sop for pathogen detection from drain point
1 OBJECTIVE
To describe the procedure for operating the pathogen detection from drain point.
2 SCOPE
This procedure is applicable to operate the pathogen detection from drain point.
3 RESPONSIBILITY
Microbiologist.
4 ACCOUNTABILITY
Head – Q. C
5 ABBREVIATIONS
SOP : Standard Operating Procedure
Q .A. : Quality Assurance
Q. C. : Quality Control
NO. : Number
SS : Stainless Steel
HLAF : Horizontal laminar air flow
XLDA : Xylose Lysine Deoxycholate Agar
RVSE : Rappaport Vassiliadis Salmonella Enrichment Broth
6 PRECAUTIONS
6.1 All equipment and material should be sterile during swab monitoring.
6.2 Sampling should be in spiral manner and cover the entire area of drain.
7 MATERIALS REQUIRED
Test tubes, conical flask, 70% Iso propyl alcohol, Dey-Engley Neutralizing broth or Soyabean Casein Digest
Medium with polysorbate 80 and Lecithin, sterile swab, normal saline 0.9% and required selective media.
8 EQUIPMENTS REQUIRED
BOD Incubators, Horizontal Laminar Air flow.
9 PROCEDURE
9.1 Prepare Normal Saline 0.9% and pour 10ml in each sterile glass tube containing swab
9.2 Prepare Dey-Engley Neutralizing Broth / Soyabean Casein Medium with plysorbate 80 and Lecithin and
selective media as per SOP No. …….
9.3 Sterilize media & swab in a validated double door autoclave at 121˚C for 20 minutes as per SOP No.
9.4 Complete the sterilization, unload from sterile side and keep for cooling at room temperature.
9.5 Transfer the swabs outside from micro sterile area through dynamic pass box.
9.6 Carry the swabs in SS container to production sterile area. Enter the material through dynamic pass box.
9.7 Enter the production sterile area as per SOP No
9.8 Monitor the required surface of drain point location by swabbing area approx 2’ x2’ (approx 25 cm2 )
Area, uniformly and horizontally in one direction . clean the swabbed area with filtered 70%Iso propyl
alcohol.
9.9 Replace the swab in the sterile Polypropylene or glass tube immediately. After swabbing operations are
over, bring all tubes containing swabs dipped in Normal saline to microbiology laboratory and transfer under HLAF for further testing.
9.10. 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.
9.11 Remove the swab stick gently under HLAF and transfer complete solution in 90ml of Dey Engley
Neutralizing broth tube, mark the sampling location &date with the help of permanent marker pen on the
tube.
9.12 Incubate the swab sample tubes in a BOD incubator at 32.5˚C ± 2.5˚C for 48 hrs.
9.13 Incubate tube for 48 hrs, if turbidity is observed further continue for pathogen testing.
9.14 Pathogen Testing
9.14.1 Test for E.coli
9.14.1.1 Primary Test : Shake the tube,transfer1.0ml Solution Dey Engley Neutralizing Broth/Soyabean
casein digest Medium with polysorbate 80 and Lecithin transfer into bottle containing 100ml of
MacConkey broth and incubate at 42˚Cto 44˚C for 24 to 48 hrs.
9.14.1.2 Secondary Test : Subculture on the surface of MacConkey agar plate and incubate at 30°C to
35°C for 18 to 72 hrs. If appearance of pinkish red color colonies on MacConkey agar indicates
the presence of E.coli.
9.14.1.3 Confirmatory Test: Indole Test:
Inoculate the suspected colonies to test tube containing 5.0 ml peptone water. Incubate the tube
at 30°C -35°C for 24 hrs, for indole production. Add 0.5 ml Kovac’s reagent shake well and allow
to stand for one minute if red colour ring is observed in the reagent layer indole is present.
MORPHOLOGICAL CHARACTERITICS OF E.coli SPECIES ON
SELECTIVE CULTURE MEDIA TABLE- I
| MEDIUM | DESCRIPTION OFCOLONY |
| Mac Conkey agar
Mac Conkey broth Kovac’s reagent |
Pinkish red colour colonies may have surrounding zone of precipitated bile
Growth present Red ring formation |
9.14.2 Test for Salmonella
9.14.2.1 Primary Test :
Add 0.1ml of enrichment culture from Dey Engley Neutralizing Broth/Soyabean casein digest Medium with polysorbate 80 and Lecithin, transfer test tube containing 10 ml Rappaport Vassiliadis
Salmonella Enrichment Broth and incubate at 30˚C to 35˚C for 24 to 48 hrs. After incubation
the tube subculture on the Xylose Lysine Deoxycholate Agar and incubate at 30˚C to 35˚C for 24 to
48 hrs. If red colonies with or without black centers possibly of salmonella.
9.14.2.2 Secondary Test:
Subculture those colonies which show the characteristics as given in table II. Subculture the triple
sugar iron agar slants by stabbing the wire well beneath the surface. Incubate the stabbed slants at
30°C to 35°C for 24 hrs. The formation of acid and gas in the stab culture (with or without Concomit –
-ant blackening) confirms the presence of Salmonella while its absence confirms the specimen or
sample to be free from Salmonella.
MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON
SELECTIVE CULTURE MEDIA TABLE-II
| MEDIUM | DESCRIPTION OF COLONY |
| Xylose Lysine Deoxycholate Agar | Red with or without black centers
|
9.14.3 Test for Pseudomonas aeruginosa
9.14.3.1 Primary Test: Streak one loop full of the enrichment culture on the surface of Cetrimide agar plate, and incubate the plates in
inverted position at 30 to 35 ˚C for 18 to 72 hrs. Greenish color colonies indicate the possibility of Pseudomonas aeruginosa.
