Laboratory Safety
General Remarks
We conducted our wet lab experiments in research laboratories in the Hugo R. Kruytgebouw, Victor J. Koningsbergergebouw and Androclusgebouw buildings, located in the Utrecht University Science Park. There, we only worked in laboratories designated for Biosafety Level 1 (BSL-1/ML-1) work, classified in accordance with the European Union's biosafety directive .
Under the assistance of our supervisors and biosafety officers, we made sure that all our work complied with the Dutch and European biosafety regulations. More specifically, we made sure to adhere to the general biosafety regulations described by the Bureau of Biosecurity (Rijksinstituut voor Volksgezondheid en Milieu) in the Netherlands . This includes both the wet lab work (i.e. plasmid design, bacteria work, zebrafish handling etc.) and the management of the waste resulting from our experiments. Therefore, all team members who did wet lab work received mandatory training on the laboratory safety regulations. This way, we were aware of the responsible lab conduct which calls for adequate clothing and behaviour (i.e. preferably long sleeves, closed-toe shoes, tied up long hair, interdiction of bringing food/drinks, etc.)
Sterile Work
In order to ensure proper sterile handling of the engineered bacteria/zebrafish larvae utilized in our project, we received training on working with GMO and handling potential incidents. This way, we were properly prepared to avoid the possible scenario of GMO leakage into the environment.
On a practical level, we were equipped with lab coats, gloves, sterilized disposables (i.e. flasks, pipette tips, etc.), Bunsen burners and biosafety cabinets. We worked carefully and made sure to disinfect the working surfaces with laboratory grade 70% ethanol. This limited and prevented the occurrence of contaminations, while ensuring that we worked in a clean, safe and controlled environment.
Figure 1: Working in the biosafety cabinet
Figure 2: Sterile handling of bacteria
Specialized Equipment
Throughout our wet lab work, we have utilized an extensive range of specialized lab equipment instruments: biosafety cabinets, HPLC machines and mass spectrometers, to name a few. The team members who worked with these instruments received training from professionals who operate the machines on a regular basis. Moreover, the usage of the HPLC/MS machines was directly supervised by these professionals to ensure a smooth, safe and reliable run of the experiments.
Zebrafish Facility
Zebrafish represented the core model organism in GutFeeling. One of our supervisors, who is highly proficient in working with this organism, demonstrated how to work with zebrafish larvae. This involved breeding the fish, harvesting and bleaching the eggs, and incubating them towards obtaining germ-free zebrafish larvae and subsequently deriving gnotobiotic zebrafish.
Specialized zebrafish work (i.e. breeding the fish) requires documentation and must be conducted by trained experts, according to article 9 of the Dutch Wet op de Dierproeven (Experiments on Animals Act) . Therefore, these specific steps were done by our supervisor in a zebrafish facility which has the corresponding documentation for such experiments.
We conducted the steps following the harvesting of the zebrafish eggs, which do not require official documentation for animal experiments work. This was done under the direct guidance of our supervisor in order to ensure a responsible and safe handling of the zebrafish larvae.
Working with Chemicals
Our work involved the use of multiple chemicals, particularly in the context of producing L-DOPA and running HPLCs for quantification. In our labs, chemicals were properly stored and sorted based on their toxicity. More specifically, hazardous chemicals were stored separately from the non-hazardous ones, in closed containers.
In order to ensure safe handling of these compounds and proper risk mitigation, we made sure to read the safety data sheets (SDS) for every chemical we worked with. These data sheets were readily available online. We worked with only one hazardous (potentially carcinogenic) chemical, catechol (1,2-dihydroxybenzene) – substrate for enzymatic L-DOPA production. After consulting with our supervisors, we made sure to always work with it under a chemical flow cabinet and wear gloves/lab coats in the process. This limited the risks of handling catechol via skin contact and vapour inhalation.
When working with agarose gels to assess our DNA constructs by size, we also made the conscious choice of using the MIDORI Green DNA stain, as an alternative to the traditional ethidium bromide-based nucleic acid stains (mutagenic and carcinogenic). This chemical is much safer and represents a safety measure taken by our team, especially since we ran a large number of gels.
Waste Disposal
Our waste disposal system worked in compliance with the Dutch and European regulations. We collected the BSL-1/ML-1 waste (plastics, glassware and liquid) separately from the uncontaminated waste. The former was labelled properly and autoclaved before its final disposal, to ensure that no GMOs were carried out of the laboratory into the environment. The specialized personnel in our buildings were in charge of these last steps.
The disposal of the leftover zebrafish larvae was done before 5 dpf (the same timeframe as the one used in experiments), as they are still not considered animals at this timepoint, according to European regulations. This was done using a filter, in order to prevent the larvae (either dead or alive) from reaching the sewers/environment.
Chemical waste disposal was also done responsibly, according to the laboratory standards. We separated the compounds based on their pH and (an)organic character, adhering to a readily available guide of our lab.
Figure 3: Disposal chart for liquids
Figure 4: Disposal chart for solids