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Autoclaving microbiological waste

Posted by Anonymous on Fri, 2018-08-03 16:53

Autoclaving microbiological waste: we have just purchased an autoclave, however it has minimal instructions and no preprogrammed settings. I have read all your related information, which has been helpful. I just want to clarify a few points:

1. Is it okay to sterilise microbiological waste in a marked biohazard bag then put it in general waste? 2. We cloned cauliflower in a growth medium, in 250ml plastic jars, which grew many organisms. We wished to re-use jars so I sterilised them at 121'c for 20min then allowed them to cool. Waste was scrapped out and double bagged, then placed in general waste. I have concerns about this procedure as a rule we never re-open petri dishes. Also I understand sterilisation times are based on clean items. Does this process mean the items are sterilised or merely decontaminated? Have the organisms (bacteria, fungi) been rendered inert? Is 121'c for 20min considered a kill cycle? 3. We always tape around our perti dishes with parafilm to prevent re-opening - will this prevent steam entering the dish and prevent sterilisation? 4. We are keeping a log of our cycles and are deciding which validation strips to purchase to suit our needs ( 121'c for 20min or 121'c for 15min).

Would greatly appreciate your advice.

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Year Level: 7 8 9 10 Senior Secondary Laboratory Technicians: Laboratory Technicians

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Autoclaving microbiological waste

Submitted by sat on 03 August 2018

1. Is it okay to sterilise microbiological waste in a marked biohazard bag then put it in general waste?

After decontamination in an autoclave, an autoclave bag depicting the biohazard symbol should not be placed directly into the general waste bin. These bags should be placed into an unlabeled strong black plastic garbage bag and sealed securely before being placed into the waste bin1. This is so that the biohazard symbol is not visible, which could cause unnecessary alarm at the contents.

It is recommended to sterilise microbiological waste in a bag that will withstand the sterilisation conditions of 15psi, 1210C for 15-20 minutes and contain the treated contents.

An alternative to an autoclave bag is an oven bag which can be purchased from supermarkets and which can also withstand the sterilization conditions. These do not have any biohazard markings on them and are commonly used in schools.

It must be remembered that effective sterilisation is also determined by the correct packing of an autoclave or oven bag. The effectiveness of autoclaving depends on steam being able to penetrate what is being autoclaved

Do not overfill bags. Loosely pack no more than 2/3 full.

Do not include any sharp objects that may puncture the bag.

Do not include any volatile chemicals.

Lids on any containers must be loosened.

Do not tightly seal the bag but loosely tape the bag shut leaving an opening of 5-6cm to allow steam penetration.

Place the bag into another container in the steriliser to capture any leaks should the bag rupture.

Do not overload the steriliser with too many bags as this will block steam circulation.

Autoclave clean items and waste separately.

Use a sterility compliance strip to verify that the sterilisation conditions have been reached.

1. We cloned cauliflower in a growth medium, in 250ml plastic jars, which grew many organisms. We wished to re-use jars so I sterilised them at 121'c for 20min then allowed them to cool. Waste was scraped out and double bagged, then placed in general waste. I have concerns about this procedure as a rule we never re-open petri dishes.

Also I understand sterilisation times are based on clean items. Does this process mean the items are sterilised or merely decontaminated? Have the organisms (bacteria, fungi) been rendered inert? Is 121'c for 20min considered a kill cycle?

You are correct that Petri dishes that have grown unknown microorganisms should never be opened prior to sterilisation, so that you don’t expose yourself or others to harmful microorganisms. All laboratory equipment, materials and wastes contaminated with microorganisms should be sterilised before being washed, stored or discarded.

Effective sterilisation of microbial cultures by autoclaving (1210C for 15-20 minutes at 15(psi) pounds per square inch) will result in the complete destruction of all living microorganisms including bacterial spores. Therefore, after sterilisation, any reusable containers and their contents would be regarded as sterile and safe to open and clean out for reuse. Autoclaved liquid cultures can be disposed of safely down the sink, whilst any autoclaved solid material can be removed from reusable containers and placed into the general waste.

Remember that sterilization can only be guaranteed when the critical parameters of temperature, steam under pressure and time are reached. It is imperative that timing only begins when the temperature and pressure conditions have been reached. Sterilisation using the autoclave will denature & coagulate proteins and other cell constituents in the bacterial cell including any spore formers. Fungal cultures including fungal spores are easily killed by heating above 80oC.

Decontamination on the other hand is defined as a process that will render an environmental area, device, item or material safe from the risk of infection transmission by removing, neutralising or destroying microorganisms. Decontamination can include disinfection, antisepsis or sterilisation.

1. We always tape around our Petri dishes with parafilm to prevent re-opening - will this prevent steam entering the dish and prevent sterilisation?

Petri dishes with sticky tape or Parafilm should be placed into the autoclave/oven bag with the tape/Parafilm left on. It will not prevent sterilisation.

