Tube culture is the conventional method used by diagnostic virology laboratories for virus isolation. Since there is no universal cell line for recovery of all clinically significant viruses, a combination of cell types is used routinely depending on the symptoms, clinical specimen type and specific viruses being sought. Shell Vials (E-Mix ) can also be adapted to extend their normal incubation times and continue as tube cultures.
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II. Reagents and Materials
Fluorescence microscope Leica DBRB with #2 filter for Rodamine/FITC Evans blue and #4 filter for FITC Evans blue or
Fluorescence microscope Leica DC300F with #3 filter for Rodamine/FITC Evans blue and #1 filter for FITC Evans blue
Inverted microscope
Control slides
Virus-specific antibody (eg. Enterovirus D3 stain, DHI)
FITC-conjugated antimouse antibody
Phosphate buffered saline (PBS)
Distilled water
Cold acetone (4oC)
Mounting fluid
Sterile pipettes
Cytospin and accessories
Vortex
Sterile freezer vial
Glass slides
Coverslips
Paper towels for blotting
Humidified chamber
III Procedure
1. Registration
i) Upon receipt in the lab, register cell culture received from the supplier in the lab information system (LIS). Refer to virology LIS manual for procedure.
ii) File the vendor QC sheet (received with the shipment) in the QC binder.
iii) Randomly, select two tubes from each lot and check the monolayer microscopically for confluent growth and quality of cells. Use these two tubes as the “unopened controls” outlined under the Quality Control section below.
iv) MRC-5, R-Mix and E-MIx cell lines are stored for 18-24 hours at 36o C in O2 (or until cell lines reach >50% confluency) and then are kept at room temperature until expiry.
2. Inoculation of cell culture shell vials
i) Aliquot 50 mL maintenance medium and allow to come to room temperature before using.
ii) Refer to the protocol for each specimen type to determine the number of tubes and types of cell lines to be inoculated. Also refer to Appendix XV if needed.
iii) Prior to inoculation, check the cell culture tubes for acceptable confluent monolayer formation and sterility.
iv) Decant the medium from the tube.
v) Using a clean, sterile pipette for each tube, add 1.5 mL of the aliquotted maintenance medium to each tube and re-cap. After set up is complete, discard any remaining maintenance medium.
vi) Inoculate 0.2 mL (4 drops) of processed specimen into each tube, recapping immediately afterward.
vii) Incubate the tubes in the roller drum at 36oC. Refer to the appropriate specimen protocol for the incubation time for each tube.
viii) Refeed MRC-5, R-Mix and E-Mix minimally once per 5 days. Shell vials showing signs of chemical toxicity (red media / sloughing cells), bacterial / fungal contamination (yellow / turbid media) or aging should be refed within the day.
III. Reading of Cultures (Shell Vials for CPE)
i) Cytopathic effect (CPE): E-Mix or other cell lines should be examined daily for CPE. Any culture demonstrating > 2+ CPE should be confirmed by staining. The cells should be scraped, a cytospin slide prepared and appropriate monoclonal antibody staining performed. If no CPE is present, refeed with corresponding maintenance medium and Reincubate.
ii) Enteroviruses (D3 enterovirus, DHI): Perform enterovirus stain when CPE is observed:
a. Prepare cytospin preparation from cell culture as outlined below:
b. Remove about 0.5 mL (leaving about 1 mL) maintenance media from the culture using a sterile pipette.
c. Scrape cells using a sterile pipette. Break up cell clumps by pipetting up and down several times.
d. Pipette 4 x 200 uL (4 x 4 drops) of scraped cells into 4 funnels on 2 double slides.
e. Cytospin at 2000 rpm (700 x g) for 5 minutes.
f. Remove slide and air dry.
g. Fix in cold acetone for 10 minutes in a coplin jar. Remove slide and air dry.
h. An enterovirus QC control slide should be stained in parallel with the patient as follows.
i. Stain the 4 wells by adding 20 mL each of Enterovirus D3, ECHO, Coxsackie B and Polio stains onto the fixed cell spots.
j. Incubate in a humidified chamber for 30 minutes at 36oC.
k. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.
l. Wipe excess PBS from the slide without touching the cell spot.
m. Add 20 mL of FITC-conjugated antibodies.
n. Incubate in a humidified chamber at 36oC for 30 minutes.
o. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.
p. Wash with distilled water for 1 minute in a coplin jar.
q. Wipe excess water from the slide without touching the cell spot.
r. Mount using coverslip and mounting fluid.
s. Read with fluorescence microscope Leica DC300F with #3 filter for Rodamine/FITC Evans blue and the 40x objective (warning:#1 filter is for FITC Evans blue only).
