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 Baker’s/Brewer’s 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).
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.
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).
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.
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.
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.
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
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: 35°C
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 Sabroud’s 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: 20-25°C
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: 35°C
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: 35°C
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 20-25°C (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
50°C.
• Using a sterile pipette, plate one mL of the milk directly onto a Petrifilm AC
plate.
• Repeat this procedure at 60 °C, 70°C and 80°C.
• 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 80°C should be sufficient to kill nearly all bacteria in milk.
• Students may also investigate the effect of heating time on bacterial populations.
For example, 60°C for 30 minutes will kill more bacteria than 60°C for 5 minutes.
• Traditionally accepted temperatures for pasteurising milk are 63°C for 30 minutes
or 72°C 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 Baker’s/Brewer’s 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 20-25°C (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 plate’s 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 plate’s 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.