Firew Tafesse (PhD)

Chapter-1
Microbiology is the science which study about the
occurrence and significance of bacteria, fungi, protozoa and
algae which are the beginning and ending of intricate food
chains upon which all life depends.
All foods carry microorganisms - from the micro-floral of
raw food or introduce during harvesting, processing,
slaughtering, storages or distribution
Micro-flora has no negative effects on consumers but
sometimes could be manifested as spoilage, food born illness,
transform food chemicals as beneficial –fermentation
 Micro-organisms and Food Materials

 Diversity of Habitat :
- Found in a very wide range of habitats ( >100 0C - spring
water or active volcano) , frozen environment –polar regions
1. Atmospheric ( Airborne fungus )
2. Soils
3. Water
4. Plants
5. Animals
 Log N
Lag phase

Time
Log phase

Stationary phase
Microbial Growth
  The lag-phase: There is no apparent growth, adjusts
to the new environment, synthesizes the required
enzymes and repairs any lesions from earlier injury,
e.g. freezing, drying, heating.
 The exponential (logarithmic phase): an increase in
cell numbers, represent the organism’s specific growth
rate.
 Stationary phase : key nutrients become depleted, or
inhibitory metabolites accumulate, and the culture

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Sources of microorganisms in food
The primary sources of microorganisms in food
include:
1. Soil and water
2. Plant and plant products
3. Food utensils
4. Intestinal tract of man and animals
5. Food handlers
6. Animal hides and skins
7. Air and dust
1.1. Food spoilage

 Food spoilage is defined as damage or injury to food

rendering in unsuitable for human consumption.
 Food must be considered spoiled if it is contaminated
with pathogenic microorganisms or various poisonous
agents, such as pesticides, heavy metals, toxins etc.
Table 1: Storage life of some foods
Food product Storage life (days) at 21oC
Raw beef and mutton 1-2
Raw fish 1-2
Raw poultry 1-2
Dried salted or smoked 360 or more
meat and fish
Fresh fruits 1-7
Dried fruits 360 or more
Leafy vegetables 1-2
Root crops 1-20
Dried seeds 360 or more
Food spoilage cont….
 In most cases there does not need to be an evident sign
of spoilage, the food might look normal and only after
eating it or by careful bacteriological and toxicological
investigation, one is able to realize the defect.
 Food decay or decomposition is implied when the
term spoiled is used.
Causes of food spoilage
• (a). Growth and activity of microorganisms
Bacteria, yeasts and molds are microorganisms that
cause food spoilage. They produce various enzymes
that decompose the various constituents of food.
• (b). Enzyme activity: Action of enzymes found
inherently in plant or animal tissues start the
decomposition of various food components after death
of plant or animal.
• (c). Chemical reactions: These are reactions that are
not catalysed by enzymes.,e.g. oxidation of fat
Causes of food spoilage cont…
• (d). Vermin. Vermin includes ants, rats, cocroaches,
mice, birds, larval stages of some insects. Vermin are
important due to:
(i). Aesthetic aspect of their presence,
(ii) Possible transmision of pathogenic agents,
(iii). Consumption of food.
• (e). Physical changes. These include those changes
caused by freezing, burning, drying, pressure, etc.
Microbial spoilage of food
 Bacteria, yeasts and molds are the major causes of food
spoilage.
 They produce various enzymes that decompose the
various constituents of food.
 Molds are the major causes of spoilage of foods with
reduced water activity e.g dry cereals and cereal
product
 Bacteria spoil foods with relatively high water activity
such as milk and products.
1.2. Factors affecting microbial growth in food

(a) Intrinsic factors:

These are inherent in the food. They include:
1.Hydrogen ion concentration (pH),
2.Moisture content,
3.Nutrient content of the food,
4.Antimicrobial substances ,
5.Biological structures.
1. Hydrogen ion concentration (PH)

