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looking at decomposers -Micro biology experiment

Submitted by sat on 14 June 2016

In brief:

School policies: Currently there are differences between the state/territory educational jurisdictional policies on whether certain microbiological activities can be carried out. Schools are advised to check what activities are permitted in their jurisdiction/school sector before proceeding to work with any microorganisms.

It is important to be aware of the possible hazards and risks, so that appropriate controls can be put into place before working with microorganisms.

Through further correspondence we have obtained further details of this activity. There are several concerns with this activity.

Collection of grass specimens: Precautions would be required for the collection of grass samples to reduce the risk of exposure to unknown microorganisms.

Unknown microorganisms: The activity you describe will produce ‘wild cultures’ which will not be identified and hence the possibility of isolating pathogens. The Petri dishes should be sealed and never opened after incubation and whilst conducting observations. Due to your tropical location you have indicated a number of potential microorganisms of concern.

Sterilising forceps: Sterilising forceps in a Bunsen flame for 1 minute will result in the forceps becoming extremely hot and the risk of severe burns. Forceps are safely sterilised by wrapping in foil and placing in an autoclave or pressure cooker at 15psi, 121oC for 15minutes.  Several forceps can be sterilised for each group at a time. An alternative is to soak the forceps in 70% alcohol for 10min, air dry and wrap in aluminium foil avoiding touching the ends.

Paper bags in a hot oven for 15 minutes: The method does not specify the temperature of the oven for sterilising. A hot air oven needs to be run at 160oC for 2-3 hours to sterilise1. The 15 minutes suggested will not be sufficient to sterilise and regular paper bags will not survive the high dry heat temperatures2. Special sterilisation paper bags are available for use in steam sterilisers.

Incubation temperature: The method specifies an incubation temperature at around 37oC. The recommended incubation temperature for schools is at temperatures of 30oC or below to avoid the growth of human pathogens.

Decontamination procedure: The method does not explain that the plates should be sterilised in an autoclave or pressure cooker prior to disposal.

Science ASSIST recommends that before schools embark on working with microorganisms they should ask the following questions and perform a site specific biological risk assessment:

Biological risk assessment: According to Biosafety in microbiological and biomedical laboratories (BMBL)5, the following five steps should be considered:

  1. Identify agent hazards and perform an initial assessment of risk.
  2. Identify laboratory procedure hazards
  3. Make a determination of the appropriate biosafety level and select additional precautions indicated by the risk assessment.
  4. Evaluate the proficiencies of staff regarding safe practices and the integrity of safety equipment.
  5. Review the risk assessment with a biosafety professional,

If after conducting a detailed risk assessment you have determined that your school can manage the risks, the following procedures are recommended to prevent the growth of any pathogenic microorganisms.

Science ASSIST has previously answered several questions relating to microbiology, see:

Inoculating agar plates and sealing them

Bacteria

Additional information:

Microbiology of soils: Soils contain a diverse range of microorganisms which include bacteria, fungi, algae and protozoa which are involved in the decomposition of plant materials as well as being involved in maintaining soil fertility and recycling nutrients. The rhizosphere (the area closely associated with the roots) of plant material is where much of the microbiological activity takes place7. The soil microbial community is influenced by many factors such as temperature, moisture, acidity or alkalinity, oxygen levels, organic matter and soil porosity.

Bacteria and fungi are the most important microorganisms involved in the decomposition process of plant materials.

Bacteria are the most predominant microorganisms present and play an important role in the early stages of decomposition of organic material, some are nitrogen fixers, some are sulfur oxidisers and  others help develop humus in soils and contribute to the smell associated with high organic matter8.  Some bacteria are very sensitive to changes in the soil environment, while others have features such as resistant spores that allow them to remain in the soil for long periods8. Some can cause disease in plants, animals and humans. Bacillus species, Pseudomonas species8 and Clostridium species are some examples of bacteria found in the soil environment. There is also the emergence of the soil bacterium Burkholderia pseudomallei which is implicated in the infectious disease of humans and animals in the tropics9. This organism is able to be cultivated on Nutrient agar10.

Fungi have roles in plant disease, organic matter decomposition and specialized functions in the rhizosphere. They dominate in the later stages of decomposition.  Examples of fungi commonly isolated from soils include Penicillium, Aspergillus, Fusarium and Mucor species 11.

