Food Microbiology Practical Manual 2020

FOOD2320/8320 Food Microbiology Practical Manual Page 1
COURSE TIMETABLE
Week Lecture topic/s Laboratory topic/s

(1) • Introduction to food microbiology Laboratory safety and microbiology basics
14 Sept • Microbial ecology of foods
(2) • Sampling and analysis Examination of food by microscopy and standard

21 Sept • Quality control and indicator organisms plate counts
(3) Contemporary analysis methods Examination of food by contemporary methods

28 Sept
(4) • Labour day public holiday

Primary beer fermentation (voluntary)
5 Oct • Food fermentation (Beer and wine)
(5) • Food Fermentation (Bread and Cheese) • Progress exam (30%)

12 Oct • Food spoilage
(6) • Secondary beer fermentation (voluntary)

19 Oct
(7) Foodborne illness (Salmonella, Escherichia, Foodborne pathogens

26 Oct Campylobacter, Vibrio)
(8) Foodborne illness (Listeria, Staphylococcus Foodborne pathogens

2 Nov and spore forming bacteria)
(9) Foodborne illness (Viruses, mycotoxins and Foodborne pathogens

9 Nov toxigenic algae)
(10) Course review • Practical exam (30%)

16 Nov • Assignment due (40%)
Gantt chart providing overview of laboratory activities

CONTENTS
Laboratory timetable 1
Objectives 3
Requirements 3
Laboratory rules 4
Course information and laboratory safety 5
Laboratory Week 1: Food microbiology - fundamental practical skills 8
Laboratory Week 2: Examination of food by microscopy and plate counts 12
Laboratory Week 3: Examination of food by contemporary methods 24
Laboratory Week 4: Primary fermentation for beer production 26
Laboratory Week 5: Progress examination 30
Laboratory Week 6: Secondary fermentation for beer production 31
Laboratory Week 7: Foodborne pathogens 36
Laboratory Week 8: Foodborne pathogens 45
Laboratory Week 9: Foodbourne pathogens 51
Laboratory Week 10: Practical examination 52
Appendices
Appendix 1 Setting up the light microscope 53

Appendix 2 Staining of bacteria 59
Appendix 3 Preparation of serial dilutions for enumeration of bacteria 61
Appendix 4 Biochemical tests 62
Appendix 5 Most probable number method 66
OBJECTIVES
Microbiological analysis is an essential component in the management of food quality by the

food industry. It is also an essential component of government departments responsible for
enforcing compliance to food regulations.
The purpose of this laboratory program is to teach you basic skills in handling
microorganisms and the microbiological examination of foods. Understanding the principles
behind the analytical processes and knowing how to interpret microbiological data are
essential parts of the analytical process.
REQUIREMENTS
Students must supply their own:
• Laboratory coat
• Safety glasses
• Permanent marker pen for labelling
LABORATORY RULES
These rules are designed for the protection of you and your fellow students. Please adhere to
them at all times.
1. Adhere to social distancing guidelines.

2. All materials and cultures used in laboratory classes should be considered as potentially
infectious and they should be handled with due care.
3. Wear appropriate clothing in the laboratory at all times, which includes a clean protective
laboratory coat and sturdy covered footwear (not water permeable), even when
performing seemingly safe tasks, such as counting plates.
4. Do not smoke, eat or drink in the laboratory or place any object on the laboratory bench
that you could later transfer to your mouth. Do not sit on laboratory benches.
5. If there is bacterial culture spill, ask a fellow student to call a demonstrator immediately.
Do not move and risk the spread of contamination.
6. When finished with class materials, they must be disposed of in the appropriate manner.
If you are unsure about how to dispose of any of the materials you are using, ask a
demonstrator or a member of the technical staff.
• Place non-disposable labware, such as glass bottles, flasks, tubes and slope cultures in
the corresponding labelled buckets provided for autoclaving.
• Discard disposable labware, such as old plate cultures, in the autoclave bags provided.
Do not overfill the bag. If the bag is about 2/3 full, notify a demonstrator, who will
replace the bag as required.
• Discard used pipette tips and your own microscope slides into the sharps container on
the bench.
• Do not discard any pre-prepared demonstration slides. Leave them on the bench.
• Never place any infectious material or toxic chemicals in the sinks. To avoid
blockages, do not drop anything solid into the sinks.
• Any chemical waste should be disposed of in the appropriately labelled containers.
7. All materials for incubation or refrigeration should be adequately labelled and given to
your demonstrator.
8. Please clean microscopes at the end of the class. Use lens tissue and alcohol to clean the
oil immersion lens and leave the low power objective in place. If the microscope has a
cover, ensure it is in place. Avoid jolts, which may damage the microscope.
9. Tidy your bench. Spray your bench with the disinfectant provided and wipe it dry with
paper towel.
10. Before you leave the laboratory, wash your hands thoroughly with disinfectant, soap and
water.
COURSE INFORMATION & LABORATORY SAFETY
OBJECTIVES
1. To learn and appreciate the rules and conduct for working in a laboratory involving
pathogenic microorganisms.
2. To be inducted in laboratory safety and good laboratory practices of Food
Microbiolgy practical classes.
COURSE INFORMATION
Your demonstrator or the academic staff in charge will outline the scope and content of the
laboratotory component of the course, explain the methods of assessment and answer any
questions you may have about them.
LABORATORY SAFETY
During this course, you will be learning standard methods and procedures for the examination
of microorganisms in foods and beverages. These methods and procedures are essentially
those recommended by Standards Australia and the International Standards Association.
Some of the microorganisms that you will be analysing are well known for their ability to
cause outbreaks of foodborne disease. That is, they are pathogenic - e.g. Listeria,
Escherichia, Staphylococcus.
Infectious (pathogenic) microorganisms have been classified into four Risk Groups by
Standards Australia and other international organisations. You will be working with the
bacteria mentioned above. They are classified into Risk Group 2 (moderate individual risk,
limited community risk).
Consequently, it is very important that you adopt and follow practices that (i) prevent you
from contaminating and infecting yourself, (ii) prevent you from contaminating and infecting
fellow students and staff, and (iii) prevent you from contaminating and infecting the wider
community. In other words, we do not want any microbiological accidents or incidents
arising from this course.
So far, the course has been operating for 29 years, and we have an incident-free record. Let's
keep it that way!!! To do this, you must follow some basic procedures with regards to safe
laboratory practices. Some basic rules are given on Page 4 of your course laboratory Manual.
Please read and follow these rules. Some additional dot points on safety and good laboratory
practice are provided over the page.
• It will benefit you to have some exposure to and training in basic microbiology and know
how to follow standard aseptic techniques for the safe handling of microbial cultures. If
you do not have this experience, let your demonstrator know so they can keep a closer
eye on you.
• It is good practice to assume that all microorganisms, whether they are pathogenic or
non-pathogenic, have the potential to cause disease. Consequently, under no
circumstances should they come into contact with your body, clothes, work manual,
pens, mobile phones, laboratory surfaces, etc. They must be contained in test-tubes,
culture flasks, petri-dishes, etc. Bottles of disinfectant are available on every bench.
• While various pathogenic species occur in foods, they are mostly present at a low, non-
infective population (cfu/g or mL). During the standard procedures of analysis, you will
culture them to much higher populations. In enrichment cultures, they are likely to be
present at populations of 107-108 cfu/mL. A single colony on an agar plate culture is
likely to contain greater than 109 cfu/mL. Consequently, these cultures are hazardous,
and must be handled with caution. The spill of a single drop of culture, presents a
significant risk to safety, and must be disinfected immediately. Report any spills to your
demonstrator immediately. Do not physically touch microbial colonies on agar plates,
except with a standard inoculating loop or wire. Organise your workspace to minimise
the risk of spillages. Under no circumstances should cultures come in contact with your
body.
• Special care is needed when transferring cultures in/out of incubators, waterbaths, and
refrigerators where there are increased risks of spillages, breakages, etc.
• All cultures must be clearly labelled with the microorganism name, current date and your
name.
• Clean-up is a very important requirement in food microbiology laboratories. When you
are finished with a culture, it must be discarded in the appropriate bucket or container for
sterilisation (autoclaving) by the laboratory staff. You will be instructed on where to put
your waste. Your work bench must be completely cleared by you at the end of the
laboratory session. Before you leave, the workbench must be thoroughly washed with
disinfectant, a bottle of which is provided on each bench. You must not discard any
material into the general garbage.
• You must thoroughly wash and disinfect your hands before leaving the laboratory.
• Students who are immunocompromised, pregnant or on special medications may be more
susceptible to microbial infection than others. If you think you are at risk due to such
conditions, please consult with the lecturer who will advise you accordingly. The
bacterial species, Listeria monocytogenes, is particularly hazardous to pregnant females.
Such students must inform the lecturer who will make other arrangements for you in
relation to undertaking the laboratory session associated with this organism.
• Read the appropriate material and plan your laboratory sessions in advance. Understand
what you are doing and why. Discuss any uncertainties or questions with the lecturer or
demonstrator.
LABORATORY INDUCTION
Your demonstrator will give you a laboratory induction, highlighting laboratory rules, safety
information and good laboratory practices. At the end of the induction, you will be asked to
sign a laboratory induction check list, demonstrating that you have read and understood the
laboratory safety information, and agree to abide by the rules at all times when in university
laboratories. You must sign this document. If you do not, you will be excluded from future
laboratory classes.
LABORATORY WEEK 1
Run sheet: 1) Group allocations

2) Laboratory safety discussion
3) Streaking plates
4) Aseptic transfers
FOOD MICROBIOLOGY - FUNDAMENTAL PRACTICAL SKILLS
OBJECTIVE
1. To establish the proficiency of students in the fundamental skills of practical (food)

microbiology
INTRODUCTION
In microbiology, including food microbiology, there are skills considered fundamental to the
competent practice of analytical procedures. These include the ability to obtain pure cultures
by plating on agar media so as to obtain isolated colonies, and the need to perform aseptic
transfers, in the dilution of samples or transfer of cultures from broth to broth, agar to agar,
and so forth.
In this practical, our aim is to develop or assess the skills you currently have in these basic
yet vital areas. Each student will subculture Escherichia coli from both plate and broth
cultures to fresh agar plates so as to obtain isolated colonies, and you will practice aseptic
transfer, using tubes of dilution fluid. You and your demonstrator will examine the plates
after incubation and your ability to perform aseptic transfer will be observed during the class.
The 16-streak technique
This is the most common method of subculture of microorganisms on agar plate media for
the purpose of obtaining isolated colonies. If the technique is performed properly, there
should be little problem in obtaining isolated colonies. The instructions presented here are
given to assist you in achieving this goal.
Observe the diagram below, which indicates how this technique is to be performed. First, an
amount of culture is transferred, using an inoculating loop, to one part of the plate. Using the
loop, the culture is spread over about 1/4 of the plate forming what is known as the pool of
inoculum. When using a 'solid' inoculum, such as that from an isolated colony on a plate, a
small amount of such material should be collected on the edge of a loop, so that it can be
transferred to a fresh agar plate. A large loopful of solid inoculum is not required as even a
small colony will contain 106 - 108 cells (or more correctly, colony forming units, cfu). On
the other hand, care must be taken to use a full loop of liquid culture, to ensure sufficient
inoculum is transferred to an agar plate. A well-grown liquid culture may contain 109 cfu/mL,
which means a loopful, approximately 10 µl, will contain about 107 cfu, equivalent to a small
colony.
A set of four streaks is then made out from the inoculum pool. It is also important that the
loop is held at an acute angle to the plate, such that the loop slides or skims over the surface
of the plate. If the loop is kept too upright in relation to the surface of the plate, the operator
loses some sense of depth, presses too hard with the loop, and is likely to dig into the agar.
The streaks should also be made using the 'edge' of the loop, so that the streak is a narrow
line. If the loop is used 'flat', such that all of the loop is in contact with the surface, it will
carry along a significant amount of inoculum, and the dilution effect of the multiple sets of
streaks will be reduced.
The first set of streaks should extend from within the pool, close to but not touching the edge
of the plate and may be made with or without flaming the loop after preparing the pool. After
completing the first set of streaks, the loop is flamed, and the second set of streaks is made.
This process is repeated until four sets of streaks have been made on the plate. Each set of
streaks should be made as close to the edge of the plate as possible, so that the use made of
the agar surface is maximal.
Figure 1.1 Schematic representation of the 16-streak technique for obtaining isolated
colonies on agar plate media.
Aseptic transfer
Another fundamental skill in microbiology is the ability to transfer material (eg. sterile
media, food sample, culture) from one vessel or culture system to another while maintaining
the integrity of such material. Transfer is made in such a way as to prevent the introduction of
other material (contaminants), and this is termed aseptic transfer. The 16-streak technique
must be performed in this way, as must the transfer of liquid from one test tube to another, a
technique used when preparing dilutions of a sample.
MATERIALS
Per pair
• 4 plates of nutrient agar

