Al-Farabi University College

Department of Biology

Fourth Class

Food Microbiology
Lecture Numbers:

(1, 2, 3, 4, 5, 6, 7, 8, 9)
Prof. Dr. Abdulwahid B. Al-Shaibani

2021 - 2022
 Syllabus of Food Microbiology
For
Students of Biology
Week No. Lecture (Number) and Title
First (1) General Introduction.
Second & Third (2) Factors influencing microorganisms of food.
Fourth & Fifth (3) Effects of Food Processing Methods on Microorganisms.
Sixth (4) Indicator Bacteria.
Seventh (5) Foodborne Diseases (Illness).
Eighth, Ninth (6) Bacteria (and their toxins) Causing Foodborne Diseases.
&Tenth
Eleventh & Twelfth (7) Fungi (and their toxins) Causing Foodborne Diseases.

Thirteenth (8) Microorganisms used in Food Industry (Lactic acid

bacteria, Yeasts, Molds).
Fourteenth (9) Probiotics.
Fifteenth (10) Microbiological Quality Control and Standard of Food.
Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 1 (6 Pages)
General Introduction

Name of Course: Food Microbiology.

Number of Credit Hours: 3 (2 hrs Theoretical + 2 hrs Lab.)
Course Objectives: To make the students acquainted with:
1-Understanding spoilage microorganisms and their effects on food.
2-Understanding how the various types of food processing affects the microflora of
food products.
3-Becoming familiar with procedures and techniques of detecting and enumerating
microorganisms in foods.
4-Be able to think critically about problems and issues concerning beneficial and
harmful microorganisms in foods.
Textbook:
Food Microbiology: An Introduction, Third Edition, Authors: Thomas J.
Montville and Karl R. Matthews. ASM Press, USA.
References:
1-Food Microbiology: An Introduction, Second Edition, Authors: Thomas J.
Montville and Karl R. Matthews.
2-Modern Food Microbiology, Seventh Edition, Authors: James M.
Jay and Martin J. Loessner
3-Food Microbiology, Fourth Edition, Authors: William C. Frazier
and Dennis C. Westhoff .

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Definition of Food Microbiology:
It is the science studies microorganisms in relation to food from the
standpoints of: (1) causing food spoilage (deterioration), (2) causing foodborne
diseases (infection or poisoning) and (3) those has beneficial use in food
processing (like baker’s yeast and probiotic microorganisms).

History of Food Microbiology:

Even though early human cultures discovered effective ways to preserve
food, they did not understood how these practices inhibited food spoilage or food
borne disease. Their ignorance was due to the belief that living things formed
spontaneously from nonliving matter (Theory of Spontaneous Generation).
Although it is extremely difficult to pinpoint the precise beginning of
human awareness of the presence and role of microorganisms in foods, the
available evidence indicate that this knowledge preceded the establishment of
bacteriology or microbiology as a science. The era prior to the establishment of
bacteriology as a science may be named as the prescientific era. This era may be
called “Food gathering period” and “Food producing period”. The first period
covers the time from human origin from over one million up to 8000 years ago,
and the second period from about 8000 to 10000 ago years (including the present
time). It is presumed that the problems of food spoilage and poisoning were
encountered early in this period.
Many early civilizations discovered and applied effective methods to
preserve and protect their food:
7000 BC –Babylonians discovered the fermentation through manufacturing beer.
6000 BC –First apparent reference to food spoilage in history was recorded.
3000 BC–Egyptians manufactured cheese.
1000 BC–Romans used snow (low temperatures) to preserve shrimp.
1683 –Leeuwenhoek examined and described bacteria through a microscope.

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1765–Spallanzani disproved the of spontaneous generation theory of life by
demonstrating that beef broth which was boiled and then sealed remained
sterile.
1795–Appert discovered food preservation by canning by placing meat in glass
bottles and boiled it.
1857-1887 Pasteur: a) proved that fermentative processes are caused by
microorganisms, b) discovered the “Pasteurization” which destroys undesirable
microorganisms in food, especially milk, c) noticed the relationship between
microorganisms in media and the chemical changes that occure in the media.
In the early 1920s, several nasty outbreaks of botulism led the canning industry
to adopt a very conservative heat treatment, known as the 12D process, that
reduces the probability of survival of the most heat resistant C. botulinum spores
to one in a billion (10-12).

Important Microorganisms of Food:

The most common microorganisms related to food (as harmful or
beneficial) are (bacteria, yeasts, molds), in addition to less common: (viruses,
protozoa and algae).
A) Bacteria:
Bacteria are the most important microorganisms to the food processor.
They are prokaryotes. Most are harmless, many are highly beneficial, some
indicate the probable presence of filth, disease organisms, spoilage and a few cause
disease.
There are thousands species bacterial, but all are single-celled and fall into
three basic shapes: spherical, straight rods, and curved (spiral) rods. They can’t
be seen with the naked eye unless with the microscope that magnifies about 1000-
fold.

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 All bacteria reproduce by the binary (transverse) fission where each parent
cell divides into two daughter cells. The two cells then divide to become 4, 4
become 8, and so forth.
Some rod-shaped bacteria are capable of existing in two forms, active
vegetative cells and dormant spores (called endospores). Vegetative cells form
spores under adverse conditions as a means of survival. Endospore forms preserve
the bacteria from starvation, drying, freezing, chemicals, and heat. When
conditions become favorable, the spores germinate, with each spore again
becoming a vegetative cell with the ability to reproduce. Among the bacteria,
sporulation is not a means of reproduction since each cell forms a single spore
which later germinates into a single cell again. Most sporulating bacteria that grow
in the presence of air belong to the Genus Bacillus, and most that grow only in the
absence of air belong to the Genus Clostridium.
B) Yeasts and Molds (Fungi):
Yeasts are oval-shaped and slightly larger than bacteria. They reproduce
most often by budding. In budding each cell can produce several buds, or
swellings, which break away to form new, fully formed daughter cells.
Molds as found on bread, fruit, or other surfaces are actually composed of
millions of microscopic cells joined together to form chains. The chains usually
have numerous branches, called hyphae. Molds can thrive in conditions too
adverse for bacteria or yeasts. They reproduce by spores that are frequently present
as green or black masses on the protruding hyphae.
As eukaryotes, yeasts and molds grow on most foods, on equipment, and
building surfaces where there are small amounts of nutrient and moisture. Since
bacteria grow faster, they greatly outnumber yeasts and molds in most foods.
However, bacteria find conditions of low pH, moisture, or temperature and high
salt or sugar unfavorable. In such environments, yeasts or molds predominate.

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Thus, they can be a problem in dry foods, salted fish, bread, pickles, fruits, jams,
jellies, and similar commodities.
C) Viruses:
Viruses are the smallest and simplest microorganisms. Unlike bacteria,
yeasts, and molds, viruses are incapable of reproducing independently. Instead,
they must first invade the cells of another living organism called the host, before
they can multiply. Hence, they are parasitic, so, viruses cannot multiply in foods.
The main mode of transmission therefore by food handlers and the use of dirty
utensils, which transfer the virus to food whereupon it is ingested by humans.

Viruses are normally specific in their selection of host cells, some infecting but
one species, while others are capable of infecting closely related species. As a
result, viruses which infect bacteria, called bacteriophages, cannot infect human
beings or other animals. On the other hand, several animal viruses, known as
zoonosis, can infect human beings.
The viruses are important to the food process in two respects:
1- Bacteriophage lactic infecting starter cultures can interfere with the manufacture
of cheese, buttermilk, pickles, and other desirable fermentative products.
2- Although viruses require a live host cell and cannot multiply in foods, they can
remain viable and infectious for long periods of time, even under highly adverse
conditions, such as drying, freezing, and pasteurization.
 Major pathogenic viruses related to the foods are:
 Rotaviruses and Norwalk virus are the major causes of gastroenteritis
 Viral hepatitis A outbreaks are mainly caused by infected food handlers.

Types of Foods According to their Shelflife:

1- Perishable foods: spoil quickly, must be preserved under convenient conditions.
Examples: milk, meats, most fruits and vegetables.

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2- Semi-perishable foods: don’t spoil quickly but not last for long, good handling
and preserving extend their shelfilfe. Examples: potatoes, onion, cauliflower.
3- Non-perishable foods: long shelfilfe, resist spoilage unless exposed to
deteriorating factors. Examples: sugar, flour, dried beans and milk.
Differences between Non-hygienic and Spoiled Foods:
The Non-hygienic food is that exposed to physical, chemical or biological
changes which making it unsafe (not healthy) for consumption. In such kind of
food spoilage signs (e.g. changes in color, taste, odor, texture) may not necessary
to appear on the food to avoid consuming it.
The Spoiled (Deteriorated) food is that where signs of spoilage changes
clearly appeared on it making it not healthy for consumption.
Major Groups of Foods:
I- Cereal grains and their products.
II- Meats, sea foods and their products.
III- Dairy (milk) and its products.
IV- Fruits, vegetables and their products.
V- Misalliances: Fats and oils, sugar, dried yeast, spices….

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Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 2 (4 Pages)
Factors Influencing Microorganisms of Food
Many factors (parameters) can affect the growth, metabolism and
survival of microorganisms of food. These factors are divided into four categories:
I-Intrinsic Factors:
1- Nutrient content.
2- pH and organic acids.
3- Redox potential.
4- Water Activity.
5- Biological constituents.
6- Antimicrobial substances.
II- Extrinsic Factors:
1- Relative humidity.
2- Temperature.
3- Gaseous Environment.
III-Implicit (‫ )ضمنية‬Factors (Growth rate, Synergism, Antagonism…….etc)
IV-Food Processing Factors (Heat treatment, Drying, Freezing, Chemical
preservation, Irradiation…..etc)
The first two categories contain the most common and important factors, so
they will be explained in some detail below:
(I) INTRINSIC FACTORS:
1-Nutrient Content: The chemical composition of a food influences the type of
microorganisms that will grow and the products that they will produce during
growth. They use organic compounds as energy and carbon sources. Food is rich in
nutrients. Meat is rich source of vitamin B and fruits are low but fruits are rich in

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ascorbic acid. Egg white contain biotin but also contains Avidin which ties up
biotin making it unavailable to microorganisms and eliminating possible spoilage
organism those which must have biotin supply.
2-pH and organic acids: Yeast and molds are more acid tolerant than bacteria.
Some foods have low pH because of inherent acidity such as fermented products
because of lactic acid during fermentation. Vegetable juices have low buffering
power so decrease in pH with production of only small amount of acid by lactic acid
bacteria. Milk is high in protein and permits growth and acid production by LAB in
the manufacture of fermented milk.