9.14.3.2 Oxidase test: Place 2 or 3 drops of Oxidase reagent (Tetra methyl para phenylene diammonium dihydro-Chloride) on a piece of
filter paper (WhatmanNo.1) and transfer the suspected colony making smear on the paper if a pink colour is produced changing to purple
within 5 –10 seconds, the test confirms the presence of P. aeruginosa.
MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON
SELECTIVE CULTURE MEDIA Table – III
| Medium | Characteristics colonial morphology | Fluorescence in UV light |
| Cetrimide agar | Generally greenish | Greenish |
9.14.4 Test for Staphylococcus aureus
9.14.4.1 Primary Test:Streak one loop full of the enrichment culture on the surface of Mannitol Salt agar incubate the plates inverted position
at 30°C to 35°C for 18 to 72 hrs. Yellow or white colonies with yellow zones indicate the possibility of presence of Staphylococcus aureus.
Proceed further for confirmatory test.
9.14.4.2 Confirmatory Test: Coagulase Test:
With the help of inoculating loop transfer representative suspected colonies from the agar surface of
Vogel Johnson agar or Mannitol salt agar to individual tube containing 0.5 ml mammalian plasma
Preferably rabbit or horse plasma with or without suitable additive. Incubate in a water bath at 30°C to
35°C and three hours and subsequently at suitable intervals up to 24hrs. along with test positive and
negative control simultaneously. If no coagulation in any degree is observed, the specimen meets the
requirements of the test of absence of Staphylococcus aureus
MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON
SELECTIVE CULTURE MEDIA TABLE- IV
| MEDIUM | DESCRIPTION OF COLONY |
| Mannitol Salt agar | Yellow colonies with yellow zones |
LIMIT OF DRAIN POINT : Pathogen should be absent in each drain.
Remark : If pathogen observed during drain point monitoring, action to be taken and Production and QA department should be inform.
NUMBER AND LOCATION OF DRAIN POINT
10. FREQUENCY
Monthly
11. ENCLOSURES
ANNEXURE -1 Pathogen Analysis from Drain Point Report Format
ANNEXURE -2 Change History Log Format
12. REFERENCE DOCUMENTS
In-house
ANNEXURE- 1
PATHOGEN ANALYSIS OF DRAIN POINT REPORT
SAMPLING DETAILS INCUBATION&INSTRUMENT DETAILS
Date of Swab Sampling/Testing : Incubation Period : 32.5 ± 2.5˚C for 2 days
Sampling done by: BOD Incubator ID No. :
Tested by : BOD Incubator ID No. :
No. of Sampling Locations: Swab Lot No.
Pretreatment of drain swab:
Shake the swabs gently under HLAF and transfer in 90ml of D/E Neutralizing broth/SCDM with Tween 80and Lecithin (Media Autoclave Lot
No.:______________)and incubate at 30to35˚C for 18to 24hours.
Pathogen Testing for E.coli:
| Media Name | Media Autocl
-ave Lot. No. |
Incuba-
tion condition |
Incub-ation Start
date |
DRAIN POINT NO. | Nega
-tive Control |
Posi
-tive Control |
Obser-ved by | |||||||||||
| T1 | T2 | T3 | T4 | T5 | T6 | T7 | T8 | T9 | T10 | T11 | MD
01 |
|||||||
| MacCo-nkey Broth | 42-44˚C for
24 t0 48 hrs |
|||||||||||||||||
| MacCo
-nkey Agar |
30-35˚C for
18 to 72 hrs |
|||||||||||||||||
Pathogen Testing for Salmonella:
| Media Name | Media Autocl
-ave Lot. No. |
Incuba-
tion condition |
Incub-ation Start
date |
DRAIN POINT NO. | Nega
-tive Control |
Posi
-tive Control |
Obser-ved by | |||||||||||
| T1 | T2 | T3 | T4 | T5 | T6 | T7 | T8 | T9 | T10 | T11 | MD
01 |
|||||||
| RVSE Broth | 30-35˚C for
24 to 48 hrs |
|||||||||||||||||
| XLDA Agar | 30-35˚C for
18 to 72 hrs |
|||||||||||||||||
Pa Pathogen Testing for S.aureus:
| Media Name | Media Autocl
-ave Lot. No. |
Incuba-
tion condition |
Incub-ation start
date |
DRAIN POINT NO. | Nega
-tive Control |
Posi
-tive Control |
Obser-ved by | |||||||||||
| T1 | T2 | T3 | T4 | T5 | T6 | T7 | T8 | T9 | T10 | T11 | MD
01 |
|||||||
| Mannit-ol salt Agar | 30-35˚C for
18 to 72 hrs |
|||||||||||||||||
Pathogen Testing for P.aeruginosa:
| Media Name | Media Autocl
-ave Lot. No. |
Incuba-tion condition | Incub
-ation Start date |
DRAIN POINT NO. | Nega
-tive Control |
Posi
-tive Control |
Obser-ved by | |||||||||||
| T1 | T2 | T3 | T4 | T5 | T6 | T7 | T8 | T9 | T10 | T11 | MD
01 |
|||||||
| Cetrimi-de Agar | 30-35˚C for
18 to 72 hrs |
|||||||||||||||||
Note: Use ‘-‘ For no growth observed and ‘+’ for growth observed.
Media Negative Control- No Growth/ Growth Observed.
Limit: Pathogen should be absent in each drain
Inference: The above Swab Sample of Drain Complies/ Does not comply as per the standard specifications of IP / BP / USP / IH ………..
Microbiologist Checked by Approved by
ANNEXURE-2
CHANGE HISTORY LOG FORMAT
| Rev. No. | Details of changes | Reason for change | Effective Date | Updated By |
|
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