Other: There is also the possibility of microorganisms being introduced via animal excretions in the area. Lymphocytic Choriomeningitis (LCM) if present may be a consideration in your situation. LCMV infections can occur after exposure to fresh urine, droppings, saliva, or nesting materials from infected rodents12.

References

1Society for General Microbiology. 2006. Basic Practical Microbiology: A Manual, Microbiology Online website, http://www.microbiologyonline.org.uk/file/ca2189fba3b39d24c5a44c1285d008...

2 ‘Sterilization – Packaging and storing’, Centers for Disease Control and Prevention website, https://www.cdc.gov/oralhealth/infectioncontrol/questions/sterilization/...(Broken link fixed: November 2017)

3 ‘Microbiology’, University of Sydney WHS website, http://sydney.edu.au/whs/guidelines/biosafety/microbiol.shtml (Accessed June 2016)

4 American Society for microbiology. 2012. Guidelines for Biosafety in Teaching Laboratories, Universitat Autònoma de Barcelona website, http://www.uab.cat/doc/teaching_lab_ASM

5 U.S. Department of Health and Human Services. 2009. Biosafety in microbiological and biomedical laboratories (BMBL) 5th Edition. 2009. Section II Biological risk assessment. Centers for Disease Control and Prevention website, https://www.cdc.gov/biosafety/publications/bmbl5/

6 'Guidelines for best practice for microbiology in Australian schools'. Science ASSIST website, https://assist.asta.edu.au/resource/4196/guidelines-best-practice-microb.... (added October 2019).

7 'Microbiology of turfgrass soils'. Grounds maintenaince website, http://grounds-mag.com/mag/grounds_maintenance_microbiology_turfgrass_soils/ (Accessed June 2016)

8 Reid, Greg and Wong, Percy. 2005. Soil Bacteria, New South Wales Department of Primary Industries website, http://www.dpi.nsw.gov.au/__data/assets/pdf_file/0017/41642/Soil_bacteria.pdf

9 ‘Melioidosis’, Centers for Disease Control and Prevention website, https://www.cdc.gov/melioidosis/ (26 January 2012)

10 Weigel, L.M and Morse, S.A. 2009. ‘Implications of Antibiotic Resistance in Potential Agents of Bioterrorism’, p 1329 in Mayers, Douglas (Ed.) Antimicrobial Drug Resistance. Volume 2 Clinical and Epidemiological Aspects. Humana Press: New York. Google Books website, https://books.google.com.au/books?id=dl_IbfXOqe4C&pg=PA1329&dq=can+Burkh...

11 ‘Soil Fungi’, Biological Sciences, University of Sydney website, https://canvas.sydney.edu.au/courses/7114.html (2004)

12 ‘Lymphocytic Choriomeningitis (LCM)’, Centers for Disease Control and Prevention website, https://www.cdc.gov/vhf/lcm/transmission/index.html (6 May 2014)

‘Dry heat sterilization’, Wikipedia website, https://en.wikipedia.org/wiki/Dry_heat_sterilization (Accessed June 2016)

Garg, Nisha and Garg, Amit. 2015. Textbook of Operative Dentistry, Jaypee Brothers Medical Publishers: New Dehli. p 180. Google Books website, https://books.google.com.au/books?id=n2TJDryohrMC&lpg=PP1&dq=Textbook%20... (Updated March, 2017)

Jenkins, Abigail. 2005. Soil fungi, NSW DPI website, http://www.dpi.nsw.gov.au/__data/assets/pdf_file/0020/41645/Soil_fungi.pdf

Standards Australia. 2010. AS/NZS 2243 Safety in Laboratories, Part 3: 2010 Microbiological safety and containment. Sydney, Australia.

‘Melioidosis’, Wikipedia website, https://en.wikipedia.org/wiki/Melioidosis (Accessed June 2016)

‘Microbiology of decomposition’, Wikipedia website, https://en.wikipedia.org/wiki/Microbiology_of_decomposition  (Accessed June 2016)

NT Government, Centre for Disease Control. 2015. Meliodosis, Northern Territory Health Department website, http://health.nt.gov.au/library/scripts/objectifyMedia.aspx?file=pdf/43/46.pdf&siteID=1&str_title=Melioidosis.pdf (December 2015)

‘Pathogenic fungus’, Wikipedia website, https://en.wikipedia.org/wiki/Pathogenic_fungus (Accessed June 2016)