Primary Inoculation of Sample extract

1.    Scope

·       This document contains the procedure that is used for inoculation of stool sample extracts in L₂₀B and RD cell lines tubes for isolation of polio and non-polio enteroviruses.

2.    Definition

·       RD       -           Rabdomyosarcoma cell.

·       L₂₀B    -           Derived from mouse L-cell.

·       CPE     -           Cytopathic effect.

·       TC tube -         Tissue culture tubes

3.    Materials

·       1ml sterile disposable pipette.

·       2 tubes of L₂₀B and 2 tubes of RD for each sample (each tube containing 1ml of maintenance medium)

·       4.5ml vial of sample extract for inoculation.

·       Discarding container with 1% Sod. Hypochlorite solution.

4.    Procedure

·       Examine the monolayered tubes (3-6 days old) to be inoculated for the cell monolayer i.e. if the sheet is healthy and 75% full.

·       Label two tubes of of L20B and RD as L1A, L1B and R1A, R1B with specimen No. (e.g. 1/12 L1A , 1/12 L1B, 1/12 R1A and 1/12 R1B) and date of inoculation and arrange them in rack as L1A, L1B, R1A and R1B. 

·       Mix the 4.5ml vial containing sample extract on vortex and allow it to settle for 5min. before inoculation.

·       Inoculate 0.2ml of sample extract from 4.5ml vial into each tube of L20B and RD cells using sterile disposable pipettes to the opposite side of the cell monolayer.

·       Both cell lines must be inoculated at the same time.

·       Discard the pipette into discarding pan containing freshly prepared 1% Sod. Hypochlorite solution.

·       After completion of work discarding pan is autoclaved at 121⁰C for 30 minutes before disposal in biomedical waste.

·       Inoculated tube rack is incubated in a stationary sloped position (5⁰) at 36⁰c in a standard incubator.

·       Examine tubes daily for 5 days using inverted microscope and record all the observations as CPE +1, 2+, 3+, 4+, toxicity, degeneration and contamination of cells in Virus Isolation Worksheet PR 13.

·       If there is 4+ CPE, freeze the tubes into -20⁰c deep freeze No.18.

Note: Toxicity – If cell degeneration appears in 24 to 48 hours then stool extract is diluted 10^-1 (0.9 ml PBS and 0.1 ml stool extract) and reinoculated with 0.2 ml of 1:10 dilution in 2 tubes of L20B and RD each. If cells are degenerated again then reinoculate with 10^-2 dilution of stool sample extract.

Contamination – If microbial contamination appears then stool extract is treated with chloroform (total volume of stool extract and 1ml chloroform) and reinoculation with 0.2 ml of stool extract is done in 2 tubes of L20B and RD each.

1.    Safety conditions

·       Stool extract to be stored only in externally threaded vials.

·       To avoid aerosol, transfer the fluid slowly in to the tissue culture tubes.

·       Avoid the finger touch on inner surface of collar area of the tubes and vials

2.    Documentation

• Virus isolation worksheet. PR-13
• Log Book of Biosafety Cabinet No. 1 PR 3, Log Book of Biosafety Cabinet No.9  PR-45

3.    References used to draw SOP

·       WHO Polio Laboratory Manual.