3-Oxidation-Reduction (Redox) Potential (Eh): The oxidation-reduction potential is

an indication of the oxidation and reducing power of the substrate. It is measured in
millivolt units, and defined as the "tendency to yield electrons (become oxidized) or
to capture electrons (become reduced)". The value of Eh at which microorganisms
grow determines whether they are aerobic or anaerobic.
The greater the degree in which a substance is oxidized, the more highly positive will
be its Eh value, and the greater the degree to which a substance is reduced, the more
highly negative will be its Eh value.
In general, aerobes require a substrate having a positive Eh, means that aerobic
microorganisms grow rapidly under a high oxidation-reduction potential. For
anaerobes it requires a substrate having a negative Eh.

The optimum Eh values for the growth of microbes vary with species. Yeasts and

molds require higher oxidation-reduction potential Eh values than most bacteria,

generally +200 to +400 millivolt.
Microorganisms that grow at:
 high Eh or +ve Eh (require oxygen) are Aerobes
 low Eh or –ve Eh (oxygen is toxic) are Anaerobes
 high and low Eh (+ve /-ve Eh) are Facultative anaerobes
 relative low Eh values are Microaerophilic
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4-Water Activity (aw): All microorganisms require free (unbounded) water in an

available form to grow and metabolize.

(aw) = Vapor pressure of food substrate/ Vapor pressure of pure water.

Water activity of pure water=1.0. The water activity of a food can be reduced by
increasing the concentration of solutes in the food.
Yeasts and molds can tolerate lower aw than bacteria. Gram-negative bacteria
require higher aw than Gram-positive bacteria.
Some of the aw values: 0.98 and above (fresh meat, fish, fruits, vegetables), 0.93-

0.98 (tomato paste, bread, cheese), 0.85-0.93 (beef, condensed milk), 0.60-0.85
(nuts, jam, jellies) and below 0.60 (chocolate, honey, potato chips, biscuits).
5-Biological Structures: Outer barriers against the invasion of microorganisms
(e.g. the skin of fruits and vegetables form a protective layer to invasion by
microorganisms). Damages during harvesting processing (peeling, skinning,
chopping) expose tissues and increase microbial loads throughout the product.
Milk has no protective barrier. Ground meat spoils faster than whole meat
cuts(grinding distributes surface microorganisms throughout). Eggs are usually
sterile inside but heavily contaminated on the shell, crack in the shell allows
microbes to enter.
6-Antimicrobial Substances: Some foods contain naturally-occurring
antimicrobial compounds that convey some level of microbiological stability to
them. There are a number of plant-based antimicrobial constituents, including
many essential oils, tannins, glycosides, and resins, that can be found in certain
foods. Specific examples include eugenol in cloves, allicin in garlic, cinnamic
aldehyde and eugenol in cinnamon, allyl isothiocyanate in mustard. Some
animal-based foods also contain antimicrobial constituents.
Examples on antimicrobial substances: lactoferrin, conglutinin, the
lactoperoxidase system in cow's milk and lysozyme in eggs and milk.

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(II) EXTRINSIC PARAMETERS:
1- Relative humidity (RH): When the food commodity of low water activity is
stored in atmosphere of high RH, water will transfer from the gas phase to the
food. Storage of fresh vegetables requires control of RH, but if the RH is too low
then the vegetables will lose water and deteriorate.
2-Temperature: It is one of the important environmental factors in controlling the
rate of microbial growth and reproduction. Microorganisms grow over a wide
range of temperatures, and according to this factor, they are divided into:
a) Psychrotrophs can grow below 7 o C with optimum at 20-30 o C.
b) Mesophiles able to grow well between 20-45 o C with optimum at 30-40o C.
c) Thermophiles able to grow well at ≥45 o C with an optimum of 55-65 o C.
3-Gaseous environment: Oxygen comprises 21% of earth and is the most
important gas in contact with food. Presence or absence of oxygen affects type of
microbial population including certain foodborne pathogens. Obligate aerobes
cannot grow under anaerobic conditions, while obligate anaerobes are unable to
grow under aerobic conditions.
Gases most related to food microbiology are:
 CO2 is the single most important atmospheric gas that is used to control
microorganisms in foods.
 O2 and O3 are important in modified atmosphere packaged (MAP) foods.
 O3 has antimicrobial properties, strong oxidizing agent, but should not be used
on high-lipid-content foods since it may increase the rancidity. It is recognized
GRAS (generally recognized as safe) in Australia, France, and Japan and USA.

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Food Microbiology Biology Dept. Al-Farabi UniversityCollege
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 3 a (7 Pages)
Effects of Food Processing Methods on Microorganisms

Microorganisms could spoil food (by changing its taste, texture, color, odor
or nutritive value) or making it unsafe for consumption (by causing fooddorne
diseases). To avoid such deterioration and diseases, growth of microorganisms
should be either delayed or inhibited through elimination, destruction (killing) or
removing.
Delaying microbial growth may be achieved by preserving food at low
temperatures (cooling and sometimes freezing), unfavorable pH or other methods.
Inhibiting microbial growth may be achieved by; pasteurization, boiling,
sterilization (canning), drying, chemical preservatives or irradiation.
Removing microorganisms from food could be performed by washing with
water or through membrane filtration.
How Food Preserving Methods Effect Microorganisms:
(1) Effect of low temperature methods:
(A) Cooling:
Preserving food at cooling temperatures delays harmful effects of
microorganisms on food by extending the Lag phase of their growth curve, so
cells will require longer time to reproduce and cause these effects.
Psychrotrophic and few mesophilic microorganisms may withstand cooling
temperatures and causing deterioration to the food; for instance, Pseudomonas
fluorescence is able to grow in refrigerators (1-4oC) and deteriorate food.

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 (B) Freezing:
While many home freezers compartments within the refrigerators are held at
-10°C, home chest freezers and commercial freezers are under -18°C. Although at
this temperature growth of microorganisms is almost stopped, deteriorative
microbial reactions will still occur, but over a much longer time.
Types of food freezing:
a) Slow freezing (0 to -10°C): takes 3-72 hours (depending on food nature).
b) Quick freezing (-18°C): takes around 30 minutes.
General effects of freezing on microbial cells:
a) Eliminating water activity due to water freezing inside the microbial cells.
b) Eliminating gases, especially O2 which effects cells metabolic activities.
c) Formation of ice crystals inside microbial cells which may rupture them.
d) Causing freeze shock and burn to the microbial cells may leads to their death.

(2) Effect of Blanching:

Blanching is a thermal process where vegetables are exposed to boiling
water or steam for a brief period (1 to 5 minutes) and then rapidly placed in cold
water to stop the action of heat.
Advantages of blanching:
1. Inactivation of most plant enzymes that may change the green color, texture or
flavor of vegetables during frozen storage.
2. Reducing the numbers of microorganisms including pathogens in the food.

3. Discarding the entrapped air in the plant tissues.

4- Helps in retarding loss of vitamins.

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(3) Effect of high temperature methods:
Treating food by high temperatures denatures protein of microbial cells.
(A) Pasteurization:
Pasteurization is the process of heating a food (usually a liquid) to or
below its boiling point for a defined period of time.
Purposes of pasteurization: (i) destroy all pathogens, (ii) reduces the number of
other microorganisms, (iii) inactivate enzymes (iv) maintain nutritive value of
food and (v) extends the shelf life of a food product.
Two groups of bacteria may not be killed by pasteurization:
Thermoduric: resist pasteurization but unable to grow on it. Ex: Lactobacillus
bulgaricus and Streptococcus thermophilus.
Thermophilic: resist pasteurization and can grow on it. Ex: Bacillus
stearothermophilus and Clostridium thermosaccharolyticum.
Types of pasteurization:
Low Temperature Long Time (LTLT): 63°C / 30 minutes.
High Temperature short Time (HTST): 72°C / 30 seconds.
Ultra-High Temperature (UHT): 139-140°C / 2-3 seconds.
(B) Canning (Sterilization):
Sterilization destroys all pathogenic and spoilage microorganisms in
foods and inactivates enzymes by heating. All canned foods are sterilized in a
retort (a large autoclave or pressure cooker). This process enables food to have
a shelf life of more than two years.
-Foods of a pH of more than 4.6, such as meat and most vegetables, must
undergo severe heating conditions to destroy all pathogens. These foods are
heated under pressure to 121°C for varying times. Sterilization is also called
“Steam under pressure” because the steam and pressure are formed inside the
retort after temperature exceeds 100°C which destructing the microorganisms.

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 -Severe conditions (sterilization) are applied to ensure that Clostridium
botulinum spores are destroyed during processing. These spores produce the
deadly botulism toxin under anaerobic conditions. The spores are destroyed by
heat or are inhibited at pH values of less than 4.6. Therefore, a food with a pH
of less than 4.6 that is packaged anaerobically, such as tomato paste, doesn't
need to undergo such a severe heat treatment.
-In food canning, commercial (not scientific) sterilization is used where
majority of the microbial population is destructed in a sufficient period of time
leaving a very few number of bacteria that has no effect on food safety and
quality when stored at normal temperature.

(4) Effect of Dehydration (Drying):

Dehydration is removing most of the water from food leading
to lower the water activity (aw) to the level below 0.86 which inhibits
or retards most microorganisms causing spoilage to the food.
-Dehydration by sun was one of the oldest methods of food
preservation. Traditionally now it is carried by other methods such as:
a) Spray-drying (by the evaporators) where the food is exposed to
high temperatures to remove the water as in the dried milk, fruits and
soups.

b) Lyophilization (by the freeze-dryers) where the food be freezing

first, then evaporated in a vacuum oven at not too high temperature
(45°C).This method keeps good quality of food as in the instant coffee.

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(5) Effect of Irradiation:
Radiation is the energy transmitted in the atmosphere. Food irradiation is
passing low doses of radiation through the particles of food. Irradiation was used
commercially for first by the Japanese in potatoes to prevent its germination.
-Radurization: A radiation treatment improving food quality and extending
shelflife by destructing most spoilage microorganisms. It is similar to pasteurization
by heat.
-Radappertization: It is the commercial sterilization of food by radiation not heat.
-Electromagnetic radiation is the most commonly used in preserving foods, and
there are three types of it:
(A) Ionic radiation: It is also called “Cold sterilization” because it doesn’t cause
any elevation in temperature of the exposed food. It ionizes particles of the material
exposed to it by the liberated ionic energy. Gamma radiation is most common and
cheapest type of ionic radiation used in preserving foods.
(B) Ultra-violet (UV) radiation: It is absorbed by the nucleic acids and protein
of the microbial cell causing various changes leading to destruct it through the fetal
mutants formed. Due to the poor penetration property, UV radiation has limited
uses (such as against oxidation, rancidity or color changing) in foods.
(C) Microwave: Its activity is due to the high temperature produced in the food
exposed to it as a result of the friction between the asymmetric particles
accumulated around the positive and negative poles. It is used in destruction of
mold in bread and in some alcoholic products.
(6) Effect of Food Preservative Agents:
A food preservative is an agent added to the food to preserve it from microbial
deterioration (spoilage) and poisoning.
-Examples on food preservatives:
(a) Organic acids and their salts: the most commonly used preservatives:
(Examples: acetic acid, benzoic acid, propionic acid, lactic acid, citric acid).
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 (b) Nitrates and Nitrites (e.g. Sodium nitrate).
(c) Sulfur dioxide and Sulfites.
(d) Epoxides (e.g. ethylene oxide and propylene oxide).
(e) Alcohols and Formaldehydes.
(f) Salts and Sugars.
(g) Spices and Condiments (e.g. garlic, mustard and clove).
(h) Antibiotics (e.g. Auromycin and Chloromycetin)
(i) Others e.g. Halogens (I, F, Br); Hydrogen peroxide; Gases (N2, CO2) .
-Mode of action of food preservative on microbial cell:
a) Destruction of cell wall by inhibiting its synthesis.
b) Effecting cytoplasmic membrane by changing its permeability.
c) Effecting cell’s proteins and enzymes by denaturation.
d) Effecting cell’s nucleic acids and reproduction.

