Indicator OrganismsIndicator OrganismsIndicator OrganismsIndicator OrganismsIndicator OrganismsIndicator OrganismsIndicator OrganismsIndicator Organisms

Indicator organismsIndicator organisms

!Group of microorganisms use to reflect thequality and safety of a process food product!Indicator organism in water

!Vibrio cholera!Salmonella typhi!Shigella spp!Coliforms and fecal coli!Hepatitis

!Indicator organism in food!Coliforms!E. coli

!Must be associated with feces

!Should not be a natural contaminant

!Easy to detect

!Test should be less complex than test for pathogen

!Growth limit under conditions

!Should not survive processing

!Processing should have same effect on pathogens

Criteria for use of an organismCriteria for use of an organismas an indicatoras an indicator

!Fecal organism

!Fecal coliform

!Escherichia coli

!Enteric bacteria

!Shigella spp.

!Salmonella spp.

Suggested indicator organismsSuggested indicator organismsSuggested indicator organismsSuggested indicator organismsSuggested indicator organismsSuggested indicator organismsSuggested indicator organismsSuggested indicator organisms

!Coliform indicates processing contamination

!Coliform can grow on food processing equipment orin environment

!Several genera are considered to be coliforms!Escherichia

!Enterobactor

!Klebsiella

!Citrobactor

!Source of coliforms!Fecal group --GI tract of humans and animals

!Non-fecal group --Soil and plant

ColiformsColiformsColiformsColiformsColiformsColiformsColiformsColiforms

!Coliform group

!Gram negative, rod shape, aerobic or facultativeaerobic

!non endospore former

!ferments lactose at 30-37 °C for 24-48 hrs

!Fecal coliform

!found only in GI tract

!can grow at elevated test temperature

!ferment LST (Lauryl Sulfate Tryptose) at 44.5 °C for 24hrs

ColiformsColiforms

!Methods for isolation of coliforms

!Selective plating

!Most probable number (MPN)

!Selective plating

!Presumptive test!Solid media

!VRBA (Violet red bile agar)

!Confirm test

!Selective and differential media

!EMB (Eosin Methylene Blue agar)

ColiformsColiforms

ColiformsColiforms

I nnoculate

PCA slant VRB EMB

Selective plating Selective plating

!Methods for isolation of coliforms

!Most probable number (MPN)

!Presumptive test

!Liquid media (MPN)

!LST (Lauryl Sulfate Tryptose)

!Confirm test

!Liquid media (MPN)

!BGLB (Brillant Green Lactose Bile broth)

!EC (EC broth) (Lauryl Sulfate Tryptose)

ColiformsColiforms

ColiformsColiforms

Most probable number (MPN)Most probable number (MPN)

101 102 103

103

I ncubate

Positive: Gas productionChange of medium color

102

101

102

101

102

101

Presumptive test Confirm test

TST medium BGLB medium EC medium

103 103

Coliform MPN/ ml

Fecal coliform MPN/ ml

!LST (Lauryl Sulfate Tryptose) or Lactose broth (LB)

!triplicate tubes for each dilution!incubate LST at 32-35 °C for 24-48 hrs

!coliform grow---turbidity and gas

!VRBA (Violet Red Bile Agar)

!pour plate method---pour VRBA on plate and overlay!incubate at 32-35 °C for 24-48 hrs

!red, purple colonies with ring of precipitate bile salt around colonies

Presumptive test of Presumptive test of coliformscoliforms

!BGB (Brilliant Green Bile Broth)

!Incubate BGB at 32-35 °C for 24-48 hrs

!Coliform reported as MPN/ml or gm

!EC (EC broth)

!Incubate EC at 44.5 °C for 24 hrs

!Fecal coliform reported as MPN/ml or gm

Confirmation of Confirmation of coliformscoliforms

!Select colony or positive tubes and streak onto PCAslants

!Identify the microorganism

!Gram stain!Biochemical test

!IMViC test!Indole test!Methyl red!Voges-Proskauer!Citrate utilization

!Selective media!EMB agar (Eosin Methylene Blue Agar)

Identification of Identification of coliformscoliforms

IMViC test: use to differentiate between thecoliform group!Indoletest

!Bacteria degrade tryptophan to indole

!Methyl red!Bacteria produce acid from glucose

!Voges-Proskauer!Detect acetylmethylcarbinol from glucose

!Citrate utilization!Bacterial utilize citrate as a carbon source

Identification of Identification of coliformscoliforms

IMViC test

Organism I ndole Methyl red Voges-Proskauer Citrate

E. coli + + - -E. aerogenes - - + +

Identification of Identification of coliformscoliforms

!Selective media

!EMB (Eosin Methylene Blue) medium!Selective and differential plating for isolation and

identification of Gram-negative enteric bacteria

!Eosin and methylene blue used as indicators

!Bacteria ferment lactose

!Sucrose used to detect coliform that ferment sucrose

!E. coli dark colony with blackcenter and green metalicsheen

!Enterobacter spp. pink clony

!Klebsiella spp. dark center colony

Identification of Identification of coliformscoliforms

ColiformsColiforms

101 102 103

103

I ncubate

Positive: Gas productionChange of medium color

102

101

102

101

102

101

Presumptive test Confirm test

TST medium BGLB medium EC medium

103 103

Coliform MPN/ ml

Fecal coliform MPN/ ml

ColiformsColiforms

I nnoculate

I MViC test

PCA slant

102

101

103

VRB

OR

BGLB/ EC

+ / - + / - + / - + / -

EMB