1205 324 Food Industrial Microbiology

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Rapid MethodDirect

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

•Pour plate•Spread plate•Drop plate

•Diluent•0.85%NaCl•0.1% peptone•Phosphate buffer

•Medium •Elective medium•Selective medium•General

•Petri dish plate•Replication

Diluent Food homogenate Dilution series Medium Plating method Incubate conditions

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Selective agentSodium telluliteLithium chloride

Elective agentSodium pyruvateGlycine

Diagnostic agentEgg yolk

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Opaque zone

Staphylococcus aureus

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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

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 green/Lactose/Bile broth

MPN of coliforms in food

Braid Parker agar Staphylococcus aureus

Thiosulfate/Bile/Citrate/agar

Vibrio sp.

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Adam and Moss (2003)

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

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

Filtration

0.45 μm

•Low number of MO. Large volume of food

Liquid food

•Count•Sterilize

Most probable number (MPN)Multiple tube techniques

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•PathogenNumber too low

• Coliform• Escherichia coli• Staphylococcus

aureus• Feacal streptococci

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 aureus

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Direct microscopic count (DMCs)Small sample (0.01 ml) & rapidOptical light microscopeTotal cell

living & dead cells

FoodsLiquidSemi-solid

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

•Howard mold

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Method Vol. of sample (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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Methylene blue

Leuco-methylene blue

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Resazurin (blue)

Resorufin (pink)

Dihydroresofin(leuco)

Triphenyltetrazolium chloride (leuco)

Formazan (red)

•Hygiene indicator•Cross contamination

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•Fresh meat•Raw milk•Pasteurized milk

ATP : Adenosine triphosphateSynthesis of new cellActive transport (uptake of materials from

environment)MovementLight production

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Luciferin + luciferase + ATP + O2

Oxyluciferin + luciferase + AMP +

light

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Mg2+

1 ATP light 1 photon

Bacteria cell1 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

Break down the non-microbial cells in food

Remove non-microbial ATP using ATPase

Release ATP from bacteria cellAddition of luciferin & luciferaseeRecord light emission (ATP

photometry)28

ApplicationFresh meat Milk Starter cultureTest UHT milk Surface contamination

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DisadvantageMixed bacteria & yeast cellDilutionRemove cell before ATP

measured FiltrationCentrifugation

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Liquid foodFilter through

membrane Acridine orange :

fluorescent dye (fluorochrome) pour through filter

Epifluorescent microscopy

Count: manual or automatic

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• Direct microscopy

• Membrane filtration

• Vol. of sample• Filter area• Area of

microscope field

• Number of field

Acridine orange bineds to:RNA --- fluorescent orangeDNA --- fluorescent green

Viable cell

RNA > DNA --- orange

Non-viable cell

DNA > RNA ---green32

RNA

DNA

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Non-viableViable

34www.teagasc.ie/.../ 4681/eopr-4681.htm A 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

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Membrane epiflurescent

Impedance : resistance

Bacteria growth ----decrease

impedance----increase

conductivity

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Con

du

cta

nce

TimeDTDetection time106-107 cells/ml

BactometerVary tempSmall volumeMany wells

Many samples

Automatic

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•Count•Growth

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Bactometer

Antigen – conjugate enzymeAntibody – conjugate enzyme

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Pathogen•Salmonella•Listeria•S. aureus

Toxin•Staphylocaccal•Botulinum toxin•Mycotoxin

AntibodyAntigen(toxin)Enzyme

Alkaline phosphatase (ALP)Horse Radish Peroxidase (HRP)

SubstrateTetramethylbenzidine (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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Colorless

antibody

SalmonellaE. coli

An

tib

od

y-c

on

jug

ate

en

zym

e

Substrate

Color

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Aflatoxin

“Aflatoxin”

Aspergillus

flavus

toxin

A. flavusA. nomiusA. tamarriA. parasiticus

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

AntibodyAntigen(toxin)Enzyme

Alkaline phosphatase (ALP)Horse Radish Peroxidase (HRP)

SubstrateTetramethylbenzidine (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

C

ppb

A= ?????? ppb B= ??????

ppb

C= ?????? ppb

Qualitative result color

Quantitative resultMicro ELISA readerSpectrophotometer Standard curve

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Ab

sorb

ant

Concentration

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PCR

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

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