A Brief Introduction to Microbiology and the Use of 3M Petrifilm Plates

Barry Marks B.App.Sc. (Micro), B.Sc. (Hons) (Chem) Special thanks to: Dr Jim Ralph (Regency TAFE) Dr Peter Ball (Southern Biological) Nicole Kyriacou (3M Microbiology) Edited by Ryan Wick

Index
1. Introduction 2. 3M Petrifilm Plates o Aerobic Count Plate (AC) o Yeast and Mould Count Plate (YM) o Coliform Count Plate (CC) o E. coli/Coliform Count Plate (EC) 3. Safety in Microbiology 4. Experiments o Yeasts and Moulds in the Air o Bacteria on Fingers (Use of Topical Antimicrobials) o Total Bacterial Population in Milk (Shelf Life Determinations) o Effect of Temperature on Bacterial Populations o Coliforms and E. coli in Ground Meat o Count of Bakers/Brewers Yeast o Bacterial Populations on Surfaces 5. Glossary of Terms 6. Appendix

Introduction
Microbiology is the study of organisms too small to see with the naked eye microorganisms . These include bacteria and fungi, which are often of prime concern to a microbiologist. Some organisms, such as mould, are visible to the naked eye, but are still considered part of microbiology because a significant phase of their life cycle is microscopic. Microorganisms are ubiquitous they are found everywhere. Throughout history, microorga nisms have been both friends and foes to humanity. Organisms with the role of foe would include Yersinia pestis (the plague), Mycobacterium tuberculosis (tuberculosis), Mycobacterium leprae (leprosy), Vibrio cholerae (cholera), Salmonella, Campylobacter, Staphylococcus and Listeria (various forms of food poisoning). As a friend, there are organisms such as Lactobacillus (fermented meats, cheeses and yoghurts), Saccharomyces cerevisae (beer, wine and bread), Acetobacter (vinegar), Propionibecterium (holes in Swiss cheese) and Penicillium (antibiotics). Most microorganisms are generally harmless, but we should always remember that life on Earth would not be possible without microorganisms. In addition, all species on Earth, including humans, have evolved from microscopic organisms that lived far in the past. While we now know much about microorganisms, this knowledge has been fairly recent. The microscope, which allows us to see microorganisms directly, was not invented until the Seventeenth Century. Even after its invention, the full ramifications of microbiology did not become apparent until the Nineteenth Century. Louis Pasteur (1822-1895) was able to use the microscope to demonstrate that in certain alcoholic fermentations (beer and wine production), the fermentation process was carried out by living microorganisms (yeasts), and that specific types of yeast produced good batches while others produced bad batches. Consequently, the concept of aetiology (study of cause and effect) has become a part of microbiology. For example, food spoils because microorganisms degrade the food. If you eliminate the microorganisms by sealing the food in a can and heating it to a high temperature, the food will last for years. The cause is the presence of microorganisms, and the effect is spoilage. Robert Koch (1843-1910) was able to demonstrate that certain bacteria caused certain diseases, including that the agent for anthrax was Bacillus anthracis. In doing so, he developed all of the basic microbiological techniques we still use to this very day. Since then, vaccines and cures have been developed for the majority of diseases in humans and other animals. We can test foods for the presence of known pathogenic (harmful) bacteria, test for other organisms that indicate the presence of pathogenic bacteria and test for spoilage organisms to see how long food will last. This manual describes some basic microbiological techniques along with safety tips for dealing with live microorganisms. It also has some fun experiments to do in the classroom to teach how microorganisms grow, how to isolate them and how to study them.

3M Petrifilm Plates
Petrifilm plates are thin film, sample -ready, dehydrated versions of the conventional Petri dish agar plate. They are ready to use immediately after taking them out of their packets and have several advantages over conventional agar plates. These include built-in biochemical confirmation, ease of preparation and use, and smaller volume requirements (10 Petrifilm plates take the same space as single Petri dish agar plate). Petrifilm plates are especially well-suited to quantitative tests in microbiology. There are four plates described in this manual, all of which are considered safe for general educational use. All plates described require a one mL sample inoculation. Other Petrifilm plates are made to isolate known pathogens, but these are unsuitable for most educational environments and are therefore not described in this manual. Petrifilm plates have international recognition by AOAC and AFNOR, and are widely used in industry in Australia and internationally.