(7) Effect of Ozone:

Ozone gas, has been used for years in applications such as treatment
water, as a food preservative for the cold storage of meats and to prevent the
growth of yeast and mold during the storage of fruits. Ozone is known as a broad-
spectrum biocide against viruses, bacteria, fungi and protozoa; none of which can
build up a resistive tolerance to ozone, because ozone disinfects and destroys
microorganisms by oxidation processes. Today, ozone technology is beginning to
be used effectively as an additional point in the food and beverage industry.
Ozone interferes with the metabolism of bacterium-cells, most likely through
inhibiting and blocking the operation of the enzymatic control system. A sufficient
amount of ozone breaks through the cell membrane leading to destruct the
bacteria.
Ozone destroys viruses by diffusing through the protein coat into the nucleic acid
core, resulting in damage of the viral RNA. At higher concentrations, ozone destroys

6
the exterior protein shell by oxidation so DNA or RNA structures of the
microorganism are affected.

(8) Effect of Hurdle Technology (Combination Processing):

The idea of hurdle technology is to use low-level combinations of existing
and novel preservation techniques ("hurdles") to eliminate the growth of
microorganisms.
-Lower-intensity individual methods can be used because of the collective effect of
the combined methods.
- Some of the more common hurdles include:
Pasteurization; Water activity (aw); Salt; Blanching; Freezing; Modified atmosphere
packaging (MAP); pH; Preservatives; Refrigeration; and irradiation.
-Examples of hurdle processing can be found in traditional and recently developed
foods, such as yoghurt where hurdles employed manufacture include:
(i) Low temperatures (ii) high acid and (iii) competitive microbial flora.

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Food Microbiology Continue Lec.3a:Effect of food processing methods on mos.

Lecture 3 b (4 Pages):
Kinetics of thermal destruction of food microorganisms
Killing microorganisms by heat is caused by denaturation of
proteins, especially through inactivation of enzymes (as proteins) required
for metabolism.
Using high temperatures and time in food preservation must by
mathematically calculated in an accurate way leads to destruct heat-resistant
spoilage and foodborne microorganisms as well as maintaining the
nutritional value of food. In this regard, Clostridium botulinum (especially
its spores), is the most concern in canning protein’s foods such as meats
and legumes.
As the most heat-resistant, bacterial spores (in suspensions) are heated
at different temperatures for periods of times, and the remaining numbers
of the heat-resistant spores after each period of time can be drawn in what so
called the survival curve. Such curve may take different patterns depending
on type of heat-resistant microorganisms or their spores.
Because of the large difference in resistance of food microorganisms
for the thermal (heat) treatment applied to the food, the thermal death for
each microorganism should be calculated.
Some terms used in thermal resistance of microorganisms of food:
1. Thermal Death Time (TDT): (or F-value) is the time (in minutes) required
to kill a given type and number of organisms (or spores) at a specified
temperatures. So, by this method, the temp. is kept constant and the time
necessary to kill all cells or spores is determined.

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2. Thermal Death Point (TDP): is the temperature required to kill a given
type and number of organisms (or spores) at a specific time period. In this
case, heating time is kept constant and temp. is determined.
3. Decimal reduction value (D-value): is the time (in minutes) required to kill
90% of a given type and number of organisms (or spores) at a specified
temperature. This value is numerically equal to the number of minutes
required for the survivor curve to traverse one log cycle.
4. Z-value: is the temperature change required to change the D value by a
factor of 10. In the illustration below the Z value is 10°C. The Z value
reflects the temperature dependence of the reaction.

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 The decimal reduction time decrease when the temp. increases, for example :
D120C = 8 min., when the temp. becomes higher 5 degree.
D125C = 2.5 min.
D130C = 0.8 min.
Much decline in the third term because in the rest of the terms, application
cannot remove all microorganisms, so it works to reduce microbial number
to keep 10% of it which usually considered as heat resistant.
D-value can be calculated through this equation:

D (n) = T (holding time) / (Log a - Log b)

T = T2 – T1 = (Final time – Initial time)
n = exposure temp.
a = viable count (initial) beginning of heat temp.
b = viable count (final) end of heat treatment.

Factors Affecting Heat Resistance of Microorganisms:

1. Temperature time relationship: the time of killing cells or spores under a
given set of conditions decrease as the temp. is increased.
2. Initial concentration of spores or cells: the more spores or cells present,
the greater the heat treatment necessary to kill all of them.
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3- Culture medium: Despite that the kind, and amount of the nutrients in the
medium vary with the organism, but in general the optimum medium for
growth, the more resistant the cells or spores to heat.
4- Temperature of incubation: In general, heat-resistant increases as the
incubation temp. raises toward the optimal for the organism.
5- Phase of growth: Bacterial cells show greatest resistance during lag phase.
6- Composition of the substrate in which microorganisms are heated:
a. Moisture: almost heat is much more effective killing agent in moist
substrate than in dry.
b. Hydrogen concentration (pH): Cells or spores are most heat-resistant in
substrate that is at or near neutrality. In general, increasing pH toward
acidity is more effective than toward the alkalinity.
c. Nature of substrate: substrates with low salt and sugar concentration
have protective effect organisms.

Effect of Temperature on the Microorganism:

1. Denaturation of proteins.
2. Inhibition of enzyme activity.
3. Destruction of nucleic acid and membranes.

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4-Water Activity (aw): All microorganisms require free (unbounded)
water in an available form to grow and metabolize.
(aw) = Vapor pressure of food substrate/ Vapor pressure of pure
water.
Water activity of pure water=1.0. The water activity of a food can be
reduced by increasing the concentration of solutes in the food.
Yeasts and molds can tolerate lower aw than bacteria. Gram-negative
bacteria require higher aw than Gram-positive bacteria.
Some of the aw values: 0.98 and above (fresh meat, fish, fruits,
vegetables), 0.93-0.98 (tomato paste, bread, cheese), 0.85-0.93 (beef,
condensed milk), 0.60-0.85 (nuts, jam, jellies) and below 0.60 (chocolate,
honey, potato chips, biscuits).
5-Biological Structures: Outer barriers against the invasion of
microorganisms (e.g. the skin of fruits and vegetables form a protective
layer to invasion by microorganisms). Damages during harvesting
processing (peeling, skinning, chopping) expose tissues and increase
microbial loads throughout the product. Milk has no protective barrier.
Ground meat spoils faster than whole meat cuts(grinding distributes
surface microorganisms throughout). Eggs are usually sterile inside but
heavily contaminated on the shell, crack in the shell allows microbes to
enter.
6-Antimicrobial Substances: Some foods contain naturally-occurring
antimicrobial compounds that convey some level of microbiological
stability to them. There are a number of plant-based antimicrobial
constituents, including many essential oils, tannins, glycosides, and
resins, that can be found in certain foods. Specific examples include
eugenol in cloves, allicin in garlic, cinnamic aldehyde and eugenol in
cinnamon, allyl isothiocyanate in mustard. Some animal-based foods
also contain antimicrobial constituents.
Examples on antimicrobial substances: lactoferrin, conglutinin, the
lactoperoxidase system in cow's milk and lysozyme in eggs and milk.

5
Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 4 (4 Pages)
Indicator Bacteria
Definition: Indicator bacteria are types of bacteria used to detect and
estimate the level of fecal contamination of water and sometimes food. They are
not dangerous to human health but are used to indicate the presence of a health
risk.

Indicator microorganisms are organisms that provide insight (‫ )رؤيا‬to the

history of a sample or to the associations with other organisms or conditions
(e.g., they can indicate the potential presence of pathogens or spoilage
organisms). Coliform bacteria have been used as indicators of unsanitary
conditions in water and foods for over a century. This concept originated in the
late 1800’s after E. coli was found to be common in feces, and it’s detection in
water was used to “indicate” an increased likelihood that pathogens such as
Salmonella typhi were in the water as well as an indicator of unsanitary
conditions. Indicators have been applied to both food and water safety and
quality.

Indicator microorganisms should be:

a- Easy distinguishable from other microorganisms common in a sample.
b- Easily detected and enumerated in a relatively short period of time.
c- Able to survive in the water or food being tested.
d- Associated, directly or indirectly, with reduced safety or loss of quality.
Each gram of human feces contains approximately ~100 billion (1×1011)
bacteria. These bacteria may include species of pathogenic bacteria, such

1
as Salmonella or Campylobacter, associated with gastroenteritis. In addition,
feces may contain pathogenic viruses, protozoa and parasites. Fecal material can
enter the environment from many sources including waste water treatment
plants, livestock or poultry manure, septic systems, sewage sludge, pets and
wildlife. If sufficient quantities are ingested, fecal pathogens can cause disease.

Types of Indicator Microorganisms:

The indicator bacteria, which are found in the intestinal tracts of warm
blooded, are:
1-Total Coliforms: Coliforms are bacteria always present in the digestive
tracts of human and animals and wastes. They are also found in plant and soil
material. Coliform bacteria include: Escherichia coli, Enterobacter
aerogenes, Klebsiella pneumoniae and Citrobacter sp; the first two are the most
common.
Coliform bacteria can be identified based on the fact that they all metabolize
the sugar lactose, producing both acid and gas as byproducts. They are most
often defined as facultative anaerobic, Gram negative, non-spore-forming,
oxidase-negative rods that ferment lactose (by β-galactosidase) to produce acid
and gas within 24-48 hours at 35-37°C. Most coliform bacteria are not
associated with foodborne illness.

2-Fecal Coliforms: Escherichia coli is the major species of fecal coliforms.

