Identification and detection of

microorganisms in foods/water/ milk

• A major problem in maintaining food safety is the need to rapidly detect
microorganisms in order to curb outbreaks that can affect large
populations.

• Standard culture techniques may require days to weeks for positive

identification of pathogens.

• Fluorescent antibody, enzyme-linked immunoassays (ELISAs), and

radioimmunoassay techniques have proven of value. These can be used to
detect small amounts of pathogen-specific antigens.

• Molecular techniques also are increasingly used in identification. These

methods are valuable for three purposes:
(1) to detect the presence of a single, specific pathogen;
(2) to detect viruses that cannot be grown conveniently, and
(3) to identify slow-growing or noncultured pathogens.
• Pathogens now can be identified by detecting specific DNA or RNA base
sequences with probes.

• Another example of the use of molecular techniques is provided by

pathogenic E. coli. Currently E. coli O157:H7 is isolated and identified
using selective culture media, rapid identification kits rapid probe-based
identification procedures,serotype-specific probes, and PCR techniques.

• These techniques also have been extended to allow the detection of a few
target cells in large populations of background microorganisms. For
example, by using the polymerase chain reaction, as few as 10 toxin-
producing E. coli cells can be detected in a population of 100,000 cells
isolated from soft-cheese samples.
 Testing Methods
Total heterotrophic
Growth on detection
plate count
media
Most Probable
Number
Culture-dependent techniques(Microbiological methods)

Sample
with Microbes

Culture-independent techniques(Molecular method)

Optical Nucleic Acid-Based

Biochemical tests Immunological

Methods
Culture-dependent techniques(Microbiological methods)

Total heterotrophic plate count

Growth on detection media

Most Probable Number

 (Total) Heterotrophic Count (TC)
General (all purpose): contains basic nutrients for
most bacteria.

• Heterotrophs are a group of microorganisms (bacteria, moulds and yeasts)

that use organic carbon sources to grow and can be found in all types of
water.

• In fact, the majority of bacteria found in drinking water systems are

considered heterotrophs.

• Heterotrophic plate count (HPC) is a method that measures colony formation

on culture media of heterotrophic bacteria in drinking water.

• Total Bacterial Count (TBC), also known as Heterotrophic Plate Count

(HPC), Heterotrophic Colony Count (HCC), Aerobic Plate Count
(APC), Total Plate Count (TPC), or Standard Plate Count (SPC), represents
the total bacterial load in a given sample.
• The cheapest and easiest technique of all.

• The TBC tests could reflect the general hygiene condition of a

sample.

• Detects only bacteria which grow fast (48 hrs at 30-37oC) on

common rich media
• won’t detect anaerobes
• won’t detect bacteria with specialized growth requirements
(e.g. Campylobacter, Listeria)
• not a Coliform test
There are 3 methods used for routine testing of heterotrophic bacteria:

1. Pour plate method

2. Spread plate method

3. Membrane filtration method

Selective medium for Pathogenic Bacteria Detection
 Detection Media

• Hundreds of different formulations available.

• Enrichment medium : extra growth factors & nutrients

allow growth of certain bacteria
– Elective enrichment (unique combination of nutrients or
physiological conditions)
– Selective enrichment (inhibitors, antibiotics)

• Isolation medium (contains nutrients for specific microbes)

• Differential medium (contains dyes/stains/indicators)

 Detection Media
• Advantages:
– Cheap.
(Molecular techniques > Detection Media> Total Heterotrophic Count$)
– Easy. Suitable for Field Testing
– OK for surveys
– Fairly informative
– Antibiotic resistance tests may detect microbes of
particular concern
 Detection Media
• Disadvantages:

– Time consuming (at least 24 hrs, more for Listeria,

Campylobacter).

– A single medium won’t support growth of all potential

problem bacteria. Not as good for surveys as THPC .

– Pre-enrichment may be required

• for dilute samples
• for complex samples (e.g. sludge, soil, bedding)
 Selective and Differential media
Selective media favor the growth of particular microorganisms .Bile salts or dyes
like basic fuchsin and crystal violet favor the growth of gram-negative bacteria by
inhibiting the growth of gram-positive bacteria without affecting gram-negative
organisms. Endo agar, eosin methylene blue agar, and MacConkey agar

Selective media allow certain types of organisms to grow, and inhibit the growth
of other organisms.

For example, organisms that can utilize a given sugar are easily screened by
making that sugar the only carbon source in the medium.