• a test tube rack with two test tubes containing distilled water (non-sterile)
• 4 ´ test tube containing sterile Nutrient Broth
Per bench
• plate culture of Escherichia coli

• broth culture of Escherichia coli
EXPERIMENTAL
A Streaking cultures to agar media
1. Using the plate culture of E. coli, streak the culture on a fresh plate of nutrient agar by the
technique described above.
2. Repeat the preparation of a streak plate using the broth culture of E. coli and a fresh plate
of nutrient agar.
3. Ensure the plates are clearly labelled then submit them for incubation at 37°C in an
inverted position (why?).
B Aseptic transfer of a sterile nutrient broth
Although the practice materials for this exercise have been provided to each pair of students,
each student must practice individually, in turn and perform an individual transfer using
nutrient broth. Demonstrators will be working their way through the class to observe you.
1. Fit a pipette tip to the pipette.
2. With the tubes containing distilled water, practice using the pipette to transfer 1 mL of
fluid from one tube/bottle to the other as demonstrated at the beginning of the class (have
you checked that the pipette is set to 1 mL?). Repeat this step as often as you wish until
you feel comfortable that you are performing the technique correctly. If unsure, ask a
demonstrator. Do not place caps on the bench or invert caps and do not touch the pipette
tip against any non-sterile surface.
3. Label two tubes of nutrient broth with your name, and label one tube "A" and the other
tube "B". Then, using a fresh, sterile pipette tip, transfer 1 mL of nutrient broth from tube
A to tube B.
4. Place the tubes in the 37°C incubator.
Checking your streaking and aseptic technique (to be performed in Week 2)
Retrieve your plates and liquids and see how well you carried out the 16 streak technique and
how sterile your liquid transfers were. Any sign of contamination? Take every opportunity to
practise throughout the term.
QUESTIONS
1. Why would you want to obtain isolated colonies on agar media? Try to think of at least
three reasons.
2. Why do we use the term “colony-forming units” instead of “cells”?

LABORATORY WEEK 2
Run sheet: 1) Observe plates and cultures (from Week 1 Pg11)

2) Set up light microscopes
2) Prepare and examine wet mounts
3) Prepare and examine stained slides
4) Observe spoiled foods by microscopy
5) Macerate and serially dilute food samples
6) Spread plate serial dilutions
EXAMINATION OF FOOD BY MICROSCOPY
OBJECTIVES
The objectives of this practical are:
1. to learn and understand the correct procedures for setting up a microscope to observe
microbial cells;
2. to prepare and examine wet mounts of living cells;
3. to prepare and examine Gram stains of microbial cells;
4. to compare the morphologies of some important food related bacteria, yeasts and moulds;
and
5. to rapidly assess the microbial ecology of a spoiled food by microscopic examination.
INTRODUCTION
The individual cells of microorganisms associated with foods are so small that they are not
visible with the naked eye. In order to reveal their many different shapes and sizes, the cells
must be examined under a microscope. Microbial morphology (cell form, such as shape and
size and, for larger organisms, even ultrastructure) may be examined using the microscope in
either of two ways: (i) by observing the living unstained organism, or (ii) by observing dead
cells stained with various dyes. Using microscopes and slides of known calibration, it
becomes possible to determine the actual size of cells and make direct microscopic counts of
organisms (see below). Using a microscope well takes practice.
Living microbial cells are almost colourless and do not have sufficient contrast against the
medium in which they are suspended to be clearly visible using normal (bright field)
microscopy. This problem can be overcome to some extent by examining living cells using
phase contrast microscopy or by staining the cells with dyes, which give the cells colour in
contrast to their surroundings. Staining procedures are particularly useful for examining
bacteria because of their smaller sizes and consequent need for better visibility. Stains also
help to classify organisms.
A good microscope is an essential part of the food microbiology laboratory. All too often
its value in the examination of foods is overlooked. Spoiled food or beverages generally
harbour 106 - 108 cells/g or mL, sufficient numbers to be seen by direct microscopic
observation. A rapid assessment of the microbial ecology of food suspected to be spoiled is

readily obtained by direct microscopic observation of a carefully taken sample.
Total cell counts
The total number of cells in a sample suspension is most quickly and directly measured by
counting the number of cells seen on examination under the microscope. There are two well-
known microscopic counting procedures.
The simplest procedure involves microscopically counting the individual cells in an

accurately determined, very small volume of the sample suspension. Such counting is done
with special microscope slides known as counting chambers. These glass slides are very
finely ruled or etched into many small squares of known area and are so constructed that a
film of suspension of known depth can be introduced between the slide and coverslip.
Consequently, the volume of liquid overlying each square is accurately known. Since this
volume is known and the number of cells within each square can be seen and counted, the
number of cells per mL of original suspension can be determined.
In the other microscopic total cell count method, use is made of a fixed, stained smear of a
sample of the cell suspension. A determined area, 10 x 10 mm, is marked out on a glass
microscope slide with a marking pen. A known volume, for example 0.01 mL, of the sample
suspension is transferred to the slide and carefully spread to cover the entire marked area. The
slide is then heat-fixed and stained with an appropriate dye, such as methylene blue.
Using the oil-immersion objective, or other objective for larger cells (such as the somatic
cells in milk), the area of a microscope field is calibrated. This is done using a special slide,
etched and calibrated with a linear scale. The diameter of the microscope field is then
measured and then the area of the field is calculated. By simple division arithmetic, the
number of microscope fields per smear area (X) is then calculated. The objective is next
focussed on the stained smear and the average number of cells per microscope field, for
several fields, is counted (Y). Multiplication of X and Y then gives the total number of cells
per entire smear area, and hence in the volume of sample suspension applied.
The drawbacks of total microscopic counts are:
• dead cells are not distinguished from living (viable) cells;

• small cells are difficult to see and possibly overlooked, giving an underestimation of
counts;
• lack of sensitivity since sample suspensions must contain at least 106 cells/mL before a
single cell can be seen in a single microscopic field (at high magnification), which means
that a cell suspension containing less than 106 cells/mL is not countable;
• a high degree of accuracy is hard to achieve;
• suspensions must be reasonably free of other particulate matter since these tend to
obscure the counting of microbial cells. This is an obvious issue with food samples.
MATERIALS
Note: Students are encouraged to bring their own spoiled food for examination.
Per bench
• TSA (plate) cultures of the following bacteria: Escherchia coli and Bacillus cereus
• MEA (plate) cultures of the yeasts Saccharomyces cerevisiae
• Nutrient Broth cultures of Escherichia coli and Bacillus cereus (10 mL)
Per class
• Microscopes
• Gram stain reagents
• samples of spoiled foods (brought by the students)
EXPERIMENTAL
A Setting up the microscope (refer to Appendix 1)
NOTE 1: There are several different models of microscope used in the teaching laboratory. A
schematic diagram of a microscope is given in Appendix 1. While the instructions given in
Appendix 1 pertain to a certain brand of microscope, they can be applied to any model of
microscope.
NOTE 2: Mishandling microscopes can cause damage to elements such as lenses, leading to
unnecessary repair and expense. These issues will be raised in class, and proper use of the
microscopes must be followed.
B Preparation and examination of wet mounts
1. Aseptically transfer a loopful of each of the broth cultures provided for examination to the
central portion of a microscope slide.
2. Carefully lower a coverslip over the loopful of suspension so as to avoid trapping air
bubbles within the film of liquid between the coverslip and slide. Avoid an excessive
amount of liquid under the coverslip, as it is likely to float and make microscopic
observations difficult. The slide is now ready for microscopic examination. If the
observation period is to be brief, the wet mount may be examined directly. If your
observation period is to be prolonged, then the heat of the microscope light on the
specimen may cause the suspension to dry out, cause convection movement and generally
making detailed observations difficult. To overcome these problems, the coverslip can be
sealed to the slide by placing molten paraffin wax around the entire edge of the coverslip
or using fingernail polish to seal the slide.
3. Examine the specimen under low power, high power and oil immersion bright field
microscopy.
4. Examine under phase contrast microscopy. Examine wet mounts of all the cultures
provided and record their morphologies. Note differences in cell sizes and shapes.
Note: To prepare a wet mount from an agar slant or plate culture, first place a loopful of
water on the slide. After flame sterilisation of the loop, transfer a minute quantity of culture
growth to the droplet of water and mix to form a uniform cell suspension. This suspension
should be only slightly turbid. If the cell concentration is too high, individual cell
morphology will be impossible to discern. Cover with a coverslip and examine.
C Preparation and examination of a stained slide
1. Aseptically transfer a loopful of the broth culture to the centre part of the slide.
Alternatively, prepare a suspension from plate cultures.
2. Spread the loopful of suspension over approximately 1-2 cm2 of slide area. If the
suspension is spread too thinly, difficulties may arise in finding cells under the
microscope. If the suspension is spread too thick then cells will be clustered and
individual cells will be hard to differentiate on microscopic examination.
3. Allow the film suspension on the slide to air dry. Passing the slide several times rapidly
through the flame of the Bunsen burner can accelerate this process.
4. Once dried, heat-fix the preparation by passing it several times through the flame of the
burner (specimen side up). The slide should not become too hot to hold. Heat fixation
ensures that the cells become affixed to the glass slide so that they do not wash away
during staining.
5. Prepare heat fixed slides of Escherichia coli and Bacillus cereus. Stain each preparation
by the Gram stain technique, for which details of the procedure are given in Appendix 2.
6. Examine the stained slides under oil immersion using bright field microscopy and record
cell morphology and Gram reaction.
D Microscopic examination of spoiled foods
1. Prepare slides (wet mounts) of the samples of spoiled food and examine under phase
contrast microscopy. Take photos, take notes. What cell shapes (cocci, rods, fungi,
yeasts, spores)? Size? Abundance? Arrangements (dispersed, clumping)? Colours?
QUESTIONS
1. What are the differences between bright field microscopy and phase contrast microscopy?
2. What are the advantages and disadvantages of using stained preparations for observing
microbial cell morphology?
3. Describe how you might quantify the numbers of microorganisms in a food/beverage

using direct microscopic observation.
REFERENCES
Zook, C.D. and F.F. Busta (2002) Total viable counts: microscopy In: Encyclopedia of Food
Microbiology, Batt, C.A., Patel, P.D. and Robinson, R.K. (eds), pp. 2176-2180. Academic
Press, London.
EXAMINATION OF FOODS BY STANDARD PLATE COUNT
OBJECTIVES
The objectives of this exercise are:
1. To gain experience in methods for total plate count determination of microbial

populations in foods.
2. To interpret in practical terms the significance of the microbiological data obtained for
each food.
3. To discuss the statistical accuracy of the data obtained.
INTRODUCTION
Microorganisms exist in foods as mixed populations consisting of many different species.