Parafilm M is a laboratory sealing film with unique properties. It is a stretchy, waxy film that is very good at moulding around tops of test tubes, bottles, flasks and around Petri dishes to provide a leakproof seal. It is gas permeable with low water permeability making it ideal for use in microbiology and cell culture applications as it does not affect oxygen or carbon dioxide permeability when used as a single layer.

Parafilm M becomes more flexible, softer and stickier at about 540C and has a melting point of 600C 2,3. So in an autoclave at 1210C it will have melted from around the Petri dish or jar no longer forming a seal, therefore allowing sterilisation to occur as steam is allowed to enter the vessel. In addition, the moisture in the agar would also convert to steam to help facilitate the sterilisation process.

Science ASSIST recommends that prior to incubation the lid and base of a Petri dish be taped with 4 pieces of sticky tape or one layer (only) of Parafilm to allow for aerobic conditions and to prevent accidental opening of the plate. Sealing with one layer of Parafilm M, completely around the circumference of the Petri dish will allow students to examine them and will prevent exposure to moisture or drips that may seep out of the Petri dish, which are potential sources of infection. 1. We are keeping a log of our cycles and are deciding which validation strips to purchase to suit our needs ( 121'c for 20min or 121'c for 15min). Would greatly appreciate your advice. Thanks

In a school setting Class 5 integrated chemical indicator strips are suitable to validate the effective operation of an autoclave. These strips are the most accurate of the internal chemical indicators and are considered comparable to Biological Indicators in saturated steam 4. They react to all the critical parameters and have the advantage of not requiring any incubation, an immediate result is produced.

An indicator strip should be placed in the centre of each load and checked after each run to ensure that the temperature and steam conditions have been met.

Generally there are several ways to determine if an autoclave or pressure cooker is working properly and sterilisation has occurred. These include physical indicators, chemical indicators and biological indicators.

Physical indicators – involves checking the steriliser gauges or digital display to determine if the correct temperature and pressure have been reached. This however does not guarantee sterilisation.

Chemical indicators – These use chemicals that change colour when exposed to high temperatures and in some cases pressure. There are 2 types of chemical indicators. Integrated chemical indicator strips generally provide a limited validation of only 1 or 2 of the critical parameters (temperature and time). Class 5 Integrated chemical sterility indicator strips, are the most accurate of the internal chemical indicators and are considered comparable to Biological Indicators in saturated steam. They react to all the critical parameters. They have an advantage in that they do not require any incubation and an immediate result is produced. These would be very suitable for the school setting.

Temperature sensitive tape in the form of autoclave tape or an indicating label on a paper or plastic pouch. These contain a heat sensitive ink and are used extensively in microbiology labs. They however only indicate that an item has gone through a heating process and not that sterility has occurred.

Biological indicators – Traditionally the most accepted means of sterilisation monitoring. They contain spores of a heat resistant bacterium such as B. stearothermophilus that will germinate if the correct sterilisation conditions are not met. This microorganism is the most resistant strain to steam autoclaving and will be inactivated under correct autoclave conditions. Biological indicators require an incubation step to obtain a result. Professional microbiology labs would regularly test their autoclave using this method. This would not generally be used in a school setting.

Both biological and chemical indicators are available from scientific suppliers. See the Science ASSIST School science suppliers list.

References and further reading:

1 Sydney University. nd. Hazardous waste. Safety Health & Wellbeing, Sydney University website, https://intranet.sydney.edu.au/content/dam/intranet/documents/employment/safety- health-wellbeing/guidelines-training/hazardous-waste-guide.pdf (Accessed July 2018)

2 Sigma-Aldrich. nd. Product Information – Parafilm® M, Sigma-Aldrich website, https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/421/110/p7543pis.pdf (Updated July 2019)

3 ‘Parafilm: Frequently asked questions’, Camblab website, https://camblab.info/parafilm- frequently-asked-questions/ (17 May 2012))

4 3M. nd. Steam Chemical Indicator Classifications, 3M website, http://multimedia.3m.com/mws/media/600678O/scic-tutorial-pdf.pdf (Accessed July 2018)

‘Autoclave Use’, Princeton University website, https://ehs.princeton.edu/book/export/html/380 (Accessed July 2018)

NSW Department of Education and Communities, Chemical Safety in Schools (CSIS) resource package, NSW DEC website, https://education.nsw.gov.au/ (DEC Intranet, login required.)

Science ASSIST. 2016. Autoclaving disposable Petri dishes, Science ASSIST website

Science ASSIST. 2017. GUIDELINES for best practice for microbiology in Australian schools, Science ASSIST website

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

Tilbrook, Dr Peta. 2015. Technical Services Manager, Department of Environment and Agriculture, School of Science, Curtin University, WA. Personal communication.

University of Adelaide. 2016. 3.14 Biological Safety Management Autoclave Information Sheet, The University of Adelaide website,

https://web.archive.org/web/20190201072339/https://www.adelaide.edu.au/hr/hsw/docs/biological- autoclave-info-sheet.pdf (Original link no longer available. This archived copy made available by the Internet Archive's Wayback Machine May 2020)

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