Environmental Monitoring Program

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Title: Procedure for Environmental Monitoring Program

Purpose :
To define procedure for monitoring microbial count.
Scope:
Applicable for environmental monitoring program.
Responsibilities:
Microbiological in charge / designate to follow the procedure.
Microbiological in charge / designate will be responsible for all kind of sampling and
will also be responsible for checking viable counts in manufacturing area.
Manager QA / designate to monitor compliance.
Quality operation Manager / designate to audit compliance.
Procedure:
Air Quality – Viable Counts Annexure-1

Surface Microbial Count Annexure-2
Validation of Disinfectants and the Floor Cleaning procedure
Annexure-3
ANNEXURE-1:
Air Quality-Viable Counts.
Note:- Sampling sites are selected on the basis of work in that area
Mark the plates of Tryptic Soya Agar and Sabrouad Dextrose Agar as per given position in the area and
expose for 1 hour.
Incubate the Tryptic Soya Agar plates at 37c for 48-72 hours
Incubate the Sabrouad Dextrose Agar plates at room temperature for 5-7 days.
Count the colonies.
Detect the pathogens from the isolated colonies at the Tryptic Soya Agar.
Select the typical colonies from the TSA plates and pick a colony with the help of a
Wire loop and inoculate into ten ml Tryptic Soya Broth and Lactose Broth.
Incubate the tubes at 37c for 24-48 hours.
Streak the Specific media plates for pathogen detection.
Streak the specific media plates from Lactose broth at given below
Eosin Methylene Blue]
MacConkeyes Agar }For the detection of E.Coli
Heckton agar.
Bismuth Sulfith Agar.
Brilliant Green Agar} For the detection of Salmonella Agar
Streak the specific media plates from Tryptic Soya Broth given below.
Vogal Johnson Agar} For the detection of Staph.aureus.
Ceteramide Agar } For the detection of Ps.aeuroginosa.
Identification of pathogens by Colonial Morphological Characteristic
E .Coli.
Gram Stain Colonial Morphological Selective Medium

Characteristic
Negative Rods Brick-Red may have

(Cocco-bacilli) surrounding zone of MacConkeyes Agar
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Precipitate-bile
Negative Rods Pink-Red color

(Cocco-bacilli) with metallic sheen Eosin Methylene blue
Morphological Characteristic of Salmonella Spp

Selective Medium Colonial Morphological Characteristic
Small, transparant, colorless or pink to white
Brilliant Green Agar
(Frequently rounded by pink to red zone)
Bismith Sulfite Agar Black or Green
If colonies of G-ve rods match the characteristic given in the table. Proceed with further identification by
transferring representative suspect colonies individually by means of inoculating wire,toa butt slant of Triple –
Sugar iron medium by first Streaking the surface of the slant and then stabbing the wire well beneath the
surface .Incubate ,if no alkaline(red) slants and acid (yellow) butts(with or without concomitant blackening of
the butt from hydrogen sulfide productive), the specimen meets the requirements of the test for the absence of
the genus Salmonella.
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Morphological Characteristic of Staphylococcus aureus on Selective Agar Media
Selective Characteristic Colonial Gram Stain
Medium Morphology
Vogal Jonhnson Black surrounded by Positive Cocci
Agar Yellow zone (in cluster)
Manitol Salt Yellow colonies with yellow zone Postitive Cocci (in Cocci)
Agar
Baird-Parker Black, Shiny, Clear Positive Cocci
Morphological Characteristic of Pseudomonas aeruginosa on Selective Agar Media

Selective Colonial Characteristic Fluorescencein Oxidase
Medium Ultraviolet Light Test
Ceteramide Generally Greenish Positive
Agar Greenish
Pseudomonas Generally to Coloreless to Yellowish Positive
Agar Medium for yellowish.
Detection of
Fluorescing
Pseudomonas Generally Greenish Blue Positive
Agar Medium for
detection of
Pyocyamin
Coagulase Test
(Staphylococcus
aureus)
With the help of inoculating loop, transfer representative colonies from the agar surface of the Vogal-Johnson
Agar medium or Manitol Salt agar to individual tubes, each contains 0.5 ml mammalian,(preferably rabbit or
horse plasma with or without suitable additives. Incubate in a water bath at 370c; examine the tubes at 3 hours
and subsequently at suitable interval, up to24 hours. Test positive and negative controls simultaneously in any
degree is observed, the specimen meets the requirements of the test for the absence of Staphylococcus
aureus.
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Oxidase and pigment Test: (Pseudomonas aeruginosa)
With the aid of wire loop, streak representative suspect colonies from agar surface of cetrimid agar medium or
the agar surface of Pseudomonas agar medium for detection of Fluorescence and Pyocyanin contain in
petridish. Incubate at 35+2oCforthree days. Examine the characteristic given in the table referred.
Pseudomonas can be confirmed by placing 2or3 drops of freshly prepared1%w/v Solution of tetra-N-P-
Phenylene –diammonium on a piece of filter paper and smear with suspected colonies, and if purple color is
produced within5-10 seconds the test is positive
Criteria and Action required
1) If limit crossed action should be taken consulting with related manufacturing personnel
and take samples on daily basis till counts of 3 Consecutive days arewithin limit
2) If high counts observed, report the section incharge.
3) If Pathogens are found in any area.
That area will be closed until disinfected and retested.

