4.3. Sterile environment

  •  Clean incubator and desiccators
  •  Sterilize equipment and water for diet preparation
  •  Sterilize rearing equipment (cups)
  •  Put plates with cups under UV

When rearing honey bee larvae in the laboratory, strict sanitation is required. A sterile environment with sterile materials and chemicals is crucial; otherwise infections from bacteria or fungi will impede experiments. We recommend laboratories to strive to achieve optimum levels of sterility using their preferred method. All equipment, such as glassware, tools, consumables and water should be autoclaved. Work areas and material for control and exposure / infection treatments should be separated. Autoclaving sugar solution should be avoided because of heat-derived formation of hydroxymethylfurfural (HMF). Two possibilities for the treatment of sugar-yeast-water solutions are recommended: first, to filter with 0.22µm pore membrane filters, and second, to use autoclaved water for the preparation.

Mask and hand disinfection are important to reduce infections. It is crucial to use incubators only for larval rearing, as any other usage needlessly increases the risk of contamination. Disinfect incubators between experiments using ethanol (>70 %) or heat (e.g. more than one hour at more than 100°C) but check operators manual of equipment. As a regular test of sterility, or for troubleshooting, we recommend incubating open agar petri dishes and analysing them to detect any possible sources of contamination. Tools such as paintbrushes (for grafting) must be washed in ethanol and rinsed in autoclaved water before and regularly throughout usage. Dental rolls can be inserted in wells of rearing plates and 500µl of 15.5 % glycerol solution added (see 5.8. Incubation conditions). Wet dental rolls and plastic cups can be put under UV-light for sterilization for at least one hour. Methyl benzethonium chloride (MBC) was proposed for the disinfection of plastic cups and also on wetted filter paper or dental roll to prevent microbial growth during incubation (Vandenberg and Shimanuki, 1987; Aupinel et al., 2005). Due to its high price, MBC is no longer in use. In early protocols, methyl-4-hydroxybenzoate was used as a fungicide in the diet (Rembold et al., 1974; Rembold and Lackner, 1981) or Nyastin was added to royal jelly by Herrmann et al. (2008). Plastic queen cups can be disinfected in a chlorine solution such as Milton® Sterilising Tablets (Brodschneider et al., 2009). To reduce bacterial infections, Penicillin G (e.g. 375 ppm) can be added to the diet on day one, most of which will degrade within a few days (Riessberger-Gallé et al., 2011).