Fecal coliforms are the group of the thermophilic total coliforms that are
considered to be present specifically in the gut and feces of warm-blooded
animals, and considered a more accurate indication of animal or human waste
than the total coliforms. Fecal coliforms are characterized by: (a) produce
acid and gas from lactose at 44.5±0.2°C within 24±2h, (b) produce indole from
tryptophan, and (c) able to produce β-glucuronidase enzyme.

2
 Despite that most coliform bacteria do not cause disease, but rare strains of E.
coli, particularly the strain 0157:H7 (others may include opportunist strains of
Enterobacter sakazakii and Citrobacter freundii). Strain 0157:H7 can cause
serious illness due to the shiga-like toxin it produces. Outbreaks of disease
caused by this strain have generated much public concern about this organism.
E. coli 0157:H7 has been found in cattle, chickens and sheep. Most of the
reported human cases were due to eating under cooked meat.

3-Fecal streptococci (FS): Gram-positive, catalase-negative cocci from

selective media (azide dextrose broth or m Enterococcus agar) that grow on bile
aesculin agar and at 45°C, belonging to the genera Enterococcus and
Streptococcus possessing the Lancefield group D antigen.

4-Enterococci: The enterococci are a subset of fecal streptococci that grow at

pH 9.6, 10° and 45°C and in 6.5% NaCl. Nearly all are members of the genus
Enterococcus which are resistance to 60°C for 30 min and ability to methylene
blue. Alternatively, enterococci can be directly identified by ability of aerobic
growth at 44±0.5°C.

5-Bacteriophages (viruses): These are bacterial viruses and are ubiquitous in

the environment. For water quality testing and to model human enteric viruses,
most interest in somatic coliphages, and phages infecting Bacteroides fragilis.

Methods of detection Indicator Bacteria:

Indicator bacteria can be cultured on specific media which allow
growth of the species of interest and inhibit growth of other organisms. They can
be detected in the following methods:

1- Conventional method: water samples are filtered through membrane

filters with small pore sizes and then the membrane is placed onto a selective

3
agar. It is often necessary to vary the volume of water sample filtered in order to
prevent too few or too many colonies from forming on a plate. Bacterial
colonies can be counted after 24 to 48 hours depending on the type of bacteria.
Counts are reported as colony forming units per 100 ml (cfu/100 ml).

2- Using chromogenic substances method: is one fast technique for

detecting indicator organisms. These substances are added to the conventional
(or newly devised) media used for isolation of the indicator bacteria. The
chromogenic compounds are modified to change color or fluorescence by the
addition of either enzymes or specific bacterial metabolites which makes it easy
for detection and avoids the need for isolation of pure cultures and confirmatory
tests.

Drinking water quality standards for bacteria:

World Health Organization (WHO) Guidelines for Drinking Water
Quality state that as an indicator organism Escherichia coli provides conclusive
evidence of recent fecal pollution and should not be present in water used for
human consumption. In the U.S., the Total Coliform Rule states that a water
system is out of acceptance if more than 5 % of its monthly water samples
contain coliforms.

Microbiological methods for testing drinking water:

1-Aerobic Plate Count (APC).
2-Most Probable Number (MPN).
3-Membrane filtration.
4-Colifoms test: Composed of three principle tests; Presumptive, Confirmed
and Completed.

4
Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 5 (2 Pages)
Foodborne Diseases (Illness)
Definition and Causes: Foodborne disease (or foodborne illness) is
any illness resulting from food spoilage, pathogenic food microorganisms
(or their toxins) and toxic materials.
Foodborne illness usually arises from improper handling, preparation,
or storage of food. Good hygiene practices before, during, and after food
preparation can reduce the chances of foodborne illness. Regular hand-
washing is one of the most effective defenses against the spread of this
illness. Foodborne illness can also be caused
by pesticides or medicines in food and naturally toxic substances such
as poisonous mushrooms.
Symptoms of foodborne illness vary depending on the cause. The
incubation period ranges from hours to days according to the cause and
on how much contaminated food was consumed. General symptoms
include; upset stomach, vomiting, fever, and may include diarrhea and
dehydration. Bouts (‫ )نوبات‬of vomiting can be repeated with an extended
delay in between, because even if infected food was eliminated from the
stomach in the first bout, microbes can pass through the stomach into
the intestine and begin to multiply in the cells lining the intestinal walls.
Some types of microorganisms stay in the intestine, some produce
a toxin that is absorbed into the bloodstream, and some can directly
invade deeper body tissues.
Food safety is the action of monitoring food to ensure that it will not
cause foodborne illness.

1
Differences between Food Poisoning and Food Infection:
In Food poisoning (also called intoxication), the toxins are released to
the food by bacteria (e.g. and the disease may occur by ingesting the food
without live bacteria (Ex: Clostridium, Staphylococcus).

In Food-infections, the live bacteria should be ingested with the food

such as, to initiate the infection in the intestines of human body. Ex:
(Salmonellosis).

In food poisoning, the microbes multiply readily in the food prior to

consumption, whereas in food infection, food is merely the vector for
viable microbes that do not grow on food substrate.

Bacteria (or their toxins) Causing Foodborne Diseases:

Bacteria are a common cause of foodborne illness. Bacterial
infections were thought to be more prevalent because few places had the
capability to test for norovirus. Toxins from bacterial infections are
delayed because the bacteria need time to multiply. As a result, symptoms
associated with food poisoning are usually not appeared until 12–72 hrs
or more after eating contaminated food. However, in some cases, such as
Staphylococcal food poisoning, the onset of illness can be as soon as 30
minutes after ingesting contaminated food.
Most common bacterial foodborne pathogens:
(Staphylococcus aureus; Clostridium botulinum; Clostridium
perfringens; Bacillus cereus, Salmonella spp (mainly S.typhimurium);
Enterohemorrhagic E. coli (EHEC); Campylobacter jejuni; Listeria
monocytogenes).
Less common bacterial foodborne pathogens:
(Escherichia coli (other virulence properties); Shigella spp.; Vibrio
cholerae; Streptococcus spp.; Vibrio parahaemolyticus).

2
 Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani
Lecture 6 (13 Pages)
Bacteria (and their toxins) Causing Foodborne Diseases
1- Staphylococcus aureus: (Incubation period: 1- 6 hrs.)
It is G (+) cocci that occurs in singles, pairs, short chains, tetrads but
mostly as grape-like clusters, mainly aerobic but can grow anaerobically,
coagulase positive. Only those strains that produce enterotoxin can cause food
poisoning. Food is usually contaminated from the infected food handlers.
Suspected foods: Bakery food filled with custard or cream, chicken, meat, milk,
fish, salads, etc.
Pathogenesis: If the food is stored for some time at room temperature, the bacteria
may multiply in the food and produce toxin called enterotoxin. S. aureus is known
to produce six serologically different types of enterotoxins (A, B, C, C2, D and E)
that differ in toxicity. Most food poisoning is caused by enterotoxin A. These
enterotoxins are heat stable, with type B being most heat resistant.
Symptoms (Clinical features): The onset is sudden (even after 30 minutes) and is
characterized by vomiting and diarrhea but no fever. Ingestion of as little as 23 µg
of enterotoxin can induce vomiting and diarrhea. The illness lasts less than 12
hours. There are no complications and treatment is usually not necessary.
Laboratory diagnosis: The presence of a large number of S. aureus organisms in
a food may indicate poor handling or sanitation. Staphylococcal food poisoning
can be diagnosed if they are isolated in large numbers from the food and their
toxins demonstrated in the food or the isolated S. aureus must be shown to produce
enterotoxins. The bacteria may be detected on Baird-Parker agar, Mannitol Salt
agar or Staphylococcus 110, while the enterotoxin can be detected and identified
by gel diffusion.
1
2- Clostridium botulinum: (Incubation period: 12-36 hrs.)
It is a Gram (+), anaerobic, sporeforming bacilli that is widely distributed in
soil, sediments of lakes and decaying vegetation.
Suspected foods: Most cases of botulism are associated with home canned or
bottled meat, beans and fish. In general, the low and medium acid canned foods are
often involved. The anaerobic environment produced by the canning process may
further encourage the outgrowth of spores.
Pathogenesis: Not all strains of C. botulinum produce the toxin botulism and cause
food poisoning. Seven immunologically distinct forms of botulism (designated A,
B, C1, D, E, F, and G) exist. If contaminated food has been insufficiently sterilized
or canned improperly, the spores may germinate and produce botulism. The toxin
is released only after the death and lysis of cells. The toxin resists digestion and is
absorbed by the upper part of the GI tract and then into the blood and blocks the
release of the neurotransmitter acetylcholine. This results in muscles paralysis.
Symptoms (Clinical features): Common features include; vomiting, thirst,
dryness of mouth, constipation, blurred-vision (‫)عذم وضوح الرؤيا‬, difficulty in
speaking, breathing and swallowing. Coma )‫ )غيبوبة‬may occur in some cases. Death
may occur due to the respiratory paralysis within 7 days.
Laboratory diagnosis: Spoilage of food or swelling of cans or presence of
bubbles inside the can indicate clostridia growth. Food is homogenized in broth
and inoculated in Robertson cooked meat medium and blood agar or egg-yolk agar,
which is incubated anaerobically for 3-5 days at 37o C. The toxin can be
demonstrated by injecting intraperitoneally the extract of food or culture into
experimental animals.

3- Clostridium perfringens: (Incubation period: 8-24 hrs.)

It is a G (+), anaerobic, sporeforming bacilli that is present abundantly in the
environment, vegetation, sewage and animal feces.

2
Suspected foods: foodborne outbreaks of C. perfringens involve meat products
that are eaten 1- 2 days after preparation. Meats that have been cooked, allowed to
cool slowly, and then held for some time before eating are commonly incriminated,
in addition to fish pastes and cold chicken.

Pathogenesis: Spores in food may survive cooking and then germinate when they
are improperly stored. When these vegetative cells form endospores in the
intestine, they release enterotoxins. The bacterium is known to produce at least 12
different toxins. Food poisoning is mainly caused by Type A strains leading to
excessive fluid accumulation in the intestinal lumen.
Symptoms (Clinical features): Illness is characterized by acute abdominal pain,
diarrhea, and vomiting. Illness is self-limiting and patient recovers in 18-24 hrs.

Laboratory diagnosis: Since the bacterium is present normally in the intestine,

their isolation from feces may not be sufficient to implicate it. Similarly, isolation
from food except in large numbers (>105 / 1 gram) may not be significant. The
homogenized food is diluted and plated on selective medium as well as Robertson
cooked meat medium and incubated anaerobically. The isolated bacteria must be
shown to produce enterotoxin.

