cotton swab across the surface to be monitored

2 Monitor an area of approximately 100 sq cm with the swab being rotated so

that the entire bud/swab head is used

3 When the area of less than 100 sq cm is monitored due note should be made

of the comparison against the general specification Such monitoring may be

required in certain critical positions within aseptic areas

4 Use the last bud/swab of every set of buds/swabs as control and cross reference

with the sample as positive and negetive control

5 Transfer the swabs/buds into 10 ml of sterile 01 % peptone water

6 Prepare uniform suspension by rotating the tubes in between palms and 1 ml of this

suspension is poured in sterile empty plate Add to it 15 to 20 ml of sterile molten

soyabean casien digest agar

7 Incubate the plates, after solidifying of the medium in bacteriological incubator for 72

hours at 35 deg C to 37 deg C

8 Record the observations in the attachment 1

READING :-

The count is done as number of colony forming units (cfu) observed on the surface of

the agar after every 24 hours till completion of 72 hours of incubation

LIMITS :-
1) Manufacturing area : wall : NMT 10 cfu Floor : NMT 20 cfu

2) Storage Area : wall : NMT 10 cfu Floor : NMT 20 cfu

3) Filling area : wall : NMT 05 cfu Floor : NMT 10 cfu

Note : In case the swab testing of the equipment is done after the sterilization
the count per swab should be Nil.

TEMPERATURE MONITORING OF DIGITAL TEMPERATURE THERMOSTATIC INCUBATORS BOD INCUBATOR AND THERMOSTATIC INCUBATORS

OBJECTIVE :- To establish a procedure for temperature monitoring of Digital

Temperature BOD Incubator and Thermostatic Incubators



UNIT OF RESPONSIBILITY :- QA Executive and Trained QA Assistants



PROCEDURE :-

1) See that the incubators are in ON position

2) Check the temperature to ascertain that it is within the specified limits

3) Check the temperature shown by digital temperature indicator of BOD and

thermostatic incubators

4) The temperature shown by the incubators should not differ by + 05 deg C from the

set temperature

5) Maintain the records of temperatures as per Attachment-I in respective QA,File


TOLERANCE LIMITS : - + 05 deg C

OUT OF LIMITS ACTION :-

If the temperature is not within the specified limits, adjust with the help of adjustment knob


MAINTAINENCE /SERVICING :- To be done as and when required

PERSONNEL TO BE REPORTED TO IN CASE OF NON CONFORMANCE /DEVIATION :

Maintenance Department


FREQUENCY OF OPERATION : Thrice a day

NO OF ATTACHMENT :

RETENTION PERIOD OF RECORDS : Five years

ENVIRONMENTAL MICROBIAL MONITORING OF PHARMACEUTICAL ASEPTIC MANUFACTURING AREA BY SETTLE PLATE METHOD

To asses the microbial load in aseptic area by means of plate count by settle plate method

UNIT OF RESPONSIBILITY:- QA Executive and trained QA Assiatants

IMPORTANT POINTS/NOTES/PRECAUTIONS:-

1 Ensure that plates are not exposed for longer period as they may get dessicated

2 The media used should be asigned a batch no

MATERIAL:-

1 Sterile Nutirent agar prepared as per sop for media preparation

2 Bateriological Incubator adjusted the temperature of 35-37 deg C

3 Autoclave

PROCEDURE:-

1 Prepare the nutrient agar media as per the media preparation SOP

2 Autoclave the media at 121 deg C ( 15 lbs ) for 20 minutes

3 After completion of autoclaving transfer the media to the microbioogy room

4 Prepare the plates by pouring the sterile molten cooled Nutrient agar medium aseptically taking care to avoid contamination from the person preparing them Cool the plates

5 The prepared plates are numbered and incubated for 24 hrs at 35 - 37 deg Cpror to sendig for exposure

6 Place the preincubated petri plates with its lid still covering the agar at predetermined positions f moinitoring

7 Remove the lid and rest it on the rim of the dish base whereby maximum exposure of
the agar surface is made

8 Leave the nutrient surface exposed for 2 hours

9 Keep one unexposed plate for negative control and one plate which is exposed in non sterile area for positive control

10 Incubate the plates an inverted position for minimum 3 days at 35 - 370C

11 Count and record the total number of colony forming units(cfu) observed on the surface of the agar after every 24 hours till completion of 72 hours of incubation

