Inoculating agar plates and sealing them

Inoculating agar plates and sealing them: Could you advise the correct procedure for innoculating agar plates, and in particular, sealing them?   I have always followed the "Microbiology - Safety Considerations"  by Sheryl Hoffmann, with tape over the edge on three sides of the plate, not sealing around the entire plate.  I have commenced at a new school this year and need help to convince and change from sealing the whole plate.

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Publication Date: 27 May 2015
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Inoculating agar plates and sealing them

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Safety issues are a significant consideration in microbiology, as there is a potential for infectious hazards. It is the strict observance of correct procedures, which enables students and staff to work safely with microorganisms. When working with microorganisms, it is best to treat them all as potential pathogens (1).


Currently in Australia, some microbiological activities are permitted in some jurisdictions and not others. Therefore, you should check the activities permitted in your jurisdiction before proceeding. Science ASSIST is in the process of consulting authorities in order to make nationally consistent sensible and workable recommendations for best practice in school microbiology.


It is important to be aware of the possible hazards and risks before working with microorganisms. Therefore, Science ASSIST recommends that a risk assessment is carried out and appropriate control measures be put into place. Science ASSIST has developed a one page risk assessment template to help with this (see Risk Assessment Template).


Inoculation techniques commonly used in schools:


The following link contains good general information on practical microbiology including detailed procedures for the following inoculation techniques.


http://www.microbiologyonline.org.uk/file/ca2189fba3b39d24c5a44c1285d008...


Microorganisms can be inoculated onto agar plates in school science laboratories by various methods.


1) Inoculation of plates via air exposure (settle plates):


Petri dishes are left open to the air in various places in the laboratory for a period of time before lids are replaced, sealed with 4 pieces of sticky tape and incubated. Plates should not be left open in areas such as toilets.


2) Inoculation by direct contact.


Microorganisms are transferred directly to an agar plate by touching the surface of the agar with an item such as a coin. Touching the agar with your finger tip is not recommended, as this could allow for the growth of pathogens.


3) Inoculation of plates with sterile swabs from environmental samples:


A sterile swab is moistened in sterile broth or water and wiped over the area to be sampled. The swab is then moved over the surface of the agar plate in a zig-zag manner (2) to transfer any microorganisms. The swab can be disposed of into the rubbish as it has not been used to swab areas that contain any pathogens. As an added precaution, the swab could be placed into a freshly prepared disinfectant solution such as 0.25% sodium hypochlorite for 2 hours before disposal into the rubbish.


4) Inoculation of plates with bacteriological loops from food samples such as yoghurt or cheese:


A heat sterilised loop can be used to sample from a yoghurt or cheese specimen. The loop is used to smear the sample either in a zig-zag manner over the surface of the agar, as described above, or placed onto a section of the surface of an agar plate and then streaked out for single colonies as described below.


Note: The above cultures are considered “wild cultures” and should not be opened or subcultured. The agar plates should be sterilised before disposal. When observations are complete, the plates should be decontaminated by sterilising in an autoclave or pressure cooker before disposal into the waste bin. Agar plates must be placed into an autoclavable bag, such as an oven bag, for sterilisation at 110 kPa/15psi, 121° C for 15–20 minutes in an autoclave or pressure cooker before disposal.


Remember to label the base of the plate around the edge (3) rather than in the middle, where any growth could be obscured, and incubate the plate upside down to prevent any condensation that forms on the lid from dripping onto the agar affecting the colonies growing on the surface.


Sealing agar plates:


The complete sealing of Petri dishes during incubation should be avoided, as this may generate an anaerobic environment inhibiting the growth of aerobic microorganisms and promoting the growth of potentially anaerobic pathogens (See below for more information). Science ASSIST recommends that the lid and base of the Petri dish be taped with 4 pieces of sticky tape (2, 3) to allow for aerobic conditions and to prevent accidental opening of the plate during incubation. Plates can be sealed with sticky tape, or preferably Parafilm, completely around their circumference prior to allowing students to examine them. This will prevent any exposure to moisture or drips that may seep out of the Petri dish, which are potential sources of infection, as well as keeping the lid securely attached to the base. All observations must occur with the Petri dish taped. Parafilm 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 much more effective than sticky tape.


