sop for Drain point of Microbiology

1.0 OBJECTIVE

1.1 To lay down the Procedure for pathogen test of Drain point of Microbiology Laboratory.

2.0 SCOPE

2.1 This procedure applicable for pathogen testing of Drain point in Microbiology Laboratory

3.0 RESPONSIBILITY

3.1 Microbiologist QC

4.0 ACCOUNTABILITY

4.1 QC Manager

5.0 PROCEDURE

5.1 Requirement
5.1.1 Test tubes, conical flask, 70% Iso propyl alcohol, Soyabean Casein Digest Medium with polysorbate 80 and Lecithin, sterile swab, normal saline 0.9% and required selective media.

5.2 PROCEDURE
5.2.1 Prepare Normal Saline 0.9% and pour 10ml in each sterile glass tube containing swab.
5.2.2 Prepare Soyabean Casein Digest Medium with polysorbate 80 and Lecithin and selective media.
5.2.3 Sterilize media & swab in a validated double door autoclave at 121˚C for 20 minutes as per SOP.
5.2.4 Complete the sterilization, unload from sterile side and keep for cooling at room temperature.
5.2.5 Transfer the swabs outside from micro sterile area through dynamic pass box.
5.2.6 Carry the swabs in SS container to production sterile area. Enter the material through Dynamic pass box.
5.2.7 Enter the production sterile area as per SOP.
5.2.8 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.
5.2.9 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.
5.2.10Shake 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.
5.2.11Remove the swab stick gently under HLAF and transfer complete solution in 90ml of SCDM broth tube, mark the sampling location & date with the help of permanent marker pen on the tube.
5.2.12Incubate the swab sample tubes in a BOD incubator at 32.5˚C ± 2.5˚C for 48 hrs.
5.2.13Incubate tube for 48 hrs, if turbidity is observed further continue for pathogen testing.
5.3 Pathogen Testing
5.3.1 Test for E.coli
5.3.2 Primary Test : Shake the tube,transfer1.0ml Solution 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.
5.3.3 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.

MORPHOLOGICAL CHARACTERITICS OF E.coli SPECIES ON
SELECTIVE CULTURE MEDIA TABLE- I

MEDIUM

DESCRIPTION OFCOLONY

Mac Conkey agar

Mac Conkey broth

Pinkish red colour colonies may have surrounding zone of precipitated bile

Growth present

5.4 Test for Salmonella
5.4.1 Add 0.1ml of enrichment culture from 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.

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

5.5 Test for Pseudomonas aeruginosa
5.5.1 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.
MORPHOLOGICAL CHARACTERITICS OF PSEUDOMONAS SPECIES ON
SELECTIVE CULTURE MEDIA Table – III

Medium	Characteristics colonial morphology	Fluorescence in UV light
Cetrimide agar	Generally greenish	Greenish

5.6 Test for Staphylococcus aureus
5.6.1 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.

MORPHOLOGICAL CHARACTERITICS OF STAPHYLOCOCCUS SPECIES ON
SELECTIVE CULTURE MEDIA TABLE- IV