2.1. Biosafety measures

In some countries, microbial species are categorized in different classes based on biosafety risk. Each biosafety risk class has its own recommendations with respect to facility design, safety equipment, and working practices (de Graaf et al., 2008). This classification mostly takes into account the risk for human health, potential for dispersal of the disease, and the potential economic impact of the disease. However, a generally accepted biosafety risk classification has not been prescribed for P. larvae. Consideration of the severity of clinical American foulbrood infections in honey bee colonies, the contagiousness, the longevity of the spores, the legal context of AFB (a notifiable disease), and the economic value of honey bee pollination services, justifies P. larvae classification as an organism with ‘high biosafety risk for animals’. Table 1 summarizes basic biosafety practices - mainly in accordance with the Belgian model (http://www.biosafety.be/) - that should be considered when manipulating P. larvae for research or diagnosis. It should be noted that P. larvae has been safely cultured in the laboratory for decades using only standard bacteriological procedures (i.e. aseptic handling techniques and careful decontamination of biological waste), and to date, no known AFB outbreaks due to intentional/accidental laboratory release of this organism have been reported.

Table 1. Biosafety rules mainly in accordance to the Belgian model; http://www.biosafety.be/.  As this model is very restrictive, we have indicated the rules that are minimally (M) required. The other biosafety rules should be considered optional depending on the demands of the responsible authority.



Facility design


- The laboratories are physically separated from other work areas in the building.
- The access to the laboratories are locked if the zone or corridor access is not reserved. The doors have automatic closing if they open directly into a public area.
- (M) The furniture has been designed to allow easy cleaning and disinfection, and easy insect and rodent control.
- (M) There is a sink for washing and decontamination of hands in the laboratory.
- (M) There are coat hooks or a dressing room equipped with protective clothing. Normal clothing and protective clothing should remain separated.
- (M) The tables are easy to clean, water impermeable and resistant to acids, alkalis, organic solvents, disinfectants and decontamination agents.

Safety equipment

- If the laboratory is equipped with a microbiological safety cabinet (MSC) of class II, it must be localized as such that it does not disturb the air flow in the room. It should be kept at a sufficient distance from windows, doors, and places with frequent passage, vents for air intake or outlet. The MSC should be checked and certified upon purchase or relocation, as well as at least once a year afterward.
- There is an autoclave available in the building if the biological waste and/or biological residues shall be inactivated by steam sterilization.

- The centrifuge that will be used is available in the containment zone.  If this is not the case, and centrifugation is done outside the containment zone, the tubes and the rotors should be free of leaks.

Working practices


- Access to laboratories is restricted to persons approved by the responsible authority, and these persons have been informed of the biological risk.

- Laboratory doors should display: the biohazard sign, the containment level (if applicable), the coordinates of the controller.

- (M) Protective clothing is worn. This protective clothing should not be worn outside the laboratory.

- There are gloves available for staff.

- The windows must remain closed during experiments.

- (M) Viable (micro-) organisms must be physically contained in closed systems (tubes, boxes, etc.), when they are not being manipulated.

- (M) Splashes or aerosols should be minimized, and their spread must be controlled by appropriate equipment and work practices.

- In no case may a horizontal laminar flow cabinet used for manipulation of pathogenic organisms.

- (M) Mechanical pipetting is required.  Pipetting by mouth is prohibited.

- (M) Drinking, eating, smoking, use of cosmetics, handling contact lenses and storage of food for human consumption is prohibited in the laboratories.

- A register of all manipulated or stored pathogenic organisms should be kept.

- The control measures and equipment should be inspected regularly and in an appropriate manner.

- (M) Hands should be washed when leaving the laboratory, when another activity is started, or when deemed necessary.

- (M) After completion of the work, or when biological material has been spilled, the work surfaces should be disinfected.

- There is a note available for the staff describing the correct use of the disinfectants. This memo specifies for a given purpose, the disinfectant that must be used, the necessary concentration and contact time.

- The staff is trained in relation to biosafety issues and is regularly monitored and retrained.

- A biosafety manual has been written and adopted.  The staff is informed of the potential risks and must read the biosafety regulations that are applicable. Instructions that must be followed in case of accident should be posted in the laboratory.

- The biohazard sign is posted in incubators, freezers, and nitrogen tanks containing biological material ‘high biosafety risk for animals’.

- An effective insect and rodent control program is applied.

Waste management

- (M) Contaminated biological waste and/or biological residues and contaminated disposable equipment should be inactivated by an appropriate, validated method before it is discharged--e.g., by autoclaving or by incineration. The incineration is performed by an authorized company.  The waste is collected in secure and hermetically sealable containers. These should be closed for transport.

- (M) Contaminated material (glassware, etc.) is inactivated by an appropriate, validated method before cleaning, reuse and/or destruction.