All products made in that area during the time of the initial test and declaration of results shall be quarantined
These products will be tested microbiologically on standard criteria..
ANNEXURE 2:
SURFACE MICROBIAL COUNT
Swabs-NonSterile area
Objective:
To establish a procedure for monitoring the bioberden at the surface of equipment
SCOPE
Manufacturing area (Tablet area, Cream area, Liquid area)
Procedure:
Preparation of Buffer 7.2 pH.
Stock Solution ;
Dissolve 34 gm of monobasic Potassium phosphate in about 500 ml of Water, contained in 1000ml
volumetric flask adjust to pH 7.2+- 0.1Nby the addition of NaOH TS about (175ml) make up the volume with the
water. Sterilize and store under refrigerator up to one month.
Working solution:
Dilute the stock solution with water into1;8 and sterilize
Dispense 5ml of diluted buffer into test tube.
Make a swab stick with tight stoppered.dip into5ml buffer , and Sterilize.
PROCEDURE
Collection of Sample:
1) Take out the swab stick and rub at the required surface of the equipment by taking the
area of 25sq.cm.
2) Dip the Stick in to test tube containing buffer.
3) Take the sample to the microbiological lab and test within one hour.
Testing of swab:
1) Take out the swab stick from the test tube ,squeeze the swab with the help of the wall of the test tube
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2) Streak the swab at the Tryptic Soya Agar plate.
3) Incubate the plate at 370c for 48-72 hours by inverting the plate.
4) Count the colonies.
5) Report the results
Criteria and active required:

Area to be thoroughly cleaned by production personnel.
If limits crossed, certify the corrective action by tacking the swab daily till 3 consequence results are within
limit.
ANNEXURE 3:
VALIDATION OF DISINFECTANTS AND THE FLOOR CLEANING PROCEDURE
Objective:
Environmental monitoring is an essential test to check the cleaning and / disinfection condition of production
area.
Cleaning/disinfection of manufacturing and other operational areas is first essential step before the
manufacturing process of a product. It keeps away the product from the accumulation of microorganism and
keeps the environment clean. The viable/ non-viable particles setting on the equipment, wall and floor may lead
to cross contamination.
Specified disinfecting/ cleaning agent should be used in order to prevent the development of essential strains
of micro organism.
PROCEDURE:
1. Perform at least three validation tests for total microbial count and pathogens before and after applying the
effective disinfectant at different interval.
1.1. Zero (0) hour
1.2. One(1) hour
1.3. Three(3) hours
1.4. Six (6) hours
2. Perform the validation test by means of

2.1. Surface microbial count/ Swab test
2.2. Air microbial count/ Direct expose plate method.
a) Surface microbial count/ Swab test.

1. Take out the swab stick from test tube containing 5 ml buffer 7.2 PH
2. Squeeze the swab with the help of the wall of the test tube and rub the surface by
taking the area about 25 sq. cm.
3. Dip the stick again in the test tube and take the sample to the microbiological section.
4. Take out the swab stick from the test tube an squeeze the swab with the help of the
wall.
5. Streak the swab at Tryptic Soya Agar plate/Sabourad Dextrose Agar.
6. Incubate the plates at 37 0C for 48-72 hours.
7. Observe the colonies.
Air microbial count / Direct expose plate method.

1) Expose the presolidifing Tryptic Soya Agar plate / Sabroud Dextrose Agar at the area where disinfectant is
applied for 1 hour
2) Incubate the Tryptic Soya agar plate at 370C for 48-72 hours.
3) Incubate the Sabroud Dextrose Agar at room temperature for 5-7 days
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The following disinfectant which are using in different time period with different concentration.
1) 1.2% of Dettol solution in the morning.
2) 1.0% of Phenyl solution in the afternoon .
3) 1.0% of Bleach solution (Sodium hypochloride) in the water at the end of work.
4) Use 2.5% Esteem solution in the morning and after noon at the end of work on alternate days Tuesday
&Thursday.
5) Evaluate the results of Microbial count & Pathogens before and after applying the above disinfection.

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Page of 6Page of 6

Title: Procedure for Environmental Monitoring Program

Air Quality – Viable Counts Annexure-1

Identification of pathogens by Colonial Morphological Characteristic

Gram Stain Colonial Morphological Selective Medium

Negative Rods Brick-Red may have

Title: Procedure for Environmental Monitoring Program

Negative Rods Pink-Red color

Morphological Characteristic of Salmonella Spp

Title: Procedure for Environmental Monitoring Program

Morphological Characteristic of Pseudomonas aeruginosa on Selective Agar Media

Title: Procedure for Environmental Monitoring Program

Criteria and Action required

That area will be closed until disinfected and retested.

Title: Procedure for Environmental Monitoring Program

Criteria and active required:

VALIDATION OF DISINFECTANTS AND THE FLOOR CLEANING PROCEDURE

2. Perform the validation test by means of

a) Surface microbial count/ Swab test.

Air microbial count / Direct expose plate method.

Title: Procedure for Environmental Monitoring Program

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