METHOD FOR THE MICROBIOLOGICAL EXAMINATION OF FOODS

1205 324 Food Industrial Microbiology

METHOD
Traditional method

Rapid Method
Direct epifluorescent filter technique (DEFT) Electrical impedance Enzyme-linked immunosorbent assay(ELISA)
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Plate counts Membrane filtration Most probable number Direct microscopic count Dye reduction tests Indicator

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
0.85%NaCl 0.1% peptone Phosphate buffer
Elective medium Selective medium General

Medium

Pour plate Spread plate Drop plate

Petri dish plate Replication

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PLATE COUNT DEPENDS ON

Diluent Food homogenate Dilution series Medium Plating method Incubate conditions

BAIRD-PARKER AGAR
Selective agent
Sodium tellulite Lithium chloride

Staphylococcus aureus

Elective agent
Sodium pyruvate Glycine

Diagnostic agent
Egg yolk
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Plate count method

Pour plate

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 L 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

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

STREAK TECHNIQUE

http://www.towson.edu/~cberkowe/medmicro/images/streak.gif 16

Filtration
0.45 m

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

Medium
Lauryl sulfate tryptose broth MacConkey purple broth EC broth Glucose azide Minerals modified glutamate medium Baird-Parker broth Organisms assessed Coliforms Coliforms Faecal coliform Faecal streptococci Coliforms Staphylococcus aureus 19

MICROSCOPIC COUNT
Direct microscopic count (DMCs) Small sample (0.01 ml) & rapid Optical light microscope Total cell Ex.
living & dead cells

Foods Liquid Semi-solid

Milk Wine Yogurt starter Tomato sauce Howard mold

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MICROSCOPIC COUNT

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COMPARISON OF SENSITIVITY OF METHOD

Method
Direct microscopy Drop plate (Miles and Misra) Spread plate

Vol. of sample (ml)

5 x 10-6 0.02 0.1

Count (cfu/g)
2 x 106 5 x 102 102

Pour plate MPN

1 3 x 10 +3x1 + 3 x 0.1

10 0.36

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DYE-REDUCTION TEST
Methylene blue
Resazurin (blue)

Leuco-methylene blue
Resorufin (pink) Triphenyltetrazolium chloride (leuco) Formazan (red) Dihydroresofin (leuco)

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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 Mg2+

Oxyluciferin + luciferase + AMP + light
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 photometry 102-103 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)

Acridine orange bineds to:

RNA --- fluorescent orange DNA --- fluorescent green

RNA
DNA

Viable cell
RNA > DNA --- orange

Non-viable cell
DNA > RNA ---green
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DIRECT EPIFLUORESCENT FILTER TECHNIQUE (DEFT)

Viable Non-viable

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Membrane epiflurescent

A rapid technique for the detection of pathogens in food products

www.teagasc.ie/.../ 4681/eopr-4681.htm

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Membrane epiflurescent

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ELECTRICAL IMPEDANCE METHOD

Conductance

Impedance : resistance Bacteria growth

----decrease impedance ----increase conductivity

DT Detection time 106-107 cells/ml

Time

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ELECTRICAL IMPEDANCE METHOD

Bactometer Vary temp Small volume Many wells
Many samples

Count Growth

Automatic

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Bactometer

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ELISA Antigen conjugate enzyme Antibody conjugate enzyme Toxin Staphylocaccal Botulinum toxin Mycotoxin
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Pathogen Salmonella Listeria S. aureus

ENZYME-LINKED IMMUNOSORBENT ASSAY

Free toxin

Antibody Antigen(toxin) Labeled toxin Enzyme

Microtitration plate

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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SANDWICH-ELISA
E. coli Salmonella
Antibody-conjugate enzyme
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antibody

Colorless

Substrate

Color

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

Aflatoxin
Aspergillus
toxin
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45 http://msa.ars.usda.gov/la/srrc/aflatoxin/afcrsp.htm

ENZYME-LINKED IMMUNOSORBENT ASSAY

Free toxin

Antibody Antigen(toxin) Labeled toxin Enzyme

Microtitration plate

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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Aflatoxin: Colorless

Coloration

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ELISA
Antibody Aflatoxin (free toxin)
E Aflatoxin-enzyme labeled (labeled toxin)

Substrate

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Direct Competitive ELISA

Conc. (ppb)

10

15

20

Aflatoxin
E E

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Direct Competitive ELISA

ppb 0 5 10 15

Standard (Aflatoxin)
A= ?????? ppb B= ?????? ppb C= ?????? ppb

Foods ample (Un-know)

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ELISA
Qualitative result Quantitative result
Micro ELISA reader Spectrophotometer Standard curve Absorbant

color

Concentration 51

PCR

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

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THE END
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