 Most bacteria grow best at neutral or weakly alkaline

pH usually between 6.8 and 7.5.
 Some bacteria can grow within a narrow pH range of
4.5 and 9.0, e.g. salmonella
 Other microorganisms especially yeasts and molds
and some bacteria grow within a wide pH range, e.g.
molds grow between 1.5 to 11.0, while yeasts grow
between 1.5 and 8.5.
Table 1: pH values of some food products

Food type Range of pH values

Beef 5.1 - 6.2
Chicken 6.2 – 6.4
Milk 6.3 – 6.8
Cheese 4.9 - 5.9
Fish 6.6 - 6.8
Oyester 4.8 - 6.3
Fruits < 4.5 (most < 3.5)
Vegetables 3.0 – 6.1
• Microorganisms that are able to grow in acid
environment are called acidophilic microorganisms.
• These microorganisms are able to grow at pH of
around 2.0.
• Yeasts and molds grow under acid conditions.
• Other microorganisms such as vibrio cholerae are
sensitive to acids and prefer alkaline conditions.
• Most bacteria are killed in strong acid or strong
alkaline environment except Mycobacteria.
 Table 2: Minimum and maximum pH for growth
of some specific microorganism
Microorganism Minimum Maximum
Escherihia coli 4.4 9.0
Salmonella typhi 4.5 8.8
All bacteria 4.0 9.0
Molds 1.5 11.0
Yeast 1.5 8.5
2. Moisture content
• The effect of moisture is in terms of water activity: -the
amount of free water in a food medium.
• The amount of free water is important for growth of
microorganisms.
• If there is lack of this free water microorganisms will
not grow.
• Water activity is defined as the vapour pressure of a
food substance to that of water at the same
temperature. (Aw = VPFood/VPWater)
Moisture content
 The water activity is therefore equal to 1.0.
 Food products have a water activity of less than 1.0.
 A saturated salt solution has a water activity of 0.75.
 Salting and drying reduces the water activity of a food
product.
Table 3: Water activity of some food products

Food Product Water activity

Raw meat and milk 0.99- 1.0

Luncheon meat 0.95

Boiled ham, sliced bacon 0.90

Dried grains 0.80

Water activity levels
 Growth of microorganisms is greatly affected by the
level of water activity(Aw) in the food.
 Inhibition of growth occurs if the water activity for
food is lowered beyond an organism’s minimum level
of water activity that is necessary for growth.
 Microorganisms have varied minimum water activity
requirements that supports their growth in food.
Table 4: Minimum water activity that supports growth
of some microorganisms
Microorganism Water activity
Clostridium botulinum, 0.95
Bacillus cereus, 0.95
Pseudmonas aeroginosa, 0.95
Salmonella spp. 0.95
Staphylococcus aureus (anaerobic), 0.90
Candida spp., Saccharomyces
Staphylococcus aureus (aerobic) 0.86
Penicillium spp. 0.82
Most spoilage yeast 0.88
Most spoilage molds 0.80
Osmotic yeast 0.70
3. Nutrients content of the food
 Microorganisms require proteins, carbohydrates,
lipids, water, energy, nitrogen, sulphur,
phosphorus, vitamins, and minerals for growth.
 Various foods have specific nutrients that help in
microbial growth.
 Foods such as milk, meat and eggs contain a
number of nutrients that are required by
microorganisms.
 These foods are hence susceptible to microbial
spoilage.
4. Antimicrobial substances
 Antimicrobial substances in food inhibit microbial
growth.
 Various foods have inherent antimicrobial substances
that prevent (inhibit) microbial attack.
 Such inhibitors are like lactinin and anti-coliform
factors in milk and lysozyme in eggs.
4. Biological structures
 Some foods have biological structures that prevent
microbial entry.
 For example, meat has fascia, skin and other
membranes that prevent microbial entry.
 Eggs have shell and inner membranes that prevent
yolk and egg white from infection.
(b). Extrinsic factors
 Are factors external to the food that affect microbial
growth. They include:
1. Temperature of storage,
2. Presence and concentration of gases in
the environment
3. Relative humidity of food storage
environment.
1. Temperature
 The growth of microorganisms is affected by the
envirnmental temperatures.
 Various microorganisms are able to grow at certain
temperatures and not others.
 Bacteria can therefore be divided into the following
groups depending upon their optimum tmperature of
growth.
(i). Pychrophilic microorganisms

