Lecture 10

Indicator Organisms
REFLECTS microbiological quality

organisms and/or their metabolic

products whose presence in given foods at certain levels may be used to assess existing quality or better, to predict product shelf life
Jay

Criteria - Indication of Quality

Present and Detectable

Correlation to product quality

Detected and distinguishable

Enumerable in short period

Not affected by food flora

Criteria -Indicators of Food Safety

Easily and Rapidly Detectable

Distinguishable
Association to Pathogen

Present when Pathogen Present

Numbers, Growth and Death Correlate to

Pathogen Absent from Foods that are Free of the Pathogen

Ideal Relationship Between a Pathogen and an Indicator Organism

Indicator

Numbers

Pathogen

Time

Indicator Organisms
Total Coliforms not all from fecal origin

Fecal Coliforms
E. coli Fecal Streptococci Enterococci

Indicator Organisms
Total Coliforms not all from fecal origin

Fecal Coliforms grow at higher temps

E. coli Fecal Streptococci Enterococci

Indicator Organisms
Total Coliforms not all from fecal origin

Fecal Coliforms grow at higher temps

E. coli best indicator of health risk Fecal Streptococci Enterococci

Indicator Organisms
Total Coliforms

Fecal Coliforms
E. coli Fecal Streptococci previously used to test

for human origin Enterococci

Indicator Organisms
Total Coliforms

Fecal Coliforms
E. coli Fecal Streptococci previously used to test

for human origin Enterococci best indicator for salt water

Escherichia coli
E. coli normally colonizes an infant's

gastrointestinal tract within 40 hours of birth, arriving with food or water or with the individuals handling the child. In the bowel, it adheres to the mucus of the large intestine. It is the primary facultative anaerobe of the human gastrointestinal tract. As long as these bacteria do not acquire genetic elements encoding for virulence factors, they remain benign commensals.

Definitions of (Total) Coliforms

Multiple-Tube Fermentation All aerobic and facultative anaerobic, Gramnegative, non-spore-forming, rod shaped bacteria that ferment lactose with gas and acid formation within 48 hr. at 35C. Membrane Filtration All aerobic and many facultative anaerobic, Gramnegative, non-spore-forming, rod-shaped bacteria that develop a red colony with a metallic sheen within 24 hr. at 35C on an Endo-type medium containing lactose.

Other Properties
-galactosidase positive Enzyme that hydrolyzes lactose into glucose and galactose Oxidase negative Indicates the absence of Cytochrome-c in the electron transport chain.

Typical Coliform Genera

Citrobacter

Enterobacter
Escherichia

Hafnia
Klebsiella Serratia

Typical Coliform Genera

Citrobacter

Enterobacter
Escherichia

Hafnia
Klebsiella Serratia

Enterobacteriacea

Coliforms

Growth Temperature (-2 to 50C) pH (4.4 to 9) Nutrients Survival (Generally die off at same rate as

fecal pathogens NOT as resistant as viruses) Distribution

E. coli Enterobacter

Traditional Methods
Multiple-Tube Fermentation The technique is based on the most probable number of bacteria present in a sample which produces gas in a series of fermentation tubes with various volumes of diluted sample.

MPN
This statistical estimating technique is based on the

fact that the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to

grow in the tubes in a dilution series.

The MPN is only a statement that there is a 95%

chance that the bacterial population falls within a certain range and that the MPN is statistically the most probable number.

MPN
The original test for coliforms that was used to meet

this definition involved the presumptive, confirmed, and completed tests.

The presumptive step is carried out by means of

tubes inoculated with three different sample volumes

to give an estimate of the most probable number (MPN) of coliforms in the water. The complete process, including the confirmed and completed tests, requires at least 4 days of incubations and

transfers.