The food microbiologist is faced with two major tasks: (i) isolation and identification of
individual species associated with the food, and (ii) enumeration of the microbial cells (more
general or more specific populations). Both of these requirements demand knowledge and
working experience in basic microbiological culturing procedures.
Microorganisms require suitable nutrients and favourable environmental conditions for

growth. Microorganisms can be grown in both liquid and solid media. Agar, a somewhat
inert and neutral heteropolysaccharide derived from seaweed, is used to solidify liquid media
into the form of a gel. Most laboratory studies in microbiology are made with pure cultures
of a single species of microorganism and a basic skill in microbiology is to be able to prepare
and sterilise cultural media and maintain it in a sterile condition. At the same time, it is
important to be able to inoculate this sterile medium with a pure culture of microorganism
without outside contamination. These requirements form the basis of aseptic technique.
Microorganisms are conveniently cultured by one of three methods. The first and most simple
method is growth in a liquid medium in a test tube where growth is recognised by a
development of turbidity due to the microbial mass. Second, microorganisms are generally
maintained in a stock form for routine use by culturing on an agar medium solidified in the
form of a slant in a test tube or small bottle. The sloped surface of such agars is easily
inoculated with a standard loop or needle. Third, solid media dispensed in the form of agar
plates in Petri dishes is widely used as the form for isolating and checking pure cultures and
for counting the numbers of individual microbial cells in a sample. In the case of agar plate
media, the microorganisms are streaked or spread over the surface of the plate in such a way
that the microbial cells initially in mixed suspension are now separated spatially as
individuals or small groups (collectively, colony forming units or cfu). On growth and
multiplication on the surface of the plate, each cfu gives rise to a visible colony which may be
either counted or isolated. A colony usually (though not always) arises from a single strain
and thus is a source of pure cultures. Colony properties such as size, texture, edge elevation,
surface and colour are quite often characteristic of a particular microbial type and therefore
aid in species identification and recognition.
The most widely used procedure for counting the number of living (viable) microbial cells in
a suspension or food homogenate is the agar plate count method. Two variations of this
method are in common use, the surface spread plate method, and the pour plate method. In
the case of the surface spread plate technique, typically 0.1 mL of the microbial suspension is
inoculated onto the surface of a prepared plate and, using a sterile, bent glass rod, is spread
over the entire surface of the plate. On incubation, colonies develop at locations all over the
surface. In the case of the pour plate procedure, typically 1.0 mL of microbial cell suspension
is pipetted into a sterile Petri dish after which 15~20 mL of molten agar medium (temperature
~48°C) is added. The plate is then carefully rotated and moved (usually with a figure-8
motion) to thoroughly mix the medium and cells before the medium solidifies. On incubation
colonies develop both on the surface and within the agar. In both procedures it is desired that
microbial cell suspensions be diluted before plating so that at least one pair of plates contains
approximately 100 colonies. Greater than 300 colonies per plate results in over-crowding and
difficulties in counting, yielding unreliable data.
Factors affecting the plate count
Some negative aspects of the agar plate count are:

• the occasional possibility of a colony arising from more than one cell, as occurs with
organisms which form cell pairs, chains or clumps. An underestimation of cell numbers
arises in this case, though this is why the term colony forming unit is used;
• the possibility that some of the cell types present in the sample will not grow on or in the
agar medium used or under the conditions of temperature, pH and oxygen tension of the
incubation adopted;
• colonies of some organisms, especially in food samples, occasionally spread over the
surface of the agar medium, thus obscuring the growth and hindering the counting of
other microbial types, and;
• there is a delay in obtaining information due to the requirement for plate incubation and
colony development, usually at least 18-24 hours.
The choice of diluent in which the food homogenate is prepared, and with which subsequent
dilutions are made, can have a significant influence on the final cell count obtained. Many
early and even recent studies have used diluents such as sterile distilled water, sterile saline
and sterile phosphate buffer. Research over the past twenty years has consistently shown that,
with food products, lower cell counts are generally obtained with such diluents when
compared with use of 0.1% peptone water or tryptone-salt solution. Thus, using the former,
an underestimation of the real or true microbial count is obtained. According to some studies,
many bacteria undergo rapid destruction when diluted in water or saline solution. Dilution in
a weak nutrient solution such as peptone evidently has some protective effect against this
destruction. However, the use of more concentrated nutrient diluents such as 1% peptone is
not recommended because of the risk of microbial growth in the diluent prior to analysis.
Peptone solution at 0.1% behaves satisfactorily as a diluent for most foods and is therefore
universally recommended. With seafood products it is suggested that 3% NaCl be added to
this diluent since many of the associated microorganisms would be of marine origin and
would have an obligate requirement for salt. Similarly, 10-20% sucrose should be added to
dilution fluid when analysing foods of high sugar concentration.
The time and temperature of incubation of agar plate counts can have a significant influence
on the final result. In general, it has been found that incubation temperatures of 25-30°C yield
significantly higher counts than at a temperature of 37°C, and that incubation times of 48
hours give higher recoveries than 24 hours. Thus, fresh foods stored at low temperatures are
more likely to encourage the growth of microorganisms best suited to low temperatures such
as Pseudomonas and Bacillus species, which do not generally respond to incubation at 37°C.
Sampling
Sampling is the first step in the identification and enumeration of microorganisms in food. It
involves two major considerations; sample selection and sample suspension. First, prior to
counting, the number of samples and the size of sample must be determined. Thus, sample
selection involves sampling plans which state how many lots or units should be examined and
the amount of the sample to be analysed. Sample suspension preparation follows, and its
major aim is to release the microorganisms from the sample into a sterile diluent, suitable for
counting. A number of different sampling methods are available but aseptic technique is of
utmost importance. Sterile sampling equipment should be used, including all the collecting
equipment and materials, to minimize any contamination and changes in microbial
populations prior to examination. The following is a brief description of some sample
suspension techniques.
1. Maceration is a procedure whereby whole or part of the product is mechanically

homogenised using a blender or stomacher.
Blending is one method of maceration conducted in a domestic food blender or Waring
blender. Blending time may vary with each food type. It is a very good method for bringing
cells associated with solid foods into suspension. Disadvantages include destruction of some
cells due to mechanical disruption, destruction of the food product and the need for sterile
blending equipment.
Stomaching counters many of the problems associated with blending. In this method, the
food sample is placed with sterile diluent into a sterile bag, which is agitated for 1 minute in a
special machine called a Stomacher. Advantages include no sterilization and little
maintenance of equipment and, in turn, considerable reduction in time and cost.
Disadvantages include breakage of the plastic bags which limits the types of foods that can be
placed into the Stomacher, for example, foods which have sharp projections or bones.
Another disadvantage is the disposal of the homogenized contents.
Some of the published literature on homogenisation indicates that the stomacher releases
significantly larger numbers of viable microorganisms than blending, while others found
slightly lower recovery. Other investigators have found that both methods gave comparable
results with no significant differences between bacterial counts. Any advantage of one
method over the other rests with the type of food sampled and the conditions of sampling.
The stomacher is a more economical and convenient means of maceration but is limited in
the types of food that can be sampled, whilst the blending method works well with generally
all foods but has its limitations in convenience and cost.
2. Rinsing - removal of organisms from surfaces of food using fluid.

In many cases the microbial flora of a product is restricted to the surface areas and may be
sampled by "whole rinsing". Such foods include cut meat, whole fish, and chicken carcasses.
In this method, the sample is placed in a sterile plastic bag with an equal w/v of sterile diluent
and shaken by hand. It is a non-destructive quick procedure, economical, and counts may be
made from the entire surface area of the product. It has been found, in some studies, to be the
most preferable method of sampling. However, not all the microorganisms may be brought
into suspension due to strong surface attachment, so that underestimation of the true
microbial population can occur.
3. Swabbing and surface contact - use of a solid object to remove surface microorganisms.
Swabbing involves sampling the surface of foods and is also employed in monitoring sanitary
conditions of food contact surfaces and eating utensils. Foods that are commonly sampled by
swabbing include meats, chicken and fish. It involves swabbing a measured surface region of
the food, transferring this swab to a sterile diluent and shaking to dislodge any
microorganisms into suspension. Normal plate counting then follows. This is a rapid,
inexpensive and convenient method, enhanced by the availability of commercial products,
such as the Millipore Swab-Membrane Filter Kit. The major restriction to this method is that
only a small area of the product under test is swabbed and not all the microorganisms may
adhere to the swab. What might be a further limitation of swabbing?
Many bacteria are spread by surface contamination. Surface contact plating or the contact
plate method is used to determine the bacterial number in this case. The surface of an agar
medium is bought into contact with the sample, and after incubation, colony counts are made.
Disadvantages include a small area of sampling and microorganisms that do not adhere to the
plate. Residue of the medium may also be left on the surface. Note that we will not be using
this technique.
4. Membrane filtration
This procedure is performed by filtering a known volume of sample through a sterile

membrane of a pore size 0.45 µm suitable to trap all the microbes. The filter is transferred to
the surface of the growth medium and incubated, after which colonies that develop on the
surface of the membrane are counted to evaluate the number of cells per mL of the original
sample. Note we will not be using this technique.
MATERIALS
Per pair
• 1 ´ 90 mL dilution fluid (unless otherwise stated, this is 0.1% tryptone + 0.85% sodium
chloride)
• 6 ´ 9 mL dilution fluid
• 6 ´ plates of Plate Count Agar
• hockey stick
Per class
• 1 ´ Stomacher and bags

• 8 ´ Large plastic bags (for rinsing)
• Instruments for sampling: forceps, scalpels, spoons, spatulas
• 2 ´ incubators, at 30°C and 37°C
EXPERIMENTAL
Each student pair will conduct a count on a sample of food purchased from a retail outlet.
Although various methods of sample preparation and enumeration are detailed below, only a
selection, as specified by the demonstrators, will be carried out.
Food sample preparation by homogenisation or rinsing
Homogenisation
1. Sterilize sampling instruments (as required; knife, forceps, spatula) by dipping into
ethanol and burning off using a low Bunsen flame. Use the instruments to take 10 g of
food sample and place into Stomacher bag. Take portions of sample from different
locations in the food.
2. Aseptically transfer 90 mL of dilution fluid from the flask into the Stomacher bag.
3. Place the bag into the Stomacher for between 30 s and 1 min.
4. Remove the bag from the Stomacher. Use the liquid to prepare dilutions as described
below. After sampling the liquid, discard the Stomacher bag containing the suspension in
biological waste bins provided.
Rinsing (for chicken only)
1. Transfer the food sample (whole chicken carcass) into a sterile plastic bag and weigh the
sample.
2. Add 250 mL of sterile dilution fluid from a flask.
3. Shake and massage the sample for 1-2 min.
Serial dilutions (decimal, 1/10, 1:9 dilutions) – see Appendix 3
1. You are provided with screw-capped bottles each containing 9 mL of dilution fluid. Label
each bottle with the dilution for which it is to be used. Note that homogenisation and
rinsing both generate a 10-1 dilution to start with, whilst beverages or liquid food samples
start undiluted (100).
2. Transfer 1 mL of the sample or sample homogenate using a 1 mL Gilson pipette and tip
to the first dilution bottle. Maintain aseptic technique. After the transfer, mix the bottle
thoroughly by drawing into and expelling fluid from the pipette used for the transfer or by
swirling.
3. Using a fresh pipette tip, dip about 1 cm into the first dilution, remove 1 mL and transfer
this to the next 9 m1 bottle. Mix thoroughly.
4. Using a fresh pipette tip each time, prepare successive dilutions.
5. Using this method prepare the following dilution series:
1/10 (10-1)
1/100 (10-2)
1/1000 (10-3)
1/10000 (10-4)
Spread plating to enumerate cells
1. Using a sterile 1 mL pipette or a 200 µl Gilson pipette and tip, transfer 0.1 mL of food,
homogenate or dilution to the surface of a pre-prepared Petri dish of PCA. Prepare plates
in duplicate for each dilution.
2. Spread the inoculum over the entire surface of the plate using a hockey stick.
3. Allow the fluid from the inoculum to be absorbed into the plates, then invert and incubate
at 30°C for 72 h.
Colony counting and calculation of results (to be performed in Week 3)
NOTE: This is a general description of the method for calculating counts from plates, and the
method differs slightly for a general count of a population compared to the count for a
specific organism. You will need to make reference back to this method when performing
future practical exercises.
For spread plates, select duplicate plates that yield between 15 and 300 colonies per plate. For
counts of specific organisms, the maximum number of colonies per plate, both typical and
atypical is 300 and the maximum number of typical colonies is 150. The minimum number of
colonies on at least one plate (whether total colonies, typical colonies or confirmed colonies,
depending on test) is 15. Remember when making the final calculation, that only 0.1 mL was
inoculated onto the plates (so multiply by 10 to get a per mL value). To obtain counts per mL
or g of the product, the count data must be multiplied by a factor of 10.
The number of organisms in a test sample, N, is calculated as a weighted mean from two
successive dilutions, using the equation:
åC
N=
V ´ [n1 + (0.1 ´ n2 )] ´ d
Where
åC is the sum of the colonies on all the plates from two successive dilutions, and where at
least one contains a minimum of 15 colonies
V is the volume of inoculum applied to each dish (in mL)
n1 is the number of dishes retained at the first dilution
n2 is the number of dishes retained at the second dilution
d is the dilution factor corresponding to the first dilution retained [d =1 when the
undiluted liquid product (test sample) is used]
Round the value obtained for N to two significant figures. If the third numeral is less than 5,
do not change the second figure; if equal to or more than 5, then round the second figure up
(e.g. 1.53 = 1.5, while 1.56 = 1.6). Present the result as a whole number with two significant
figures (e.g. 150) or as a number between 1.0 and 9.9 multiplied by a power of 10 (e.g. 1.5 ´
105). Report the result as the number of colony-forming units per gram or milliliter of
product.
Example calculation
This example is taken from ISO7218 (ISO, 2001)
Counts at 10-2, 168 and 215, counts at 10-3, 14 and 25.
åC 168 + 215 + 14 + 25 422