 These grow best at about 20oC but also down to -10oC

in unfrozen media.
 Psychrophilic bacteria can cause food spoilage at low
temperatures.
 Several of the microorganisms found in the soil and
water belong to this group.
(ii). Mesophilic bacteria
 These organisms grow between 25oC and 40oC,
with an optimum growth temperature close to
37oC
 Some such as Pseudomonas aeroginosa may
grow at even lower temperatures between 5-
43oC
 None of the mesophilic bacteria are able to
grow below 5oC or above 45oC.
 Most pathogenic bacteria belong to this group.
(ii). Thermophilic bacteria.
 These grow at temperatures above 45oC. Often their
optimum growth temperatures is between 50oC and
70oC.
 Growth of some bacteria occur at 80oC.
 Bacteria in this group are mainly spore formers and are
of importance in the food industry especially in
processed foods.
2. Concentration of gases in the environment
 This relates to the presence and concentration of gases
in the food environment.
 Various microorganisms require for growth, either
high oxygen tension (aerobic), low oxygen
tension(microaerobic) or absence of oxygen
(anaerobic).
 Some microorganisms may grow either in high oxygen
tension, or in the absence of oxygen (facultative
anaerobes).
3. Relative humidity
 Relative humidiy is the amount of moisture in the
atmosphere or food environment.
 Foods with low water activity placed at high humidity
environment take up water, increase their water
activity and get spoiled easily.
 For example, dry grains stored in a environment with
high humidity will take up water and undergo mold
spoilage.
 1.3. Food preservation
 Food preservation is a process through which
physical and /or chemical agents are used to
prevent microbial spoilage of food.
 Food preservation aims at treating food in a
manner to prolong its storage life
 In food preservation, efforts are made to
destroy organisms in the food,or
 Increase the period taken by microorganism to
adapt to the food environment before they start
to spoil the food.
Food preservation principles
 Two general principles are employed in food
preservation.
 (1). Inhibition priciple
 (2). Killing principle
 (1). Inhibition principle
 In this principle, food preservation is achieved by
inhibition of growth and multiplication of
microorganisms.
 The inhibition principle can be achieved by any of the
following methods:
 (a). Reduction of water activity e.g. By drying and
salting
 (b). Reduction in pH e.g. by fermentation and addition
of acids.
 (c). Use of preservatives, e.g. sodium benzoate
 (d). Use of low temperatures (chilling or freezing)
 (e). Smoking – which has a drying and preservative
effect
Inhibition methods
 Preservation of food by inhibition methods does not
necessarily imply the destruction of organisms,
 On removal of the inhibiting influence, the food will
undergo spoilage as the microorganism present will
grow and multiply to cause spoilage.
 Food preservation by lowering pH
 Many food products can be preserved by lowering
pH so that the growth of spoilage and pathogenic
bacteria is prevented.
 The lowering of pH can be achieved by addition of
acids and fermentation
 Fermentation is the breakdown of carbohydrates
under anaerobic conditions into alcohol or lactic
acid and carbon dioxide.
Food preservation by lowering water activity

Lowering of water activity can be achieved by:

 Addition of high content of salt: Sodium chloride and
sometimes nitrats and nitrites
 Addition of high content of sugar
 Drying: sun/air drying; electrical drying or freeze
drying.
The salting procedure
The salting procedure can be performed in four
ways:
1. Dry cure in which the meat or fish is rubbed with
salt
2. Pickling: The products are immersed in pickle of
brine, usually containing about 15% salt.
3. The injection cure: concentrated salt injected to
muscles
4. Direct addition method
 Preservation of food by addition of high content of
sugar
 Monosaccharides such as glucose(dextrose) and
fructose are more effective in reducing the water
activity than disaccharides like sucrose.
 Thermophiles are more susceptible to the action of
sugar than than other bacteria.
 Osmophilic yeasts are able to tolerate very high
concentrations of sugar and cause food spoilage.
Food preservation by use of low temperatures