Essential Background Information:


Facilities:


School science laboratories are classified as (PC1) Physical Containment level 1, if they comply with the requirements of AS/NSZ 2243.3-2010. Safety in laboratories. Part 3. Microbiology safety and containment. PC1 laboratories are suitable for work with risk group 1 microorganisms only. These are infectious microorganisms that are “unlikely to cause human, plant or animal disease” and “where laboratory personnel can be adequately protected by Standard Laboratory practices(4).


Anaerobic microorganisms:


The growth of anaerobic microorganisms, those that cannot be grown in the presence of oxygen, should be avoided in school laboratories. Anaerobes are widely distributed in nature and are potentially pathogenic (capable of causing disease) when removed from their normal environments (5). Most anaerobes fall under Risk Group 2 or 3 microorganisms which are not to be handled in PC1 laboratories and are associated with many illnesses, for example, dental infections, abscesses, pneumonia and appendicitis. It is important that procedures and techniques that are used when dealing with microbes in the school laboratory do not produce anaerobic conditions that may select for the growth of pathogenic anaerobic organisms.


Procedures to prevent the growth of pathogens:


In addition to not completely sealing agar plates during incubation, there are other procedures that should be carried out in school laboratories in order to prevent the growth of pathogenic organisms.


  • When handling microorganisms, it is important to use aseptic techniques at all times. A significant risk associated with microbiology is the generation of microbial aerosols, where fine droplets of water containing cells and/or spores are released into the air.
  • Aseptic technique is a fundamental skill in microbiology:
    • to avoid the contamination of culture media with unwanted microbes,
    • to prevent contamination of personnel and work surfaces, and
    • to prevent microbes from being accidently released into the environment.
  • The type of media used should not select for pathogens.
    • Nutrient agar is a simple media which supports the growth of a wide variety of bacteria and moulds and is suitable for use in school laboratories.
    • Media designed to select for more fastidious microorganisms and pathogens such as Blood and MacConkey Agar should not be used.
  • When culturing from the environment, samples should not be taken from areas likely to contain organisms harmful to humans, for example: body surfaces, coughs, sneezes, animal sources such as bird cages and unsanitary environments such as drains and toilets.
  • Cultures should be incubated at temperatures of 30° C or below to avoid the growth of potential human pathogens that are adapted to human body temperature.

Precautions when inoculating agar plates.


  • Wash hands with soap and water before and after working with microorganisms.
  • Cover any cuts with a waterproof dressing and consider wearing disposable gloves.
  • Make sure work surfaces are decontaminated before and after working with microorganisms with 70% ethanol.
  • Make sure inoculating instruments (loops, swabs, pipettes and spreaders) are sterilised prior and after use.
  • Make sure that inoculating instruments containing microbiological samples are not allowed to touch any surface other than the agar that requires inoculation.
  • Flame the mouth of all test tubes and bottles both when the cap is removed and before it is replaced.
  • Plates should be open for a minimum amount of time to minimise the risk of introducing any contaminants from the air.
  • Inoculation should be carried out as quickly as possible to minimise introducing any contaminants.
  • Work close to the Bunsen flame, as it provides an updraught that carries air away from the workspace, so reducing contamination from the air.
  • Have a bacterial spills kit available (freshly prepared 1% sodium hypochlorite, bucket ,mop, plastic waste disposal bags, paper towel, disposable gloves, safety glasses, mask, plastic disposable apron).

Subculturing:


Science ASSIST is currently seeking advice regarding the following activites in schools. Currently they are permitted in some jurisdictions and not others. Teachers supervising students carrying out these activities should have microbiological training.


