In brief: This is a complex activity which involves a number of chemical hazards as well as microbiological hazards. We have not performed this activity, however we can make the following comments.
Risk Assessment: It is very wise of you to investigate this before proceeding with ordering chemicals and other resources. It is important to undertake a risk assessment of both the chemicals/substances used as well as the processes involved in an activity prior to ordering the chemicals and equipment. We have developed a Risk Assessment Template that you may find helpful.
Toluene (methylbenzene): This is a hazardous chemical, which we have not to date included in our List of recommended chemicals for science in Australian schools. However, we could consider it for inclusion where there is a curriculum requirement. Toluene is highly flammable, produces harmful vapours and is suspected of damaging fertility and the unborn child. There is also evidence of health effects due to long term exposure and it is toxic to aquatic organisms. It requires storage separate from incompatible substances in a flammable liquids cabinet. It should be handled in a fume cupboard and waste should not be allowed to go down the sink, but collected for disposal via a waste contractor. Controls to minimise the risk of exposure to this chemical include:
- using it in an operating fume cupboard;
- wearing Personal Protective Equipment (PPE) i.e. safety glasses, PVA gloves (such as viton or PVA. Note: if no PVA gloves are available, one can double glove with nitrile gloves, but remove the gloves if they become contaminated), laboratory coat or apron;
- using small quantities;
- restricting the use to Year 11 and 12 students and staff only.
β-galactosidase is an enzyme that catalyzes the breakdown of the substrate lactose (a disaccharide sugar) into two monosaccharide sugars: galactose and glucose. An initial search for this enzyme confirms your comment regarding the high cost of reagents(1).
Enzymes are biologically active proteins. All enzymes in powder form are considered hazardous, due to the potential inhalation of enzyme dust or aerosols, which can lead to sensitisation and allergic reactions. They are also irritating to the eyes, respiratory system and skin. Always use practices that do not generate dust or aerosols. However, in dilute aqueous solutions they are generally considered to be a low hazard. It is also important to understand that enzymes, when dissolved into solution, are much less stable than in powder form and lose their activity quickly. Therefore, it is best to prepare only what is required just before use, and recognise that the solution may need to be kept at a particular temperature during an activity. For more details see our previous question Enzyme preparation for experiments.
ONPG (2-Nitrophenyl β-D-galactopyranoside) is a colourimetric substrate for the detection of β galactosidase activity. Whilst not considered hazardous, it is still recommended not to generate any dust and wear appropriate PPE such as gloves, safety glasses and if required a dust mask.
The activity requires a high level of staff training in microbiology as well as extremely close supervision of students.
- The activity involves the subculturing of a Risk Group 1 microorganism (E. coli K–12) and would require laboratory facilities that comply with Physical Containment Level 1. Subculturing is a specialised technique requiring sound knowledge and expertise to minimise the risks involved. It is a skill developed with much practice. This procedure is not permitted in some jurisdictions so your school would need to find out and comply with your school jurisdiction or governing body regarding this type of activity. When working with microorganisms, it is best to treat them all as potential pathogens(2). The E. coli K–12 strain, which is a Risk Group 1 microorganism, is not considered pathogenic to healthy individuals, but may present a higher risk to people who may be immunosuppressed or immunocompromised.
- The type of media used in schools should not select for pathogens such as selective or enriched agars. Nutrient broth or agar are simple media which support the growth of a wide variety of bacteria and moulds and are suitable for use in school laboratories. In this activity, the addition of lactose to the growth medium provides an additional carbon source and induces the gene that produces β galactosidase in the E.coli.
- The recommended temperature for the incubation of microorganisms in schools is at room temperature or up to a maximum of 30° C to minimise the likelihood for growth of potential human pathogens that are adapted to human body temperature. The growth rate of E.coli will increase in response to an increase in temperature in the 25–37° C range, with 37° C being the optimum (3,4). Whilst incubating E.coli at 37° C may be acceptable where good aseptic technique is used, it is difficult to guarantee good aseptic technique in a school setting, therefore we recommend incubating at 30° C, which will produce growth at a slower rate and reduce the risk of encouraging the growth of human pathogens.
- The use of a pressure cooker or autoclave is required for the sterilisation of bacterial cultures before disposal.
Science ASSIST is in the process of developing guidelines for microbiology in Australian schools. The guidelines are due for release late 2016.
In the meantime, we recommend that before schools embark on working with microorganisms, they should ask the following questions and perform a site-specific biological risk assessment.
- What microorganism is being used? Is it a Risk Group 1 microorganism?