12 Maintain the records as per the attachment
ACTION PROCEDURE :

If the number of organism recorded is higher than the agreed action limit, get the area / machine throughly cleaned as per the standard operating procedure for cleaning In case of mould counts get the area / machine cleaned with 70 % IPA and fumigate the area

HOW TO CHECK THE EFFICACY OF DISINFECTANT

OBJECTIVE :-

Disinfectant used in the aseptic area and sterility testing area should be effective enough

to kill the organisms in that particular area



UNIT OF RESPONSBILITY :- QA Executive and trained QAAssistants



NOTE :-

a) Disinfectant should be used alternatively

b) Disinfectant should be checked for its effectivity, for every consignment and every six months to ensure its effectivity

PROCEDURE :-

A) Material Requirement

1) Sterile Soyabean casien digest agar plates

2) Sterile distilled water

3) Sterile pipettes, test tubes and petri plates

4) Disinfectant to be tested

B) METHOD

1) Prepare 10 ml of different dilutions or different concentrations of disinfectant to be tested using
sterile distilled water

2) In each dilution, or different concentrations of disinfectant, add 1 ml of 24 hours old test culture

3) Keep the tubes at room temperature for 30 minutes

4) From each dilution of disinfectant or different concentration of disinfectant, a loopful of suspension
is streaked on sterile soyabean casien digest agar plates

5) Incubate the plates at 35 - 370C for 72 hours in inverted position and observe the plates for presence

6) Positive and negative control are kept along with test

7) Maintain the records terms of cfu in the given format Attachment-I



OTHER RELEVANT DETAILS:-

Disinfectants used are

1) Lysol solution

2) Dettol solution

3) Benzylalkonium chloride solution

Limits :- No growth of microorganisms should be observed in the selected range of disnfectant

PERSONNEL RO BE REPORTED IN CASE OF NON COMPLIANCE :- QA Executive

FREQUENCY OF OPERATION :- Every consignment and to be revalidated every six months

NO OF ATTACHMENTS:- One

RETENTION OF RECORDS:- 5 Year

HANDLING AND MAINTAINENCE OF MICROBIAL CULTURES

OBJECTIVE :-

Cultures of microorganisms are frequently used in microbiological laboratory for

routine microbiological work It is important that microorganisms in frequent use

do not become genetically altered as a result of sequential transfer and do not get

contaminated due to improper aseptic techniques A safe handling of all microorganisms

including non pathogens is of prime importance as all microorganisms may cause

certain amount of health and environment hazards



UNIT OF RESPONSIBILITY:- QA Executive and trained QA Assistants



IMPORTANT POINT/NOTE/PRECAUTIONS:-

SAFETY:



The type of culture handled in the microbiology lab are in the form of slants, stabs,

liquid culture and viable spores

1 The personnel handling the culture must protect themselves with suitable

means such as use of gloves and mask and any spillage of accidental

contamination that may occur during the handling must be taken care of, properly

2 The gloves and the mask should be removed and disinfected using a

disinfectant and or by sterilizing by autoclaving

3 The hand or any other portion of the body exposed to contamination must

be washed thoroughly with soap water and disinfected immediately



PROCEDURE :-

a) MAINTAINENCE OF CULTURE

1 The culture recieved from the source like FDA, or any other authentic source

should be labelled as mother culture and suitably batch numbered and stored

in a cool condition at a temperature of 8-15 deg C for not more than six months

2 Mother culture should be checked for purity and for identity as per SOP for indentification

of micro organisms and stored for not more than six months It should be subcultured

in duplicate

3 One of the two mother cultures should be used for preparation of working culture and

stock culture The other is intended as reserve in the event of first mother culture getting

contaminated

4 Inocula from mother culture must not be used directly in microbiolgoical testing

Prepare a stock culture and a working culture and label that as generation 1

5 After 15 days subcultrue from the generation 1, prepare stock culture and working

culture label them as generation 2 (Detailed outline for maintenance of culture - Refer

6 Prepare a routine culture from the working cultures for the microbiological purpose


7 If all the cultures become contaminated or fail to grow it is necessary to revert to the

mother culture for preparation of new working culture


8 The culture slants should be labelled as follows:

Name of the Organisms with classified codes:

Batch No / Generation no of culture

Date of Subculturing

Use before date

9 All the working cultures must be stored at a temperature of 8-15 deg C in a covered

container

b) METHOD OF SUBCULTURING

1 Clean the area where subculturing is to be done with the suitable disinfectant

(70 % isopropyl alcohol)

2 Wear previously sterilised gloves and nose mask

3 Wipe out the outside of the old culture tube with cotton wool soaked in 70 % IPA soln

4 Get fresh slants/stab culture of maintainence medium and wipe out outside of

tube with cottom wool soaked in 70 % IPA solution

5 Hold on the extreme end and heat the nichrome wire loop on the reducing flame till

red hot

6 Allow it to cool to 40-45 deg C and slowly open the cottom plug of old culture tube

and warm the mouth of the tube

7 Pick up the smear with the help of the loop after having removed cottom plug of the

test tube



8 Plug tube again still holding nichrome wire between two fingers and thumb

9 Open plug of the slant/stab tube Warm the mouth of the tube holding in other hand

and carefully transfer the culture that is held on the loop

10 Spread the culture spirally on the surface of the slant if the organism is aerobic and

stab the medium if the organism is anaerobic in nature

11 Warm mouth of the tube and immediately plug the cotton into the mouth

12 Label tubes with the name of the organism Classified code,Generation number, Batch No

and date of subculturing

13 Incubate fresh subcultured organism tube at 30-35 deg C for 18-24 hrs and then

store it in refrigerator

14 Each generation of subcultured organism should be manitained for months





c) DESTRUCTION:

1 Follow the SOP Disposal of Microbial Waste Material

Destruction pattern for stock culture generation

The subculturing data and the destruction should be recorded as per attachment No 1



PERSONNEL TO BE REPORTED IN CASE OF NON COMPLIANCE:- QA Executive



FREQUENCY OF OPERATION :- Fortnightly



NO OF ATTACHMENTS:-



RETENTION OF RECORDS :- 5 Years

FUMIGATION OF ASEPTIC AREA (STERILITY TESTING ROOM)

OBJECTIVE : To maintain the microbial load to a minimum of sterility testing room by fumigation by use of formalin and potassium permagnate powder

UNIT OF RESPONSIBILITY :- QA Executive and trained QA Assistants

IMPORTANT POINTS /NOTES /PRECAUTIONS :-

1 Handle the formalin solution carefully as it is corrosive in nature

2 Use hand gloves while handling the formalin


MATERIAL :-

1 Formalin solution 37 % ( 50 to 75 ml )'

2 Potassium paermagnate powder

3 Seprtating flask 125 ml capacity

4 Petridishes and Tripod stand


PROCEDURES :


1 Before entering into the airlock remove the footwear outside the room

2 Ensure that the door closes completely on releasing when you enter the airlock

3 Remove the normal clothing and put it on the hanger

4 Take one set of sterile uniform Wear sterile uniform as per the instructions given below

a) Put on the headgear

b) Put on the bushshirt and then pyjama

c) Headgear to be tucked into the bushshirt and the in turn to be tucked in the pyjama

d) Put on the footwear and tie it properly

e) Take the pair of sterile gloves of proper size and wear them properly

5 Rinse the gloved hands with 70 % Isopropyl alcohol

6 Open the door with care while entering the sterility testing room

7 Before the start of fumigation ensure that the Air handling unit is shut off to prevent

the fumes of formalin to enter the other areas through Air handling unit

8 Fill the seperator with suffficient quantity of 50 ml of formalin ( 1 : 1 )

9 Take around 25 gm of potassium permagnate powder in dry pertridish without lid

Keep the seperator filled with formalin on a tripod stand and the pertridish with potassium

below it at the centre of the area which is to be fumigated

10 Loosened the stopcock so that formalin will fall drop by drop on the potassium permagnate

powder

11 Immediately come out of the room, lock the door

12 While coming out remove the sterile uniform in the reverse order, that is fisrt gloves,

second footwear, third pyjama, and forth bushshirt and lastly the headmask

13 Put your normal clothing kept on the hanger Before opening the door of the airlock corfirm

that door of sterility room is closed

14 These uniform gloves are to be washed and resterilised before using for next operation

15 The fumigation record is recorded in the ataachment 1

PERSONNEL TO BE REPORTED IN CASE OF NON COMPLIANCE :- Department head


FREQUENCY OF OPERATION :- Weekly


NO OF ATTACHMENT :-


RETENTION OF RECORDS :- 5 Years

DETERMINATION OF PARTICLE SIZE OF RAW MATERIAL AND FINISHED PRODUCTS IN PHARMACEUTICALS BY THE USE OF MICROSCOPE

OBJECTIVE :- To determine the particle size of the raw material that is used as the active ingrediants in pharmaceutical product and also the finished product microscopically