N= = -2
= = 19182 = 1.9 ´ 10 4
V ´ [n1 + (0.1 ´ n2 )] ´ d 1 ´ [2 + (0.1 ´ 2)] ´ 10 0.022
Report as 1.9 ´ 104 cfu /g or /mL of product.
QUESTIONS
1. With respect to microbiological methods, define the terms: (i) accuracy, (ii)
reproducibility/precision, (iii) sensitivity, (iv) specificity or selectivity.
2. Using the class data, briefly evaluate the accuracy and reproducibility of the various
techniques used in sample preparation.
3. Why should you use a high concentration of sugar in the dilution fluid when testing
products such as sugar syrups?
Standard Plate Count Data
Record your observations as detailed on the following record sheet.
Stomaching
Dilution Counts/plate cfu/g or cfu/mL
-1
-2
-3
Rinsing
-1
-2
-3
REFERENCES
International Organization for Standardization (2001) International Standard ISO7218. Microbiology

of food and animal feeding stuffs – general rules for microbiological examinations. International
Organization for Standardization, Switzerland.
Standards Australia (2004) Australian Standard AS5013.1 – 2004. Food Microbiology. Method 1:
Examination for specific organisms – Standard plate count. Standards Australia International Ltd,
Sydney.
Standards Australia (2004) Australian Standard AS5013.5 – 2004. Food Microbiology. Method 5:
Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of
microorganisms – colony count technique at 30°C. Standards Australia International Ltd, Sydney.
Standards Australia (2004) Australian Standard AS5013.11.1 – 2004. Food Microbiology. Method
11.1: Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination – General rules for the preparation of the initial
suspension and decimal dilutions. Standards Australia International Ltd, Sydney.
Cox, J.M. and G.H. Fleet (2003) Chapter 5. New directions in the microbiological analysis of foods.
Pages 103-161. In Foodborne Microorganisms of Public Health Significance. Sixth Edition. Hocking,
A.D. (Editor-in-Chief). Australian Institute of Food Science and Technology (NSW Branch) Food
Microbiology Group, Sydney.
LABORATORY WEEK 3
Run sheet: 1) Collect and collate standard plate count data (Week 2 pg21)
2) Observe industry demonstrations
3) Ask questions and take notes
EXAMINATION OF FOOD BY CONTEMPORARY METHODS
OBJECTIVES
The aims of this laboratory exercise are to:
1. Expose students to a range of modern and/or rapid methods for the detection, enumeration,
identification or differentiation of microorganisms or their toxins associated with foods.
2. Provide hands-on practical experience of selected techniques.
INTRODUCTION
Traditionally, microorganisms have been detected, enumerated, identified and characterised using
conventional microbiological procedures. These utilise the test tubes, Petri dishes, and relatively
long incubation times you are now all familiar with. The techniques for detection and
enumeration have relied on growth and typically, as an endpoint, the presence, absence or
number of a specific type or the total number of visible colonies on an agar plate medium. Over
the last 30 years, an extensive and diverse range of techniques has been developed to facilitate
and accelerate these procedures.
Facilitated cultural techniques, such as Petrifilm, still rely on detection and enumeration of
colonies, after incubation of media for similar times, they ease the process of culture through
provision of preformed media. Such techniques do not generally accelerate the testing process.
On the other hand, a range of methods both facilitates and accelerates detection or enumeration.
Many of these techniques rely no longer on the observation of colonies. Some rely on the
detection of specific parts of an organism, such as the detection of an antigen in an immunoassay,
or a specific gene in the PCR. Others are based on the relative enumeration of organisms through
detection of a universal molecule, such as ATP in the bioluminescence assay. Still others rely on
the indirect detection of a specific organism or a population of organisms through their activities,
such as the changes in electrical properties brought about in culture media during the growth of
microorganisms.
In addition, a range of methods has been developed to identify and/or characterise microbial
isolates. Once again, these have moved from phenotypic tests performed using various culture
media, through the facilitation of miniaturised biochemical test kits, to the chemotypic methods
of today, which rely on analysis of cell components, in particular the nucleic acids.
While it is useful as students of food microbiology to know and understand the principles on
which these methods are based, as future professionals in the food industry it is just as important,
if not more so, to consider the ‘real-world’ issues associated with these techniques. Is the
technique under consideration accurate, precise, robust? Does it offer the desired level of
sensitivity of limit of detection? Is it specific? Is it appropriate for use in your specific context or
operation? Will it work with your food product? How much does the instrument cost? How much
does it cost per test? What technical support is offered? What are the service arrangements if the
equipment breaks down? These are just a few questions that have to be asked when trying to
select a method from the myriad available today.
In this practical, you will have the opportunity to observe a number of technologies as well as
meet and discuss with industry representatives their specific kits or systems. It is important that
you not only use this exercise to see how tests work, thereby reinforcing the theory provided in
lectures, but to also try to answer, through your observations, experiences and discussions some
of those practical questions listed above.
MATERIALS AND EXPERIMENTAL
As the methods to be demonstrated or made available as hands-on practical exercises may vary
from year to year, an accurate list of materials and experimental protocol is impossible to prepare.
Make your own notes on materials used, how techniques were performed, the results you
obtained, and any interpretation of those results.
LABORATORY WEEK 4
Run sheet: 1) Head to Food Laboratory SEB123

2) Sanitise fermentation vessel
2) Mix the wort
4) Inoculate the fermentation
5) Measure specific gravity
FERMENTATION FOR BEER PRODUCTION
OBJECTIVES
1. To consider the contributions of yeasts to the fermentation of alcoholic beverages

2. To ferment wort to produce beer
INTRODUCTION
The production of alcoholic beverages is a giant international industry of substantial significance

to the economics of most countries. The microbiology of alcoholic beverages products is complex
and fascinating (Fleet 1998) and has been studied since the pioneering contributions of Louis
Pasteur in the 1850-1860s in France. The yeast species, Saccharomyces cerevisiae, is the key
microorganism in all fermentations of alcoholic beverages, but other species can make a
contribution (sometimes particular species of bacteria are involved). The key process, is the yeast
metabolism of sugars into principally ethanol and CO2, plus small amounts of secondary
products that add flavour. Also, microorganisms can play a major role in spoiling alcoholic
beverages.
Beer is one of the more popular and widespread alcoholic beverages. It is prepared by fermenting
the extracts of cereal grains, especially malted barley (Campbell, 1997). The basic raw materials
for beer brewing are malted barley (which provides the sugars for conversion into alcohol), hops
(for flavour), water and yeast (to catalyse fermentation reactions, almost always Saccharomyces
cerevisiae). In addition, other cereals (eg wheat, corn, oats, sorghum) and sugar (collectively
called adjuncts) can be used in place of a portion of the malted barley. The malting process
determines the colour of the malt, light (amber, brown, black) and hence the colour of the beer.
The first main process in brewing is mashing where cracked or milled malted barley is mixed
with water and incubated at controlled temperatures for 2-3 hours. The temperature is regulated
to maximize the activities of enzymes (amylases, proteases, -gluconases) that originate in the
barley malt and are responsible for hydrolysis of malt starch to maltose. Maltose is then
fermented into ethanol, the final concentration of which depends on the initial concentration of
fermentable sugars.
In this laboratory exercise we will make beer using fermentation and then bottle the product for
tasting later in the session. The fermentation will be carried out in Chemical Engineering
SEB123. The class will split into two brewing shifts made up of two demonstrator groups each.
Barle
y
Maltose is a disaccharide of glucose
derived from starch in barley through
mashing. Maltos
e
MATERIALS
Fermentation reactor (25 L) with airlock

Sanitiser
Dextrose (D isomer of glucose) (1 kg)
Malt extract with added hops (lagar or pale ale)
Long handle spoon
Can opener
Yeast sachet (lagar yeast or pale ale yeast)
Hydrometer
Measuring cylinder
Water
EXPERIMENTAL
Fermentation will be carried out in Chemical Engineering SEB123. Each laboratory group
(approximately 15 students) will prepare a 23 L beer fermentation. We will produce four different
beers. Two lagers and two pale ales. One lager will be fermented with a lager specific yeast and
the other will be prepared with the pale ale specific yeast. One pale ale will be fermented with the
pale ale specific yeast and the other with the lager specific yeast. This will enable us to explore
the impact of wort and yeast on the resulting beer.
Sanitising the fermentation vessel
1. Turn the fermentation vessel tap to the off position.

2. Add approximately 2 L of hot water to the fermentation vessel.
3. Add 5 mL of sanitizer solution.
4. Secure the lid to the fermentation vessel and swirl the solution around inside to ensure contact
with all surfaces. CAUTION: Be careful not to let any hot water escape through the airlock.
5. Empty the fermentation vessel through the tap into a sink to ensure sterility of the tap.
Remove the lid and pour out remaining solution. If foam remains, give the vessel a final rinse
with hot water.
Mixing the wort
1. Remove the plastic cap from the extract can and let it stand in bucket filled with hot tap water
for 10 minutes.
2. Caution: Hot Water! Add 2 L of boiling hot water into the fermentation vessel. Consider the
consequence and likelihood of the hazard (scalding) manifesting. Use common sense.
3. Open the bag of dextrose and pour the contents into the fermentation vessel.
4. Stir the solution with the long handle spoon until the dextrose is dissolved.
5. Open the extract can with the can opener and pour the contents into the fermentation vessel.
Use the spoon to scrape out the remaining wort.
6. Stir the contents until the extract dissolves.
Fermentation
1. Top the fermentation vessel up to the 23 L mark with room temperature water.
2. Check that the temperature is in the 18-24 ˚C range using the sticker on the side of the vessel.
3. Open the sachet of yeast and sprinkle the contents into the top of the fermenter vessel. No
need to mix.
4. Replace the lid.
5. Take a sample from the tap and measure specific gravity using the hydrometer. It should have
a specific gravity around 1.045.
6. Add approximately 5 mL of water to the airlock.
7. Incubate the fermenter in the dark at approximately 20 ˚C for approximately two weeks.
Bottling: secondary fermentation (to be performed in Week 6)
After 24 hours the airlock should start releasing gas (what gas?) up to 5-10 times a minute. The
frequency of gas release can be used to monitor the rate of fermentation. Once the gas release
drops to less than once a minute the fermentation is more or less complete. You can also use the
hydrometer to determine when the fermentation is complete. It should have a specific gravity
around 1.010. Then the beer can be bottled. Let your demonstrator know if you would like to
follow the fermentation and help out with the bottling. Alternatively, the teaching staff will take
care of it for you.
1. Prime the 750 mL PET bottles with 2 carbonation drop. This contains sugar that will be
fermented to produce carbon dioxide giving the beer effervescence.
2. Taking care not to disturb the fermentation vessel (avoid suspended the yeast biomass) place
the PET brewing bottles in contact with the tap at an angle. Open the tap and slowly pour the beer
down the side of the bottle. Leave a small headspace.
3. Cap the bottles with screw caps and incubate for an additional two weeks at approximately 20
˚C in the dark. Longer incubation is fine also.
4. Refrigerate (4 ˚C) before consumption.
Later in the session we will have a tasting. Did the yeast strain influence the outcome? Consider
what flavours are imparted by the wort and what flavours are related to the yeast.
REFERENCES
1. Campbell, I. (1997) Beer. In: Food Microbiology, Fundamentals and Frontiers edited by
M.P. Doyle, L.R. Beuchat, T.J. Montville, pp662-670. ASM Press, Washington DC.
2. Fleet, G.H. (1998) Microbiology of alcoholic beverages. In: Microbiological Fermented
Foods 2nd edition, edited by B.J. Wood, pp 217-262. Blackie, Academic and Professional.
3. Priest, F.G. and Campbell, I. (1996) Brewing Microbiology second edition, Chapman and
Hall.
LABORATORY WEEK 5
Run sheet: 1) Sit progress examination

