Lab Safety

1. Laboratory Overview

The wet lab members of our team conduct experiments in the Department of Biotechnology's Project Lab, which is designated as Biosafety Level 2 (BSL-2). Our laboratory adheres to the biosafety and biosecurity guidelines outlined by the Indian government and the Centre of Bioethics at Manipal Academy of Higher Education (MAHE).

2. Training and Risk Assessment

All members of the wet lab subsystem have received extensive training in safe laboratory practices from our Principal Investigator (PI), Dr. Ritu Raval, and our advisors prior to commencing work in the lab. This training includes:

• Risk Assessment: An assessment of the reagents in use.
• Waste Management: Proper identification, management, and safe disposal of biological and chemical hazardous waste and lab consumables.
• Equipment Familiarization: Training on the functioning and safe usage of all available equipment, including centrifuges, UV-visible spectrophotometers, and autoclaves.

3. Safety Equipment and Emergency Protocols

Lab members have been made familiar with:

• Emergency Equipment: The locations of safety showers, eyewash stations, first aid kits, fire extinguishers, and emergency evacuation routes.
• Biosafety Cabinet Training: Training sessions conducted by advisors and PhD scholars Mr. Rajesh M. Gowda and Mrs. Atheena PV on working within the biosafety cabinet to avoid contamination.

Standard Operating Procedures (SOP's)

Our team drafted this document that outlines the Standard Operating Procedures (SOPs) for all our laboratory instruments to ensure safety, consistency, and compliance in all activities. These guidelines provide clear, step-by-step protocols for conducting tasks efficiently while minimizing risks and errors. By adhering to these SOPs, we aim to maintain a safe, structured, and high-quality research environment that upholds the highest standards of scientific practice.

4. Safety Practices in the Laboratory

The following safety practices are strictly enforced within the laboratory:

• Prohibitions: Eating and drinking are strictly prohibited in the lab.
• Personal Protective Equipment (PPE): All members are required to wear lab coats, protective goggles, and nitrile gloves as necessary while working.
• Sterilization: Proper sterilization protocols are followed before working in the BSL-2 biosafety cabinet.
• Equipment Maintenance: All laboratory equipment, including centrifuges, spectrophotometers, cold storage units, and autoclaves, are handled with care and regularly maintained.
• Fumigation: The laboratory is fumigated every 3-4 weeks for additional safety.

5. Use of Harmful Reagents and Procedures

While some experimental procedures involve harmful chemicals and reagents, all necessary safety measures are taken to prevent any harm. Below are the details of specific hazardous chemicals and their implications:

a. Human Health and Safety Hazards:

• Highly Flammable Chemicals:
  • Ethyl Alcohol: Used as a solvent and for sterilization; poses a fire hazard during storage and handling.
  • Isopropanol: Highly flammable and used for sanitation; must be stored away from fire sources.
• Acids and Corrosive Chemicals:
  • Hydrochloric Acid: Strong acid that can cause severe burns; risk of skin, eye, or respiratory irritation during buffer preparation or pH adjustments.
• Other Toxic Chemicals:
  • Iturin A: Maximum of 5 mg used; negligible toxicity at low concentrations.
  • Acetonitrile: Extremely toxic; used as a solvent for HPLC. Inhalation can cause severe symptoms, while ingestion is highly unlikely.

b. Environmental Hazards

• Hemileia vastatrix: Pathogenic spore-forming fungus; potential risk of spores dispersing outside the lab.
• Candida albicans: Can contaminate surfaces and enhance resistance to antifungal agents.

6. Antifungal Safety Assessment

Iturin A is not a hazardous substance or mixture according to Regulation (EC) No 1272/2008 (Sigma Aldrich) [1]. A preclinical safety assessment of the consumption of Iturin A conducted acute (7 days, 5000mg/kg BW) and subacute (28 days, 200mg/kg BW) toxicity tests through daily intragastric administrations of the stipulated amounts. The results showed no significant damage to the small intestine, liver or kidney [2]. In a second pre-clinical study, the injection of 150 mg /kg BW of Iturin A into the bloodstream of the test mice caused lethality in all of them [3]. Doses of up to 50mg/kg BW caused mild anemia and damage to only the liver and spleen, with the effects being completely reversible when exposure was discontinued in the test mice, reflecting the dose dependent hemolytic effects of iturin that were previously reported [3]. Another study compared the effect of Iturin A in comparison to amphotericin B in treating 24-hour old Candida albicans infections via intraperitoneal injections. Treatment with 15mg/kg BW of Iturin per day led to the survival of all infected mice throughout the six-day duration of the study, as opposed to amphotericin B which killed all mice that it was administered to [4]. Initial clinical trials on animals and humans showed that Iturin A was effective as an antifungal, with low allergic effects and no discernable toxic effects from topical applications [5][6]. In the case of any overdose on Iturin A, potential treatments can be provided before permanent damage occurs through adding magnesium ions to or raising cholesterol levels in the bloodstream to inhibit Iturin A's activity [7][8].