 Two methods are employed to arrest microbial growth

and multiplication.
 These are chilling (cold storage above their freezing
point, typically 0–5 C) and freezing.
 Chilling is keeping food at temperatures between 0-
15oC. The commom chilling temperatures ranges
between 4-5oC.
 Freezing is keeping food at temperatures between 0oC
and -35oC.
Effect of low temperatures
 Low temperatures are used to retard chemical
reactions and actions of food enzymes and to slow
down or stop the growth and activity of
microorganisms in the food.
 A low enough temperature will prevent growth of any
microorganisms.
 Spores are not usually injured at all by freezing.
However, most parasites are killed by freezing.
(2). Killing principle
 In this principle, spoilage microorganisms are
destroyed (Killed) in the food, and the food protected
against subsequent contamination by being enclosed
in an air tight container.
Methods employed to achieve the killing principle
1. Heat treatment: through pasteurization or
sterilization
2. Irradiation with either ionizing or
electromagnetic radiation e.g gamma rays, cobalt
60 radioactive particles. Radiations kill
microorganisms by destruction of DNA and by
creating toxic reactive compounds in a medium
and in microbial cells
3. Use of gases: by use of ethylene oxide or ozone.
The gases destroy both vegetative cells and
spores.
Pasteurization
 Is the process of heat treatment at specific
temperatures and times.
 Pasteurization is aimed at destroying all pathogenic
microorganisms without affection the nutritive value
of the food.
Three methods of pasteurization
a. Low temperature long time( LTLT) (63oC for 30 min)
b. High Temperature short time ( HTST)(72oC for 15
seconds)
c. Flash method (80oC for 1-2 seconds)
Sterilization
 Is the use of physical or chemical means to destroy all
microorganisms that are present in the food.
 Sterilization can be achieved by:
a. Heating at high temperatures, e.g. 100-
140oC
b. Irradiation:Irradiation kills bacteria,
spores, and insects as well as inactivates
enzymes.
Applications (combinations)
 In pracice, often a combination of inhibition and
killing principles and the various methods are used
depending on the food type. e.g.
 use of pasteurization and chilling of milk,
 lowering of water activity and low
temperature storage,
 use of preservatives and low temperature
etc.
Important terminologies on use of heat in
food preservation

D- value
Z- value
F-value
Decimal reduction Time (D-Value)
 Is the time required at any temperature to destroy
90% of the spores or vegetative cells of a given
organism.
 The higher the temperature, the faster is the rate
of destruction and the shorter it takes to kill 90%
of the cells.
 Example: D-value for Clostridium sporogenes in a
given food at
 120oC is 1 minutes,
 115oC is 4 minutes,
 110oC is 10 minutes.
D- value
A D value can be obtained from a plot of log10
survivors versus time
D-Value cont..
 a plot of the log of the number of surviving cells at a
given T against time will give a straight line with
negative slope, k
 As the temperature increases, so the slope of the
survivor curve increases
 Example: if the D72 of Salmonella Senftenberg 775W
in milk is 1.5 s, then HTST pasteurization (15 s at 72 C)
will produce a 10D reduction in viable numbers
D-Value cont..
 The larger the initial number of vegetative cells
or spores, the longer it will take to destroy 90 %
of the cells at a given temperature.
 D- value is numerically equal to the number of
minutes required for the survivor curve to
trasverse one log cycle.
Z-value
 The Z value: Is the number of degrees the
temperature has to be increased in order to reduce the
thermal death time tenfold.
 The z value is relatively constant and depends very
little upon the environment.
 For spores of bacteria, the z - value used is 10oC.
 Generally psychrotrophs are less heat resistant than
mesophiles, which are less heat resistant than
thermophiles;
 Gram-positives are more heat resistant than Gram-
negatives
Z-value
•As the T is increased so the D value decreases. so that plotting log
D against temperature gives a straight line, this would give us
another parameter- Z