MPN table. MPN tables enable us to calculate for a sample the microbial numbers that are statistically likely to lead to such a result. The number of positive tubes is recorded for each set: in the shaded example. 5. 3, and 1. We look up this combination in an MPN table. we find that the MPN index per 100 ml is 110. Statistically, this means that 95% of the water samples that give this result contain 34250 bacteria, with 110 being the most probable number.

Fecal coliform
Unfortunately the coliforms include a wide range of

bacteria whose primary source may not be the intestinal tract.

To deal with this difficulty, tests have been

developed that allow waters to be tested for the

presence of fecal coliforms.
These are coliforms derived from the intestine of

warm-blooded animals, which can grow at the more restrictive temperature of 44.5C.

P-A test
More simplified tests for detecting coliforms and fecal

coliforms are presence-absence test (P-A test)

This is a modification of the MPN procedure, in which a

larger water sample (100 ml) is incubated in a single culture bottle with a triple-strength broth containing lactose broth, lauryl tryptose broth, and bromcresol purple indicator.
The P-A test is based on the assumption that no coliforms

should be present in 100 ml of drinking water. A positive test

results in the production of acid (a yellow color) and constitutes a positive presumptive test requiring confirmation

Defined Substrate test

A water sample of 100 ml is added to a specialized

medium containing o-nitrophenyl--D-galactopyranoside

(ONPG) and 4-methylumbelliferyl- -D-glucuronide

(MUG) as the only nutrients.
If coliforms are present, the medium will turn yellow

within 24 hours at 35C due to the hydrolysis of ONPG, which releases o-nitrophenol.
To check for E. coli, the medium is observed under long-

wavelength UV light for fluorescence.

The Defined Substrate Test. This much simpler test is now being used to detect coliforms and fecal coliforms in single 100 ml water samples. The medium uses ONPG and MUG (see text) as defined substrates. (a) Uninoculated control. (b)Yellow color due to the presence of coliforms. (c) Fluorescent reaction due to the presence of fecal coliforms.

Defined Substrate test

When E. coli is present, the MUG is modified to yield a

fluorescent product.
If the test is negative for the presence of coliforms, the

water is considered acceptable for human consumption.

The main change from previous standards is the

requirement to have waters free of coliforms and fecal coliforms.

If coliforms are present, fecal coliforms or E. coli must

be tested for.

Traditional Methods
Membrane-Filtration suitable for highly dilute samples prone to interference eliminates injured cells

Membrane filtration

Enzymatic Methods
Coliform specific enzymes -Galactosidase (lacZ) total coliforms
Ability to breakdown lactose

-D-Glucuronidase (uidA) fecal coliforms

Ability to breakdown complex sugars

Molecular Methods
Nucleic Acid based techniques Polymerase Chain Reaction (PCR)
phylogenetic primers enzyme specific primers detection limit viability

Molecular Methods
Nucleic Acid based techniques In Situ Hybridization
radioactive vs. fluorescent very high-tech

Metabolites as Indictors
The diamines (cadaverine and putrescine), histamine, and polyamines have been found to be of value for several. Diacetyl indicates the quality of frozen orange juice

concentrates
Ethanol has been suggested as a quality index for canned salmon where 25-74 ppm were associated with "offness," and levels higher than 75 ppm indicated spoilage

Metabolites as Indicators
Ethanol was found to be the most predictive of several alcohols

in fish extracts stored at 5c.

Lactic acid was the most frequently found organic acid in spoiled canned vegetables, and a rapid (2-hour) silica-gel plate method was developed for its detection. The production of trimethylamine (TMA) from trimethylamineN oxide by fish spoilers has been used by a large number of investigators as a quality or spoilage index.

Enterococci
Fecal Material

Plants
Salt-Tolerant

Resistant to Freezing
Few reasons to specify presence of

Enterococci within coliform group

Special Considerations
Vegetables Enterobacter a natural inhabitant Must look at E. coli Meats Aeromonas and psychrotrophic enterics are coliforms Must look at E. coli Cooked Products No Coliforms should be present Pasteurized milk