UNIT OF RESPONSIBILITY :- QA Executive and trained QA Assistants

IMPORTANT POINT/ NOTE/ PRECAUTIONS : -

1 Ensure the complete cleanliness of the microscope eyepiece and the objective before its use

2 Handle the Instruement carefully

PROCEDURE :-

1 Calibrate the eye piece micrometer scale by aligning with the stage micrometer (graduated in 10 u increment) under 10 X magnification so that they are parellel

2 Spread evenly over the cleaned microscopic slide, the material or the powder to be tested for particle size

Add a drop of liquid paraffin over it in case of powder to be tested and prepare the smear In case of finished product spread the material over the microscopic slide and cover with cover slip to prepare the smear

3 Adjust the microscope in such a way that the particles are clearly visible

4 Count about 100 particles

5 Record the count as per the limits specified in the individual monograph

6 Record the count as per the observations in the attachment No 1


OTHER RELEVANT DETAILS :

1 Limits as specified in the individual monograph


PERSONNEL TO BE REPORTED IN CASE OF NON COMPLIANCE :- Department Head


FREQUENCY OF OPERATION :- For every lot of raw material as well as finished product
NO OF ATTACHMENTS :- NOs

RETENTION OF RECORDS :- 5 Years

STERILTY TESTING OF STERILE PHARMACEUTICAL TOPICAL PREPARATIONS

OBJECTIVE :- To check whether the product to be tested is sterile

UNIT OF RESPONSIBILITY :- QA Executive and trained QA assistants

IMPORTANT POINT/ NOTE/ PRECAUTIONS :-

1 Follow proper entry/exit procedure

2 Follow proper aseptic technique while testing the sample

3 Do not work in laminar air flow unit when UV lamp is on
MATERIAL :-

a) Media : 1) Sterile Soybean Casien Digest Medium with ploysorbate 80 ( if specified in monograph

2) sterile Fluid Thioglycollate Medium with ploysorbate 80 (if specified in monograph)

b) Sterile Isopropyl Myristate obtained by filteration through 02 u membrane filter

c) 01 % w/v sterile peptone water with 2 % v/v polysorbate 80 (if specified in monograph)

d) Manifold unit for 3 or more holder membrane filter funnel with suitable vaccum flask

e) Sterilised membrane filter of 045 u and 02 u

f) Sterile forceps and scissors wrapped in parchment paper/butter paper

g) Sterile 10 ml pipettes

h) sterile empty flasks

PROCEDURE :-

I) METHOD:-

a) MEMBRANE FILTERATION METHOD :-

1 Follow the entry procedure in the aseptic area as mentioned in the SOP
2 Dissolve not less than 1 gm of ointment from not less than 20 tubes/ containers in not
less than 100 ml of sterile isopropyl myristate with a pH of water extract not less than 65, which previously has been rendered sterile by filteration through 02 u membrane filter
3 Warm the sterilised solvent, and if necessary the test material, to not more than 44 degC
( just prior to use)

4 Swirl the flask to dissolve the topical preparation, taking care to expose a large surface of the material to the solvent

5 Following dissolution aseptically transfer the mixture in membrane filter funnel and immediately pass the mixture through the membrane filter of 045 u with the aid of vacuum

6 Keep the filter membrane covered with liquid throughout the filteration for maximum efficiency of the filter Following filteration of the liquid wash the membrane filter with 300 ml of sterile 01 % w/v peptone water

7.Aseptically remove the membrane filter from the filter holders, cut the membrane in two halves immerse one half of the membrane filter with the aid of sterile forceps in 100 ml of Soybean Casien Digest Medium and the other half in 100 ml of sterile Fluid Thioglycollate Medium

