To isolate the viral genomic material the bee samples, which were frozen at -20°C, had to be treated as described in the following section:

To ensure successful experiments all the required material like cups and mortars was treated with NaOH to destroy all remaining genomic material and enzymes from previous experiments. Afterwards we rinsed everything with DEPC-treated (di-ethyl-pyro-carbonate) water. To protect ourselves we used strict safety regulations like gloves and masks.

DEPC inactivates RNases, abundant enzymes, which would destroy the viral RNA and thus falsify our results. DEPC is a dangerous chemical that has to be incubated with water for 12 hours at 37°C. To inactivate DEPC itself the mixture is autoclaved and DEPC is degraded to harmless CO2 and ethanol.

Before autoclaving, which also serves for sterilization, all the material was wrapped in aluminium foil. To sterilize the tweezers they were soaked in ethanol and breamed.

For each bee sample that has to be analyzed we took 20 individual bees and collected them in a sterilized cup.

To breakup the bees, they were treated with liquid nitrogen. Within seconds the bees are totally frozen and they can easily be disrupted with a mortar into single cells.

Liquid nitrogen has a temperature of -196°C, which means that we had to work very carefully. Injuries due to contact with liquid nitrogen show similar symptoms like burns, with the difference that the affected body parts totally freeze off.

The bee homogenate was then dissolved in DEPC-treated water. For storage this solution was frozen at -20°C in sterile tubes until the molecular biological analysis.