Iturin does not affect D. melanogaster (common fruit fly) [9], honeybees [10], and other organisms commonly found in the coffee microbiota, and it causes no harm to coffee plantations. It is larvicidal to Aedes aegypti mosquito larvae [11].

7. Regulatory Compliance

Our project complies with the following regulations:

• The Environment (Protection) Act, 1986: Provides guidelines for disposal of synthetic biology-related products and handling of hazardous substances.
• Rules for the Manufacture, Use/Import/Export, and Storage of Hazardous Micro-Organisms/Genetically Engineered Organisms or Cells (Rules 1989): Oversee safe practices in experimentation and waste disposal.

8. Laboratory Facilities and Safety Equipment

We have access to the following facilities:

• BSL-1 Laminar Air Flow Hood
• BSL-2 Biosafety Cabinet
• Chemical Fume Hood

Emergency installations include eyewash stations, full-body showers, and fire extinguishers to minimize risks to lab members.

9. Waste Disposal and Inactivation

Waste management protocols include:

• Waste Segregation: Different colored bins for general waste, plastic/gloves, tissue paper, and glass syringes/needles.
• Liquid Chemical Disposal: Reserved containers for liquid chemicals.
• Decontamination Procedures: Used media and glassware are carefully packaged and autoclaved. Lab technicians ensure proper disposal and cleaning before reusing glassware.

References

1. Iturin | Sigma-Aldrich. (2024). Accessed: September 8, 2024. Retrieved from https://www.sigmaaldrich.com/IN/en/search/iturin?focus=products&page=1&perpage=30&sort=relevance&term=iturin&type=product
2. Zhao, H., Xu, Y., Zha, J., & Chen, W. (2018). Potential of iturins as functional agents: safe, probiotic, and cytotoxic to cancer cells. Food Function, 9(11), 5580–5587. doi: 10.1039/C8FO01523F
3. Dey, G., Ramkumar, P., Subramanian, S., & Ghosh, R. (2016). Pre-clinical risk assessment and therapeutic potential of antitumor lipopeptide ‘Iturin A’ in an in vivo and in vitro model. RSC Advances, 6(75), 71612–71623. doi: 10.1039/C6RA13476A
4. Lei, S., Liu, Y., & Wang, C. (2019). Capability of iturin from Bacillus subtilis to inhibit Candida albicans in vitro and in vivo. Applied Microbiology and Biotechnology, 103(11), 4377–4392. doi: 10.1007/S00253-019-09805-Z
5. Clairbois, J. P., & Delcambe, L. (1958). Clinical and biological trials on iturin, a new antifungal drug. Archives Belges de Dermatologie et de Syphiligraphie, 14(1), 63–82.
6. Blocquiaux, S., & Delcambe, L. (1956). Treatment of dermatomycoses with iturin. Archives Belges de Dermatologie et de Syphiligraphie, 12(2), 224–227.
7. Besson, F., Peypoux, F., Michel, G., & Delcambe, L. (1978). Mode of action of iturin A, an antibiotic isolated from Bacillus subtilis, on Micrococcus luteus. Biochemical and Biophysical Research Communications, 81(2), 297–304. doi: 10.1016/0006-291X(78)91532-2
8. Quentin, M. J., Besson, F., Peypoux, F., & Michel, G. (1982). Action of peptidolipidic antibiotics of the iturin group on erythrocytes. Biochimica et Biophysica Acta (BBA) - Biomembranes, 684(2), 207–211. doi: 10.1016/0005-2736(82)90007-4
9. Assié, L., Ricci, P., & Boulanger, E. (2002). Insecticide activity of surfactins and iturins from a biopesticide Bacillus subtilis Cohn (S499 strain). Mededelingen van de Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, 67(3), 647–655. Accessed: October 1, 2024. Retrieved from https://orbi.uliege.be/handle/2268/4444
10. Hertlein, M. B., Haff, R. F., & O’Neill, S. L. (2016). Biological role of paenilarvins, iturin-like lipopeptide secondary metabolites produced by the honeybee pathogen Paenibacillus larvae. PLoS One, 11(10), e0164656. doi: 10.1371/JOURNAL.PONE.0164656
11. Revathi, K., Chandrasekaran, R., Thanigaivel, A., Kirubakaran, S. A., Sathish-Narayanan, S., & Senthil-Nathan, S. (2013). Effects of Bacillus subtilis metabolites on larval Aedes aegypti L. Pesticide Biochemistry and Physiology, 107(3), 369–376. doi: 10.1016/J.PESTBP.2013.10.005