Z = (t2-t1)/(log D2-log D1)

Z-value
Microbial heat resistance

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Z- value
 The spore killing effect of a heat treatment can be
expressed as a function of temperature and the time
the material has been exposed to that heat.
 For example, when it takes;
 1 min to kill 90% of the remaining spores at 120oC,
 10 min to obtain the same effect at 110oC
F-value
 F-value. The F-value express the time taken to expose
food to the same amount of heat required to destroy
spores and vegetative cells of a particular organism using
different temperatures.
 Example: A process may have an F121 value of say 4, which
means that its particular combination of times and
temperatures is equivalent to instantaneous heating to
121 C, holding at that temperature for four minutes and
then cooling instantly, it does not even necessarily imply
that the product ever reaches 121 C.
F-value
 The F value will depend on the z value of the organism of
concern;
 if z=10 C then 1 minute at 111 C has an F121=0.1,
 if z=5 C then the F121 value will be 0.01.
 This means that one can obtain the same killing effect at
lower temperature, provided the time of exposure is
longer.
 It is therefore necessary to specify both the z value and
the temperature when stating F.
 For spores z is commonly about 10 C and the F121
determined using this value is designated F0.
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 Typical F values for some canned foods

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Foodborne Illness
 Commonly referred to as food poisoning
 Occurs when a pathogen or its toxin is consumed
 Consumers must employ sound preserving, preparation and
cooking techniques to avoid hazards of food products
 Estimated millions of cases of food poisoning occur each
year
 Vast majority could have been prevented
Foodborne Illness
 Food intoxication
 Illness resulting from consumption of an exotoxin
produced by organisms growing in food product
 When food is ingested it is the toxin responsible for illness not
organism
 Common causes of foodborne intoxication are
• Staphylococcus aureus
• Clostridium botulinum
Foodborne Illness
 Staphylococcus aureus
 Produces toxin that causes nausea and vomiting

 Thrives in moist, rich foods in which other organisms

have been killed or inhibited
 Survives well in unrefrigerated foods with high salt content

 Source of S. aureus generally human carrier

 Organism is inoculated into food during preparation

 Food left at room temperature allows organism to grow and
produce toxin
 Toxin is heat stable and not inactivated by cooking
Foodborne Illness
 Botulism
 Paralytic disease caused by ingestion of a neurotoxin

 Produced by Clostridium botulinum

 Growth of organism or production of toxin may not result in

changes in taste or appearance of food
 Canning process designed to destroy endospores

 Processing errors can allow germination of endospores

 Errors extremely rare in commercial canning

 Home canned foods should be boiled for 10 to 15 minutes

immediately before consumption
 Heat destroys toxin
Foodborne Infection
 Foodborne infection requires consumption of living
organisms
 Symptoms do not appear for at least one day after
ingestion
 Major symptom usually diarrhea
 Thorough cooking of food immediately before consumption
will kill organisms
 Prevent infection
 Foodborne illness commonly caused by
• Salmonella
• Campylobacter
• Escherichia coil O157:H7
Foodborne Infection
• Salmonella and
Campylobacter
 Commonly associated with
poultry products
 Inadequate cooking can result
in foodborne infection
 Cross-contamination can
result in transfer of pathogens
to other foods
 Cutting boards and knives often
become contaminated
Foodborne Infection
• Escherichia coil O157:H7
 Causes bloody diarrhea

 Sometimes develops into

hemolytic uremic syndrome
(HUS)
 Life threatening

– E. coli O157:H7 responsible for

several large food poisoning
outbreaks

 Ground meats are troublesome

source of foodborne infection
 ground meat should be cooked
thoroughly through
69
Why Microbial examinations?
To check the safety and quality of food ( Standard)
General Procedures for microbial examinations
a. Enrichment :
b. Preparation of dilution
c. Isolation
d. Quantification : characteristic and countable
numbers of colonies (15 – 300 colonies) colonies are
considered in Petri dishes.
e. Conformation and identification

