Microbiology involves working with bacteria that are too small to see in normal circumstances. As a result, special techniques, methods and apparatus are usually required. In particular, special agar gel substrates are often employed to grow microorganisms. Following is information on using 3M Petrifilm® to make student experiments easier to perform by not requiring specially made agar filled petri dishes.

What is Petrifilm?

The Petrifilm plate is a thin film, sample ready, dehydrated version of the conventional Petri dish agar plate. It is often referred to as the Post-it® Note of microbiology.  

Petrifilm is widely used in the food and beverage industry throughout the world to monitor the quality of products and to audit cleaning processes. Foods, beverages and surfaces can be tested for the presence of harmful bacteria (pathogens), indicator bacteria (that indicate the possible presence of pathogens), and spoilage organisms that can affect the shelf-life for products.

 

Advantages of Petrifilm 

The advantages of Petrifilm include:

§          Ready to use – no preparation required for samples.

§          Can be used for direct environmental testing.

§          Wide range of official approvals.

§          Long shelf life when shielded from moisture and humidity.

§          Simple to use and interpret.

§          Built-in biochemical confirmation.

§          Smaller (thinner) and less likely to be damaged than agar plates if dropped.

§          Require less space for storage, incubation or disposal. 

 

Types of Petrifilm

There are many types of Petrifilm, each formulated and designed for a specific purpose. The type of plate required depends on the aim of the experiment. 

§          Aerobic Count (AC): Designed to enumerate common aerobic bacteria in 48 hours. The AC plate contains Standard Methods nutrients, a cold water gelling agent and triphenyltetrazolium chloride (TCC), an indicator that colours bacterial colonies red.

§          Aerobic Count (AC) with Lactic Acid bacteria: Designed to enumerate common aerobic bacteria. Incubate the AC plate with anaerobic conditions.

§          Coliform Count (CC): Designed to enumerate coliform bacteria in 24 hours. Coliforms are members of the family Enterobacteriacea which ferment lactose to produce gas. Coliforms are an indicator of faecal contamination, particularly in dairy products. The CC plate contains violet red bile (VRB) lactose nutrients, a cold water gelling agent and triphenyl tetrazolium chloride (TTC), an indicator that colours bacterial colonies red. Gas produced by coliforms are trapped between the two sheets of film, providing a confirmation test.

§          E. coli/Coliform Count (EC): Counts all coliforms in a sample and differentiates E. coli as a subset of the total. Results in 24 to 48 hours depending on food tested. The EC count is an indicator of faecal contamination, particularly in meat products. The EC plate is essentially the same as the CC plate, except that it contains the BCIG chromogenic indicator which colour the E. coli bacteria a blue colour.

§          Enterobacteriacea Plate (EB): Enumerates coliforms plus potential pathogens; it provides a broader picture of potential contamination in 24 hours. Enterobacteriaceae Count Plate is a sample-ready culture medium system which contains modified Violet Red Bile Glucose (VRBG) nutrients, a cold-water-soluble gelling agent, and a tetrazolium indicator that facilitates colony enumeration. On the plate, Enterobacteriaceae will appear as red colonies with yellow zones and/or red colonies with gas bubbles with or without yellow zones.

§          Staphylococcus Express (ST): Selective and differential for Staphylococcus aureus. This next generation Staph test provides confirmed results in as few as 22 hours. Staph Express Plate is a sample-ready-culture-medium system that contains a chromogenic, modified Baird-Parker medium and a cold-water-soluble gelling agent. The test requires only one incubation temperature and is equivalent to the BAM three-plate Baird-Parker agar and single tube-coagulase method. A chromogenic indicator in the Petrifilm Staph Express Count Plate identifies Staphylococcus aureus by producing red-violet colonies. When only red-violet colonies are seen, count the colonies and report as S. aureus; the test is complete. If you encounter background flora in your Staph testing, the Petrifilm Staph Express Disk may be used to identify S. aureus from all other suspect colonies. The Petrifilm Staph Express Disk contains toluidine blue-O to facilitate the visualization of deoxyribonuclease (DNase) reactions. The Petrifilm Staph Express Disk should be used whenever colonies other than red-violet are present on the plate.

§          Environmental Listeria (EL): Designed to detect Listeria species from environmental samples in only 29 hours with no enrichment step required. Petrifilm EL Plate is a sample-ready culture medium that allows you to detect the most prevalent environmental Listeria species, including Listeria monocytogenes. It may be used as a qualitative, semi-quantitative, or quantitative test.

§          Yeast and Mould Count (YM) Plate: Designed to count all common yeast and mould species in a sample in 3-5 days. The YM plate contains modified Sabroud’s Dextrose nutrients in a cold water gelling agent, a dye to enhance the visualization of growth on the plate (colours all yeasts aqua green), and two broad spectrum antibiotics to suppress bacterial growth. Yeast are typically small, raised, blue-green colonies, with defined edges. Molds are often larger, variably colored, flat colonies with diffuse edges and central foci. 

 

Using Petrifilm

Petrifilm is supplied in sealed packs that should be kept cool and dry. Storage in the dry atmosphere of a refrigerator is recommended to achieve the labeled shelf-life.  When using for testing, allow the pack to warm up to room temperature before opening to avoid condensation forming. Take out just the number of plates you will need for your experiment, then reseal the pack. Do not put it back in the refrigerator as the moisture will affect the plates. 

For detailed descriptions of sample preparation, inoculation methods, incubation conditions and interpretation of results, visit www.arrowscientific.com.au then follow the links type of Petrifilm Plates used. 

 

Testing Liquids

Liquids such as orange juice, milk or water can be tested directly by inoculating the Petrifilm plate with 1mL and incubating. If the count is too high, you should dilute the sample then correct for the dilution factor when expressing the result.  