- Do the school facilities comply with the requirements of Physical Containment Level 1 laboratories?
- Does the school have the necessary equipment for sterilisation and decontamination procedures?
- Do the staff have training in microbiological skills?
- What manipulations are being performed with the microorganism? Are methods being used to eliminate or minimize exposure to potentially infectious material via aerosols, splashes, ingestion, absorption and accidental inoculation?
- Are any staff or students wishing to participate in microbiological activities immunocompromised or immunosuppressed (include those who are pregnant or may become pregnant, or are living with or caring for an immunocompromised individual)? These individuals are more prone to infections. If so, then they should consult a doctor to determine whether their participation is appropriate.
In addition, the answer to the following question also contains links to earlier questions and other resources:
The activity that you refer to cites another activity considering the enzyme β-galactosidase(5). Depending upon the desired learning outcome, this activity could be a suitable alternative, which does not require the use of toluene or the culturing of a microorganism. However, it does still involve the significant cost of purchasing the enzyme.
Alternatively, other enzyme activities using less expensive enzymes could be conducted.
(1) 'β-Galactosidase from Escherichia coli', Sigma-Aldrich website, http://www.sigmaaldrich.com/catalog/product/sigma/g5635?lang=en®ion=US (Accessed September 2016)
(2) University of Sydney. 2013. ‘Microbiology’, http://sydney.edu.au/whs/guidelines/biosafety/microbiol.shtml (Accessed September 2016)
(3) Farewell, A. and Neidhardt, F.C. 1998. Effect of Temperature on In Vivo Protein Synthetic Capacity in Escherichia coli, Journal of Bacteriology, 180. 4704-4710. https://jb.asm.org/content/180/17/4704.full.pdf+html
(4) 'B1 - Escherichia coli, K-12 strain, live broth' Southern Biological Website, https://www.southernbiological.com/specimens/microbiological-specimens/bacteria-risk-group-1-suitable-for-schools/b1-escherichia-coli-k-12-strain-live-broth/ (Accessed October 2016)
(5) 'Investigating the effect of competitive and non-competitive inhibitors on the enzyme B-galactosidase' Science and Plants for Schools website, http://www.saps.org.uk/attachments/article/95/SAPS%20-%20Inhibitors%20on%20enzyme%20beta-galactosidase%20-%20Scottish%20Highers.pdf (Accessed September 2016)
‘2-Nitrophenyl β-D-galactopyranoside’ Safety Data Sheet, Sigma-Aldrich website, http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=AU&lang... (June 2014)
'Aseptic techniques', Nuffield foundation website, http://www.nuffieldfoundation.org/practical-biology/aseptic-techniques (Accessed September 2016)
'Biology – Genetic control', BBC Higher Bitesize website, http://www.bbc.co.uk/bitesize/higher/biology/control_regulation/genetic_control/revision/1/ (Accessed September 2016)
'Gene induction: β-galactosidase in E. coli', Nuffield Foundation website, http://www.nuffieldfoundation.org/practical-biology/gene-induction-%C3%9F-galactosidase-e-coli
'Maintaining and preparing cultures of bacteria and yeasts', Nuffield foundation website, http://www.nuffieldfoundation.org/practical-biology/maintaining-and-preparing-cultures-bacteria-and-yeasts (Accessed September 2016)
'Practical Biology Student Sheet: Gene induction: β–galactosidase in E. coli', Nuffield Foundation website, www.nuffieldfoundation.org/sites/default/files/PB_gene-induction-galactosidase-in-e-coli-ss.doc (Accessed September 2016)
‘Regulation of the Lactose System’ in Griffiths A.J.F., Gelbart W.M., Miller JH, et al. 1999. Modern Genetic Analysis, W.H. Freeman: New York. National Center for Biotechnology Information website, https://www.ncbi.nlm.nih.gov/books/NBK21402/
'Standard Health and Safety guidance', Nuffield foundation website, http://www.nuffieldfoundation.org/standard-health-safety-guidance (Accessed September 2016)
'Toluene (methylbenzene)', Australian Government National Pollutant Inventory website, http://www.npi.gov.au/resource/toluene-methylbenzene (Accessed September 2016)
‘Toluene’ Safety Data Sheet, Chem Supply website, https://www.chemsupply.com.au/documents/TA0141CH75.pdf (August 2013)
'Working with enzymes', Southern Biological website, http://file.southernbiological.com/Assets/Products/Chemicals/Enzymes/EnzymeOverview.pdf (Accessed September 2016)