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Quantification
 The total number of aerobic mesophilic bacteria must
not be greater than 103 cfu/g in a food.
 N= np/ R where
 N- Number of microorganisms (cfu/ml, cfu/g)
 Np -average number of colonies
 R -dilution of food sample by which colonies are
counted

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 Methods
Traditional method Rapid Method
• Plate counts  Direct
• Membrane filtration epifluorescent
• Most probable number
• Direct microscopic filter technique
count (DEFT)
• Dye reduction tests  Electrical
• Indicator impedance
 Enzyme-linked
immunosorbent
assay(ELISA)
72
 1.Plate count method
Standard plate count (SPC)
Aerobic plate count (APC)

Total bacteria count (TBC)

Total viable count (TVC)

“Live”
73
 Plate count method
•Diluent
•0.85%NaCl
•0.1% peptone
•Phosphate buffer
•Medium
•Elective medium
•Selective medium
•Pour plate
•General
•Spread plate
•Petri dish plate
•Drop plate •Replication
74
Plate count depends on
 Diluent
 Food homogenate
 Dilution series
 Medium
 Plating method
 Incubate conditions

75
Plate count method

76
Pour plate

77
 Spread plate

Number of
colony forming units (cfus)
?????
78
 Drop plate
•Small vol.
≈ 20 μL

79
Application of plate count
 Check quality
 Check condition hygiene
 Estimate storage life of products
 Determine
 Production
 Transport
 Storage
 Determine pathogens

80
 Selection of media in food microbiology
Medium Use
Plate count agar Aerobic mesophilic
count
MacConkey broth MPN of coliforms in
water
Brilliant green/Lactose/Bile MPN of coliforms in
broth food
Braid Parker agar Staphylococcus
aureus
Thiosulfate/Bile/Citrate/agar Vibrio sp.
Adam and Moss (2003)
 Streak technique

http://www.towson.edu/~cberkowe/medmicro/images/streak.gif
 2.Filtration
0.45 μm
Liquid food
•Low number of
MO.
Large volume of
food

•Count
•Sterilize

http://www.biology.lsu.edu/webfac/rgayda/biol1011/Lecture_notesF2004/lecture8.pdf 83
3.Most probable number
Most probable number (MPN)
Multiple tube techniques

• Pathogen
 Number too low
• Coliform
• Escherichia coli
• Staphylococcus aureus
• Feacal streptococci
84
 Most probable number
Medium Organisms assessed

Lauryl sulfate tryptose broth Coliforms

MacConkey purple broth Coliforms

EC broth Faecal coliform

Glucose azide Faecal streptococci

Minerals modified Coliforms

glutamate medium
Baird-Parker broth Staphylococcus
aureus
4. Microscopic count
Direct microscopic count (DMCs)
Small sample (0.01 ml) & rapid
Optical light microscope
Total cell Ex.
living & dead cells •Milk
Foods •Wine
•Yogurt starter
Liquid •Tomato sauce
Semi-solid •Howard mold
86
Microscopic count

87
Comparison of sensitivity of method
Method Vol. of Count
sample (ml) (cfu/g)
Direct microscopy 5 x 10-6 2 x 106

Drop plate 0.02 5 x 102

(Miles and Misra)
Spread plate 0.1 102

Pour plate 1 10

MPN 3 x 10 0.36
+3x1
+ 3 x 0.1
5. Dye-reduction test
Methylene blue
Resazurin (blue)
Leuco-methylene blue
Resorufin (pink)
Triphenyltetrazolium
chloride (leuco) Dihydroresofin
(leuco)
Formazan (red)

89
6.Indicators
• Hygiene indicator
• Cross contamination

•Fresh meat
•Raw milk
•Pasteurized milk
90
1. ATP photometry
ATP : Adenosine triphosphate
Synthesis of new cell
Active transport (uptake of materials from environment)
Movement
Light production