LABORATORY WEEK 5
Run sheet: 1) Bottle your brew in the food labs (from Week 4 Pg29)
LABORATORY WEEK 7
Run sheet: 1) Establish secondary enrichment for Listeria

2) Homogenise and dilute samples for coliforms and Escherichia
3) Inoculate Most Probable Number assay for coliforms
4) Inoculate Pertifilm for Escherichia
EXAMINATION OF FOOD FOR FOODBORNE PATHOGENS
From Week 7 to Week 9 we will be examining food samples for the presence of three of the most
significant foodborne pathogens (Listeria, Escherichia and Staphylococcus). This is a mainstay of
food microbiology so make sure you keep track of the three different pathogen pracs. The
following table gives an overview.
Overview of examination for foodborne pathogens

EXAMINATION OF FOODS FOR LISTERIA SPECIES.
SAFETY WARNING
Listeria monocytogenes is a pathogen of some concern to susceptible populations and is a major

health risk in pregnancy. As cultures of this pathogen may be used in the class, or may be
produced during the culture process, you MUST notify the lecturers/demonstrators if you are
pregnant. While not wishing to invade privacy, it is imperative that you are protected from the
risk of contracting listeriosis.
OBJECTIVES
The objectives of this laboratory exercise are:
1. To learn, understand and perform methods for the isolation and confirmation of Listeria spp.
2. To discuss the significance of Listeria species as foodborne pathogens.
INTRODUCTION
Listeria monocytogenes is a relative newcomer to the ranks of foodborne pathogens, only

assuming significance with the widespread use of refrigeration in the food industry. It is one of
the psychrotrophic pathogens, growing well at refrigeration temperatures, although its optimum
temperature for growth is considerably higher.
Unlike a number of other foodborne pathogens, derived from raw animal foods, Listeria is
ubiquitous in the food production environment and can be considered an 'industrial' foodborne
pathogen, frequently contaminating product at post-safety process stages of production. It is also
relatively resilient when compared to Gram-negative foodborne pathogens, surviving well in the
food production environment.
The organism does not cause serious disease in the general population, infection in this group
leading classically to a flu-like syndrome. More recently, there have been a number of outbreaks
of gastrointestinal disease associated with L. monocytogenes, including several in New South
Wales in the last few years. More serious forms of disease are associated with pregnancy,
infection leading to severe negative outcomes such as abortion, stillbirth and neonatal sepsis and
meningitis. The dose needed to cause disease is still unknown, although early beliefs that the dose
was very low (as little as a few cells), even in susceptible hosts, have been revised and it is now
considered that up to 104 cells may be required to cause disease.
Because of the significance of L. monocytogenes in relation to susceptible populations, and the

possibility that low doses only may be required for infection, a qualitative approach to detection
(i.e. presence or absence in a given amount or product) is taken, in much the same way as that for
Salmonella. Unlike Salmonella, which is more prone to injury, a non-selective enrichment is not
used. Rather, two phases of selective enrichment are used to minimise the outgrowth of non-
Listeria, which might produce Listeria-like colonies on selective differential plating media.
Selective enrichment media consist of a rich nutrient base and incorporate a wide range of
selective agents, including lithium chloride, antibiotics such as nalidixic acid, polymyxin and
ceftazidime, dyes such as acriflavine, and in some cases, a differential system, based on the
ability of Listeria to hydrolyse the glycoside aesculin.
Plating media in common use are of similar composition to that of enrichment media and may
contain blood for the detection of haemolytic activity.
Flow diagram for the isolation of Listeria spp. from foods.

MATERIALS
Per pair
• 1 ´ 90 mL Half Fraser Broth (Week 6)

• 1 ´ sterile test tube (Week 6)
• 2 ´ 10 mL Fraser broth (Week 7)
• 2 ´ PALCAM agar plates (Week 8)
Per bench
• 1 ´ cultures of L. monocytogenes on PALCAM agar (Week 7)

• 2 ´ 10 mL semi-solid motility tube stab inoculated with L. monocytogenes (Week 9)
• 1 ´ TSA-YE agar plates inoculated with L. monocytogenes (Week 9)
• 1 ´ 10 mL Brain Heart Infusion broths inoculated with L. monocytogenes (Week 9)
Per class
• Incubator, 30°C (Week 6)

• Incubator, 37°C (Week 7 & 8)
• 3% hydrogen peroxide (Week 9)
• Gram stain reagents (Week 9)
EXPERIMENTAL
Sample preparation and primary enrichment (this will be done for you)
1. For most foods, prepare a homogenate of a 10 g sample in 90 mL Half Fraser Broth (HFB) by
stomaching (NOTE: standard method involves using 25g/25 mL sample in 225 mL HFB). In
the case of poultry carcasses, the rinse technique must be used. After sample preparation,
transfer 5 mL of HFB into a sterile test tube. This constitutes the primary enrichment.
2. Incubate the primary enrichment for 24 h at 30ºC.
Secondary enrichment (to be performed in Week 7)
3. Transfer 0.1 mL of the primary enrichment to 10 mL of Fraser Broth, and incubate for 24 h at
37ºC. This is secondary enrichment. Inoculate a second tube of Fraser broth with the L.
monocytogenes plate culture.
Isolation of Listeria on PALCAM (to be performed in Week 8)
4. Streak a loopful of the secondary enrichment onto a plate of PALCAM agar. Repeat with the
reference culture of Listeria. Incubate plates at 37ºC for 24-48 h. NOTE: the Standard method
requires plating to PALCAM and Oxford agars.
Confirmation of isolates as Listeria (to be performed in Week 9)
Observe plates for colonies typical of Listeria spp. After 48 h, the colonies are approximately 2
mm in diameter, grey-green in colour with a dark sunken centre, surrounded by a black halo
against the cherry-red background colour of the medium.
Gram stain
Perform the Gram stain as described in Appendix 2. Listeria spp. usually appears as short Gram-
positive rods.
Catalase reaction – see Appendix 4
1. Streak up to three typical colonies onto plates of Tryptone Soya Agar with 0.6% yeast extract
(TSA-YE). Streak the reference culture to TSA-YE. Incubate all plates at 37ºC for 24-48 h
(this culture will be provided to you).
2. Emulsify one or two small colonies from the TSA-YE plates in a small drop of water on a
glass slide.
3. Add a drop of 3% hydrogen peroxide. Production of the enzyme catalase by the organism is
indicated by the immediate production of bubbles of oxygen. Listeria spp. are catalase-
positive.
Other confirmation tests for Listeria include motility, the Methyl Red/Voges Proskauer test,
carbohydrate fermentation, haemolysis and CAMP reaction. Refer to Appendix 4 for details.
QUESTIONS
1. Do you think it would be appropriate to use direct plating to quantify (enumerate) Listeria?
Give reasons for your answer.
REFERENCES
Standards Australia (1998) Australian/New Zealand Standard AS1766.2.16.1: 1998. Food

