Virus Isolation

Protocol for inoculating rubella specimens onto Vero cells

1. Preferred specimens are throat or nasal swabs in 3 - 4 mls virus transport medium (VTM - see below for recipes) or phosphate buffered saline (PBS). Agitate swabs for 2 minutes in VTM, squeeze liquid out against side of tube. Discard swab. (Swabs can also be broken off and left in tube.) VTM can be stored frozen or inoculated immediately. Urine can also be used - dilute 1:1 with PBS.
2. Draw 0.5 mls of sample into 1 cc syringe. Attach 0.2um syringe filter (such as a Acrodisc 13) and pass sample through filter directly onto cells. This is the preferred method because it prevents contamination better than antibiotics and takes less time. The drawback is that some samples will not pass through the filter.
   Alternate method of inoculation:
   If sample will not go through the filter, to 1 ml of virus transport medium (after removal of swab) add 0.1 ml of a solution containing 1000 ug/ml of gentamicin, 0.1 ml of a solution containing 100 ug/ml of fungizone, and 5 ul of a 1000x solution of Penn/Strep. Incubate at 4^oC for 30 minutes. Add 0.5 mls to 35 mm plate of Vero cells.
3. Inoculate the sample directly onto a 35 mm tissue culture plate or well of six well plate containing confluent Vero cells (can wash cells with PBS before adding virus if desired). Incubate at 35^oC for 1 hour. Remove inoculum and replace with 2 mls of Dulbecco's Minimal Essential Media (DMEM), supplemented with 5% fetal calf serum (FBS). Return plates to 35^oC incubator.
4. At 7 days post-inoculation, transfer 0.5 ml of culture medium to new plate of Vero cells, adsorb for 1 hour and replace with DMEM, as above. Repeat again after 1 week, if tests for rubella virus are negative.

Viral Transport Mediums

1. From Schmidt, Nathalie and Emmons, Richard (Eds), Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections
   American Public Health Association, Washington, D.C., 1989. Pg. 10.

   HEPES acid 0.953 g

   HEPES Na salt 1.562 g

   Tryptose broth 26.0 g

   Gelatin 5.0 g

   Phenol red 0.02 g

   Distilled water to 1000 ml

2. The chemicals in the order listed are dissolved in 700 ml of distilled water and the remainder of the water is added to a volume of 1 L. The medium is dispensed in 3.5 ml volumes in screw-capped vials and autoclaved at 15 lbs pressure for 15 min. The pH should be approximately 7.2 after autoclaving. The mediumis stored at room temperature.

3. From Leland, Diane, Clinical Virology. WB Saunders Co. Philadelphia, 1996. Pg.190.

   Hanks BSS 500 ml

   FBS 10 ml

   gentamicin sulfate 1 ml (of 50 mg/ml stock)

   fungizone 1 ml (of 250 ug/ml stock) Amphotericin B

4. To bottle of Hanks BSS add FBS and antibiotics and mix. Label the bottle:
   2%FBS, 100 ug/ml genamicin, 0.5 ug/ml fungizone.
   Expiration date is one year from date prepared.

   Aliquot 3 ml of medium into sterile screw-capped tube. Close tightly and store at 4^oC. Do not freeze.

Rubella Laboratory, CDC January, 2002

Use of trade names and commercial sources in this manual does not imply endorsement by the Centers for Disease Control and Prevention.

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