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1205 324 Food Industrial Microbiology…1/48..Thidarat
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1205 324 Food Industrial Microbiology…1/48..Thidarat
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1205 324 Food Industrial Microbiology…1/48..Thidarat
Traditional method Rapid Method
• Plate counts Direct
• Membrane filtration epifluorescent
• Most probable
number filter technique
• Direct microscopic (DEFT)
count Electrical
• Dye reduction tests impedance
• Indicator Enzyme-linked
immunosorbent
assay(ELISA)
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 Plate count
method
Standard plate count (SPC)
Aerobic plate count (APC)

Total bacteria count (TBC)

Total viable count (TVC)

“Live” 14
 Plate count
method
•Diluent
•0.85%NaCl
•0.1% peptone
•Phosphate
buffer
•Medium
•Elective
•Pour plate medium
•Spread plate •Selective
medium
•Drop plate •General
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Plate count
method

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Pour plate

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 Spread plate

Number of
colony forming units (cfus)
?????
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 Spread plate

Number of
colony forming units (cfus)
?????
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 Drop plate

Sample:
•Small vol. ≈ 20 μL
Cost

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Drop plate

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 Check quality of RM & final products
 Check condition hygiene
 Estimate storage life of products
 Determine
 Production
 Transport
 Storage
 Determine pathogens
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Medium Use
Plate count agar Aerobic
mesophilic count
MacConkey broth MPN of coliforms
in water
Brilliant MPN of coliforms
green/Lactose/Bile broth in food
Braid Parker agar Staphylococcus
aureus
Thiosulfate/Bile/ Vibrio sp.
Citrate/agar
Adam and Moss (2003
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http://www.towson.edu/~cberkowe/medmicro/images/streak.gif
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 Filtrati
on
0.45 μm
Liquid food
•Low number
of MO.
Large volume
of food

•Count
•Sterilize

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http://www.biology.lsu.edu/webfac/rgayda/biol1011/Lecture_notesF2004/
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Most probable number (MPN)
Multiple tube techniques
• Pathogen
 Number too low
• Coliform
• Escherichia coli
• Staphylococcus
aureus
• Feacal streptococci 29
 Presumptive test.

 Confirmatory test.

 Completed test

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1205 324 Food Industrial Microbiology…1/48..Thidarat
Medium Organisms
assessed
Lauryl sulfate tryptose Coliforms
broth
MacConkey purple broth Coliforms
EC broth Faecal coliform
Glucose azide Faecal streptococci
Minerals modified Coliforms
glutamate medium
Baird-Parker broth Staphylococcus
aureus
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Direct microscopic count (DMCs)
Small sample (0.01 ml) & rapid
Optical light microscope
Total cell Ex.
living & dead cells •Milk
•Wine
Foods
•Yogurt
Liquid starter
Semi-solid •Tomato
sauce 34
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 Method Vol. of Count
sample (cfu/g)
(ml)
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

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 Methylene blue
Resazurin
Leuco-methylene blue (blue)

Triphenyltetrazolium Resorufin (pink)

chloride (leuco)
Dihydroresofin
Formazan (red) (leuco)

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• Hygiene indicator
• Cross contamination

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

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Luciferin + luciferase + ATP + O2

Mg2+

Oxyluciferin + luciferase + AMP +

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

fg = femto gram = 10-15 g

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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)
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Application
Fresh meat
Milk
Starter culture
Test UHT milk
Surface contamination
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Disadvantage
Mixed bacteria & yeast cell
Dilution
Remove cell before ATP
measured
Filtration
Centrifugation
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Liquid food • Direct
Filter through microscopy
membrane • Membrane
Acridine orange : filtration
fluorescent dye • Vol. of sample
(fluorochrome) pour • Filter area
through filter
• Area of
Epifluorescent
microscope
microscopy
field
Count: manual or
• Number of
automatic
field 46
Acridine orange bineds to: RNA
RNA --- fluorescent orange
DNA --- fluorescent green
DNA
Viable cell
RNA > DNA --- orange
Non-viable cell
DNA > RNA ---green
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Viable Non-viable

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 Membrane epiflurescent

A rapid technique for the detection of pathogens in food products

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 Membrane
epiflurescent

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Impedance :

Conductance
resistance
Bacteria growth
----decrease
impedance
----increase
conductivity Time
DT
Detection time
106-107 cells/ml
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Bactometer
Vary temp
Small volume •Count
Many wells •Growth
Many samples
Automatic

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Bactometer

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Antigen – conjugate enzyme
Antibody – conjugate enzyme

Toxin
Pathogen •Staphylocacc
•Salmonella al
•Listeria •Botulinum
•S. aureus toxin
•Mycotoxin 58
 Free toxin
Antibody
Antigen(toxin)
Labeled toxin Microtitration plate
Enzyme
Alkaline phosphatase (ALP)
Horse Radish Peroxidase (HRP)
Substrate
Tetramethylbenzidine (TMB) + 30% H2O2
Azinobis sulphonic acid (ABTS)
o-phenylinediamine (OPD)
p-nitrophenyl phosphate
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“ELISA”

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 E. coli Salmonella
antibody

Antibody-conjugate
enzyme
Colorless Substrate

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

“Aflatoxin”
Aspergillus toxin

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

E E

Aflatoxin: Colorless Coloration 65

 “ELISA”

Antibody

Aflatoxin (free toxin)

E
Aflatoxin-enzyme labeled (labeled toxin)

Substrate

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

Aflatoxin

E E

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Direct Competitive ELISA
ppb 0 5 10 15 A B C

Standard Foods ample

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

Absorbant
Standard curve

Concentration69
 PCR

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home.nvg.org/~forthun/ cdr-lenker.html
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