Aerobic Count Plate (AC)

The AC plate counts nearly all aerobic and facultative anaerobic bacteria in a sample. The AC plate contains : plate count nutrients the coloured dye triphenyl tetrazoliumchloride (TTC) which colours all bacterial colonies red a cold water-soluble gelling agent Incubation time: two days Incubation temperature: 35C Terms previously used for this plate are total viable count (TVC), standard plate count (SPC) and plate count (PC). While yeasts and moulds are capable of growing on this plate, they generally do not appear within the two day incubation time, and they are easily distinguished from bacteria since they do not reduce the TTC to produce a red colour. When quantifying bacterial growth, count all red colonies regardless of their size or intensity.

Count all red colonies visible on the AC plate.

Yeast and Mould Count Plate (YM)

The YM plate counts nearly all common yeast and mould species in a sample. The YM plate contains : modified Sabrouds dextrose nutrients two broad spectrum antibiotics to suppress bacterial growth an alkaline phosphatise indicator which colours all yeasts aqua green a cold water-soluble gelling agent Incubation time: three to five days Incubation temperature: 20-25C Yeasts appear as small, regularly-shaped, aqua green colonies. Moulds appear as larger, variable coloured colonies with diffuse edges and a central focal point. Mould colonies will have a furry appearance.

Yeas t colonies are characterized by an aqua green colour.

Mould colonies are characterized by diffuse edges and a central focal point.

Coliform Count Plate (CC)

The CC plate counts all coliforms within a sample without differentiating between genera. Coliforms are the members of the family Enterobacteriaceae which ferment lactose to produce gas. This count has been used as a measure of faecal contamination in dairy products and other foods. The CC plate contains: lactose nutrients violet red bile to select for the family Enterobacteriaceae TTC indicator to assist in visualising colonies a cold water-soluble gelling agent Incubation time: 24 hours Incubation temperature: 35C Colonies of organisms which ferment lactose to produce gas will have gas bubbles trapped in the gel next to the colony. When quantifying coliform growth, count all red colonies which are associated with gas bubbles.

Coliform colonies have gas bubbles trapped next to the colony.

Colonies without gas bubbles are not coliforms.

E. coli/Coliform Count Plate (EC)

The EC plate counts all coliforms in a sample and differentiates Escherichia coli from other coliforms. E. coli is used as an indicator of faecal contamination in meat products and other foods. The EC plate contains: lactose nutrients violet red bile to select for the family Enterobacteriaceae TTC indicator to assist in visualising colonies the BCIG indicator which colours E. coli colonies blue a cold water-soluble gelling agent Incubation time: 24 hours Incubation temperature: 35C When quantifying growth, count all blue colonies which are associated with gas bubbles as E. coli and all red colonies which are associated with gas bubbles as other coliforms.

E. coli colonies have gas bubbles and are coloured blue.

Colonies of other coliforms have gas bubbles and are red.

Colonies without gas bubbles are not coliforms.

Safety in Microbiology
Since experiments described in this manual deal with live microorganisms, it is essential that caution be exercised. When plates are inoculated prior to incubation, they may contain only a few microorganisms per plate. After incubation, each single microbial cell will have multiplied to over 1,000,000 cells, and at that level may present a risk. Plates presented to the class for examination and counting should either be taped shut or placed in a zipper storage bag so they cannot be opened. Plates with viable colonies must be disposed of in a responsible way. Autoclaving, soaking in an appropriate disinfectant, or using a contract collection service such as Stericorp are all acceptable means of disposal. Adequate antibacterial hand wash and hand rub solutions must be provided so that all students may wash their hands prior to leaving the class.