Microbiology. Method 2.16.1: Examination for specific organisms-Food and animal feeding
stuffs–Horizontal method for the detection and enumeration of Listeria monocytogenes-Detection
method. Standards Australia International Ltd, Sydney.
Sutherland, P.S., D.W. Miles and D.A. Laboyrie (2003) Chapter 13. Listeria monocytogenes.
Pages 381-444. In Foodborne Microorganisms of Public Health Significance. Sixth Edition.
Hocking, A.D. (Editor-in-Chief). Australian Institute of Food Science and Technology (NSW
Branch) Food Microbiology Group, Sydney.
THE EXAMINATION OF FOODS FOR COLIFORMS AND ESCHERICHIA
OBJECTIVES
The objectives of this practical are:
1. To learn, understand and perform the correct procedures for the isolation and enumeration
of coliforms and Escherichia coli using the Most Probable Number (MPN) and the
Petrifilm technique.
2. To evaluate the microbiological significance of indicator organisms in foods.
INTRODUCTION
Foods may be contaminated with pathogenic microorganisms from faeces of humans, animals
and birds, either directly, or indirectly, from other sources such as sewage. Serious bacterial
diseases that may be food-borne include typhoid fever (Salmonella typhi) and other
salmonelloses, cholera (Vibrio cholerae) and bacillary dysentery (Shigella dysenteriae). The viral
agents of infectious hepatitis and poliomyelitis may also find their way into foods and people
suffering form these diseases, excrete high populations of these organisms in their faeces.
The quality and safety of food may be compromised if processes designed to render these
outcomes are not achieved. Such processes include heat treatment, or adequate cleaning and
sanitation, preventing in particular post-process contamination. In instances where processes have
been performed inadequately, specific spoilage or pathogenic organisms may be present in foods,
although under these circumstances they often exist in an injured state, and in small numbers.
While some foodborne pathogens or specific spoilage organisms are easily detected, it is not
always economically or technically feasible to examine water/foods for the presence of specific
organisms, such as viruses, on a routine basis. Furthermore, pathogenic species may be present in
samples in only small numbers and hence may be difficult to demonstrate in culture.
Instead, foods are often analysed for marker organisms. These organisms, which can be easily
tested for, indirectly suggest the presence of other organisms or the failure of a process. When
used in the former sense, as a marker for other specific organisms, they are referred to as index
organisms, while markers used to measure the adequacy of a process are termed indicator
organisms. In some cases, they are one and the same thing.
The Standard Plate Count is an example of a marker test, providing some indication of the overall
quality of a food product, based on the number of organisms detected. Other important marker
organisms include the coliform group, of which E. coli is a member. Coliforms are defined as a
physiological rather than a taxonomic group of organisms, although in reality they are closely
related. Coliforms are non-sporulating, facultative anaerobic, Gram-negative, rod-shaped bacteria
which ferment lactose to acid and gas in the presence of bile salts or their analogues. While E.
coli is usually associated with the human intestine and passes into water/food via faeces, other
coliforms exist more commonly in the open environment.
Coliforms are usually employed as indicator organisms, their presence indicating procedural
failures, such as poor hygiene and sanitation, or inadequate processing, especially heat processing
of foods. The presence of faecal (thermotolerant) coliforms and in particular E. coli in a raw/fresh
food is a good (but not absolute) indicator of recent direct or indirect faecal contamination, while
the detection of E. coli serves as an index of the presence of enteric bacterial pathogens. Many
countries have microbiological standards for the presence of coliforms, faecal coliforms and E.
coli in foods and these may have important implications in securing or maintaining export
markets. The use of enteric marker organisms in food microbiology has been reviewed
extensively (Craven et al. 2003).
Thus, microbiological examination is often based upon determining the presence of specific
marker organisms, such as the total coliform group, or E. coli, as an index of faecal
contamination and a specific indicator of the presence of enteric bacterial pathogens. The
presence of coliforms, faecal coliforms, and E. coli is easily determined. In Europe in particular,
testing for the entire family Enterobacteriaceae is favoured over the coliform group.
The test for coliforms is performed by inoculating a sample (or dilutions thereof) of water/food
into test tubes of a broth containing lactose, a surface-active agent such as bile salts, and a small
inverted tube (known as a Durham tube). After 24-48 h of incubation at 37°C the tubes are
examined for production of acid and gas. Other than coliforms, few organisms can ferment
lactose rapidly with the production of gas under these test conditions.
Positive results at this stage are not conclusive of the presence of coliforms and are only
considered presumptive. It is necessary to confirm that coliforms are present by inoculation of
each positive broth culture onto or into another medium. Traditionally this has involved plating to
an agar medium, usually eosin methylene blue or EMB agar, which permits, through colony
morphology, the easy recognition of coliforms. However, the current standard method uses a
second liquid medium for confirmation.
As many coliforms are of non-faecal origin, their presence gives a false positive assessment of
faecal contamination. So-called faecal coliforms (better termed thermotolerant coliforms) can be
differentiated from typical environmental coliforms by introducing an extra set of lactose
fermentation tubes and incubating at the higher temperature of 44-45°C. Under these conditions,
only thermotolerant coliforms, considered to be of faecal origin, are able to produce gas from
lactose. It is important to note that some thermotolerant coliforms of non-faecal origin may still
give a false-positive result. Nevertheless, these results give a quite reliable index of faecal
contamination and the test is referred to commonly as the faecal coliform test. As with total
coliforms, gas production at the higher temperature is only considered a presumptive result.
Traditionally, the presence of so-called faecal coliforms was confirmed by plating to EMB agar.
Positive ‘faecal’ coliform tests may be confirmed as E. coli by taking colonies typical of E. coli
from EMB agar and performing an indole test at 44°C. As most (>90%) ‘faecal’ coliforms are
confirmed as E. coli, the current standard simply requires confirmation, using the production of
indole from tryptophan, of the culture from the second lactose-containing broth.
Coliform, faecal coliform and E. coli tests can be made quantitative by doing them using the most
probable number (MPN) procedure as described in the current standard methods. Due to time
constraints, however, the MPN procedure is employed here to enumerate coliforms only.
The standard MPN procedure for E. coli requires several days to complete and the result is a
statistical approximation of the numbers in the sample. Over the years, many rapid methods have
been developed to overcome the short comings of the conventional method. Among these, the 3M
Petrifilm method is one of the most widely used in the industry, and has been accepted by many
authoritative organisations, such as AOAC, for enumerating coliforms and E. coli in foods. The
Petrifilm consists of two sheets of film that carry a dehydrated selective medium and an indicator
to assist with colony identification. The film for coliforms incorporates lactose and tetrazolium
salts, while the E. coli film contains an additional substrate for β-D-glucuronidase, an enzyme
that is produced by a large proportion (>96%) of E. coli strains. Coliforms appear as red colonies
while E. coli colonies are blue, with both accompanied by gas bubbles trapped by the top film, as
a result of lactose fermentation.
In this exercise, we will use the Petrifilm method to enumerate E. coli in foods.
MATERIALS
Per pair
• 4 ´ 9 mL dilution fluid (Week 7)
• 1 ´ 90 mL dilution fluid (Week 7)
• 15 ´ 10 mL lauryl tryptose (LT) broth in test tubes with Durham tubes (Week 7)
• 3 ´ Petrifilm E. coli/coliform (Week 7)
• 10 ´ EMB agar (Week 8)
Per class
• Petrifilm spreader (Week 7)
• incubator, 37°C
• positive control organism, Escherichia coli (Week 7)
• negative control organism, Salmonella typhmurium (Week 7)
Flow diagram for the most probable number (MPN) determination of coliforms, faecal coliforms
and Escherichia coli in foods and water. Note that this diagram approximates the approach taken in
the exercise as it represents the method published in the previous Standard method.
EXPERIMENTAL
Food sample homogenization and dilution (to be performed in Week 7)
1. For solid food samples, weigh 10 g into 90 mL sterile dilution fluid and macerate in a
stomacher for 30 sec. The homogenate will represent a 10-1 dilution of the food. For liquid
foods such as pasteurised milk, use the undiluted product (100), as well as dilutions of the
product as described below.
2. Prepare 10-1, 10-2, 10-3 and 10-4 dilutions of homogenate using dilution fluid.
Inoculation of Most Probable Number (MPN) assay (to be performed in Week 7)
1. Check that Lauryl Tryptose (LT) broths do not contain gas bubbles in the Durham tubes
(small inverted tube inside the bottle).
2. For food samples, aseptically inoculate 1 mL of the four sample dilutions into LT broth in
triplicate (three bottles per dilution = 12 bottles). For beverage samples, aseptically inoculate
1 mL of the four sample dilutions plus the undiluted sample into LT broth in triplicate (three
bottles per dilution plus undiluted = 15 bottles). Inoculate two additional sets of triplicates
with 1) 1 mL of the positive (known coliform) and 2) 1 mL of the negative (known non-
coliform) control cultures provided into LT broth. Label appropriately. Submit the tubes for
incubation at 37°C for 24 h.
Inoculate Petrifilm for Escherichia coli (to be performed in Week 7)
1. Label Petrifilm plates for 10-1 to 10-4 dilutions.
2. Place Petrifilm plate on level surface. Lift top film.
3. With pipette perpendicular to Petrifilm plate, place 1 mL of sample onto center of bottom
film.
4. Carefully ROLL top film down to avoid entrapping air bubbles. Do NOT let top film drop.
5. With FLAT side down, place spreader on top film over inoculum. GENTLY apply pressure
on spreader to distribute inoculum over circular area before gel is formed. Do not twist or
slide the spreader. Lift spreader. Wait a minimum of one minute for gel to solidify.
6. Stack plates with clear side up in stacks of no more than 20.
7. Submit plates for incubation at 35 °C for 24 h.
Calculate most probable number of coliforms (to be performed in Week 8)
1. Record gas formation in the Durham tube. A positive result is taken as any tube in which
there is sufficient gas production to fill the concave part of the Durham tube. Incubate the
tubes that do not show gas production for a further 24 h and then record the results. As carbon
dioxide (the main gas produced) is quite water-soluble, the liquid in the LT bottle may be
supersaturated with the gas, and there may be no bubble in the Durham tube. Before reading,
it is advisable to tap each bottle down gently on the bench to release gas from solution, which
may then yield a positive result. Do not invert tube or vortex. A demonstrator will show how
to perform this technique.
2. Calculate the MPN of coliforms in the original food sample as follows. Select the highest
dilution in which all three tubes are positive for acid and gas production after 48 h AND at
least one tube in the next two dilutions is negative. Record the positive tubes in those three
dilutions. If this is not possible because none of the dilutions yielded three positive tubes,
select the first three dilutions and record the number of positive tubes in each dilution. If all
tubes are positive, select the last three dilutions. See Appendix 5 for the McCrady statistical
tables from which the number of cells/mL of water or per gram of food can be calculated.
These tables are not exhaustive and only provide the most common combinations of positive
tubes for the three-tube MPN.
Inoculate EMB plates for coliform confirmation (to be performed in Week 8)
Pick 8 positive tubes and streak a loopful from each of the tubes onto EMB agar plates. Use
negative tubes if you do not have enough positive tubes. Also streak the positive and negative
control onto EMB agar. Submit the EMB plates for ncubation 37°C for 24 h.
Examine Petrifilms for Coliforms and Escherichia coli (to be performed in Week 8)
Examine the plates. Coliforms appear as red colonies with gas bubbles. E. coli appears as blue
colonies with gas bubbles. Count the coliform and E. coli colonies.
Examine EMB plates to confirm coliforms (to be performed in Week 9)
The following week (Week 9) record the presence or absence of typical coliform colonies on
EMB agar. Coliform colonies ferment the lactose in EMB and therefore typically have a deep
purple to dark red colour. Some coliforms (e.g. Klebsiella) appear as pink, mucoid (slimy)
colonies. Record the types of colony forms you observe.
QUESTIONS
1. What kinds of errors are associated with the MPN technique?

2. How may these errors be minimised?
3. How can the accuracy of the MPN method be improved?
4. E. coli is usually used as an index of the presence of enteric bacterial pathogens. Do you think
it would be a suitable index of presence of (a) other bacterial pathogens, and (b) enteric viral
pathogens?
REFERENCES
Craven, H., M.J. Eyles and J.A. Davey (2003) Chapter 6. Enteric indicator organisms in food.
Pages 163-194. In Foodborne Microorganisms of Public Health Significance. Sixth Edition.
Hocking, A.D. (Editor-in-Chief). Australian Institute of Food Science and Technology (NSW
Branch) Food Microbiology Group, Sydney.
Standards Australia International (2004) Australian Standard 5013.3-2004. Food microbiology.
Method 3: Microbiology – general guidance for the enumeration of coliforms – most probable
number technique. Standards Australia International, Sydney.
Standards Australia International (2006) Australian Standard 5013.15-2006. Food microbiology.
Method 15: Microbiology of food and feeding stuffs–horizontal method for the detection and
enumeration of presumptive Escherichia coli–Most probable number technique. Standards
Australia International, Sydney.
RECORD SHEET
Examination of foods/water for coliforms, faecal coliforms and E.coli
Presumptive total coliforms
Gas production in LT broth at 37ºC.

Indicate gas production with a '+' or '-'.
LT broth
Dilution 24 hours 48 hours
1 2 3 1 2 3
-1
-2
-3
-4
Coliform morphology on EMB
Record here your descriptions of colonies typical of coliforms on EMB agar.

Enumeration of Coliforms and Escherichia coli
Coliforms
Dilution Counts/plate cfu/g or cfu/mL MPN
-1
-2
-3
-4
Escherichia coli
-1
-2
-3
-4
LABORATORY WEEK 8
Run sheet: 1) Isolation of Listeria on PALCAM (from Week 7 Pg35)

2) Inoculate EMB plates for coliform confirmation (from Week 7 Pg43)
3) Examine Petrifilms for Coliforms and E. coli (from Week 7 Pg43)
4) Sample homogenisation, dilution and plating for Staphylococcus
EXAMINATION OF FOODS FOR STAPHYLOCOCCUS AUREUS
OBJECTIVES
The objectives of this exercise are:
1. To learn, understand and perform the correct procedures for isolating and enumerating
Staphylococcus aureus from foods.
2. To interpret and discuss the significance of Staphylococcus aureus in foods.
INTRODUCTION
Staphylococcus aureus is a Gram-positive coccus in which the cocci are characteristically

arranged in grape-like clusters but occasionally in short chains or pairs. S. aureus lives in close,
stable association with the human body. A large percentage of the population (on average 30% of
healthy adults) carries the organism as part of the normal microflora of the skin, nose and throat.
It is an important pathogen to humans causing a wide variety of diseases, including skin lesions
and respiratory infections, as well as gastroenteritis. Approximately one third of all microbial
food poisoning cases in Australia and the USA can be attributed to S. aureus. In Australia,
prawns and chickens are the common vehicles of transmission although meats, dairy and bakery
products are also involved. The foods are most often contaminated through the hands of
individuals processing or handling the food. Improper storage of the food then results in
multiplication of the cells to an infective level.
Although S. aureus is a relatively hardy organism, it is a poor competitor with other