Interpretation of Results
Positive for enterovirus: Green fluorescence
Negative: Dull-red counterstained cells with no apple-green fluorescence.
Invalid: If no counterstain is visible, repeat staining
QC slide failed, report to senior/charge
If positive, record in freezer program and freeze cells and supernate. Refer to Appendix X and XII for procedure.
iii) Confirmation by PHL: Any culture demonstrating CPE for which a virus cannot be detected using monoclonal antibodies or other in-house methods and for which toxicity has been ruled out (see below) should be referred to the Public Health Laboratory (PHL) for further work-up. Pass cells to a new culture before sending. Scrape and add 0.2 ml (4 drops) of scraped cells to a fresh culture containing 2 mL of fresh maintenance media (1:10 dilution). Consult the charge/senior technologist or medical microbiologist before referring the specimen to PHL.
iv) Culture Toxicity: If chemical toxicity is suspected in a culture (rounding of cells, sloughing of cells, granular cytoplasm of cells or unusual CPE, consult senior/charge technologist if unsure), proceed as follows:
v) Pass cells by scraping and adding 0.2 ml (4 drops) of these scraped cells to a fresh culture containing 2 mL of fresh maintenance media (1:10 dilution). Proceed with tube culture method as outlined above.
vi) The effects of chemical toxicity would be reduced by dilution whereas the effects of CPE (caused by viral replication) would be the same, if not accelerated on passage. If CPE is suspected, identify virus by antibody stains. If chemical toxicity is suspected, continue to incubate (may need further refeeding to reduce toxicity). If unsure of cell toxicity or CPE , refer to the charge/senior technologist for review.
vii) Contaminated Culture: If the culture appears visibly contaminated (eg. cloudy and/or yellow medium) and thus uninterpretable, proceed as follows:
a. On 1st or 2nd reading - change the maintenance medium, and reincubate.
b. On 3rd or later reading or recurrence - issue a final report stating:
“Virology culture: Specimen is heavily contaminated with bacteria and/or fungus. Unable to interpret virology culture.”
c. Replant if specimen is from a sterile site or contamination is attributed to the lab. If multiple specimens are contaminated, report to senior/charge.
IV. Quality Control (Tube Cultures are not in routine use)
Record all results of QC in LIS (or log). Refer to virology LIS manual for procedure. Report any abnormal results to charge/senior technologist.
Five tubes are reserved from each lot of cell cultures received, and used as controls as follows:
i) Negative controls: (tubes labelled N1, N2, N3)
On Wednesday, Friday and Monday, an uninoculated tube from each cell line used that day is placed in the roller drum with the inoculated specimens. These tubes are incubated, read and refed with the patient inoculated cultures to monitor the monolayer quality, medium toxicity/contamination. They can also be used
to provide a baseline for comparison for inoculated cultures when reading for CPE. HFF, CMK, HEp-2 and RD tubes are kept for 5, 2, 2 and 1 weeks respectively.
ii) Unopened Controls: (2 tubes labelled C and V respectively)
These tubes are not opened. One tube is kept at 36o C in O2 in the clean room (C)
and one is placed on the roller drum (V) at 36o C in O2. These tubes are observed
for 1 week to identify toxicity and contamination originating with the vendor.
iii) Positive Controls:
Each week HSV-1 ATCC strain # VR- 539 is scraped from the previous week’s positive control tube and used to inoculate a fresh HFF tube. If the control fails to propagate, a new vial can be retrieved from liquid N2 tank MINS shelf 6.
Additional positive controls may be set up for the following reasons:
- · Low isolation rates
- · Comparison of cell lines
- · Vendor changes
- · Proficiency test failures
- · Training
- · Continuing problems with negative controls
- · Preparation of QC material (i.e. positive control slides)
- Consult a charge/senior technologist to determine the cell lines and viruses to be set up.
V. Reference
1) Isenberg, H.D. 1992. Clinical Microbiology Procedures Handbook. Vol. 2. ASM
Press.
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