bacterial count (1)

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METHOD FOR THE MICROBIOLOGICALEXAMINATION OF FOODS

1205 324 Food Industrial Microbiology

1


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METHOD

Rapid MethodDirect

epifluorescent

filter technique(DEFT)

Electrical

impedanceEnzyme-linkedimmunosorbentassay(ELISA)

Traditional method Plate counts Membrane filtration

Most probable number Direct microscopic

count Dye reduction tests

Indicator

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3

Plate count method

Standard plate count (SPC)

Aerobic plate count (APC)

Total bacteria count (TBC)

Total viablecount (TVC)

Live


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4

Plate count method

Pour plateSpread plate

Drop plate

Diluent0.85%NaCl

0.1% peptone

Phosphate bufferMedium

Elective medium

Selective medium

GeneralPetri dish plate

Replication


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PLATE COUNT DEPENDS ON

Diluent

Food homogenate

Dilution series

Medium

Plating method

Incubate conditions

5


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Plate count method


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Pour plate


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9

Spread plate

Number of

colony forming units (cfus)

?????


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10

Spread plate

Number of

colony forming units (cfus)

?????


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DISADVANTAGE OF PLATE COUNT ???

???????

11


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12

Drop plate

Sample:Small vol. 20 L

Cost


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13

Drop plate


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APPLICATION OF PLATE COUNT

Check quality of RM & final products

Check condition hygiene

Estimate storage life of productsDetermine

Production

TransportStorage

Determine pathogens

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SELECTION OF MEDIA IN FOOD

MICROBIOLOGY

Medium UsePlate count agar Aerobic mesophilic

count

MacConkey broth MPN of coliforms inwater

Brilliant green/Lactose/Bile

broth

MPN of coliforms in

food

Braid Parker agar Staphylococcus

aureus

Thiosulfate/Bile/Citrate/agar Vibrio sp.

15 Adam and Moss (2003)


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STREAK TECHNIQUE

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


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17http://www.biology.lsu.edu/webfac/rgayda/biol1011/Lecture_notesF2004/lecture8.pdf

Filtration0.45 m

Low number of

MO.Large volume of

food

Liquid food

CountSterilize


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MOST PROBABLE NUMBER

Most probable number (MPN)

Multiple tube techniques

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PathogenNumber too low

Coliform

Escherichia coli

Staphylococcus aureus

Feacal streptococci


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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

glutamate medium

Coliforms

Baird-Parker broth Staphylococcus

aureus19


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MICROSCOPIC COUNT

Direct microscopic count (DMCs)

Small sample (0.01 ml) & rapid

Optical light microscopeTotal cell

living & dead cells

FoodsLiquid

Semi-solid20

Ex.Milk

WineYogurt starterTomato sauceHoward mold


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MICROSCOPIC COUNT

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COMPARISON OF SENSITIVITY OF METHOD

Method Vol. ofsample (ml) Count(cfu/g)

Direct microscopy 5 x 10-6 2 x 106

Drop plate(Miles and Misra)

0.02 5 x 102

Spread plate 0.1 102

Pour plate 1 10

MPN 3 x 10

+ 3 x 1

+ 3 x 0.1

0.36

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DYE-REDUCTION TEST

Methylene blue

Leuco-methylene blue

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Resazurin (blue)

Resorufin (pink)

Dihydroresofin

(leuco)

Triphenyltetrazolium

chloride (leuco)

Formazan (red)


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INDICATORS

Hygiene indicator

Cross contamination

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Fresh meatRaw milk

Pasteurized milk


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ATP PHOTOMETRY

ATP : Adenosine triphosphateSynthesis 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

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Mg2+

1 ATP light 1 photon


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ATP PHOTOMETRY

Bacteria cell

1 fg of ATP

Yeast cell100 fg of ATP

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fg = femto gram = 10-15 g

Limit of ATP

photometry102-103 fg ATP/ml


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ATP PHOTOMETRY

Break down the non-microbial cells in

food

Remove non-microbial ATP usingATPase

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 milkSurface contamination

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ATP PHOTOMETRY

Disadvantage

Mixed bacteria & yeast cell

Dilution

Remove cell before ATP measured

FiltrationCentrifugation

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DIRECT EPIFLUORESCENT FILTER TECHNIQUE (DEFT)