Experiments
Yeasts and Moulds in the Air
Students may use this experiment to qualitatively demonstrate the presence of yeast and mould spores in the air. They may also quantify the number of spores detected and test different areas for a comparison. Equipment: 3M Petrifilm YM plates Sterile diluent Sterile pipettes Tape Procedure: Rehydrate as many Petrifilm YM plates (with a sterile diluent and pipette) as are required for the class, and allow to gel for at least one hour. The exact procedure is described in the Environmental Monitoring Procedures manual and can be sourced from www.3M.com/microbiology or from Southern Biological. Peel back the top film without touching the rehydrated culture media, and expose the plate to the air for precisely five minutes. Reserve one or two plates to use as controls. Hydrate these plates but do not expose them to the air. Use double-sided tape to hold the plates open for the duration of their exposure. Fold a piece of single-sided tape onto itself to make it double-sided. Incubate the plates for 3-5 days at 20-25C (ambient temperature will suffice). Count the colonies as described in the YM plate section. The YM plate has an area of 30 square cm. Since both the plate and the top film are exposed to the air, the total exposure area is 60 square cm. The resultant count should be exp ressed as cfu/square cm/minute. Notes: Petrifilm plates can be rehydrated and stored in a refrigerator for up to two weeks prior to use. Placing plates in front of air conditioners or air vents will guarantee a high count.

Bacteria on Fingers (Use of Topical Antimicrobials)

This experiment will clearly demonstrate the benefits of washing and sanitising hands. Specific variables may be tested using this procedure. For example, students may test: volume of antibacterial rub, brand of antibacterial rub, time passed after use of bacterial rub, etc.

Equipment: 3M Petrifilm AC plates Sterile diluent Sterile pipettes Topical antibacterial rub (e.g. Avagard, chlorhexidine/alcohol) Procedure: Rehydrate as many Petrifilm AC plates (with a sterile diluent and pipette) as are required, and allow to gel for at least one hour. The exact procedure is described in the Environmental Monitoring Procedures manual and can be sourced from www.3M.com/microbiology or from Southern Biological. Using a marker pen, divide the plate in two by marking the top film with a line. Label one side unwashed and the other side washed. Peel back the top film and touch the three middle fingers directly on the gel on the inside of the top film (touch only on the unwashed side). Return the top film to the plate when finished. Sanitise both hands with the antibacterial rub and allow to air dry. Pay particular attention to sanitising the finger tips. Repeat the inoculation procedure using the sanitised fingers on the washed side of the top film. Incubate the plates for two days at 35 C. Notes: Petrifilm plates can be rehydrated and stored in a refrigerator for up to two weeks prior to use. After bacterial colonies have grown under incubation, the plates may be refrigerated for up to two weeks and still show typical colonies. They may also be frozen after growth and will show typical colonies almost indefinitely.

Total Bacterial Population in Milk (Shelf-Life Determinations)

By law, pasteurised milk must have a bacterial count of fewer than 50,000 cfu per mL prior to leaving the factory. Otherwise, it will fail to last the two weeks until its use-by date. By allowing fresh milk to sit at room temperature for 8 to 24 hours, the bacterial population will increase substantially, thus guaranteeing a reasonable count. This experiment may be conducted in conjunction with the following experiment, Temperature Effects on Bacterial Populations. Equipment: 3M Petrifilm AC plates 9 mL bottles of sterile diluent Sterile pipettes Milk Procedure: Prepare temperature-abused milk by leaving a container of milk at room temperature for 8 to 24 hours. Using a sterile pipette, add 1 mL of temperature-abused milk to a 9 mL container of sterile diluent. Mix well and discard the pipette. Repeat this procedure three more times, each time sampling from your most recent dilution with a fresh sterile pipette. You should now have a 1:10, 1:100, 1:1000 and 1:10000 dilution of your milk. This is called a serial dilution and allows us to reduce the bacteria to a countable level. Take a fresh sterile pipette and plate 1 mL of the highest dilution (1:10000) to an AC plate. Using the same pipette, plate 1 mL of the next highest dilution (1:1000) to a second AC plate and then plate 1 mL of the 1:100 dilution to a third AC plate. Incubate the plates for two days at 35 C. Count the colonies as described in the AC plate section. Calculate the cfu per mL of milk by multiplying the plate count by the dilution factor of that plate. For example, if the 1 in 1000 dilution plate had 56 colonies, then the count for the undiluted milk would be 56 x 1000 = 56,000 cfu/mL. It is common to use scientific notation in microbiology, so the result would be expressed as 5.6 x 104 cfu/mL.