91
 ATP photometry
Luciferin + luciferase + ATP + O2

Mg2+

Oxyluciferin + luciferase + AMP + light

1 ATP light 1 photon

92
ATP photometry
Bacteria cell
1 fg of ATP •Limit of ATP
Yeast cell photometry
102-103 fg ATP/ml
100 fg of ATP

fg = femto gram = 10-15 g

93
ATP photometry
Break down the non-microbial cells in
food
Remove non-microbial ATP using ATPase
Release ATP from bacteria cell
Addition of luciferin & luciferasee
Record light emission (ATP photometry)

94
ATP photometry
Application
Fresh meat
Milk
Starter culture
Test UHT milk
Surface contamination
95
ATP photometry
Disadvantage
Mixed bacteria & yeast cell
Dilution
Remove cell before ATP measured
Filtration
Centrifugation

96
Direct epifluorescent filter technique (DEFT)
Liquid food • Direct
Filter through membrane microscopy
Acridine orange : • Membrane
fluorescent dye filtration
(fluorochrome) pour • Vol. of sample
through filter
• Filter area
Epifluorescent microscopy
• Area of
Count: manual or microscope field
automatic
• Number of field

97
Direct epifluorescent filter technique (DEFT)

Acridine orange bineds to: RNA

RNA --- fluorescent orange
DNA --- fluorescent green
Viable cell DNA
RNA > DNA --- orange
Non-viable cell

DNA > RNA --- green

98
Direct epifluorescent filter technique (DEFT)

Viable Non-viable

99
 Membrane epiflurescent

A rapid technique for the detection of pathogens in food products

100
www.teagasc.ie/.../ 4681/eopr-4681.htm
Membrane epiflurescent

101
Electrical impedance method
Impedance : resistance

Conductance
Bacteria growth
----decrease impedance
----increase conductivity

Time
DT
Detection time
106-107 cells/ml
102
Electrical impedance method
Bactometer
Vary temp
Small volume
•Count
Many wells
Many samples •Growth
Automatic

103
Bactometer

104
ELISA
Antigen – conjugate enzyme
Antibody – conjugate enzyme

Toxin
Pathogen •Staphylocaccal
•Salmonella •Botulinum toxin
•Listeria •Mycotoxin
•S. aureus
105
Enzyme-linked Immunosorbent Assay
Free toxin
Antibody
Antigen(toxin)
Enzyme Labeled toxin Microtitration plate
Alkaline phosphatase (ALP)
Horse Radish Peroxidase (HRP)
Substrate
Tetramethylbenzidine (TMB) + 30% H2O2
Azinobis sulphonic acid (ABTS)
o-phenylinediamine (OPD)
p-nitrophenyl phosphate

106
107
“ELISA”

108
 Sandwich-ELISA
E. coli Salmonella
antibody

Antibody-conjugate
enzyme
Colorless Substrate

Color
109
 Aflatoxin
A. flavus
A. nomius
flavus A. tamarri
A. parasiticus

“Aflatoxin”
Aspergillus toxin

110
http://msa.ars.usda.gov/la/srrc/aflatoxin/afcrsp.htm
111
Enzyme-linked Immunosorbent Assay
Free toxin
Antibody
Antigen(toxin)
Enzyme Labeled toxin Microtitration plate
Alkaline phosphatase (ALP)
Horse Radish Peroxidase (HRP)
Substrate
Tetramethylbenzidine (TMB) + 30% H2O2
Azinobis sulphonic acid (ABTS)
o-phenylinediamine (OPD)
p-nitrophenyl phosphate

112
 E E

E E

Aflatoxin: Colorless Coloration 113

 “ELISA”

Antibody

Aflatoxin (free toxin)

E
Aflatoxin-enzyme labeled (labeled toxin)

Substrate

114
 Direct Competitive ELISA
Conc. 0 5 10 15 20
(ppb)

Aflatoxin

E E

115
 Direct Competitive ELISA
ppb 0 5 10 15 A B C

Standard Foods ample

(Aflatoxin) (Un-know)
A= ?????? ppb
B= ?????? ppb
C= ?????? ppb
116
 ELISA
Qualitative result color
Quantitative result
Micro ELISA reader
Spectrophotometer
Standard curve

Absorbant

Concentration
117
PCR

118
119