Liquid foodFilter through membrane

Acridine orange :fluorescent dye(fluorochrome) pourthrough filter

Epifluorescent

microscopyCount: manual or

automatic

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Directmicroscopy

Membranefiltration

Vol. of sample

Filter area

Area of

microscope field

Number of field


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Acridine orange bineds to:

RNA --- fluorescent orange

DNA --- fluorescent greenViable cell

RNA > DNA --- orange

Non-viable cell

DNA > RNA ---green

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RNA

DNA


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Non-viableViable


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34www.teagasc.ie/.../ 4681/eopr-4681.htmA rapid technique for the detection of pathogens in food productsA rapid technique for the detection of pathogens in food productsA rapid technique for the detection of pathogens in food products

Membrane epiflurescent
http://www.teagasc.ie/research/reports/foodprocessing/4681/eopr-4681.htmhttp://www.teagasc.ie/research/reports/foodprocessing/4681/eopr-4681.htmhttp://www.teagasc.ie/research/reports/foodprocessing/4681/eopr-4681.htmhttp://www.teagasc.ie/research/reports/foodprocessing/4681/eopr-4681.htm


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Membrane epiflurescent


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ELECTRICAL IMPEDANCE METHOD

Impedance :

resistance

Bacteria growth

----decrease

impedance

----increase

conductivity

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Conductance

TimeDT

Detection time

106-107 cells/ml


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ELECTRICAL IMPEDANCE METHOD

Bactometer

Vary temp

Small volumeMany wells

Many samples

Automatic

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CountGrowth

B t t


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Bactometer


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ELISA

Antigen conjugate enzyme

Antibody conjugate enzyme

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Pathogen

SalmonellaListeria

S. aureus

ToxinStaphylocaccal

Botulinum toxinMycotoxin


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ENZYME-LINKED IMMUNOSORBENT ASSAY

Antibody

Antigen(toxin)

Enzyme

Alkaline phosphatase (ALP)

Horse Radish Peroxidase (HRP)

Substrate

Tetramethylbenzidine (TMB) + 30% H2O2Azinobis sulphonic acid (ABTS)

o-phenylinediamine (OPD)

p-nitrophenyl phosphate

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Microtitration plate

Free toxin

Labeled toxin


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ELISA


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SANDWICH-ELISA

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Colorless

antibodySalmonellaE. coli

Antibo

dy-conjug

ate

enzyme

Substrate

Color


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44

Aflatoxin

Aflatoxin

Aspergillus

flavus

toxin

A. flavusA. nomius

A. tamarri

A. parasiticus


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


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ENZYME-LINKED IMMUNOSORBENT ASSAY

Antibody

Antigen(toxin)

Enzyme

Alkaline phosphatase (ALP)

Horse Radish Peroxidase (HRP)

Substrate

Tetramethylbenzidine (TMB) + 30% H2O2Azinobis sulphonic acid (ABTS)

o-phenylinediamine (OPD)

p-nitrophenyl phosphate

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Microtitration plate

Free toxin

Labeled toxin


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E E

EE

Aflatoxin: Colorless Coloration


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Antibody

Aflatoxin (free toxin)

Aflatoxin-enzyme labeled (labeled toxin)

Substrate

E

ELISA


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Aflatoxin

0 5 10 15 20

EE

Direct Competitive ELISA

Conc.(ppb)


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Direct Competitive ELISA

Standard

(Aflatoxin)Foods ample

(Un-know)

0 5 10 15 A B Cppb

A= ?????? ppb

B= ?????? ppbC= ?????? ppb


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ELISA

Qualitative result color

Quantitative result

Micro ELISA reader

Spectrophotometer

Standard curve

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Absorbant

Concentration


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PCR

home.nvg.org/~forthun/ cdr-lenker.html
http://home.nvg.org/~forthun/cdr-lenker.htmlhttp://home.nvg.org/~forthun/cdr-lenker.htmlhttp://home.nvg.org/~forthun/cdr-lenker.htmlhttp://home.nvg.org/~forthun/cdr-lenker.htmlhttp://home.nvg.org/~forthun/cdr-lenker.htmlhttp://home.nvg.org/~forthun/cdr-lenker.html


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bacterial count (1)

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