TUBE CULTURE (not in routine use) or SHELL VIALS for CPE PROCEDURE (Appendix III)

I. Introduction

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.

http://bahankuliahkesehatan.blogspot.com/

 

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 (4^oC)

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 36^o 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 36^oC. 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 36^oC.

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 36^oC 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 1^st or 2^nd reading - change the maintenance medium, and reincubate.

b. On 3^rd 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 36^o C in O₂ in the clean room (C)

and one is placed on the roller drum (V) at 36^o 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.

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/tube-culture-not-in-routine-use-or.html

Read More

Appendix XIII PRESERVATION OF CELL CULTURE MONOLAYERS & Appendix XIV QUALITY CONTROL OF CELL CULTURES USED FOR ROUTINE VIRUS ISOLATES

1. Aspirate the medium from the cell culture tubes to be preserved.

2. Add 8 mL buffered formaldehyde preservative medium to each tube.

3. Record the following information on the tube:

- virus

- number of days incubation

- lab number

4. Store preserved culture at room temperature.

Formaldehyde Preservative

100 mL Formaldehyde Solution (37 - 40%)

900 mL Distilled Water

20 mL Phenol Red (0.5%)

4.0 g NaH₂PO₄ - H₂O

6.5 g Na₂HPO₄

Materials

Vortex

Sterile pipettes

10 - 100 uL Eppendorf pipette

Humidified chamber

Coplin jars

Fluorescent microscope

 

Appendix XIV

QUALITY CONTROL OF CELL CULTURES USED FOR ROUTINE VIRUS ISOLATES

Tube Culture (Not in routine use)

Upon receipt of cell culture tubes, the record of the date received, vendor, lot number, and passage number is kept in the QC binder for cell lines. The monolayer is checked microscopically for sterility and appearance of an acceptable confluent monolayer.

 

A. Uninoculated Negative Controls:

Reserve 4 tubes of each lot cells for use as controls and label as follows:

N1, date;

N2, date;

N3 date;

C, date;

V, date

 

a. Negative Controls with refeed: (3 tubes, N1; N2; N3)

Select one tube each on Wednesday Friday and Monday to set up along with inoculated specimens. Incubate, observe and refeed these tubes in parallel with patient inoculated cultures to monitor monolayer quality, toxicity and sterility. They can also be used to provide a baseline for comparison of inoculated cultures when reading for CPE and immunostaining. CMK and HFF tubes are kept for 3 weeks, HEp-2 and RD for 2 weeks

b. Unopened Negative Controls without refeed: (2 tubes, C; V)

These tubes are left unopened and observed to identify toxicity and contamination originating with the vendor. All tubes, CMK, HEp-2 , HFF and RD tubes are kept for only one week. One tube (C) is kept in the clean room and one (V) is placed on the Virology drum.

 

Record sterility and cell appearance for these tubes into the LIS Manual (REGISTRATION OF TUBE CULTURE MEDIA)

B. Positive Controls:

Each week scrape from an HFF tubes containing the following QC strains of HSV-1 HSV-2 and CMV to propagate the QC strains in the new lot of HFF tubes, Use CMK tubes for RSV and nfluenza A. Examine microscopically for CPE and record the results into the LIS as growth control. At the same time also inoculate MRC-5 and R-Mix shell vials to perform quality control for MRC-5 shell vials (see II. Shell Vial Cell Lines (MRC-5 cell suspension below)

HSV-1 (ATCC VR-5539)

HSV-2 (ATCC VR-540)

CMV (ATCC VR-807)

RSV (ATCC VR-284)

Influenza A (ATCC VR-544)

The MRC-5 shell vials are then stained with HSV-1, HSV-2 and CMV IEA monoclonal antibodies at 24 hours, R-Mix shell vials with RS. Record the results into the LIS as shell vial growth control as well as stain controls.

Each month (and whenever necessary), remove from the liquid nitrogen storage, cryovials of above strains to propagate the QC strains in HFF tubes.

Additional positive controls may be set up with the following strains and for the following reasons:

Coxsackie B1 (ATCC VR-28)

Parainflunza 3 (ATCC )

Varicella-zoster (ATCC VR-1367)

i) Low isolation rates

ii) Comparison of cell lines

iii) Vendor changes

iv) Proficiency test failures

v) Training purposes

vi) Consistent problems with negative controls

vii) Preparation of QC material (i.e. Positive control slides)

Consult a senior technologist to determine the cell lines and viruses to be set up.

II. Shell Vial Cell Line (Suspension seeding is not in routine use)

Upon receipt of a shipment of cells, initial and date the record sheet accompanying the shipment. The record should contain vendor, lot number, passage number and QC data. File in the QC binder for cell lines.

Before seeding the shell vials, aspirate about 30 mL of MRC-5 cell suspension into a 125 cm² tissue culture flask, label on the side of flask with “MRC-5, date and ‘Pre’”.

After seeding shell vials, aspirate about 30 mL of MRC-5 cell suspension into another 125 cm² tissue culture flask, label on the side of flask with “MRC-5, date and ‘Post’”.

Reserve 6 shell vials for use as negative and positive controls as follows:

 

A. Negative Controls for this week, also become Positive Old Lot for following week (3 Vials)

These are incubated at 36^oC and observed daily for one week or more to identify toxicity and contamination originating with the vendor. Results are recorded on the QC chart.

Three shell vials are reserved to QC the next shipment in parallel after the cells are added.

 

B. Positive Controls (3 Vials)

Each week, usually 2-3 days after seeding the shell vials, HFF tubes containing HSV-1 (ATCC VR-5539), HSV-2 (ATCC VR-540) and CMV (ATCC VR-807) are scraped from and used to inoculate 6 MRC-5 shell vials, 3 from the current lot and 3 from the previous lot (the same 3 vials were used as Negative Controls for a week). The shell vials are then stained with HSV-1, HSV-2 and CMV IEA monoclonal antibodies after 1 day incubation. Record the results into the LIS daily QC under the following codes:

a. VHSV1D HSV1 daily SLIDE SV QC

b. VHSV1D HSV1 daily SLIDE SV QC

c. VCMV-D CMV-IE daily SLIDE SV QC

d. VQCSV Shell Vial MRC-5 Quality Control

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/appendix-xiii-preservation-of-cell.html

Read More