microorganisms and is rapidly overgrown and inhibited. Consequently, foods which are at
greatest risk are those where the natural flora has been removed or destroyed by processing or
cooking procedures. Cooked, peeled, frozen prawns/shrimps, and sliced delicatessen meats are
often contaminated with S. aureus.
The pathogenicity of Staphylococcus aureus depends upon its ability to produce any one of
several serologically related extracellular enterotoxins as a result of multiplication in the food. It
is the consumption of this toxin which causes the poisoning syndrome and approximately 106
cells/g are required to produce a toxic dose. The toxins are proteins and very heat stable, not
being destroyed by usual domestic cooking and even more severe heat processes, such as
canning. Both viable cells of S. aureus and enterotoxin may be present in the food, although S.
aureus cells may grow in a food, produce enterotoxin, and then be killed by processing, heating
or autolysis, leaving only active enterotoxin. In such cases, examination for viable organisms
would not indicate the hazardous nature of the food, bearing in mind that the enterotoxins are
very heat stable. Not all strains of S. aureus produce enterotoxin. However, the presence of high
populations of non-toxigenic strains in foods certainly indicates unsanitary handling at some
point. Enterotoxin production by strains of S. aureus is affected by a variety of factors such as
phase of growth, food composition, pH, temperature and presence of salts.
The ultimate assessment of the safety of a food product with regard to S. aureus should be based
upon freedom from enterotoxin. Hence, the ability to detect enterotoxin in a food becomes
important. Reliable immunological techniques such as ELISA tests are available for the detection
of staphylococcal enterotoxin but foods are still routinely checked for the presence of viable cells
of S. aureus. The most widely used detection technique involves direct plating onto Baird-Parker
(BP) agar medium. This medium consists of a variety of selective and differential agents (refer to
medium description below). These compounds are tolerated by Staph. aureus at the
concentrations used. Differentiation is based upon the ability of Staph. aureus to produce black
colonies as a result of tellurite reduction to elemental tellurium, and to enzymatically degrade egg
yolk lipoprotein to produce a halo of clearing in the medium surrounding the colony. Some recent
modifications to this medium include the use of sulphamezathine to suppress the growth of
Proteus, and use as a pour plate to increase sensitivity. Baird-Parker's medium has performed
satisfactorily in a number of comparative trials with other plating media. Alternative media are
available, including those incorporating some from of plasma, enabling direct detection of
coagulase activity. While direct plating is most often used, detection of low numbers in long
shelf-life products may be necessary, so enrichment or an MPN method can be used.
Typical S. aureus colonies appearing on BP agar are confirmed by a range of tests including:
1. coagulase production, though it is known that a small percentage of enterotoxin producing,

non-coagulase forming strains occur;
2. DNAse production, which may be affected by environmental conditions, and;
3. mannitol fermentation and colony pigmentation on media containing 7% NaCl. Mannitol non-
fermenting strains may occur, and colony pigmentation, even on specialised media, may vary
from white, through cream, to golden yellow.
It is noteworthy that the production of a heat stable DNAse (thermonuclease) seems to correlate
best with enterotoxin production. Further details on S. aureus in foods and media used for its
isolation and identification may be found in Mossel and Van Netten (1990) and Stewart (2003).
Flow diagram for the detection of coagulase-positive staphylococci from foods.

MATERIALS
Per pair
• 7 ´ Baird-Parker agar plates

• hockey sticks
Per class
• Food sample, Stomacher and bags

• S. aureus cultures
• Incubator, 37°C
EXPERIMENTAL
Food sample homogenization, dilution and plating (to be performed in Week 8)
1. Homogenise 10 g of food sample in 90 mL of dilution fluid.
2. Prepare serial dilutions to 10-3.
3. Spread 0.1 mL of each dilution onto duplicate plates of Baird Parker (BP) agar.
4. Streak a positive control culture of S. aureus onto a plate of BP agar.
5. Submit the plates for incubation at 37°C for 48 h.
Confirm Staphylococcus and calculate abundance (to be performed in Week 9)
6. Examine for colonies typical of S. aureus. Typical colonies are black and convex, surrounded
by a double zone in the medium, an inner zone on the surface of the medium which appears
opalescent in reflected light, and a wider (outer) zone of clearing, due to hydrolysis of egg
yolk components.
7. Count the colony number and calculate Staph. aureus cfu/g of food, using the method
described for the Standard Plate count.
8. Confirm S. aureus cellular morphology and Gram reaction by staining and microscopic
observation. Note that the Australian Standard method involves determination of coagulase-
positive staphylococci, rather than the species S. aureus, and confirmation of the former
involves use of the coagulase test. In this case, both typical and atypical colonies are tested, as
atypical colonies may still be coagulase-positive.
QUESTIONS
1. Heat processed canned mushrooms have caused an outbreak of Staphyloccus aureus food
poisoning. No visible colonies of Staph. aureus were produced after plating mushroom
sample onto Baird Parker Agar. Standard ELISA tests did not reveal the presence of
enterotoxin. How can you explain this result?
2. Why is it not necessary to undertake a selective enrichment step of the food sample before
plating onto Baird-Parker agar? Do you think sublethally injured cells might be inhibited on
Baird-Parker agar?
REFERENCES
Jablonski, L.M. and Bohach, G.A. (1997) Staphylococcus aureus. In Food Microbiology:
Fundamentals and Frontiers. Doyle, M.P., Beuchat, L.R. and Montville, T.J. (eds.). ASM Press,
Washington DC.
Mossel, D.A.A., and Van Netten, P. (1990) Staphylococcus aureus and related staphylococci in
foods: ecology, proliferation, toxigenesis, control and monitoring. J. Appl. Bacteriol. 69
(Symposium Supplement), 123S-145S.
Standards Australia International (2004) Australian Standard 5013.12.1-2004. Food

microbiology. Method 12.1: Microbiology of food and animal feeding stuffs horizontal method
for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other
species) – technique using Baird-Parker agar medium. Standards Australia International, Sydney.

species) – technique using rabbit plasma fibrinogen agar medium. Standards Australia
International, Sydney.

species) – detection and MPN technique for low numbers. Standards Australia International,
Sydney.
Stewart, C.M. (2003) Chapter 12. Staphylococcus aureus and staphylococcal enterotoxins. Pages
359-380. In Foodborne Microorganisms of Public Health Significance. Sixth Edition. Hocking,
A.D. (Editor-in-Chief). Australian Institute of Food Science and Technology (NSW Branch)
Food Microbiology Group, Sydney.
RECORD SHEET
Examination of foods for Staphylococcus aureus
Populations of S. aureus on Baird-Parker agar
Dilution Counts/plate cfu/g or mL
-1
-2
-3
LABORATORY WEEK 9
Run sheet: 1) Confirm Listeria using Gram stain and catalase (from Week 8 Pg36)
2) Examine EMB plates to confirm coliforms (from Week 8 Pg43)
3) Staphylococcus confirmation and quantification (from Week 8 Pg49)
LABORATORY WEEK 10
Run sheet: 1) Take practical examination

APPENDIX 1
SETTING UP THE LIGHT MICROSCOPE
BRIGHT FIELD MICROSCOPY
The most common way in which a microscope is used in routine food microbiology is for the
examination of stained specimens by bright field microscopy. A microscope correctly configured
for the performance of bright field microscopy provides light to the specimen by what is known
as Köhler illumination. This form of illumination is defined in the following way. If an image of
a light source is conjugate with the rear focal plane of an objective and the field iris diaphragm of
the light source is conjugate with the field of view of the objective/eyepiece combination,
maximum light intensity of the light source is obtained and the field of view is free from
illumination irregularities, irrespective of the shape of the light source.
Setting the microscope for Köhler illumination, or optimal bright field microscopy
The Department of Food Science and Technology possess a range of microscopes in the teaching
laboratory. While they all operate in the same fundamental way, their configuration does vary to
some extent, in terms of the location of some levers, knobs and so forth. You should consult the
schematic diagrams on the following pages when setting up the particular microscope you may be
using on any given day. It is worthwhile familiarising yourself with each type of microscope, as
you may not always be able to use the same microscope every time, both during this class, and in
your future career. The instructions below are written to with respect to the older model of
Olympus microscopes, and some steps may be omitted when using another type of microscope.
1. Set the "High-Low" selector knob to the "L" position.
2. Switch on the lamp and adjust the rheostat to give a light of moderate intensity (between
settings 3 and 5).
3. Focus an object (a cross or line made with a marker pen on a glass slide will suffice) with the
x10 (low-power) objective.
4. Stop down the condenser iris diaphragm (aperture stop) and the field (lamp) iris diaphragm
and adjust the condenser lens to a position where the image of the light source is sharply
focused.
5. If necessary, centre the image at the light source using the centering screw attached to the
source.
6. Open the lamp diaphragm slowly, to expose the full field of view.
7. Remove the eyepiece, and looking through the eyepiece tube, slowly open the aperture iris
diaphragm until approximately 2/3 of the back focal plane of the objective is illuminated.
8. Centre the bulb filament.
9. Replace the eyepiece and the microscope is ready for use.
Use of the oil immersion objective
1. Adjust the illumination as described above.
2. Using the low power objective, select a suitable area of the specimen for examination.
3. Place a drop of immersion oil on this area. Swing to the oil immersion lens (100x). Most
microscopes are parfocal but, if it is necessary to adjust the focus, do so by racking the fine
focus knob upwards.
PHASE CONTRAST MICROSCOPY
The relatively low contrast of living cells, as viewed by conventional bright field microscopy, can
be greatly enhanced by using a microscope with a modified optical system, known as the phase
contrast microscope. Phase contrast microscopy is based on the fact that the rate at which light
travels through objects is inversely related to the refractive index of those objects. Since the
wavelength of light is independent of the medium through which it travels, the phase of a light
ray passing through an object of higher refractive index than the surrounding medium will be
relatively retarded. A system of rings or annuli in the condenser and the objective separate the
light rays diffracted by the specimen from those which are not. The two sets of rays are
recombined, but only after the diffracted rays have been passed through a glass ring, which
introduces an additional phase difference. Through this optical mechanism, the degree of contrast
between cells and their surrounding medium that differ only slightly in their refractive index is
greatly increased.
Correcting faults during bright field microscopy

Apparent fault Possible causes Correction
Field dark Light not directed into barrel With the lamp diaphragm
open, alter mirror angle.
Condenser and/or lamp Open
diaphragm closed
Rheostat faulty or Replace or
Set too low Increase current
Lamp filament burnt out Replace
Lamp filament grossly Adjust as for step1 above
non-centred
Colour of objects indistinct Condenser diaphragm closed Open

too far
Condenser unfocussed See steps 7-9 above
Lamp filament off-centre See steps 1 and 2 above
Resolution poor Condenser closed Open
Edges seem unduly Illumination system Check that filament is

contrasted and fuzzy due to incorrectly adjusted. focussed at condenser fringes
diffraction
Turn to low power objective.

Close lamp diaphragm.
Check that image of lamp
aperture is sharp - condenser
focussed
Check that colour rings

uniform (ie filament centred).
Unable to focus an object Cover-slip too thick.

Slide upside down
Focussing attempts too rapid Correct orientation
Use fine focus and adjust
more slowly.
Objective used has Try a higher power to make
insufficient resolving power object visible
and/or magnification
Objective covered with oil – Clean with lens tissue and

frequently dried on from solvent (check with
previous misuse demonstrator before using
solvent).
APPENDIX 2
STAINING OF BACTERIA
Preparation of bacterial smears
Bacteria are normally stained in a dried, fixed condition.
1. Use a clean slide, completely free from grease.
2. In the case of a liquid culture or sample, apply a small loopful to a microscope slide. If too
few organisms are present in a single loopful, further material can be added. For cultures from
solid media, place two loopfuls of water onto a slide. Add only a small amount of growth
using the edge of an inoculating loop and emulsify in the water. If the suspension is prepared
too thickly, individual cells will not be visible.
3. Spread over the slide to provide a shallow, uniform and slightly turbid layer.
4. Air-dry the smear. Passing the slide quickly through the flame of the Bunsen burner can
accelerate the drying of the smear.
5. Once dry, fix the smear by passing the slide rapidly through the Bunsen flame two or three
times.
6. Allow the slide to cool, then apply the stain.
7. Examine without a cover slip with direct application of immersion oil.
8. Since fixing and staining does not always kill organisms, slides should be placed in the
container provided for sterilization when examination is completed.
Simple stains
These simply make the organism visible without giving any additional information about it.
Stains such as dilute carbol fuchsin or safranin are most commonly used for this purpose.
1. Place slide with heat fixed smear, organism side upwards on the staining rack.
2. Cover smear with stain.
3. Leave 30 seconds.
4. Wash off stain
5. Air dry and examine.