4- Bacillus cereus:
Bacillus cereus is a G (+), aerobic, sporeforming bacilli. It is found
abundantly in environment and vegetation.
Suspected foods: Commonly associated with rice and vegetables.
Incubation period: 1-6 hrs in short-incubation form and 8-16 hrs in long
incubation form.
Pathogenesis: During the slow cooling, spores germinate and vegetative bacteria
multiply resporulate again. Sporulation is also associated with toxin production.
The toxin is heat-stable, and can easily withstand the brief high temperatures used
to cook rice. The short-incubation form is most often associated with rice that has
been cooked and then held at warm temperatures for several hours. Long-

3
incubation food poisoning is frequently associated with meat or vegetable-
containing foods after cooking. The short-incubation form is caused by a
preformed heat-stable enterotoxin, while the long incubation form is mediated by
a heat-labile enterotoxin which activates intestinal adenylate cyclase and causes
intestinal fluid secretion.
Symptoms (Clinical features): Bacillus cereus causes two types of foodborne
intoxications. The „emetic-type‟ or the short incubation type (1-6 hrs.). It is
characterized by nausea, vomiting and abdominal cramps. The second type is the
long incubation type manifested primarily by abdominal cramps and diarrhea
(light or heavy and watery) within (8-16 hrs.). This type is referred to as the "long-
incubation" or diarrheal form of the disease and it resembles food poisoning caused
by Clostridium perfringens. In either type, the illness usually lasts less than 24
hours after onset.
Laboratory diagnosis: The short-incubation or emetic form of the disease is
diagnosed by the isolation of B. cereus from the suspected foods. The long-
incubation or diarrheal form is diagnosed by isolation of the organism from stool
and food. Isolation from stools alone is not sufficient because 14% of healthy
adults have been reported to have gastrointestinal colonization with B. cereus.

5- Salmonella enteritidis: (Incubation period: 12-36 hrs).

These are Gram negative rod shaped bacteria that are classified under family
Enterobacteriaceae. This species does not occur normally in humans but several
animals act as reservoirs.
Suspected foods: Mainly Chicken infected with Salmonella, which find its way
into its feces or eggs, in addition to Milk and its products including ice creams.
Pathogenesis: Organism penetrates and passes through the epithelial cells lining of
the small intestine where LPS is released and fever caused. Prostaglandins cause
electrolytes to flow into lumen of the intestine. Water flows into lumen in response
to osmotic imbalance resulting in diarrhea.

4
Symptoms (Clinical features): Sudden onset of abdominal pain, nausea,
vomiting and diarrhea, which may be watery, greenish and foul smelling. This may
be preceded by headache and chills. Later, prostration, muscular weakness and
moderate fever. In most cases the symptoms resolve in 2-3 days without any
complications.
Laboratory diagnosis: Homogenized food is cultured in Selenite F broth and then
sub-cultured on deoxycholate citrate agar. Plates are incubated at 37o C overnight
and growth identified by biochemical tests and slide agglutination test.
6- Enterohemorrhagic E. coli (EHEC): (Incubation period: 72-120 hrs).
Escherichia coli are Gram negative enteric bacilli that are carried normally in
the intestine of humans and animals. E. coli O157:H7 is the most common serotype
of EHEC.

Suspected foods: Cattle are the main source of infection. This disease is often
associated with ingestion of inadequately cooked hamburger meat, raw milk,
cream and cheeses made from raw milk.

Pathogenesis: EHEC strains may produce one or more types of cytotoxins, which
are collectively referred as Shiga-like toxins (SLT) since it is similar to Shiga
toxin produced by Shigella dysenteriae. SLTs were previously known as verotoxin.
The toxins kill colonic epithelial cells.
Symptoms (Clinical features): Initial symptoms may be diarrhea with abdominal
cramps, which may turn into bloody diarrhea in a few days. No fever occurs.
Laboratory diagnosis: Laboratory diagnoses involve culturing the feces on
McConkey's agar or on sorbitol McConkey's agar, where they don't ferment
sorbitol. Strains can then be identified by serotyping using specific antisera. SLTs
can be detected by ELISA.

5
7- Campylobacter jejuni: (Incubation period: 2 to11 days).
These are small, curved-spiral Gram negative bacilli with polar flagella,
appearing mainly in comma shape. Campylobacter are harbored in reproductive
and alimentary tracts of some animals.
Suspected foods: Transmission to humans occurs via a fecal-oral route, originating
from farm animals, birds, dogs, and processed poultry, with chicken preparation
comprising 50-70% of all campylobacter infections. The organism is transmitted to
man in milk, meat products and contaminated water. Undercooked poultry and
unpasteurized dairy are most often implicated as a source of it.
Pathogenesis: As few as 500 organisms can cause enteritis. The organism is
invasive but generally less so than Shigella. Campylobacter produces adenylate
cyclase-activating toxins same as of E .coli LT and cholera.
Symptoms (Clinical features): Abdominal pain and cramps, diarrhea, headache,
and usually fever. Typically the diarrhea is watery, but in severe cases bloody
diarrhea may occur. Diarrhea may last 2-7 days and the bacteria may stay in the
patients stool for up to 2 months. Bacteremia is observed in a small cases.
Laboratory diagnosis: The feces may be inoculated in enrichment medium or on
selective media such as Campy BAP or Skirrow's medium. The plates are
incubated in microaerophilic conditions at 42o C for 2-5 days.

8- Listeria monocytogenes: (Onset time 2-20 days)

This species is the only one out of the ten spscies causing foodborne
illness called listeriosis. The bacterium is a G (+) rods, facultative anaerobe
(capable of surviving in the presence or absence of oxygen), able to grow as low as
0 °C permits multiplication at typical refrigeration temperatures, motile with
peritrichous actin flagella at room temp.(about 25 °C and below), but not at body
temp.(37 °C). It can grow and reproduce inside the host's cells and is one of the
most virulent foodborne pathogens with a fatal risk of 20 to 30%. In the US and

6
EU (European Union), listeriosis is the third-leading cause of death among
foodborne bacterial pathogens, with mortality rates exceeding
even Salmonella and Clostridium botulinum. However, L. monocytogenes was not
identified as a cause of foodborne illness until 1981.

Growth conditions: Temperature: range: 1-45°C and optimum 30-37°C. pH

range: 4.5-9.6. Salt Tolerance: 25.5%. Environment: Prefers 10% CO2.

Sources of the organism: Intestinal tracts of animals and humans, contaminated

water, soil, manure (‫)مخلفات الحيوانات‬.

Suspected foods: Unpasteurized milk, soft cheeses made from unpasteurized milk,
raw fruits and vegetables, ready-to-eat meats and salad

Pathogenicity: Listeriosis is a potentially fatal disease which occurs most often in

immunocompromised individuals and elderly. Pregnant women and their unborn
fetus are at the greatest risk. Meningitis in newborns from mothers, so pregnant
mothers are often advised not to eat soft cheeses, which may be contaminated with
the bacteria. Meningitis and septicemia may result in spontaneous
abortion or stillbirths. Listeria monocytogenes can infect the brain, spinal cord
membranes and/or the bloodstream of the host.
Symptoms (Clinical features): Meningitis, abortions and stillbirths, diarrhea,
nausea, vomiting, fever, confusion, septicemia. The onset time to serious forms of
listeriosis may range from a few days to three weeks. The onset time to
gastrointestinal symptoms is unknown but probably exceeds 12 hours.
Laboratory diagnosis: The Anton test is used to identify of L. monocytogenes;
instillation of a culture into the conjunctival (‫ )الملتحمة‬sac of a rabbit or guinea pig
causes severe kerato-conjunctivitis within 24 hours. Listeria species grow on
media such as Mueller-Hinton agar. The organism is a facultative anaerobe,
produces acid but not gas, hemolysin producer, catalase-positive and motile.

7
Treatment: Reports of successful treatment with parenteral
(‫ )بالحقن‬penicillin or ampicillin exist. Trimethoprim-sulfamethoxazole is used.

Some other Bacteria Causing Food Illness (less common):

9- Enterotoxigenic Escherichia coli (ETEC)
Enterotoxigenic E coli (ETEC) are bacteria that cause disease in humans
and domestic animals by producing a heat labile or heat stable toxin or both.
It is an important cause of bacterial diarrheal illness. Infection with ETEC is
the leading cause of travelers' diarrhea and a major cause of diarrheal disease in
lower-income countries, especially among children. ETEC is transmitted by food
or water contaminated with animal or human feces.
Suspected foods: Fresh fruits and vegetables (especially lettuce), parsley, basil,
scallops, shrimp, crabs, tuna paste, salads, and soft cheese. Contaminated water
and food are vehicles for transmission of ETEC infection. Infection can be
prevented by avoiding or safely preparing foods and beverages that could be
contaminated with the bacteria, as well as washing hands with soap frequently.
Symptoms of ETEC: The main characteristic of the disease is a watery diarrhea.
It begins about 14-50 hours after ingesting the bacteria. The diarrhea may be mild
and last only a few days or it may become severe and prolonged, lasting up to three
weeks. Generally the diarrhea does not contain blood or mucus. Other symptoms
include: Abdominal cramps, Low-grade fever, Nausea, Headache and Muscle
aches. The pathogenesis of ETEC-induced diarrhea is similar to that of cholera and
includes the production of enterotoxins and colonization factors.
Clinical symptoms of ETEC infection: Range from mild diarrhea to a severe
cholera-like syndrome. The effective treatment of ETEC diarrhea by rehydration is
similar to treatment for cholera, but antibiotics are not used routinely for treatment
except in traveler's diarrhea. The frequency and characterization of ETEC on a

8
worldwide scale are inadequate because of the difficulty in recognizing the
organisms; no simple diagnostic tests are presently available.
10- Vibrio parahaemolyticus
It is a Gram-negative curved rod, facultative aerobic, non-spore forming,
oxidase positive bacterium. It is motile by one polar flagellum. Vibrio
parahaemolyticus is a self-limiting, enterotoxic bacterium, typically causing acute
gastroenteritis in humans. More severe cases of infection can occur in immune-
compromised individuals, which can lead to septicemia and death, although this is
very rare. Moderate to severe skin infections can also result from open wound
exposure to V. parahaemolyticus in warm seawater, Vibrio parahaemolyticus is
typically transmitted to human hosts through the consumption of raw and
undercooked shellfish including clams (‫)رخويات‬, mussels (‫)المحار‬, and oysters, or
drinking contaminated water.
11- Shigella toxins (heat stable and heat labile).
Shigella is a genus belongs to Enterobacteriaceae family that causes
disease in humans and animals. The disease caused by the ingestion of Shigella
bacteria is referred to as Shigellosis, or „bacillary dysentery‟ which is most
typically associated with diarrhea (often bloody) and other GI symptoms.
Compared to other bacteria, Shigella is easily spread person-to-person because:
(1) its relatively tiny infectious dose (Infection can occur after ingestion of fewer
than 100 bacteria). (2) it thrive (‫ )يزدهرنموها‬in the human intestine and can commonly
spread either by person-to-person contact or through food contamination.
Shigella transmit through a fecal-oral route. Foods that come into contact with
human or animal waste can transmit Shigella. Thus, handling baby diapers, eating
vegetables from a field contaminated with sewage, or drinking pool water are all
activities that can lead to shigellosis.