Questions for students: Why is it necessary to use a fresh pipette during each stage of the serial dilution? Why is it acceptable to reuse the same pipette when inoculating the AC plates, starting with the most dilute and ending with the least dilute? Notes: A count of 25-250 bacterial colonies is ideal when quantifying growth on AC Petrifilm plates. By conducting a serial dilution and using multiple dilutions to inoculate pla tes, we increase our chances of having one plate in this ideal range.

Effect of Temperature on Bacterial Populations

Heating is one of the principle ways in which microorganisms can be killed. This experiment investigates the temperatures necessary to kill bacteria in milk. This experiment is ideally conducted in conjunction with the previous experiment, Total Bacterial Populations in Milk. Equipment: 3M Petrifilm AC plates Sterile pipettes Milk Hot plate Beaker Test tubes Procedure: Conduct the previous experiment to prepare temperature-abused milk and determine its bacterial population. Prepare a water bath by putting a beaker of water with a thermometer on a hot plate. Place a test tube of the milk into the water bath, and slowly heat the water to 50C. Using a sterile pipette, plate one mL of the milk directly onto a Petrifilm AC plate. Repeat this procedure at 60 C, 70C and 80C. Incubate the plates for two days at 35 C. Count the colonies as described in the AC plate section. Compare the population of the milk before heat treatment to the populations after being heat treated to the different temperatures. Notes: A temperature of 80C should be sufficient to kill nearly all bacteria in milk. Students may also investigate the effect of heating time on bacterial populations. For example, 60C for 30 minutes will kill more bacteria than 60C for 5 minutes. Traditionally accepted temperatures for pasteurising milk are 63C for 30 minutes
or 72C for 15 seconds.

Coliforms and E. coli in Ground Meat

This experiment is similar to the Total Bacterial Populations in Milk experiment, but it requires a different method of processing because the tested food is solid. Additionally, this experiment tests specifically for E. coli and other coliforms, bacteria species that are the principle indicators of faecal contamination in the food industry. The Meat Standards Committee has set the following microbiological limits for E. coli in raw meat: Meat Quality Excellent Good Acceptable Marginal E. coli cfu per gram 0 1-10 10-100 100-1000

Equipment: 3M Petrifilm EC plates 90 mL bottles of sterile diluent Sterile pipettes Sterile stomacher bags Sterile spoon Raw minced meat (any kind) Procedure: Place approximately 10 grams of raw minced meat in a sterile stomacher bag with a sterile spoon and add 90 mL of sterile diluent. Mix the contents of the bag by mashing the mixture with your hands from the outside of the bag for at least 30 seconds. This effectively washes the bacteria into the diluent. Using a sterile pipette, plate 1 mL of the liquid from the bag onto a Petrifilm E. coli/Coliform Count plate. Incubate the plates for two days at 35 C. Count the colonies as described in the EC plate section. Calculate the E. coli and coliform counts per gram of meat. Because the dilution factor used was 10, the plate counts must be multiplied by 10. Use the table above to determine the microbiological quality of the meat according to the Meat Standards Committee. Notes: This experiment may be conducted using Petrifilm CC plates instead of EC plates, but it will then not be possible to distinguish between E. coli colonies and other coliform colonies.