8 Keep the positive control by inoculating the culture in Fluid thioglycollate medium and Soybean casien digest medium

9 Keep the negative control by filtering autoclaved 100 ml of 01%w/v peptone water,cut the membrane filter in two halves & immerse one half of the filter in Soybean casien Digest Medium and the other half in sterile Fluid Thioglycollate Medium

10 Incubate the tubes at 20 - 25c and 30 - 33c for growth of fungus and bactreia respectively for not less than 7 days as per IP and 14 days as per USP and BP
11Observe the tubes for growth in terms of turbidity daily till the completion of 7 days as per IP and 14 days as per USP & BP
12 If none of the tubes shows the growth in terms of turbidity within 7days as per IP and as per USP and BP within 14 daysof observation then the test passes for sterility

14 If any of the tubes shows growth in terms of turbidity than preserve the tubes and carry
out the investigation by identifying the organism
15 Repeat the test using double quantity of tubes/ containers and if no growth observed the
test passes for sterility and if growth observed then the test fails for sterility

b) DIRECT INOCULATION METHOD :-

1 Quantity to be used is 1 to 10 gm

2 Quantity to be used for each culture medium 05 to 10 gm

3 Follow the entry procedure in the aseptic area as mentioned in the SOP NO QA/SOP/304/98

4 Take about 20 Samples of topical preparation and take out about 05 gm from each sample into a flask containing 100 ml of 01 % w/v sterile peptone water with 2 % w/v polysorbate 80 and mix the ointment by swirling the flask

5 Transfer aseptically 10 ml of the diluted media to each 100 ml of sterile Soybean casien Digest Medium and 100 ml of sterile Fluid Thioglycollate medium Incubate the media at 20 - 25 deg C and 30 -35 deg C for not less than 14 days for fungus and bacteria respectively

6 Observe the tubes for growth in terms of turbidity daily till the completion of 14 days

7 Keep the positive control by inoculating the culture in Fluid thioglycollate medium and Soybean casien digest medium and Negative control by inoculating an autoclaved 01% peptone water

8 If none of the tubes shows the growth in terms of turbidity within days of observation then
the test passes for sterility

9 If any of the tubes shows growth in terms of turbidity than preserve the tubes and carry
out the investigation by identifying the organism

10 Repeat the test using double quantity of tubes/ containers and if no growth observed the
test passes for sterility and if growth observed then the test fails for sterility

II) GROWTH PROMOTION TEST:-

To each autoclaved load of each lot of medium for its growth promotion qualities by
seperately inoculating test containers of each medium with about 100 vaible micro
organisms of each strain as listed below and incubating according to the conditions
specified The test media are satisfactory if clear evidence of growth appears in all
inoculated media within 7 days as per IP and 14 days as per USPand BP
If the freshly prepared media are not used within 2 days then store them in dark
preferably at 8 - 15 deg CThe sterility is considered invalid if the test medium
shows inadequate growth response

Medium -----------------------------------Test Microorganism ----------Temp------- Condition

Fluid Thioglycollate Medium ------B.subtilis ATCC 6633/S.aereus NCTC 7447---30-35 C-- -Aerobic/Anaerobic

Fluid Thioglycollate Medium ------ -----S.aereus NCTC 7447------------------------30-35 C-- --Aerobic

Soybean Casien Digest Medium -------C.albicans ATCC 10231-----------------------30-35 C-------Aerobic

III) PRODUCT POSITIVE CONTROL TEST :-

After tge sterility testing is over, add to each media tubes of the particular batch tested
around 100 cfu/ml of B subtilis ATCC 6633 and C albicans ATCC 10231 resp in
Fluid thioglycolate medium and Soybean casien Digest medium, incubate furthur for
minimum of 24 hrs at 30-35 deg C and 20-25 deg C resp Observe for growth in the
inoculated media tubes to verify that the media does not contain bacteriostatic or
fungistatic activity

IV) DESTRUCTION :-
After the testing is over the media is destroyed by autoclaving in a pressure cooker
for 20 minutes This destroyed media is discarded into the drain
The samples after testing are destroyed by sweezing the ointment out and incernating it
All the observations , results and destruction records are recorded in the attachment 1

PERSONNEL TO BE REPORTED IN CASE OF NON COMPLIANCE :- Department head

Production Manager


FREQUENCY OF OPERATION :- Batchwise