9
12-Yersinia enterocolitica:
Yersinia enterocolitis is a Gram negative rod bacterium belong to the
Enterobacteriaceae family. It causes an infectious disease called Yersiniosis.
Common symptoms of Yersiniosis in children are: fever, abdominal pain,
and diarrhea, which is often bloody. Symptoms typically develop 4 to 7 days after
exposure and may last 1 to 3 weeks or longer. In older children and adults, right-
sided abdominal pain and fever may be the predominant symptoms and may be
confused with appendicitis. Yersiniosis is most often acquired by eating
contaminated food, especially raw or undercooked pork products. The preparation
of raw pork intestines may be particularly risky. Drinking contaminated
unpasteurized milk or untreated water can also transmit the infection.

The chart below includes foodborne disease-causing organisms that

frequently cause illness in the United States.
Onset
Time
After
Organism Common Name of Illness Ingesting Signs & Symptoms Duration Food Sources

Bacillus cereus B. cereus food poisoning 10-16 hrs Abdominal cramps, 24-48 Meats, stews,
watery diarrhea, hours gravies, vanilla
nausea sauce

Campylobacter Campylobacteriosis 2-5 days Diarrhea, cramps, 2-10 days Raw and
jejuni fever, and vomiting; undercooked
diarrhea may be poultry,
bloody unpasteurized
milk,contaminated
water

Clostridium Botulism 12-72 Vomiting, diarrhea, Variable Improperly canned

botulinum hours blurred vision, double foods, especially
vision, difficulty in home-canned
swallowing, muscle vegetables,
weakness. Can result fermented fish,
in respiratory failure baked potatoes in
and death aluminum foil

Clostridium Perfringens food 8–16 hours Intense abdominal Usually Meats, poultry,
perfringens poisoning cramps, watery 24 gravy, dried or
diarrhea hours precooked foods,

10
 Onset
Time
After
Organism Common Name of Illness Ingesting Signs & Symptoms Duration Food Sources

time and/or
temperature-abused
foods

Cryptosporidium Intestinal 2-10 days Diarrhea (usually May be Uncooked food or

cryptosporidiosis watery), stomach remitting food contaminated
cramps, upset stomach, and by an ill food
slight fever relapsing handler after
over cooking,
weeks to contaminated
months drinking water

Cyclospora Cyclosporiasis 1-14 days, Diarrhea (usually May be Various types of

cayetanensis usually at watery), loss of remitting fresh produce
least 1 appetite, substantial and (imported berries,
week loss of weight, relapsing lettuce, basil)
stomach cramps, over
nausea, vomiting, weeks to
fatigue months

E. coli E. coli infection 1-3 days Watery diarrhea, 3-7 or Water or food
(Escherichia (common cause of abdominal cramps, more contaminated with
coli) “travelers‟ diarrhea”) some vomiting days human feces
producing toxin

E. coli O157:H7 Hemorrhagic colitis 1-8 days Severe (often bloody) 5-10 days Undercooked beef
or E. coli O157:H7 diarrhea, abdominal (especially
infection pain and vomiting. hamburger),
Usually, little or no unpasteurized milk
fever is present. More and juice, raw
common in children 4 fruits and
years or younger. Can vegetables (e.g.
lead to kidney failure. sprouts), and
contaminated water

Hepatitis A Hepatitis 28 days Diarrhea, dark urine, Variable, Raw produce,

average jaundice, and flu-like 2 weeks- contaminated
(15-50 symptoms, i.e., fever, 3 months drinking water,
days) headache, nausea, and uncooked foods
abdominal pain and cooked foods
that are not
reheated after
contact with an
infected food
handler; shellfish
from contaminated
waters

Listeria Listeriosis 9-48 hrs Fever, muscle aches, Variable Unpasteurized

monocytogenes for gastro- and nausea or diarrhea. milk, soft cheeses
intestinal Pregnant women may made with

11
 Onset
Time
After
Organism Common Name of Illness Ingesting Signs & Symptoms Duration Food Sources

symptoms, have mild flu-like unpasteurized milk,

2-6 weeks illness, and infection ready-to-eat deli
for can lead to premature meats
invasive delivery or stillbirth.
disease The elderly or
immunocompromised
patients may develop
bacteremia or
meningitis.

Noroviruses Variously called viral 12-48 hrs Nausea, vomiting, 12-60 hrs Raw produce,
gastroenteritis, winter abdominal cramping, contaminated
diarrhea, acute non- diarrhea, fever, drinking water,
bacterial gastroenteritis, headache. Diarrhea is uncooked foods
food poisoning, and food more prevalent in and cooked foods
infection adults, vomiting more that are not
common in children. reheated after
contact with an
infected food
handler; shellfish
from contaminated
waters

Salmonella Salmonellosis 6-48 hours Diarrhea, fever, 4-7 days Eggs, poultry,
abdominal cramps, meat, unpateurized
vomiting milk or juice,
cheese,
contaminated raw
fruits and
vegetables

Shigella Shigellosis or Bacillary 4-7 days Abdominal cramps, 24-48 hrs Raw produce,
dysentery fever, and diarrhea. contaminated
Stools may contain drinking water,
blood and mucus. uncooked foods
and cooked foods
that are not
reheated after
contact with an
infected food
handler

Staphylococcus Staphylococcal food 1-6 hours Sudden onset of severe 24-48 Unrefrigerated or
aureus poisoning nausea and vomiting. hours improperly
Abdominal cramps. refrigerated meats,
Diarrhea and fever potato and egg
may be present. salads, cream
pastries

Vibrio V. 4-96 hours Watery (occasionally 2-5 days Undercooked or

parahaemolyticus parahaemolyticusinfection bloody) diarrhea, raw seafood, such

12
 Onset
Time
After
Organism Common Name of Illness Ingesting Signs & Symptoms Duration Food Sources

abdominal cramps, as shellfish

nausea, vomiting,
fever

Vibrio vulnificus V. vulnificusinfection 1-7 days Vomiting, diarrhea, 2-8 days Undercooked or
abdominal pain, raw seafood, such
bloodborne infection. as shellfish
Fever, bleeding within (especially oysters)
the skin, ulcers
requiring surgical
removal. Can be fatal
to persons with liver
disease or weakened
immune systems.

13
Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 7 (8 Pages)
Mycotoxins
Mycotoxins are natural chemical substances (secondary metabolites) produced
by fungi (molds) growing as contaminants on some food crops, in particular
cereals, nuts and fruits. Mycotoxicoses is the poisoning by mycotoxin.
Mycotoxins are toxic and undesirable if found in foods and animal feeds due to
causing various adverse effects on humans and animals health. They can affect
the immune system, nervous system, liver, kidneys, and blood, and some
mycotoxins are known to be carcinogens (cancer-causing). A wide range of
adverse and toxic effects in animals are produced by mycotoxin in addition to
being food borne hazards to humans.
There is a major difference between the toxic metabolites of fungi and those
of most bacteria associated with food poisoning. Despite that the chemistry of
fungal toxins may be very complex, they are relatively low molecular weight
compounds, while the bacterial toxins are macromolecules (such as polypeptides,
proteins or lipopolysaccharides) with high molecular weight.
Occurrence and Exposure to Mycotoxins:
The Food and Agriculture Organization (FAO) estimates that 25% of
the overall world’s food crops (including cereals, nuts, fruit and vegetables) are
affected by mycotoxins. Many foods and feeds including corn, wheat, barley,
rice, oats, nuts, milk, cheese, peanuts and cottonseed can become contaminated
with mycotoxins since they can form in commodities before harvest, during the
time between harvesting and drying, and in storage.

1
 Mycotoxins can enter the human and animal food chains by direct
contamination when the food has been contaminated by toxigenic fungi while
growing or after harvest, or indirect contamination, for example in milk from
cows fed with contaminated food. Exposure of consumers to mycotoxins is
mainly via plant foods. However, an additional potential exposure may be via
foods of animal origin such as milk, cheese and meat, as a result of consumption
of contaminated feed by food animals. This illustrates the need to control levels
of mycotoxins in animal feed as well as food.
The chemical and biological properties of the mycotoxins and their toxic
effects are extremely variable. Mycotoxins have been reported to be
carcinogenic, tremorogenic, haemorrhagic, teratogenic (‫ تسبب تشوهات خلقية‬,‫)ماسخة‬,
and dermatitis to a wide range of organisms and to cause hepatic carcinoma in
humans. More than a 100 species of filamentous fungi are known to cause toxic
responses under naturally occurring conditions by producing mycotoxins.
Most Common Types of Mycotoxins:
More than 300 mycotoxins are known, of which about 20 are serious
contaminants of crops used in human foods and animal feeds. Mycotoxin
contamination of foods and feeds depends highly on environmental conditions
that lead to mold growth and toxin production.
The most common mycotoxins are aflatoxins, ochratoxin A, T-2 toxin and
zearalenone. Some mycotoxins or their derivatives are used as antibiotics, growth
promotants, and other kinds of drugs (used as chemical warfare agent due to their
pharmacological activity).
1-Aflatoxin (It is the most common mycotoxin):
Aflatoxins B1, B2, G1 and G2 are produced by three species of the mold
genus Aspergillus (A. flavus /A+fla+toxin), A. parasiticus and A. nomius), and

2
some species of mold genra (Penicillium, Rhizopus, Mucor) in addition
Streptomyces, which contaminate plants and plant products.
Milk contamination by Aflatoxin M1 (AFM1) might occur in 2 ways, directly
due to intake of contaminated feeds by animals that might pass into the milk, or
indirectly following contamination of milk and milk products with fungi.
However, it should be noted that Aflatoxin M1 is a metabolite of Aflatoxin
B1, and therefore the possibilities of any direct carryover of AFM1 from feed to
milk could be ruled out.
Significance: Aflatoxins are of economic and health importance due of their
ability to contaminate foods and feeds, particularly cereals, nuts and oilseeds.
Significant (Pathogenicity) of Aflatoxins are:
(1) Reducing the immune system.
(2) Increasing the chances of infection.
(3) Targets the liver reducing its function causing death.
Symptoms of Aflatoxins are:
(1) Reducing feed intake.
(2) Reducing milk production.
(3) Increasing somatic cell counts.
2-Ochratoxin-A (OTA):
Ochratoxin-A (OTA; molecular weight 403.8) is the 2nd most important
mycotoxin produced by the fungi Aspergillus ochraceus and Penicillium
verrucosum. Isolates of Aspergillus niger and A. carbonarius are capable of
producing OTA. OTA generally appear during storage of fresh produce (in
cereals, coffee, cocoa, dried fruit, spices) and occasionally in the field on grapes.
It may also be present in some of the internal organs (particularly blood and
kidneys) of animals that have been fed on contaminated feeds.