For information on serial dilution, download the “Tips on Sample Preparation – Serial Dilution” sheet from our website. 

Products such as as fruit juice or yoghurts may need the pH of the sample adjusted to bring it to a testing range of 7.00 pH. 

Testing potable water can be problematic because standards require no E.coli per 100mL of water. In practical terms, this means you would have to perform 100 tests with EC plates because each plate uses only 1mL of water. It is possible to use a sterile filtration system to concentrate a volume of water for testing, but for student demonstrations, it is more usual to dose a water sample with an E.coli culture to ensure a positive result. 

Testing Solids

Solids need to be macerated in sterile liquid to extract microorganisms before they can be tested.

Use a plastic gas with 25g of solid sample added to 75mL of sterile water. The plate can be inoculated with 1mL of the extract. One needs to account for the dilution by multiplying the answer by 3. 

Testing Surfaces

Two approaches are possible when using Petrifilm to test for the presence of microorganisms on surfaces.

One method is to swab an area, and then to extract the swab with sterile liquid that can be tested. This is useful when testing irregular surfaces such as a tap, door handle, light switch or hand. The 3M Quick Swabis a self-contained ready-to-use surface swab system, otherwise you can use a sterile cotton wool or rayon swab held with sterile forceps. 

The second method is direct contact with a Petrifilm plate. This is suitable for smooth regular surfaces such as bench tops and floors, and even the surface of the skin.

In order to activate the plates, they need to be hydrated with 1mL of sterile liquid before contact. Hydrated plates should be allowed to gel for at least one hour, but can be stored under refrigeration for up to a week before being used totest a surface. For more information, visit our website.

                                         

Testing Air

By taking a hydrated plate and peeling back the protective film, you can expose the activated surface of the plate to the air to check for the presence of airborne microorganisms. For example, compare different air conditioner outlets.  

Student Experiments

Petrifilm plates are suitable for many student experiments because of their ease of use and versatility. As well as meeting outcomes in the field of microbiology, the use of Petrifilm touches on areas such as maths (dilution and correction factors, calculations, scaling and statistics), scientific experimental design, and the safe and proper use of laboratory equipment.  

Sterile Technique

All microbiological procedures require sterile methods to avoid contamination that would interfere with the determinations being made.  Basic measures include:

§          Wash hands thoroughly before and after each procedure.

§          Tie back hair and use a lab coat.

§          Swab the work surface with 70% ethanol before starting work. Leave wet and allow to evaporate dry.

§          Use a Bunsen flame to form a heat curtain to shield the work area from airborne microorganisms.

§          Use “economical” movements and minimize the time that surfaces and samples are exposed. When mixing or diluting samples, it is acceptable to use well boiled water that has been allowed to cool in a sealed contained.  

For macerating food samples, you can get good results in a fresh polythene lunch bag. Provided they haven’t been opened, these bags can generally be considered as sterile for the purpose of school experiments. 

To check for the presence of a background level of bacteria in the water and/or bag, run a control that substitutes sterile liquid for the test substance. 

 

Safety and Disposal

Following inoculation, plates should be taped shut or placed in a press-seal bag to keep them isolated.  Follow good laboratory practice and have students thoroughly wash their hands after handling microbiological samples and equipment.

Adequate antibacterial hand wash and hand rub sanitizer solutions should be provided. Treat plates with viable colonies as you would deal with cultures on conventional Petri dishes.

Autoclave or soak in a suitable biocide, e.g. 10% bleach solution.

Alternatively, you can use a contract waste collection service such as that provided by Stericorp. 

 

Trouble Shooting

The most common problems we have seen tend to occur with novice users of Petrifilm, but they are easily avoided: 

§          Dispensing more than 1mL of liquid: If you accidentally draw more than 1mL of liquid into the pipette and dispense it onto the Petrifilm plate, it will overflow off the plate when the spreader is applied. Be careful to apply only 1mL.

§          Having the liquid roll off the plate as it is being applied: This can occur if you hold the pipette at a low angle. Place the Petrifilm plate on a flat surface and hold the pipette perpendicular to the surface, in the central region. Dispense the liquid onto the plate with care.

§          Waiting too long: Have the plate, spreader and sample to be tested ready, and apply the spreader without delay after dispensing the sample onto the plate. Proper spreading may not occur if you wait too long between dispensing the liquid and applying the spreader.

§          Trapping bubbles on the plate: First of all, ensure the sample does not have entrained air bubbles. Allow it to stand if necessary to allow bubbles to clear. As a further measure, use the recommended method to lower the protective film back onto the plate after dispensing the sample. For the AC and YM plates, allow the film to drop back into place. For the CC and EC plates, roll the film gently back into place.

§          Using the wrong spreader: Ensure you use the correct spreader. For YM plates, use only the special (larger size) Yeast and Mould Plate Spreader. For AC plates, use the General Purpose Spreader with the ridge side down (smooth side up). For CC and EC plates, use the General Purpose Spreader with the smooth side down (ridge side up).

§          Cross contamination: Avoid contamination by planning your work and using good aseptic technique and  sterile equipment. This applies to all microbiological work, not just Petrifilm.

§          Correct sample collection: When using Petrifilm plates for environmental sampling, be sure to use thiosulfate-free letheen broth if sanitizer may be present. Letheen broth neutralizes halogen, quaternary ammonium, and acid sanitizers. Do not use diluents containing thiosulphate or sodium citrate with Petrifilm plates  

 

Further Information

The key to good results with microbiology experiments is familiarity with the equipment, confidence in the techniques, and respect for the safety aspects. Arrow Scientific offers a range of other products that can be used in teaching microbiology: 

§          Safety in the Food Microbiology Lab

§          GlitterBug Potion for Handwashing technique training

§          GlitterBug Powder for teaching cross-contamination.

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