sop for Environmental monitoring of all the Classified area

1.0 OBJECTIVE
1.1 To describe a procedure for Environment monitoring to minimize the risks of particulate or microbiological contamination of the product or material being handled.

2.0 SCOPE
2.1 This procedure is applicable for environmental monitoring of all the Classified area

3.0 RESPONSIBILITY
3.1 Microbiologist QC

4.0 ACCOUNTABILITY
4.1 Q.C. Manager

5.0 PROCEDURE
5.1 Material Required : Soybean casein digest agar media, Petri- plates (90 mm), Sterile RODAC plates (55mm), Sterile cotton swab,

Normal Saline, SS container, SS stand.
5.2 Equipment’s Required: Double door autoclave, Centrifugal air sampler, Particle counter, BOD incubator (22.5˚C+2.5˚C), BOD

incubator (32.5˚C+2.5˚C), Laminar Air Flow, Colony counter.

5.3 Microbial Contamination Monitoring
5.4 Settle plate method-
5.4.1 Sterilize the Petri plates in double door autoclave at 121˚C for 30 minutes
5.4.2 Prepare SCDA media required for plate exposure as per SOP for media preparation.
5.4.3 Pour 15ml to 20 ml of liquified SCDA media cooled to 45˚C into the sterilized Petri plates under HLAF.
5.4.4 Cover the Petri plates and allow the poured media to solidify under HLAF. After solidification, pre-incubate all the media containing

Petri plates in a BOD incubator (32.5˚C + 2.5˚C) for 24 hours in inverted position for detecting any contamination during plate pouring operations.
5.4.5 After incubation, examine all the Petri plates for microbial growth. Select media petri plates without contamination, for plate exposure operation.
5.4.6 Take SS container and spray properly with filtered 70% IPA.
5.4.7 Take all pre-incubated media containing plates in a SS container in area.
5.4.8 Expose the Petri plates at each location during production ( plate exposure locations Annexure ), By removing the upper lid of the

Petri plate and placing the media containing part on the upper side of SS plate holder angle of 45 ˚C.
5.4.9 Place the lid of Petri plate on the lower side of the SS plate holder. Expose the Petri plates for 4 hours.
5.4.10For micro lab expose the plate at each location as per the Annexure.
5.4.11After completion of 4 hours of exposure, close the Petri plate with lid, collect all the petri plates in same SS container and bring the all exposed plates to laboratory.
5.4.12 Incubate all exposed Petri plates in BOD incubators at 22.5˚C + 2.5˚C for 3 days and at 32.5˚C +2.5˚C for 2 days in inverted position.
5.4.13 After incubation, count the number of colony forming units (cfu) per plate with the help of Colony Counter.
Fill the results in the report format.
Exposure Frequency: Daily

Area Classification

Hours of Exposure

Limits

Alert

Action

Acceptance/ Specification limit

Class 100 (Class A)

Class 1000 (Class B)

Class 10,000 (Class C)

Class 1,00,000 ( Class D)

4 Hours

4 Hour

<1 cfu

3 cfu

30 cfu

60 cfu

<1 cfu

4 cfu

NMT 40 cfu

NMT 80 cfu

<1 cfu

5 cfu

NMT 50 cfu

NMT 100 cfu

5.5 Personnel Monitoring / Dab Test:
5.5.1 Take sterile RODAC (contact) plates (55mm).
5.5.2 Prepare SCDA(lecithin polysorbate) media required for personnel monitoring as per SOP for media preparation.
5.5.3 Pour approx.. 5 ml of liquified SCDA media cooled to 45˚C into the sterilized RODAC plates under HLAF.
5.5.4 Cover the plates and allow the poured media to solidify under HLAF. After solidification, pre incubate all the media containing

RODAC plates in a BOD incubator (32.5˚C ± 2.5˚C) for 24 hours in inverted position for detecting any contamination during plate pouring operations.
5.5.5 After incubation, examine all the RODAC plates for microbial growth. Select media RODAC plates without contamination, for personnel monitoring.
5.5.6 Take all pre-incubated media containing plates in a SS container (mop properly with filtered70% IPA).
5.5.7 Take pre-incubated RODAC (contact) plates (55 mm diameter) and open & touch the agar surface on different location of body

of the operators & other persons inside the sterile area (Armpit, Chest, and Forehead).
5.5.8 Open the lid of the media plates and take the finger dab of the right hand and left hand five fingers of the operators working in sterile area.
5.5.9 Close the lid of the Petri plates and place them in the SS container and bring all the plates to the laboratory.
Incubate all exposed Petri plates in BOD incubators at 22.5˚C + 2.5˚C for 3 days for Fungal growth and at 32.5˚C +2.5˚C for 2 days for Bacterial in inverted position.
5.5.10 After incubation, count the number of colony forming units (cfu) per plate with the help of Colony counter.
5.5.11The plate counts should not cross the Alert Limits.