3
Significance: Ochratoxin A (OTA) is a nephrotoxic (‫)سام للكلية‬, hepatotoxic and
teratogenic mycotoxin produced by storage molds of a variety of commodities.
Exposure to low concentrations of this toxin causes morphological and
functional changes in kidney and liver of several domestic and experimental
animals.
3-Fusariotoxins (Fusarium toxins):
Fungi belonging to the genus Fusarium are associated with the production
of toxic substances of considerable concern to livestock and poultry producers
called Fusariotoxins; they include mainly Deoxynivalenol, T-2toxin, Zearalenone
HT-2 toxin and Diacetoxyscirpenol (DAS).
Zearalenone (ZEN), also known as RAL and F-2 mycotoxin, is a
potent estrogenic metabolite produced by some species of Fusarium (F.
graminearum, F. culmorum, F. cerealis) and Gibberella species. Zearalenone is
heat-stable and is found in a number of cereal crops, such
as corn, barley, oats, wheat, rice, and sorghum. Zearalenone is the primary toxin,
causing: (1) Infertility, (2) Abortion or (3) Other genetic problems.
4-Alternaria toxins:
They are mycotoxins produced by species of the mold Alternaria. They
commonly occur during the pre-and post-harvest stages of fruits and vegetables.
The most important toxin-producing species is Alternaria alternata, which
usually contaminates cereals, sunflower seeds, olives, and fruits. Some
Alternaria species are well known for the production of toxic secondary
metabolites, some of which are powerful mycotoxins that have been implicated in
the development of cancer in mammals.

4
5-Ergot Toxin: (Produced by Claviceps purpurea):
- It is the first mycotoxins recognized as affecting human beings. Many
Russians died during World War II from eating moldy grains.
- Sclerotia of species belong to genus Claviceps produce ergot alkaloids.
- A sclerotium (is a dark-colored, hard mycelial mass that establishes itself on
the seed or kernel of the plant).
- Ergot reduces the yield because seeds or kernels are replaced by sclerotia.
- Ergot is produced by the mold Claviceps purpurea, which can cause ergotism
(a disease causes severe liver damage, hemorrhaging, and some fatalities) in
humans and other mammals who consume grains contaminated with its fruiting
structure (ergot sclerotium)..
-Apart from Claviceps, ergot alkaloids are also produced as secondary
metabolites by fungal species belonging to Penicillium, Aspergillus, and
Rhizopus).

Claviceps purpurea
6-Patulin
It is a mycotoxin produced by a variety of molds, in particular,
Penicillium expansum. Despite that patulin is most commonly found in rotting

5
apples, it is also in other foods such as grains, fruits, and vegetables. While not
considered a particularly potent toxin, a number of studies have shown patulin to
be genotoxic, which may lead to cancer. Patulin has shown antimicrobial
properties against some microorganisms. It is heat-stable, so it is not destroyed
by pasteurization or thermal denaturation. Patulin (molecular weight: 145.1) is
mainly associated with surface-injured fruits, and has also become important to
apple processors as a method for monitoring the quality of apple juices and
concentrates. The presence of high amounts of patulin indicates that moldy
apples were used in the production of the juices. Patulin is being considered as a
“possible toxin” in Europe and New Zealand and is regarded as the most
dangerous mycotoxin in fruits, particularly apples, pears, and their products.
7-Citrinin (Produced by Penicillium citrinum):

Citrinin acts as a nephrotoxin .It causes mycotoxic nephropathy in

livestock and has been implicated as a cause of Balkan nephropathy (referred to
as "yellow rice disease") in humans. Citrinin is used as a reagent in biological
research. It induces mitochondrial permeability pore opening and inhibits
respiration. It has been found on human foods, such as grains and cheese. Citrinin
(molecular weight: 250.25) often occurs as a common contaminant of food and
feed (fruits, barley, corn, cheese, dietary supplements). Barley, as well as other
cereals employed for producing beer has been reported to be a good substrate for
the growth of many toxigenic fungi capable of producing Citrinin.
Production of Mycotoxins:
Several factors influence mold development, including moisture,
temperature, aeration and substrate. Moisture is probably the most important of
these factors but the type of mold and whether or not a toxin is produced will
depend on the interplay of all these factors. Physical damage, such as breakage

6
and stress cracks in grain, will increase the likelihood of fungal growth. High
moisture corn, especially if ground, is highly susceptible to fungal invasion and
toxin formation. To prevent production of the toxin on harvested products, care
should be taken to prevent physical damage at harvest and to reduce the moisture
level soon after harvesting. Rapid ensiling (‫ )تخزين األعالف‬or the addition of organic
acids will aid in preventing the formation of additional mycotoxin.

Diagnosis of Mycotoxins:
Fungal growth may occur, without the production of mycotoxins. A
“blacklight” can be used to detect the presence of mold growth on grain and is
satisfactory for use as an initial test (for aflatoxin). It must be realized, however,
that this checks only for the presence of mold growth and even if this test is
positive, it doesn’t mean that a toxin is present. Culture of mold or even isolation
of specific molds from feeds means very little. The specific toxin must be
isolated and/or its toxicity demonstrated.
Mycotoxin assays are generally directed toward specific compounds. Toxins
that are not specifically looked for in the testing will be missed. The diagnostic
problem is further aggravated by wide variations among samples of the same feed
and sensitivities of animals. There are also interactions between the mycotoxins
and other stresses on the animals. These factors make it very difficult to
recommend “safe” concentrations in the feed.
Some mycotoxins can be identified in the rumen contents or urine. But often
guidelines for diagnostic levels have not been established. The suspect feed is a
better source for sampling but recognize that mold
growth (and toxin production) can be very spotty.

7
 Alternaria
Aspergillus flavus

Penicillium Aspergillus niger

Fusarium

8
Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 8 (4 Pages) Probiotics

Probiotics are defined as: “Live microorganisms (mostly bacteria) which
when administered in adequate amounts confer a health benefit on the host”.
-Probiotics (derived from Latin and Greek) means „„for life‟‟.
-Most commonly used probiotics are Lactic acid bacteria (LAB) and
bifidobacteria.
-Probiotics colonize the gut; within our gut there are over 500 different types of
beneficial bacteria as a normal microflora. These bacteria have several beneficial
functions such as inhibiting the growth of pathogenic bacteria, aiding in
digestion, and B vitamin synthesis.
-There are some substances (normally oligosaccharides) called prebiotics which
serve as substrate for growth of probiotics. They are found in breast milk and
some vegetables.
Prebiotics are defined as “Substances that are not used or metabolized by non-
probiotic bacteria”
Types of probiotics:
There are different types of microorganisms which are used as probiotics
(figure below). Commonly used bacteria include Lactobacilli and Bifidobacteria,
commonly used yeast is Saccharomyces boulardii. These bacteria show symbiotic
relationship with human. They are present in the mucus membrane of epithelial
cells of the gut where they inhibit the growth and attachment of harmful bacteria
by producing bactericidal agents against these bacteria. With the development of
evidences regarding usefulness and safety of probiotics, these bacteria are
replacing the traditional prophylactic and treatment regimes.

1
Sources of probiotics
Yogurt is the most common source of probiotics. Yogurt consists of milk
(usually from the cow, goat or sheep) fermented by bacteria that modify lactose
into lactic acid which is responsible to give the yogurt its characteristics (sharp
taste) and also denatures and precipitates casein, resulting in a semisolid
consistency.
-‘‘Bioyoghurts’’ are produced in a similar way, but bacteria used for fermentation
are of different strains, usually L. acidophilus.
-Fermented milk and fortified fruit juice are common sources of probiotics.
-Probiotics are also available in supplements consisting of freeze dried bacteria in
tablets, capsules and powders. Selection of probiotic product depends on type of

2
bacteria and type of beneficial effect expected. There are thousands of strains of
probiotics and all of them show different beneficial effects.
Regulation of probiotics:
Food and Drug Administration (FDA) has established a regulatory authority
to regulate probiotics production, manufacturers, labeling and safety of products.
These regulations depend on intended use of the product (indicated on the Label).
FDA proposed four probiotics regulatory categories differed in their
requirements as follow:
(1) Drug or biological products, (2) Dietary supplements,
(3) Food or food ingredient and (4) Medical food.
Probiotics are different from other types of bacteria in that they are considered
“good” bacteria or non-pathogenic in healthy people.

Differences between Prebiotics and Probiotics:

Prebiotic provides the non-digestible carbohydrate fibers, called
oligosaccharides, for probiotics. The Prebiotics are non-digestible foods that make
their way through our digestive system and help beneficial bacteria to grow and
flourish. The oligosaccharides remain in the digestive tract where they stimulate
the growth of beneficial bacteria. Sources of oligosaccharides include fruits,
legumes, and whole grains.
-Below is a list of foods that research has confirmed to be prebiotic: Bananas,
Onions, Garlic, Leeks, Asparagus, Whole wheat, Barley and Rye.

Prebiotic + Probiotic = Symbiotic

A food is considered synbiotic when contains a prebiotic and a probiotic
ingredient. Below are some examples of how to incorporate to create synbiotic
foods.
-Eating yogurt with live active cultures with a sliced banana.

-Consuming fruit-fortified yogurt.

3
Diseases may get benefits of Probiotics:
Diabetes, Hypertension, Urogenital health care, Lactose intolerance, Colon
cancer Inflammation, Immune function and infections, Peptic ulcer, Liver diseases,
Food allergy, Upper respiratory tract infection and Oral health.
Harmful effects of probiotics:
Normally probiotics are considered as non-harmful microorganisms that
exert health benefit to the host. This is true in most cases. Lactobecillus are used
for a long times due to their safety. Probiotics which are mostly considered as safe
are Lactococcus and Lactobacillus. Enterococcus, Streptococcus and Lactobacillus
contain some opportunistic bacteria. Probiotics are also grouped into two classes
based on their risk to health. Risk group 1 (No risk) consists of Lactobacillus and
Bifidobacteria. Risk group 2 (small risk) contains Lb. rhamnosus and
Bifidobacterium dentium. Pharmaceutical companies should keep in mind the
potential of health risk of probiotics before adding these into particular products.
Before launching any probiotic product, the safety profile of that particular
probiotic species should be assessed to avoid incidence of any unexpected harmful
effect.

