BriefIntroMicro3MPetrifilmPlates

8/6/2019 BriefIntroMicro3MPetrifilmPlates

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A Brief Introduction toMicrobiology 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


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Index

1. Introduction2. 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 Microbiology4. Experiments

o Yeasts and Moulds in the Airo Bacteria on Fingers (Use of Topical Antimicrobials)o Total Bacterial Population in Milk (Shelf Life

Determinations)

o Effect of Temperature on Bacterial Populationso Coliforms and E. coli in Ground Meato Count of Bakers/Brewers Yeasto Bacterial Populations on Surfaces

5. Glossary of Terms6. Appendix


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Introduction

Microbiology is the study of organisms toosmall to see with the naked eye

microorganisms. These include bacteria andfungi, which are often of prime concern to a

microbiologist. Some organisms, such asmould, are visible to the naked eye, but are

still considered part of microbiologybecause a significant phase of their life cycle

is microscopic.

Microorganisms are ubiquitous they arefound everywhere. Throughout history,

microorganisms have been both friends andfoes to humanity. Organisms with the roleof foe would include Yersinia pestis (theplague), Mycobacterium tuberculosis

(tuberculosis), Mycobacterium leprae(leprosy), Vibrio cholerae (cholera),

Salmonella, Campylobacter, Staphylococcusand Listeria (various forms of foodpoisoning).

As a friend, there are organisms such as

Lactobacillus (fermented meats, cheeses andyoghurts), Saccharomyces cerevisae (beer,

wine and bread), Acetobacter (vinegar),Propionibecterium (holes in Swiss cheese)

and Penicillium (antibiotics).

Most microorganisms are generallyharmless, but we should always remember

that life on Earth would not be possiblewithout microorganisms. In addition, all

species on Earth, including humans, haveevolved from microscopic organisms that

lived far in the past.

While we now know much aboutmicroorganisms, this knowledge has beenfairly recent. The microscope, which allows

us to see microorganisms directly, was notinvented until the Seventeenth Century.

Even after its invention, the fullramifications of microbiology did not

become apparent until the NineteenthCentury.

Louis Pasteur (1822-1895) was able to use

the microscope to demonstrate that in certainalcoholic fermentations (beer and wine

production), the fermentation process wascarried out by living microorganisms

(yeasts), and that specific types of yeastproduced good batches while others

produced bad batches.

Consequently, the concept of aetiology(study of cause and effect) has become apart of microbiology. For example, foodspoils because microorganisms degrade the

food. If you eliminate the microorganismsby sealing the food in a can and heating it to

a high temperature, the food will last foryears. The cause is the presence ofmicroorganisms, and the effect is spoilage.

Robert Koch (1843-1910) was able to

demonstrate that certain bacteria causedcertain 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 beendeveloped for the majority of diseases in

humans and other animals. We can testfoods for the presence of known pathogenic

(harmful) bacteria, test for other organismsthat indicate the presence of pathogenic

bacteria and test for spoilage organisms tosee how long food will last.

This manual describes some basicmicrobiological techniques along with safety

tips for dealing with live microorganisms. Italso has some fun experiments to do in the

classroom to teach how microorganismsgrow, how to isolate them and how to studythem.


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3M Petrifilm Plates

Petrifilm plates are thin film, sample -ready,dehydrated versions of the conventionalPetri dish agar plate. They are ready to use

immediately after taking them out of theirpackets and have several advantages over

conventional agar plates. These includebuilt-in biochemical confirmation, ease ofpreparation and use, and smaller volumerequirements (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 thismanual, all of which are considered safe forgeneral educational use. All plates

described require a one mL sampleinoculation. Other Petrifilm plates are made

to isolate known pathogens, but these areunsuitable for most educationalenvironments and are therefore notdescribed in this manual. Petrifilm plates

have international recognition by AOACand AFNOR, and are widely used in

industry in Australia and internationally.


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Aerobic Count Plate (AC)

The AC plate counts nearly all aerobic andfacultative anaerobic bacteria in a sample.

The AC plate contains:

plate count nutrients the coloured dye triphenyl

tetrazoliumchloride (TTC) whichcolours all bacterial colonies red

a cold water-soluble gelling agentIncubation 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 withinthe two day incubation time, and they are easily distinguished from bacteria since they do notreduce 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.


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Yeast and Mould Count Plate (YM)

The YM plate counts nearly all common yeastand mould species in a sample.

The YM plate contains:

modified Sabrouds dextrose nutrients two broad spectrum antibiotics to

suppress bacterial growth

an alkaline phosphatise indicatorwhich colours all yeasts aqua green

a cold water-soluble gelling agentIncubation 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.

Yeast colonies are

characterized by an

aqua green colour.

Mould colonies arecharacterized by

diffuse edges and a

central focal point.


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Coliform Count Plate (CC)

The CC plate counts all coliforms within asample without differentiating betweengenera. Coliforms are the members of thefamily Enterobacteriaceae which fermentlactose to produce gas. This count has been

used as a measure of faecal contamination indairy products and other foods.