Frequency- Daily

Working Area	Location	Alert Limits	Action Limits	Acceptance/ Specification limit
1,000	Armpit, Chest, Forehead	03 cfu	4 cfu	5 cfu
1,000	Finger Dab	<1 cfu	<1 cfu	<1 cfu
10,000	Armpit, Chest, Forehead	05 cfu	8 cfu	10 cfu
10,000	Finger Dab	<1 cfu	<1 cfu	<1 cfu

5.6 Swab test:
5.6.1 Take sterile swabs in Polypropylene tubes and pour 10ml Sterile Normal saline in each of the required swab tubes aseptically.

Mark the swab tubes with the respective sampling locations with the help of marker pen.
5.6.2 Monitor the required surface of pre defined location by swabbing an area of approx 2’ x 2’ inch (i.e. approx. 25 cm2) area.

Uniformly and horizontally in one direction. Clean the swabbed area with filtered 70% IPA solution.
5.6.3 Replace the swab in the sterile Polypropylene tube immediately. After swabbing operations are over, bring all tubes

containing swabs dipped in Normal saline to the microbiology laboratory and transfer under the HLAF for further testing.
5.6.4 Now shake the normal saline of each tube reciprocally or vortex so that any particle stuck on the surface should get dispersed properly in the normal saline.
5.6.5 Now remove the swabs gently under HLAF and pour 1.0 ml of normal saline on the sterile Petri plate marked with sampling location with marker pen.
5.6.6 Pour gently approx. 15ml to 20 ml of autoclaved Liquified SCDA media on the plate and rotate the plate in order to mix the contents.

Proceed similarly for all other swab sampled tubes.

5.6.7 Allow the plates to solidify under HLAF and after solidification transfer all the solidified plates representing each swabbed sample

location for incubation in inverted position in a BOD incubator at 22.5˚C + 2.5˚C for 3 days for Fungal growth and then incubate in

BOD incubator at 32.5˚C + 2.5˚C for 2 days for Bacterial growth.

Acceptance Criteria: in operation

	Alert Limits	Action Limits	Acceptance/ Specification limit
Class 100 (A)	< 01 cfu	< 01 cfu	< 01 cfu
Class 1000 (B)	3 cfu	04 cfu	05 cfu
Class 10,000 (C)	10 cfu/25cm²	20 cfu/25cm²	25 cfu/25cm²

5.7 Air Sampling: – By Microbiological Air Sampler
5.7.1 Remove the Impeller Assembly of the sampler carefully and sterilize at 121°C for 30 minutes.
5.7.2 Wear hand gloves and mop with 70% Isopropyl Alcohol. Switch on the instrument. Insert the media Plate with the Agar surface facing towards upwards.
5.7.3 Press the start button & collect the air sample from the sampling point.
5.7.4 Switch off after the sampler has stopped running.
5.7.5 Remove the media plate & cover it with lid.
5.7.6 Incubate the plates at 22.5˚C + 2.5˚C for 3 days for fungal growth. Observe and continue further Incubation at 32.5˚C + 2.5˚C for 2 days

for Bacterial growth. Observe the plates and report the results.
5.7.6 Frequency: Production – Daily
Micro Lab – Daily

Acceptance Criteria: in operation

Area	Alert Limit	Action Limit	Acceptance/ Specification limit
Class 100 (A)	Less than 01 cfu/m³	NMT 1 cfu/m³	NMT 1 cfu/m³
Class 1000 (B)	5 cfu/m³	8 cfu/m³	10 cfu/m³
Class 10,000 (C)	50 cfu/m³	NMT 80 cfu/m³	NMT 100 cfu/m³
Class 100,000 (D)	100 cfu/m³	NMT 160 cfu/m³	NMT 200 cfu/m³

6.0 ABBREVIATIONS

Abbreviation used	Full form of abbreviation used
SOP	Standard Operating Procedure
IPA	Iso Propyl Alcohol
QA	Quality assurance
QC	Quality Control
RODAC	Replicate organism detection and count
SS	Stainless steel
CFU	Colony forming unit
ML	Milliliter
SCDA	Soyabean casein digest agar