Differential media are media that distinguish between different groups of bacteria
and even permit tentative identification of microorganisms based on their
biological characteristics.
• On the other hand, selective inhibition of some types of
microorganisms can be achieved by adding dyes, antibiotics,
salts or specific inhibitors which affect the metabolism or
enzyme systems of the organisms.
EOSIN METHYLENE BLUE AGAR (EMB agar)

• EMB is an undefined selective/differential medium. It

contains aniline dyes (methylene blue and eosin), which
inhibit the growth of Gram-positive bacteria selecting for
Gram-negative bacteria.

• EMB also contains lactose which makes the media

differential based on an organisms ability to ferment
lactose.

• Sucrose is also included in the medium because certain

members of the Enterobacteria or coliform group ferment
sucrose more readily than they ferment lactose. These
sugars provide favorable conditions for the growth of
fecal coliforms.
• Lactose and sucrose
fermenters will grow as dark
colonies accompanied by a
metallic green sheen (E.
coli).

• Organisms that slowly

ferment lactose will appear
as pink colonies
(Enterobacter aerogenes are
usually mucoid and much
larger than colonies of E.
coli).
 EMB agar

Non-fermenters lactose or sucrose will

remain colorless or take on the color of the
medium such as Salmonella (one of the
causative agents of food poisoning).
Note: This media is used to confirm the
presence of E. coli in water samples
contaminated with sewage or fecal
material.
You will use this agar again with in the
water sampling experiment to differentiate
between E. coli and Enterobacter spp.
 Mannitol Salt Agar (MSA) for pathogenic
Staphylococcus aureus
Mannitol salt agar is a selective medium used for the
isolation of pathogenic staphylococci.
• The medium contains mannitol, a phenol red indicator,
and 7.5% sodium chloride.
• Ferment mannitol (differential):
• Phenol red = pH indicator
• Pink : pH >7.4 (basic)
• Orange: pH = 7 (neutral)
• Yellow: pH <6.8 (acidic)
• Selective: The high salt concentration inhibits the
growth of most bacteria other than staphylococci.

• Note: The high salt content does not kill Gram negative bacteria, it
just inhibits growth.

Mohammed laqqan
On MSA, pathogenic Staphylococcus aureus
produces small colonies surrounded by yellow
zones. The reason for this change in color is
that S. aureus ferments the mannitol,
producing an acid, which, in turn, changes the
indicator from red to yellow.
Other Staphylococcus don’t ferment mannitol
don’t produce a color change from the normal
red-pink color of the medium.
The growth of other types of bacteria is generally
inhibited. S. epidermidis
M. luteus

1. S. aureus = salt tolerant; ferments mannitol

(yellow)
2. S. epidermidis = salt tolerant; does NOT
ferment (red)
3. M. luteus = not salt tolerant; does NOT grow
on mannitol
S. aureus

Mohammed laqqan
 Problems in Culture Methods for Bacterial
Pathogens
• Inefficient growth (low plating efficiency),
• Slow growth rates
• Overgrowth by other non-target bacteria.

Efforts to improve culture and reduce or eliminate non-target

bacteria:
• antibiotics
• physical (heat) treatments
• chemical (acid) treatments
• specialized plating: e.g., dual media plating
 Colilert
Add your sample into the bottle with the buffers+
substrates

Pour into the plate. Seal ⎯

Incubate

yellow = total coliforms,

fluorescent = E.coli

http://www.idexx.com/water/colilert18/index.jsp
Colilert®
Coliform/E. coli Results in 24 Hours
IDEXX Colilert reagent is used around the world for the
detection of coliforms and E. coli in water. Colilert, with its
patented Defined Substrate Technology® (DST®), is used in
over 90% of all U.S. State labs. Colilert is, in fact, used more
than all other methods combined (U.S., Canada and Japan
drinking water markets).
 ONPG Positive Reaction
Colilert & Colilert-18

Coliforms have an enzyme galactosidase and if present will eat the

glactopyranoside (sugar molecule) of the ONPG. o-nitrophenyl is then
hydrolzed and yields a yellow color indicating the presence of coliforms.
 MUG Positive Reaction
Colilert & Colilert-18