The CC plate contains:

lactose nutrients violet red bile to select for the family

Enterobacteriaceae

TTC indicator to assist in visualisingcolonies

a cold water-soluble gelling agentIncubation time: 24 hoursIncubation temperature: 35C

Colonies of organisms which ferment lactose to produce gas will have gas bubbles trapped in thegel next to the colony. When quantifying coliform growth, count all red colonies which areassociated with gas bubbles.

Coliform colonies

have gas bubblestrapped next to the

colony.

Colonies without

gas bubbles are

not coliforms.


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E. coli/Coliform Count Plate (EC)

The EC plate counts all coliforms in asample and differentiates Escherichia colifrom other coliforms. E. coli is used as anindicator of faecal contamination in meatproducts and other foods.

The EC plate contains:

lactose nutrients violet red bile to select for the

family Enterobacteriaceae

TTC indicator to assist invisualising colonies

the BCIG indicator which coloursE. coli colonies blue

a cold water-soluble gelling agentIncubation time: 24 hoursIncubation 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 gasbubbles and are red.

Colonies without

gas bubbles are

not coliforms.


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Safety in Microbiology

Since experiments described in this manualdeal with live microorganisms, it is essential

that caution be exercised.

When plates are inoculated prior toincubation, 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 atthat level may present a risk.

Plates presented to the class for examination

and counting should either be taped shut orplaced in a zipper storage bag so they cannot

be opened.

Plates with viable colonies must be disposedof in a responsible way. Autoclaving,soaking in an appropriate disinfectant, orusing a contract collection service such as

Stericorp are all acceptable means ofdisposal.

Adequate antibacterial hand wash and handrub solutions must be provided so that allstudents may wash their hands prior toleaving the class.


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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 arerequired for the class, and allow to gel for at least one hour. The exact procedureis 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 exposethe plate to the air for precisely five minutes.

Reserve one or two platesto use as controls.Hydrate these plates butdo not expose them to the

air.

Use double-sided tape tohold the plates open forthe duration of theirexposure. Fold a piece of

single-sided tape ontoitself 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 expressed as cfu/square cm/minute.Notes:

Petrifilm plates can be rehydrated and stored in a refrigerator for up to two weeksprior to use.

Placing plates in front of air conditioners or air vents will guarantee a high count.


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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 ofantibacterial 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 inthe Environmental Monitoring Procedures manual and

can be sourced from www.3M.com/microbiology orfrom Southern Biological.

Using a marker pen, divide the plate in two by markingthe top film with a line. Label one side unwashed andthe other side washed.

Peel back the top film and touch the three middle fingersdirectly on the gel on the inside of the top film (touchonly on the unwashed side). Return the top film to theplate when finished.

Sanitise both hands with the antibacterial rub and allowto air dry. Pay particular attention to sanitising thefinger tips.

Repeat the inoculation procedure using the sanitisedfingers on the washed side of the top film.

Incubate the plates for two days at 35C.Notes:

Petrifilm plates can be rehydrated and stored in a refrigerator for up to two weeksprior to use.

After bacterial colonies have grown under incubation, the plates may berefrigerated for up to two weeks and still show typical colonies. They may alsobe frozen after growth and will show typical colonies almost indefinitely.


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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-bydate. 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 roomtemperature for 8 to 24 hours.

Using a sterile pipette, add 1 mL of temperature-abused milk to a 9 mL containerof sterile diluent. Mix well and discard the pipette.

Repeat this procedure threemore times, each timesampling from your most

recent dilution with a freshsterile 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 toreduce the bacteria to a

countable level.

Take a fresh sterile pipette and plate 1 mL of the highest dilution (1:10000) to anAC plate.

Using the same pipette, plate 1 mL of the next highest dilution (1:1000) to asecond AC plate and then plate 1 mL of the 1:100 dilution to a third AC plate.

Incubate the plates for two days at 35C. 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.


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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 ACPetrifilm plates. By conducting a serial dilution and using multiple dilutions to

inoculate plates, we increase our chances of having one plate in this ideal range.


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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. Thisexperiment 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 anddetermine its bacterial population.

Prepare a water bath by putting a beaker of water with a thermometer on a hotplate.

Place a test tube of the milk into the water bath, and slowly heat the water to50C.

Using a sterile pipette, plate one mL of the milk directly onto a Petrifilm ACplate.

Repeat this procedure at 60C, 70C and 80C. Incubate the plates for two days at 35C. 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 minutesor 72C for 15 seconds.


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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 E. coli cfu per gramExcellent 0

Good 1-10Acceptable 10-100

Marginal 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 witha sterile spoon and add 90 mL of sterile diluent.

Mix the contents of the bag by mashing the mixture with your hands from theoutside 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 35C. 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 meataccording 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.


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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 experimentinvolves 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 thepackaging, 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 itdirectly 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 dehydratedyeast cells and that each cfu is a single yeast cell. Determine the number of yeastcells 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 afermentation. 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.


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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 consideredacceptable 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 mayuse 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 adequately transfer microorganisms to the Petrifilm plate ifthe surface is uneven. In this circumstance, the use of a 3M Quick Swab is preferred:

Snap the ampoule of a Quick Swab andsqueeze the bulb so that all of the diluentis pumped into the barrel.

Remove the moistened swab from thebarrel and swab the area to be tested.

Put the swab back into the barrel andshake 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.


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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 lifecycle. 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.


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