1. Streak-plate method:


The principle of this method is the gradual dilution of an inoculum of bacteria over the surface of an agar plate to produce single isolated pure colonies. A small amount of sample is placed onto a section of the surface of an agar plate with either a sterile swab or flamed inoculating loop. This is called the initial inoculum. The loop is sterilised and used to spread out the initial inoculum in one direction to make several streaks. This is referred to as the first set of streaks. The loop is sterilised again by flaming and the streaking process repeated 2–3 more times, each time going back into the previous set of streaks. Following incubation, single colonies should be seen in the final set of streaks. Each colony is produced from a single bacterial cell as it multiplies.


2. Inoculation of plates with pipettes and glass spreaders to produce lawn plates:


Sterile cotton-wool-plugged graduated or Pasteur pipettes can be used to deliver 2–3 drops of bacteria from nutrient broth cultures on to the surface of an agar plate. A sterile glass spreader is then used to spread the drops evenly over the surface of the agar. This technique is used to prepare a lawn or spread culture, where the entire surface of the plate is uniformly covered with bacteria. It is commonly used for testing antimicrobial substances such as antibiotics and disinfectants, or for performing colony counts. The pipette and spreader should be decontaminated by autoclaving or placing into a disinfectant solution immediately after use and before cleaning.


Glossary:


Anaerobic microorganisms:


Microorganisms that do not require oxygen for growth. Oxygen is not used to obtain energy and is toxic to these microorganisms. They are often called strict or obligate anaerobes.


Facultative anaerobic microorganisms:


Microorganisms that grow in either the absence or the presence of oxygen. They can metabolise energy aerobically or anaerobically. Oxygen is not toxic to these microorganisms.


Pathogen:


A microorganism that is capable of causing disease.


Subculture:


The aseptic transfer of a microorganism from one agar plate to another fresh agar plate to allow it to continually grow.


References:


(1) ‘Microbiology’ University of Sydney WHS website October 2013. http://sydney.edu.au/whs/guidelines/biosafety/microbiol.shtml


(2) Hoffmann, Sheryl K. 1999. Microbiology Safety Considerations. Southern Biological website http://file.southernbiological.com/Assets/Products/Specimens/Microbiological_Specimens/MicrobiologySafetyConsiderations.pdf


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


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


(5) Hentges, David J. ‘Anaerobes: General Characteristics’ in Baron S, (Editor) 1996. Medical Microbiology. 4th edition. University of Texas Medical Branch: Galveston (TX). National Center for Biotechnology Information website https://www.ncbi.nlm.nih.gov/books/NBK7638/


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.


‘Microbes all around us’ Nuffield Foundation website https://www.nuffieldfoundation.org/practical-biology/microbes-all-around-us (Accessed May 2015)


‘Aseptic Techniques’ Nuffield Foundation website https://www.nuffieldfoundation.org/practical-biology/aseptic-techniques (Accessed May 2015)



(4) This extract from AS/NZS 2243 Safety in Laboratories, Part 3: 2010 Microbiological safety and containment has been reproduced with permission from SAI Global Ltd under Licence 1407-c117

Inoculating agar plates and sealing them

For full details of Inoculating agar plates and sealing them, please see the answer posted on 03/06/2015


Further information on the gas permeability data for Parafilm M indicates that when used as a single layer it will allow sufficient oxygen exchange to promote the growth of aerobic microorganisms and inhibit the growth of potential anaerobes.  Petri dishes should only be wrapped with a single layer and a small overlap of Parafilm during incubation to allow adequate gas exchange. Wrapping Parafilm around a Petri dish more than once should be avoided and will be sufficient to stop any gas exchange creating an undesirable anaerobic environment. (1)


The product information sheet states the gas permeability (rates corrected to 760 mm pressure) to be:


  • Oxygen: 150 cc/m2 /24 hours
  • Carbon dioxide: 400 cc/m2 /24 hours (both at about 22.8 °C, 50% relative humidity (R.H.)
  • Water vapour: 0.88 g/m2 /24 hours (37.8 °C and 90% R.H. vs desiccant).[2]

 


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

[2] Sigma-Aldrich, Product Information  – Parafilm® M, Sigma-Aldrich website https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/p7543pis.pdf (Accessed June 2015)

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