Blind Passage

Scope

·       This document contains the procedure that is used for second passage of virus in another set of L₂₀B and RD tubes for isolation of polio and non-polio enteroviruses.

1.     Definition

·       RD                   -           Rabdomyosarcoma cell.

·       L₂₀B                -           Derived from mouse L-cell.

·       CPE                 -           Cytopathic effect.

·       TC tubes         -           Tissue culture tubes.

·       ⁰C                    -           Degree Centigrade.

2.    Materials

·       1ml disposable pipette.

·       L20B and RD tubes-containing 1ml of maintenance medium in each tube.

·       Discarding container with 1% Sod. Hypochlorite solution.

·       Completely thawed tubes for blind passage.

3.    Procedure

·       Two persons should check the labels on the thawed tubes and tubes to be inoculated.

·       Transfer 0.2ml of harvested material from primary inoculation tubes to another set of L20B and RD tubes (blind passage) as per following protocol.

·       If no CPE appears in primary L20B and  RD tubes, passage in corresponding tubes as

• L1A to L2A                L1B to L2B                
• R1A to R2A                R1B to R2B

• Each tube is observed for 5 days for appearance of CPE. If no CPE appears after 5 days, report this specimen as negative. If CPE appears in any of the tubes then they are processed according to the following schemes.
• If two tubes of primary L20B are positive (at least 3+ CPE) then freeze and thaw the tubes. Pool the harvested material and inoculate 0.2 ml into two RD tubes containing fully grown sheet and label as LR1 and LR2

• LR tube is incubated at 36⁰ C and observed for appearance of CPE.
• When at least 3+ CPE appears freeze the tube and prepare two vials with lable having sample no. and cell line (L+R+). Send one vial for ITD and preserve one vial for storage.

·       If only one L20B tube is positive inoculate two RD tubes as LR! and LR2 as explained in above step and negative tube is inoculated to same cell line - L20B and again observed for the appearance of CPE for five days.

·       If blind passage tubes of L20B are positive (at least 3+ CPE), then 0.2 ml of frozen and harvested material is inoculated from this material to fully grown two RD tubes as LLR1 and LLR2.

• When at least 3+ CPE appears -freeze the tube pool the harvested material and prepare two vials with lable having sample no. and cell line (LL+R+) send one vial for ITD one vial preserve for storage.

• If two RD tubes of primary inoculation are positive (at least 3+ CPE) then pool the harvested material of RD and inoculate 0.2 ml into L20B tube as RL

• RL tube is observed for 5 days for appearance of CPE.
• If no CPE appears after 5 days and L20B (primary and blind passage) is negative, the specimen is reported as NPEV.
• When 4+ CPE appears in RL tube, freeze the tube and pass 0.2 ml into fully grown RD tube as RLR. Observe this tube for appearance of CPE.
• If CPE appears – freeze and thaw the tube and prepare vials. If L20B is negative then send RLR vial for ITD. If L+R+ is positive and already sent for ITD then preserve RLR vial.

·       If two tubes of primary RD are negative, blind pass is done in RD from tube to tube and observed for appearance of CPE for five days.

• If CPE appears then tubes are frozen and harvested material is pooled.
• 0.2 ml is inoculated from this material to L20B tubes as RRL.
• If no CPE appears after five days and L20B (primary and blind passage) is negative, the specimen is reported as NPEV.
• If CPE appears then tube is frozen and harvested material is passaged in fully-grown RD tubes as RRLR.

• When at least 3+ CPE appears --freeze the tube, prepare vial and if L+R+ or LL+R+ is already sent for ITD then preserve vial for storage. If there is no CPE in L20B then send RR+L+R+ for ITD.

• Discard the pipette into the discarding tray containing freshly prepared 1% Hypochlorite solutions.

• After completion of discarding pan is autoclaved at 121c for 30 minutes before disposal in biomedical waste.

·       Possible outcomes and reporting of virus isolation results

1. Negative

L20B Positive Isolates (Vaccine and Wild) are retained in laboratory for three months and discarded after autoclaving. NPEV isolates are retained in laboratory for one year. Discarding is documented in virus inventory register

L20B POSITIVE VIALS (L+R+, LL+R+, R+L+R+, RR+L+R+) ARE SENT FOR ITD.

     

• If both the arms are positive then only LR positive tubes are sent for ITD.
• If only L20 B arm is positive then LR positive tube is sent for ITD.
• If only RD arm is positive then RLR or RRLR positive tubes are sent for ITD.

Vials of L20B positive isolates (LR/LLR/RLR/RRLR) that are sent for ITD are transported in a mini cooler.

1.    Safety conditions

• The utmost care should be taken to avoid cross contamination of cultures during primary inoculation and blind passage procedures.

o   Medium should never be decanted from inoculated tubes. It should be transferred with pipette.

o   Care should be taken to avoid aerosol creation during vigorous pipetting and spilled droplets must be immediately cleaned with 1% Sod. Hypochlorite solution.

2.    Documentation

• Virus isolation worksheet PR-13
• Map of rack for Blind passage. PF-1
• Log Book of Biosafety Cabinet  No. 2 PR-80
• NPEV, Vaccine, Wild Virus vial storage register PR-19

3.    References used to draw SOP

·       WHO Polio Laboratory Manual.