The Gram stain
This is the most important differential stain in microbiology. All bacteria can be classified as
Gram-positive or Gram-negative, a characteristic produced by marked differences in cell wall
structure and correlates with many other characteristics of the organism.
1. Fix smear by heat in the usual way.
2. Stain with crystal violet for 1 minute.
3. Wash well with tap water - drain slide.
4. Add Grams iodine, drain off and allow to stand for 1-1.5 minutes.
5. Wash well under the tap and drain off the excess water.
6. Decolourise with alcohol or alcohol-iodine until the violet colour ceases to run from the smear.
This can most readily be seen if the slide is held against a white background - Usually 30
seconds. Wash well in water.
7. Counterstain with safranin or dilute carbol fuchsin for 15-20 seconds.
8. Wash, blot dry and examine with the oil immersion objective. (No coverslip required).
Gram-positive organisms may appear ‘black’, though typically a deep violet, while Gram-
negative organisms appear red to pink. Note that some organisms, while having a typical
Gram-positive wall structure and appearing Gram-positive in young cultures, may become
Gram-negative with age as the wall becomes fragile. These are termed Gram-variable.
APPENDIX 3
PREPARATION OF SERIAL DILUTIONS FOR ENUMERATION OF BACTERIA
1 mL
1 mL 1 mL 1 mL
99 mL 9 mL 9 mL 9 mL
10-2 10-3 10-4 10-5
0.1 mL
1 mL 2 drops /
quarter
SPREAD PLATE
cfu x 103 (df) x 10 = cfu/g or
mL POUR PLATE
g or mL cfu x 104 (df) x 10 = cfu/g or
mL DROP PLATE
g or mL cfu x 105 (df) x 10 = cfu/g or
mL
g or mL
df = dilution factor
APPENDIX 4
BIOCHEMICAL TESTS
1. Catalase test
Introduction
The enzyme catalase is present in most aerobic cells where its function is to remove the highly
toxic peroxide (H2O2) which is formed in small amounts during passage of electrons down the
electron transport chain to oxygen. Catalase catalyses the reaction 2H2O2 ð 2H2O + O2. Catalase
activity may be detected by observing for the appearance of gas bubbles after hydrogen peroxide
is added to a culture.
Medium
Any medium supporting growth can be used to obtain cultures for the catalase test with the
exception of those containing blood. Blood agar may contain residual catalase sufficient to give a
false positive result.
Method
The catalase test can be performed in several different ways as outlined below:
(a) Place one drop of H2O2 (10 vols.) on a glass slide and emulsify in it a colony from an
agar plate or slope.
OR
(b) Fill a capillary tube with H202 and place over the colony observe for the production of
bubbles.
Interpretation
Positive results - immediate production of
bubbles of oxygen
Negative result - no bubbles formed

2. Oxidase test
Introduction
The production of oxidase is one of the most significant tests we have for differentiating certain
groups of bacteria. For example all the Enterobacteriaceae are oxidase negative and most species
of Pseudomonas are oxidase positive.
In the positive oxidase or cytochrome c-test, the cytochrome c must be oxidized being haem-
containing proteins of high molecular weight and are involved in respiratory metabolism.
The iron in the haem-moiety of cytochromes is responsible for the reduced or oxidized state of
the cytochrome.
cytochrome-Fe+++ + e- > cytochrome-Fe++
In the oxidase test, tetramethyl-p-phenylene diamine is oxidized by oxidized cytochrome c to

form a purple substance.
Materials
Oxidase test reagent - C1% solution of dimethyl-p-phenylenzdiamine hydrochloride).

Whatman No. 2 filter paper
Petri dish
Procedure: On a piece of Whatman No. 2 filter paper in a petri dish place several drops of the
oxidase test reagent. Remove a loopful of the organism from one of the colonies and smear the
organisms over a small area of the paper.
Positive: Dark purple to black within 15 seconds
Negative: No colour change or faintly purple
3. Sugar fermentation (sugar peptone water)
Used to determine whether an organism is able to utilise specific substrates (usually sugars but
may be any material which can be fermented) by fermentative pathways. The end products
usually contain a high proportion of acidic molecules thus lowering the pH of the medium - Gas
may or may not be produced. The medium is dispensed in tubes with an inverted Durham tube
included for gas detection.
(a) Inoculate tube of sugar peptone water

(b) Incubate until growth is apparent
(c) Examine
Interpretation:
Fermentation acid production, i.e. medium yellow

Gas gas apparent in inverted Durham tube
4. MRVP test
Methyl Red (MR) Test
Introduction
The Embden-Meyerhof pathway sugar fermentation products vary. One mode of sugar
breakdown is the mixed-acid fermentation which yields lactic, acetic and succinic acids, formic
acid (or CO2 and H2) and ethanol. This fermentation is characteristic of the genera Escherichia,
Salmonella, Shigella, Proteus, Yersinia, Vibrio and Photobacterium and occurs in some
Aeromonas species.producing a pH of 4.2 or less and maintain this for at least 4 days.
The ability to carry out this fermentation is assessed by growing the organism in a medium
containing glucose, and testing the pH after 4 days by using the pH indicator methyl red (red at
pH 4.2, yellow at pH 6). The test is usually done in conjunction with a Voges-Proskauer test
which tests for 2,3 butylene-glycol fermentation, the alternative pathway.
Materials
Glucose phosphate peptone water
Methyl Red indicator
Procedure
Inoculate the organism into glucose phosphate peptone water (MRVP medium, Oxoid)
Incubate for 4 days at required temperature
Add a few drops of methyl red indicator
Interpretation
Note colour. A positive resaction is red while a negative is yellow.

5. VOGES-PROSKAUER (VP) TEST
Some organisms of the enteric and related groups, ferment sugars by a route alternative to the
mixed acid fermentation. Glucose is fermented by the organisms to pyruvic acid in the usual
way. From here it may continue in a mixed acid pathway or yield acetylmethylcarbinol (acetoin)
and/or 2,3 butanediol (2,3 butylene glycol). Which of these reactions predominates depends on
the organisms and the pH of the medium. The production of these non-acid end products results
in less lowering of the pH in MRVP medium causing the methyl red test to be negative.
Unfortunately there is no satisfactory test for 2,3-butaneaiol; however, acetoin is easily detected.
Since acetoin and 2,3 butanediol are always simultaneously present, the test is valid. This
indirect method of testing is called the Voges-Proskauer test.
Materials
MRVP medium
5% w/v alcoholic a naphthol solution and 3 mL of 40% w/v KOH solution (Barritt's method)
OR
2 drops of a 0.3% w/v creatine solution and 5 mL of 40% KOH solution (O'Meara's method).
Procedure
Inoculate organism into MRVP medium
Incubate at required temperature for prescribed period of time.
Add 3 mL of 4.2.2 or 4.2.3
Shake vigorously to aerate.
Interpretation
Positive: Pink colour
Negative: Colourless
APPENDIX 5
MOST PROBABLE NUMBER METHOD
The precision of the plate count method is greater than the standard dilution method, but the latter
can be used in circumstances where plate counts are impractical or unfeasible. The basis of the
dilution technique is to determine the presence or absence of growth at each dilution. Estimation
of the total number of organisms likely to be present per gram (g) or millilitre (mL) sample is
determined from probability tables - this estimation is termed the Most Probable Number or
MPN.
The MPN test and related probability tables can make use of either three or five tubes; Table 1
shows the MPN values for a 3-tube test.
To calculate the MPN/g or mL, under the appropriate dilution heading find the number of
positive tubes that corresponds to your experimental findings and read off the MPN i.e. if there
were 2 positive tubes at 10-1, 1 positive for 10-2 and no positive tubes at 10-3, the MPN per g
food is 15.
Table 1 Most Probable Number of bacteria; 3 tubes at each dilution
Number of positive tubes Confidence Levels

_______________________________ __________________________________
10-1 10-2 10-3 MPN per g 99% 95%

0 1 0 3 <1 23 <1 1
1 0 0 4 <1 28 1 21
1 0 1 7 1 35 2 27
1 1 0 7 1 36 2 28
1 2 0 11 2 44 4 35
2 0 0 9 1 50 2 38
2 0 1 14 3 62 5 48
2 1 0 15 3 65 5 50
2 1 1 20 5 77 8 61
2 2 0 21 5 80 8 63
3 0 0 23 4 177 7 129
3 0 1 40 10 230 10 180
3 1 0 40 10 290 20 210
3 1 1 70 20 370 20 280
3 2 0 90 20 520 30 390
3 2 1 150 30 660 50 510
3 2 2 210 50 820 80 640
3 3 0 200 <100 1900 100 1400
3 3 1 500 100 3200 200 2400
3 3 2 1100 200 6400 300 4800

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FOOD2320/8320 Food Microbiology Practical Manual Page 1

Week Lecture topic/s Laboratory topic/s

(2) • Sampling and analysis Examination of food by microscopy and standard

(3) Contemporary analysis methods Examination of food by contemporary methods

(4) • Labour day public holiday

(5) • Food Fermentation (Bread and Cheese) • Progress exam (30%)

(6) • Secondary beer fermentation (voluntary)

(7) Foodborne illness (Salmonella, Escherichia, Foodborne pathogens

(8) Foodborne illness (Listeria, Staphylococcus Foodborne pathogens

(9) Foodborne illness (Viruses, mycotoxins and Foodborne pathogens

(10) Course review • Practical exam (30%)

Gantt chart providing overview of laboratory activities

Course information and laboratory safety 5

Laboratory Week 1: Food microbiology - fundamental practical skills 8

Laboratory Week 2: Examination of food by microscopy and plate counts 12

Laboratory Week 3: Examination of food by contemporary methods 24

Laboratory Week 4: Primary fermentation for beer production 26

Laboratory Week 5: Progress examination 30

Laboratory Week 6: Secondary fermentation for beer production 31

Laboratory Week 7: Foodborne pathogens 36

Laboratory Week 8: Foodborne pathogens 45

Laboratory Week 9: Foodbourne pathogens 51

Laboratory Week 10: Practical examination 52

Appendix 1 Setting up the light microscope 53

Microbiological analysis is an essential component in the management of food quality by the

Students must supply their own:

1. Adhere to social distancing guidelines.

COURSE INFORMATION & LABORATORY SAFETY

Run sheet: 1) Group allocations

FOOD MICROBIOLOGY - FUNDAMENTAL PRACTICAL SKILLS

1. To establish the proficiency of students in the fundamental skills of practical (food)

The 16-streak technique

• 4 plates of nutrient agar

• plate culture of Escherichia coli

A Streaking cultures to agar media

B Aseptic transfer of a sterile nutrient broth

1. Fit a pipette tip to the pipette.

4. Place the tubes in the 37°C incubator.

Checking your streaking and aseptic technique (to be performed in Week 2)

2. Why do we use the term “colony-forming units” instead of “cells”?

Run sheet: 1) Observe plates and cultures (from Week 1 Pg11)

EXAMINATION OF FOOD BY MICROSCOPY

The objectives of this practical are:

observation. A rapid assessment of the microbial ecology of food suspected to be spoiled is

Total cell counts

The simplest procedure involves microscopically counting the individual cells in an

The drawbacks of total microscopic counts are:

• dead cells are not distinguished from living (viable) cells;

A Setting up the microscope (refer to Appendix 1)

B Preparation and examination of wet mounts

C Preparation and examination of a stained slide

D Microscopic examination of spoiled foods

3. Describe how you might quantify the numbers of microorganisms in a food/beverage

EXAMINATION OF FOODS BY STANDARD PLATE COUNT

The objectives of this exercise are:

1. To gain experience in methods for total plate count determination of microbial

Microorganisms exist in foods as mixed populations consisting of many different species.

Microorganisms require suitable nutrients and favourable environmental conditions for

Factors affecting the plate count

Some negative aspects of the agar plate count are:

1. Maceration is a procedure whereby whole or part of the product is mechanically