Processing of stool samples

1.    Scope

·       This document contains the procedure that is used in processing of stool samples to isolate polio and non-polio enteroviruses.

2.    Definition

• PBS     -           Phosphate buffered saline with calcium and magnesium Ions.
• ⁰C        -           Degree Centigrade.
• RPM    -           Rotation per minute.

3.    Materials

·       Gloves( medium or small)

·       15ml Polypropylene centrifuge tubes (Chloroform resistant)

·       Sterile plastic pipettes-10ml, 1ml

·       Sterile glass pipette.

·       Sterile glass beads-3mm

·       PBS(with Ca⁺⁺  and Mg⁺⁺)

·       Chloroform

·       Fecal specimens to be processed.

·       Specimen storage vials- 1.8ml and 4.5ml externally threaded, screw capped.

·       Extra specimen containers and labels.

·       Discarding container with freshly prepared 1% Sod. Hypochlorite solution.

·       Cotton swabs soaked with 1% Hypochlorite solution.

·       Mechanical Shaker

·       Refrigerated centrifuge.

4.    Procedure

·       Stool processing to be done inside the biosafety cabinet class II in virology room No2.

·       Switch on the refrigerated centrifuge.

·       Follow all the precautions for handling infectious materials(Biosafety precaution)

·       Before starting sample processing keep an absorbent bench pad inside the cabinet.

·       Take 15ml Polypropylene centrifuge tubes and add 2gm glass beads in each tube.

·       Add 9ml PBS with Ca⁺⁺ and Mg^++.

·       Add 1ml chloroform with a glass pipette to each tube.

·       Label the centrifuge tubes with lab no. of stool samples.

·       The stool container to be opened carefully without touching collar area.

·       Transfer approximately 2gm of fecal sample using the attached spoon inside the specimen Container or sterile Pasteur pipette in to the appropriately labeled centrifuge tube without spoiling the mouth of the tube. Close the lid tightly.

·       Carefully close the lid of the specimen container and keep it back in the polythene bag. Apply rubber band and put the sample in the labeled storage box.

·       If any spillage occurs at this time discard the absorbent bench pad in to the discarding bucket containing 1% Sod. Hypochlorite solution.

·       If the gloves are contaminated during any steps immediately remove the gloves and discard in to 1% Sod. Hypochlorite solution and wear new gloves.

·       Retain the original stool sample at -20⁰C in deep freeze no.7. Tighten the cap of centrifuge tube properly.

·       Tighten all the centrifuge tubes in a thermocol rack and fix it securely horizontally in the shaker. Shake it for 20min at 200 motions per minute.

·       After that place the centrifuge tubes in the rotor of the refrigerated centrifuge and balance all the tubes.Close the lid of the rotor firmly and spin for 30 min. at 3500 RPM at 4⁰C.

·       After 30 min., allow the centrifuge to completely stop. Open front cover of machine and open the lid of rotor carefully. Take out all the centrifuge tube without disturbing.

·       Label 2 vials (1.8ml and 4.5ml) for each sample. Match the no. on the centrifuge tube and on the vials carefully. With sterile Pasteur pipette take 4 ml supernatant into the 4.5ml vial which is to be used for inoculating cell culture tubes and take 1.8ml of the supernatant into the 1.8 ml vial which serves as untouched back up and preserve it in -20 ⁰C in Deep Freeze No. 4 without disturbing the sediments.

·       Disturb the sediments of centrifuge tube with the same Pasteur pipette. Discard the pipette and centrifuge tube with slightly loosened cap into the discarding pan containing 1% hypochlorite solution.

·       Cover the discarding pan with aluminum foil and send for autoclaving.      

·       Clean the BSC and switch off. Enter the details of the specimen processed, the names of the staff involved in the work, date of processing and batch of PBS used in Log Book of Biosafety Cabinet No. 1 PR 3 or Log Book of Biosafety Cabinet No.9  PR-45

·       After completion of work, discarding pan is autoclaved at 121⁰c for 45 minutes before disposal in biomedical waste.

Original stool samples are retained in the laboratory for a period of six months from the date of receipt. Discarding is documented in Stool Sample Receiving Register and stool sample box register. Stool sample extracts are retained for a period of three months in the laboratory from the date of receipt.Discarding is documented in Sample Extract Vial Register. Stool samples and sample extracts are discarded after autoclaving at 121°C for 45 minutes.

1.    Safety conditions

·       Take precaution during handling of stool samples.

·       To prevent cross contamination, if there is any spillage clean it with cotton soaked in 1% Sod. Hypochlorite solution.

2.    Documentation

·       Stool Sample Receiving Register PR-1

·       Log book of Biosafety Cabinet No.1 PR-3

·       Log book of Refrigerated Centrifuge No.1 PR-6 & No.3 PR-7

·       Stool Sample Box Register-PR-11

·       Sample Extract Vial Register-PR-12

3.    References used to draw SOP

·       WHO Polio Laboratory Manual.