Method for the Microbiological Examination of Foods

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Method for the Microbiological Examination of
Foods

• 1205 324 Food Industrial Microbiology

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Method

• Rapid Method
• Direct epifluorescent filter technique (DEFT)
• Electrical impedance
• Enzyme-linked immunosorbent assay(ELISA)

• Traditional method
• Plate counts
• Membrane filtration
• Most probable number
• Direct microscopic count
• Dye reduction tests
• Indicator

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Plate count method
Standard plate count (SPC) Aerobic plate count
(APC)
Total bacteria count (TBC) Total viable count
(TVC)
Live
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Plate count method

• Diluent
• 0.85NaCl
• 0.1 peptone
• Phosphate buffer
• Medium
• Elective medium
• Selective medium
• General
• Petri dish plate
• Replication

• Pour plate
• Spread plate
• Drop plate

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Plate count depends on

• Diluent
• Food homogenate
• Dilution series
• Medium
• Plating method
• Incubate conditions

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Baird-Parker Agar

• Selective agent
• Sodium tellulite
• Lithium chloride
• Elective agent
• Sodium pyruvate
• Glycine
• Diagnostic agent
• Egg yolk

Staphylococcus aureus
Opaque zone
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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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Disadvantage of plate count ???

• ???????

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Drop plate

• Sample
• Small vol. 20 µL
• Cost

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Drop plate
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Application of plate count

• Check quality of RM final products
• Check condition hygiene
• Estimate storage life of products
• Determine
• Production
• Transport
• Storage
• Determine pathogens

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Selection of media in food microbiology
Adam and Moss (2003)
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Streak technique
http//www.towson.edu/cberkowe/medmicro/images/st
reak.gif
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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
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Most probable number

• Most probable number (MPN)
• Multiple tube techniques

• Pathogen
• Number too low

• Coliform
• Escherichia coli
• Staphylococcus aureus
• Feacal streptococci

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Most probable number
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Microscopic count

• Direct microscopic count (DMCs)
• Small sample (0.01 ml) rapid
• Optical light microscope
• Total cell
• living dead cells
• Foods
• Liquid
• Semi-solid

• Ex.
• Milk
• Wine
• Yogurt starter
• Tomato sauce
• Howard mold

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Microscopic count
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Comparison of sensitivity of method
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Dye-reduction test

• Methylene blue
• Leuco-methylene blue

Resazurin (blue) Resorufin (pink) Dihydroresofin
(leuco)
Triphenyltetrazolium chloride (leuco) Formazan
(red)
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Indicators

• Hygiene indicator
• Cross contamination

• Fresh meat
• Raw milk
• Pasteurized milk

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ATP photometry

• ATP Adenosine triphosphate
• Synthesis of new cell
• Active transport (uptake of materials from
  environment)
• Movement
• Light production

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ATP photometry

• Luciferin luciferase ATP O2
• Oxyluciferin luciferase AMP light

Mg2
1 ATP light 1 photon
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ATP photometry

• Bacteria cell
• 1 fg of ATP
• Yeast cell
• 100 fg of ATP

• Limit of ATP photometry102-103 fg ATP/ml

fg femto gram 10-15 g
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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)

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ATP photometry

• Application
• Fresh meat
• Milk
• Starter culture
• Test UHT milk
• Surface contamination

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ATP photometry

• Disadvantage
• Mixed bacteria yeast cell
• Dilution
• Remove cell before ATP measured
• Filtration
• Centrifugation

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Direct epifluorescent filter technique (DEFT)

• Liquid food
• Filter through membrane
• Acridine orange fluorescent dye (fluorochrome)
  pour through filter
• Epifluorescent microscopy
• Count manual or automatic

• Direct microscopy
• Membrane filtration

• Vol. of sample
• Filter area
• Area of microscope field
• Number of field

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Direct epifluorescent filter technique (DEFT)
RNA

• Acridine orange bineds to
• RNA --- fluorescent orange
• DNA --- fluorescent green
• Viable cell
• RNA gt DNA --- orange
• Non-viable cell
• DNA gt RNA ---green

DNA
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Direct epifluorescent filter technique (DEFT)
Non-viable
Viable
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Membrane epiflurescent
A rapid technique for the detection of pathogens
in food products
A rapid technique for the detection of pathogens
in food products
A rapid technique for the detection of pathogens
in food products
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Membrane epiflurescent
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Electrical impedance method

• Impedance resistance
• Bacteria growth
• ----decrease impedance
• ----increase conductivity

Conductance
Time
DT Detection time 106-107 cells/ml
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Electrical impedance method

• Bactometer
• Vary temp
• Small volume
• Many wells
• Many samples
• Automatic

• Count
• Growth

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Bactometer
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ELISA

• Antigen conjugate enzyme
• Antibody conjugate enzyme

• Toxin
• Staphylocaccal
• Botulinum toxin
• Mycotoxin

• Pathogen
• Salmonella
• Listeria
• S. aureus

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Enzyme-linked Immunosorbent Assay
Free toxin Labeled toxin

• Antibody
• Antigen(toxin)
• Enzyme
• Alkaline phosphatase (ALP)
• Horse Radish Peroxidase (HRP)
• Substrate
• Tetramethylbenzidine (TMB) 30 H2O2
• Azinobis sulphonic acid (ABTS)
• o-phenylinediamine (OPD)
• p-nitrophenyl phosphate

Microtitration plate
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(No Transcript)
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ELISA
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Sandwich-ELISA
Salmonella
E. coli
antibody
Antibody-conjugate enzyme
Colorless
Substrate
Color
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Aflatoxin

• flavus
• A. nomius
• tamarri
• A. parasiticus

flavus
Aflatoxin
toxin
Aspergillus
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http//msa.ars.usda.gov/la/srrc/aflatoxin/afcrsp.h
tm
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Enzyme-linked Immunosorbent Assay
Free toxin Labeled toxin

• Antibody
• Antigen(toxin)
• Enzyme
• Alkaline phosphatase (ALP)
• Horse Radish Peroxidase (HRP)
• Substrate
• Tetramethylbenzidine (TMB) 30 H2O2
• Azinobis sulphonic acid (ABTS)
• o-phenylinediamine (OPD)
• p-nitrophenyl phosphate

Microtitration plate
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E
E
E
E
Aflatoxin Colorless
Coloration
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ELISA
Antibody Aflatoxin (free toxin) Aflatoxin-enzyme
labeled (labeled toxin) Substrate
E
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Direct Competitive ELISA
Conc. (ppb)
0
5
10
15
20
Aflatoxin
E
E
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Direct Competitive ELISA
ppb
A
B
C
0
5
10
15
Standard (Aflatoxin)
Foods ample (Un-know)
A ?????? ppb
B ?????? ppb
C ?????? ppb
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ELISA

• Qualitative result color
• Quantitative result
• Micro ELISA reader
• Spectrophotometer
• Standard curve

Absorbant
Concentration
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PCR
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(No Transcript)
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THE END