E.coli has a specific enzyme glucuronidase and will start to eat the sugar
part (glucuronide) of the MUG. When this happens, the 4-methylumbelliferyl
is hydrolized and under a UV light, will fluoresce.
 Multiple-Tube Fermentation Technique,
Total Coliform Procedure, SM 9221B
• Presumptive medium – LTB
• Incubation
– 35 ± 0.5oC
– 24 ± 2 hours, examine and re-incubate negative tubes
• Use brilliant green lactose bile broth
(BGBB) for confirmed phase
• Incubation
– 35 ± 0.5oC
– 24 ± 2 hours
• Use EC (for FC) or EC-MUG for E.coli
• Incubation
– 44.5 ± 0.2oC
– 24 ± 2 hours
• Calculate results using SM Table 9221.IV.
Coliform Test
 Most Probable Number
• Advantages:
– simple. Cheap.
– suitable for field tests
– can be combined with detection media to better ID
the organisms
• for enterics:
 Most Probable Number
• Disadvantages:
– sterile technique is crucial under field
conditions
– when bacteria aggregate, MPN is wildly
inaccurate. Vibrio, Pseudomonas, older or
stressed cultures aggregate easily
 Optical
Microscopic Morphology of cell

 shape,

 size,

 Gram stain,

 special structures e.g. endospores, and capsule

can be used to give an initial putative identification.

Biochemical Tests
Biochemical Tests (1)
Biochemical Tests (1)
Confirmatory biochemical tests

Acid production from sugar

Catalase production (Staphylococcus epidermidis)

Coagulase test (Staphylococcus aureus)

Gram reaction (Staphylococcus epidermidis)

Haemolysis (Listeria monocytogenes)

Hydrogen sulphide test (Pseudomonas aeruginosa)

Indole test (E. Coli)

Oxidase test (Aeromonas hydrophila)

Urease test (Proteus rettgeri)

Methyl Red test (E. Coli)

 Immunological Methods

• Immuno-assays are based on a quantitative reaction of an antigen (bacterial

metabolite, e.g., toxin) with its antibody.

• Therefore, they are suited for detection of microorganisms based on their

production of specific antigens and for quantitative detection of bacterial
toxins.
• The ELISA has been used as a diagnostic tool.

• Antigens from the sample are attached to a surface. Then, a further specific
antibody is applied over the surface so it can bind to the antigen.

• This antibody is linked to an enzyme, and, in the final step, a substance containing
the enzyme's substrate is added.

• The subsequent reaction produces a detectable signal, most commonly a color

change in the substrate.
Different Immunological methods:

 Enzyme-linked immuno sorbent assays (ELISA)

 Direct and Indirect ELISA, Sandwich ELISA,

 Immunoblot /western blot

 Immunoprecipitation

These can be used to detect small amounts of pathogen-specific

antigens.
 MOLECULAR
IDENTIFICATION:
 Nucleic Acid-Based
 Polymerase Chain Reaction (PCR)-Based Pathogen
Detection.
PCR is an exponentially progressing synthesis of the defined target
DNA sequences in vitro.
It was invented in 1983 by Dr. Kary Mullis, for which he received the
Nobel Prize in Chemistry in 1993.

Real time PCR

 The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the
sequence to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA

building blocks.
4) Thermostable DNA Polymerase - enzyme
that catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic
strength of the reaction solution suitable for
the activity of the enzyme
 The Reaction

PCR tube THERMOCYCLER

 Denature (heat to
95oC)

Lower temperature to
56oC Anneal with primers

Increase temperature to
72oC DNA polymerase +
dNTPs
 Target Microorganisms for
Molecular-Based Testing
• Those that are difficult or time-consuming to
isolate
– e.g., Mycobacteria

• Hazardous organisms
– e.g., Histoplasma, Coccidiodes

• High-volume tests
– e.g., S. pyogenes, N. gonorrhoeae, C. trachomatis
 Applications of Molecular Based
Testing in Food Microbiology
• Rapid or high-throughput identification of
microorganisms
• Detection and analysis of resistance genes
• Genotyping
• Classification
 Specimen Collection

• Preserve viability/nucleic acid integrity of target

microorganisms
• Avoid contamination
• Appropriate time and site of collection
• Use proper equipment (coagulant, wood, or
plastic swab shafts)
• Commercial collection kits are available
• The Clinical and Laboratory Standards Institute
(CLSI) has guidelines for proper specimen
handling
 Sample Preparation
• Consider the specimen type
• More rigorous lysis procedures are required to
penetrate cell walls
• Consider the number of organisms in the sample
• Inactivate RNases
• Inactivate DNases
PCR Detection of Microorganisms:
Quality Control
• PCR and other amplification methods are
extremely sensitive and very specific. For
accurate test interpretation, use proper
controls.
– Positive control: positive template
– Negative control: negative template
– Amplification control: omnipresent
template unrelated to target
– Reagent blank: no template present