4
Food Microbiology Biology Dept. Al-Farabi University College
(Forth Class) Prof. Dr. Abdulwahid Al-Shaibani

Lecture 9 (8 Pages)
Detection and Enumeration of Microbes in Foods
Depending on purpose of the testing, there are several methods to enumerate
and detect microorganisms in food. Enumeration methods are classified as follow:
1-Direct Enumeration:
These methods include a rapid estimation of all viable and nonviable cells by
using Direct Microscopic Counts (DMC). In addition, through the DMC methods,
bacteria can be also identified through staining and observation of morphology.
2-Viable Enumeration:
The use of Standard Plate Count/SPC (Aerobic Plate Count/APC), Most
Probable Number/MPN, Membrane Filtration/MF. Plate Loop and Spiral
plating methods allows the estimation of only viable cells. These methods can be
used in the food industry to enumerate fermentation, spoilage, pathogenic, and
indicator microorganisms.
3-Metabolic Activity Measurement:
An estimation of metabolic activity of the total cell population is possible
using dye (stain) reduction tests such as resazurin or methylene blue dye
reduction (as below), acid production, electrical impedence (‫ )مقاومة‬and others.
The level of bacterial activity can be used to assess the keeping quality and
freshness of milk. Microbial toxin levels can also be measured, indicating the
presence of toxin producing pathogens.
4-Cellular Constituents Measurement:
Using the luciferase test to measure ATP is one example of the rapid and
sensitive tests available that will indicate the presence of even one pathogenic
bacterial cell.

1
(I) Enumeration of Microorganisms:
Determination of the safety of many food and drug products depends on
evaluating the levels of microorganisms in those products by using a variety of
developed enumeration methods. These methods measure cell numbers, cell mass,
or cell constituents that are proportional to cell number.
The four general approaches used for estimating the sizes or intensity of
microbial populations are: a) direct count and b) indirect count of cells and c)
direct measurement and d) indirect measurement of microbial biomass. Each
method will be described in more detail below.

A) Direct Count of Cells:

Cells are counted directly under the microscope or by an electronic particle
counter. Two of the most common procedures used in microbiology are:
a) Using Counting Chamber:
Direct microscopic counts are performed by spreading a measured volume
of sample over a known area of a slide, counting representative microscopic
fields, and relating the averages back to the appropriate volume-area factors. In
addition to the Breed slide, there are specially constructed counting chambers,
such as the Petrhoff-Hauser and Levy counting chambers, simplify the direct
counting procedure because they are made with depressions in which a known
volume overlies an area or a chamber that is ruled into squares. The ability to
count a defined area and convert the numbers observed directly to volume
makes the direct enumeration procedure relatively easy. Direct counting
procedures are rapid but have the disadvantage that they do not discriminate
between living and dead cells. This method is used to assess the sanitation level
of a food product and in performing blood cell counts in hematology.

2
 b) Using Fluorescent Dyes:
Fluorescent dyes are becoming more used in recent years for a variety
of procedures as for count bacteria. These dyes can stain all species, a
particular species of interest (in an environmental sample) or even a specific
component of cells.
The most widely used fluorescent dye for counting the number of
bacterial cells is acridine orange which stains both living and dead cells by
interacting with DNA and protein components of cells. The stained cells
fluoresce orange when excited near ultraviolet light. The procedure is widely
used in marine microbiology where population levels are often low and
where viable plate counts are known to severely underestimate total number
of bacteria. Typically, the viable count is less than 1% of the direct count for
marine samples. In this procedure the bacteria in a known volume of sample
are stained with acridine orange and the sample is then filtered through a 0.22
µm filter. The bacteria are trapped on the filter that is then examined under a
fluorescence microscope. The bacteria in a defined area of the filter are
counted and the concentration in the original sample is then calculated.
Other fluorescent dyes that are also gaining popularity are cyanoditolyl
tetrazolium chloride (CTC), auramine and rhodamine. CTC binds to
respiration proteins in the cell and thus can demonstrate live cells. Auramine
and rhodamine bind to cell wall of Mycobacteria and emit bright yellow or
orange color under a fluorescent microscope. These latter stains are gradually
replacing the acid-fast stain.
B) Indirect Count of Cells:
Microorganisms in a sample are diluted or concentrated and grown on
a suitable medium; the development of growing microorganisms (colonies
formation) is then used to estimate the numbers of microorganisms in the
original sample. Some of the common indirect counting methods are:

3
a) Viable Count:
It is the most commonly used procedure for the enumeration of bacteria.
Basic concepts of this method:
1 It is difficult to expect how many bacteria are in a sample, so it is necessary
to prepare a serial of dilutions, usually from 10-1 (1/10) to 10-8
(1/100,000,000), then plating each onto a suitable growth medium. The sample
suspension is either spread onto the surface of agar plates (spread plate
method), or is mixed with molten agar, poured into plates, and allowed to
solidify. The plates are then incubated inversely, under conditions that permit
microbial colonies to be seen without the aid of a microscope.
2- It is assumed that each bacterial colony arises from a single bacterial cell.
3- In general, the dilution which contains 25-250 colonies should be selected
and used for calculating the number of colony forming units (cfu) per 1 ml, 1
gm or 1cm2 according following equation:
Number of cfu /1 ml (gm or1cm2) = Colonies Number X Dilation factor
Disadvantages of viable count method:
1-The single colony may not initiate from only one bacterial cell. Because
some organisms exist as pairs or groups or because mixing and shaking of the
sample does not always separate all the cells.
2-The medium composition and pH in addition to the nature of growth
conditions such as temperature, determine which bacteria in a mixed
population can grow. Since there is no universal set of medium and conditions
that permits the growth of all microorganisms, it is impossible to enumerate all
microorganisms by viable plating.
b) Most Probable Number (MPN):
It is usually used to estimate numbers bacteria in the very low-
contaminated samples such as estimating coliforms in water, milk, and other

4
 foods (Coliforms are G (-) small rods bacteria that reside in the intestine of
warm-blooded mammals and are regularly excreted in the fece).
The MPN procedure: is a statistical method based upon the probability
theory. Samples are serially diluted to the point of extinction, that is, to a point
where there are no more viable microorganisms. To detect the end point,
multiple serial dilutions are inoculated into a suitable growth medium, and the
development of some recognizable characteristic, such as acid production or
turbidity, is used to indicate growth (the presence of at least one viable
microorganism in the diluted sample). The pattern of positive tests (growth) in
the replicates and statistical probability tables are used to determine the
concentration (most probable number) of bacteria in the original sample.
Statistical MPN tables are available for replicates of 3, 5, and 10 tubes of each
dilution. The more replicate tubes used, the greater the precision of the estimate
of the size of the bacterial population.
C) Direct Measurement of Microbial Biomass:
Cell mass is determined directly by weighing whole cells; biomass can be
correlated with cell numbers by reference to a standard curve. Wet weight or dry
weight of bacteria may be used for estimation of cell numbers.
D) Indirect Measurement of Microbial Biomass:
Microbial biomass is estimated by measuring relatively constant
biochemical components of microbial cells, such as protein, ATP,
lipopolysaccharides, peptidoglycan, and chlorophyll; biomass can also be
indirectly estimated by measured turbidity that can then be correlated with cell
numbers by reference to a standard curve.
Various procedures based on the detection of specific microbial
macromolecules or metabolic products can be used to estimate numbers of
microorganisms. For example, peptidoglycan can be quantified, and because this
biochemical occurs exclusively in the cell wall of bacteria, the concentration of
5
 peptidoglycan can be used to estimate bacterial numbers. Such biochemical
approaches for determining bacterial numbers depend on the development of
analytical chemical procedures for quantifying the particular biochemical and
determining what proportion of bacterial cell is composed of the specific
biochemical constituent.

Coulter counter; A Coulter counter, which is named after its inventor Wallace
H. Coulter, is a device that is used to measure the number of cells in a certain
volume of a sample suspension. The counter achieves this enumeration by
monitoring the decrease in electrical conductivity that occurs when the cells pass
through a small opening in the device. While originally developed for use with
blood cells, the Coulter counter has found great use in a diverse number of
disciplines, including microbiology, where it is used to determine the total number
of bacteria in samples. Because the device operates on the physical blockage of
electrical conductivity by particles in a sample, the Coulter counter cannot
distinguish between living and dead bacteria. An indication of the total number of
bacteria (alive, dormant, and dead) is provided. The number of living bacteria can,
however, usually be easily determined using another volume from the same
sample. A bacterial suspension is best analyzed in the Coulter counter when the
suspension has been thoroughly shaken beforehand to disperse the bacteria. Most
bacteria tend to aggregate together in a suspension. If not dispersed, a clump of
bacteria passing through the orifice of the counter could be counted as a single
bacterium. This would produce an underestimate of the number of bacteria in the
suspension.
(II) Isolation and detection of microorganisms:
Isolation is an important preliminary step in the detection or identification
of food spoilage and pathogenic microorganisms. Isolation can be performed by
using a simple “streak plate method” where the suspected food sample is cultured
by streaking on surface of the agar medium in a Petri dish.
Detection or identification of food microorganisms can be done by several
methods such as:
1-Conventional method:
It is composed of three steps as follow:

6
Step 1: Cultural characterization: which is done either a) on a solid medium by
plating food sample on surface of medium and describing grown colonies (after
incubation) for their shape, size, color, margin ….etc, or b) in a liquid medium by
inoculating the sample into the medium and describing the growth (after
incubation) as ring forming, precipitation, dispersed, flocculation ….etc.
Step 2: Microscopic characterization: by taking sample from the grown culture and
examined it under the compound microscope after staining by Gram staining
method to describe cell shape, grouping, and Gram reaction….etc.
After performing the first two steps, genus of the bacteria may be identified.
Step 3: Biochemical (Physiological) characterization: which identifies the species
of bacteria. Several tests are used in this step depending of the type of bacteria.
Some of these are; carbon sources fermentation, type of N2 utilization, production
of enzyme such as coagulase, catalase, gelatinase, urease….etc.
2- API Kit method:
API system is the well-established method for manual microorganism
identification to the species-level identification of Gram positive and Gram
negative bacteria and yeast. The API system utilizes a plastic strip with 20
separated compartments. Each compartments consists of a depression, or cupule,
and a small tube that contains a specific dehydrated medium. After inoculating
each compartment with an unknown bacteria, a reaction will occur within 24
hours. The results of each reaction are tabulated and then given a number based on
the results. The unknown bacteria is identified by searching the number
corresponding to the unknown through a leaflet attached with the kit.
3-VITEK 2 instrument method:
The VITEK 2 Compact system is a device used for fast and accurate microbial
identification. The innovative This technology includes an expanded identification
database, the most automated platform available, rapid results, improved
confidence, with minimal training time. The rapid response time means results can

7
be provided more quickly than with manual techniques. VITEK® 2 technology uses
Advanced Colorimetry; an identification technology that enables identification of
routine isolates. Advanced Colorimetry provides:
 High discrimination between species
 Low rate of multiple choice and misidentified species
 Minimal number of off-line tests