Count of Bakers/Brewers Yeast

The yeast Saccharomyces cerevisiae is widely used in industry for brewing alcoholic beverages and baking bread, and it is the main ingredient in Vegemite. This experiment involves a large serial dilution and allows for the quantification of yeast sold in sachets. Equipment: 3M Petrifilm YM plates 9 mL bottles of sterile diluent Sterile pipettes Sachet of dehydrated yeast Electronic balance Procedure: Determine the mass of the dehydrated yeast in a sachet. It may be printed on the packaging, or else you may weigh the dried yeast on an electronic balance. Take a very small pinch of the yeast and determine its mass by either weighing it directly or reweighing the remaining yeast to find the difference. Add the pinch of yeast to a 9 mL bottle of sterile diluent and mix thoroughly. Perform a serial dilution to achieve dilution factor of 1,000,000. Plate the last three dilutions (10,000; 100,000; and 1,000,000) onto Petrifilm YM plates. You may use the same pipette if you start from the most dilute and work your way towards the least dilute. Incubate the plates for 3-5 days at 20-25C (room temperature). Count all aqua green colonies as described in the YM plate section. Questions for students: Assume that the contents of the yeast packet are entirely made up of dehydrated yeast cells and that each cfu is a single yeast cell. Determine the number of yeast cells in your pinch. Use your results to calculate the average mass of a yeast cell. Use your results to calculate the number of yeast cells in a sachet. Commercial yeast makers produce yeast in fermentation vessels that are 10 metres in diameter and four stories high. Do some calculations to estimate the number of yeast cells that would be present in such a vessel at the end of a fermentation. Note the assumptions made during your estimate. Why is it acceptable to reuse the same pipette when inoculating the YM plates, starting with the most dilute and ending with the least dilute? Notes: Dehydrated yeast sachets are cheaply available from any supermarket.

Bacterial Populations on Surfaces

Microorganisms can be found on almost all surfaces. The CSIRO has guidelines on acceptable limits for work surfaces in the food industry. General surfaces are considered acceptable if they have less than six cfu per square cm. Easily cleaned surfaces are considered acceptable if they have less than one cfu per square cm. This experiment may use either AC plates to count bacteria or YM plates to count fungi. Equipment: 3M Petrifilm AC or YM plates Sterile diluent Sterile pipettes 3M Quick Swab Procedure: Rehydrate as many Petrifilm AC or YM plates (with a sterile diluent and pipette) as are required for the class, and allow to gel for at least one hour. The exact procedure is described in the Environmental Monitoring Procedures manual and can be sourced from www.3M.com/microbiology or from Southern Biological. Peel back the top film without touching the rehydrated culture media. Press the inside surface of the top film onto the surface to be tested. Ensure that all of the film has touched the area to be tested by gently smoothing down the outside part with your fingers. Return the top film to the plate when finished. Incubate the plates and count the colonies as described in the used plates section. Calculate the cfu per square cm tested. The area of the AC plate is 20 square cm. The area of the YM plate is 30 square cm. The above technique may not adequate ly transfer microorganisms to the Petrifilm plate if the sur face is uneven. In this circumstance, the use of a 3M Quick Swab is preferred: Snap the ampoule of a Quick Swab and squeeze the bulb so that all of the diluent is pumped into the barrel. Remove the moistened swab from the barrel and swab the area to be tested. Put the swab back into the barrel and shake the swab vigorously for at least 15 seconds. This will wash the microbes captured on the swab into the diluent. Remove and discard the swab. Plate the contents onto an AC or YM plate. Incubate the plates and count the colonies as described in the used plates section. Calculate the microbiological level by counting the colonies per plate, then dividing by the area tested.

Glossary of Terms
AC plate Aerobic Count Plate. aseptic Sterile. Performed in a manner to keep microorganisms out. CC plate Coliform Count Plate. cfu Colony forming unit. diluent Sterile fluid used to dilute a sample or re-hydrate a plate. Usually 0.1% peptone solution or sterile water. EC plate E. coli/Coliform Count Plate. inoculate To apply a sample containing microorganisms to the test media. microbiology The study of all forms of microorganisms. microorganism An organism not visible to the naked eye for at least part of its life cycle. This includes bacteria, fungi (yeasts and moulds) and viruses. pathogen A biological agent that causes disease or illness to its host. serial dilution Repeated dilutions of a sample to achieve high levels of dilution. Tenfold dilutions are preferred because they make calculations relatively easy. YM plate Yeast and Mould Count Plate.

Appendix
Relevant websites: o www.3m.com/microbiology This site allows the user to access information on Petrifilm products including interpretation guides and specific microbiological applications. o www.southernbiological.com This site has information on Petrifilm plates as well as microbiological experiments suitable for the classroom. In addition, there is a range biological and scientific products and information.