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5 Basic Principles of

Microbiology

Keith R. Schneider

Email: keiths29@ufl.edu

Tel: 352-294-3010

September 17th, 2015

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5 Overview

• Farmers and processors have

numerous programs that require

testing

– GAPs, HACCP, FSMA

– Primus, GFSI, ISO

• Types of organisms:

– Indicators, E. coli, pathogens

• Tests will you be conducting:

– Plate counts (CFUs),

MPNs, PCR, ELISA, PFGE,

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Why Should People take Care?

Every year foodborne illnesses result in an estimated:

48 million cases of foodborne illness.

325,000 people hospitalized for foodborne illness.

3,000 deaths each year.

Economic losses between 10-83 billion dollars.

Scallan, E., et al. 2011

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The Programs

• GAPs (Good Agricultural Practices)

– Updating to the Produce Safety Rule

• HACCP (Hazard Analysis Critical

Control Point)

– Was only for meat, juice and seafood

– Similar program under FSMA called

Preventive Controls (for Human and

Animal Food)

• FSMA (Food Safety Modernization Act)

– These and many more

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Rule

2015

2016 2017 2018 2019 2020 2021 Aug Oct

Produce

Safety

Preventive

Controls

Human

Preventive

Controls

Animal

Foreign

Supplier

Verification

Program

Final Large Small V. Small Water

FSMA – It‟s Here and it‟s Time to get Ready

Slide provided by Dave Gombas United Fresh

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Rule

2015 2016

2017 2018 2019 Oct Mar May

Intentional

Adulteration

Sanitary

Transportation

3rd Party Cert.

Final Large Small V. Small

FSMA – It‟s Here and it‟s Time to get Ready

Slide provided by Dave Gombas United Fresh

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Microbial Review: The Organisms

There are

three

common

types of

biological

hazards

associated

with food:

Bacteria (E. coli, Salmonella, Shigella, Listeria, etc.)

Viruses (Hepatitis A, Norovirus)

Parasites (Cyclospora, Giardia,Cryptosporidium, etc.)

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Microbes are small, really small…

Can you guess

what this is a

picture of?

http://mimp.mems.cmu.edu/~ordofmag/BACTERIAMAGNIFICATION.htm

A needle

(and bacteria)

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…and microbes are found

everywhere

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Growth of Bacterial Cells

• Bacteria can “growth” or “multiply”

divide every 20 to 30 minutes.

• Thus a single bacteria reach counts

exceeding 1,000,000,000 in 24

hours

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A brief word on indicators:

Coliforms and E. coli

• Sometimes searching for pathogens is difficult,

expensive and time consuming

• Indicators are useful as an assessment of the

overall quality of hygienic conditions present during

production

• Total coliform counts can give an indication of the

sanitary condition of a work surface or product

• E. coli is regarded is the „gold standard‟ for fecal

contamination in water, raw food and environmental

swabs.

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The Tests

• There many micro testing methods

– Bacterial Enumeration

• Serial Dilution

• Plate counts/CFUs

• MPN

– Presence/Absence (though can be used to

estimate counts)

• ELISA

• PCR

– Genetic Finger Printing

• PFGE

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Bacterial Enumeration:

Direct Microscopic Counts

• Method of determining total count of

microorganisms (dead and living)

using a slide that holds a known

volume of culture

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Bacterial Enumeration:

Serial dilutions

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Bacterial Enumeration:

Serial dilutions

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Bacterial Enumeration:

Plate counts/Colony Forming Units (CFUs)

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Bacterial Enumeration:

Most Probably Number/MPN

• Uses a series of dilutions (typically in

groups of 3 or 5)

• Unlike plate counts, MPN do not

provide a direct measurement of the

bacterial count

• MPNs allow for the enumeration of very

low levels of bacteria (less than 10

viable cells per ml or gr)

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MPN (Most Probable Number)

Assume Red tubes are positive

5

3

1

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Take combination of positive tubes and

interpret using a MPN statistical table

110 MPN/100 ml

95% confidence

levels - The true

number of cells lies

within these limits

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SimPlate

Other types of MPNs

Quantitray

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Presence/Absence

Cultural Methods

Colilert

Coliform/E. coli

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Presence/Absence

Antibody-Based Methods

• Uses antibodies that bind specific cellular

structures called antigens

– Unique to a particular microbial group

• Was one of the the most commonly used

techniques for water and food

• Many are commercialized ELISA and

immuno-based tests

– Dynal Beads for Salmonella

– Pathatrix for E. coli O157:H7

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Presence/Absence

Immunological Testing: Enzyme Linked

Immunosobent Assay (ELISA)

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Presence/Absence

Genetic Testing: Polymerase

Chain Reaction (PCR)

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Genetic Fingerprinting:

Pulsed-field gel electrophoresis

(PFGE)

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Take Home Points

• There are many ways to test water,

product and the environment

– Plate counts (CFU) and MPN are the

most common

– Presence/Absence may be appropriate

depending on your needs

• E. coli is regarded as the

„gold standard‟ of indicators

• Use approved/recommended

methods

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Questions

Thanks to D. Gombas and M. Danyluk for letting steal some of their slides