The document discusses methods for examining food and water microbiologically. There are three main methods discussed: culture media methods, immunoassay methods, and polymerase chain reaction (PCR) methods. Culture media methods involve culturing samples on selective and differential media to isolate and identify microorganisms. Immunoassay methods like ELISA use antigen-antibody binding to detect toxins and microbes. PCR methods amplify and detect targeted nucleic acid sequences to identify pathogens. Examining food and water microbiologically is important to ensure safety and determine processing parameters needed to meet standards.

1. Microbiological examination of
food and water
Prepared by
Samira fattah
Assis. Lec.
College of health sciences-HMU
Lecture 11

2. Why we need to examine food and water?
1. to know the level of sanitation used during
handling.
2. to know whether the food / ingredient meet
the normal standard/ guidines.

3. 3. The examination of raw ingredients also
provide information about heat processing
parameters that would be necessary to meet
the microbiological standards.
4. To determine the source / load/ type of
microorganisms in foods and water.

4. Criteria for choice of methods
1. Accuracy
2. Sensitivity
3. Easy of implementation
4. Equipment/ operating cost
5. speed

5. Common Test Methods
• Although a wide range of technologies are used for the
identification and verification of microorganisms, but among
these technologies, three types of method are commercially
popular.
These are:
• Culture Media,
• Immunoassay
• Polymerase chain reaction.

6. Culture Media
• Traditionally, cultural techniques have been the tests of choice
for both ready-to-eat foods and fresh produce.
• However, today immunoassay and PCR methods are more
accepted than cultural methods, because recent
developments of newer testing methods and validation
studies have demonstrated that cultural methods aren't
suitable for all food groups.

7. • The traditional way of detecting and identifying
bacteria from food, or other samples, is based
on:
- Culturing
- Enumeration
- isolation of presumptive colonies for further
identification analysis.
• If necessary, the food sample must be
homogenized, concentrated, and/or
pre-enriched prior to culturing.

8. • Bacterial cells can become injured or viable but
nonculturable (VNC) due to the sublethal stressors, such as:
- Heat
- cold
- acid
- osmotic shock during the food processing steps.
• These bacterial cells still pose a threat in the food industry
and therefore, methods to improve the detection levels of
these injured cells have been developed.

9. The pre-enrichment of the bacteria in a
food sample can be performed by a:
- non-selective
- or selective broth culture
- or by the selective agar overlay
technique to resuscitate the injured cells.

10. • Another manner in which the detection levels of
viable cells can be increased is by concentration of
the food sample by filtration or centrifugation prior
to plating.

11. • The pre-treated food sample can then be plated on:
- non-selective
- selective
- differential media
• Non-selective media or standard methods agar, such as the
aerobic plate count, can be used to detect and count the
amount of bacteria in the sample.

12. • Selective medium contains a compound, such as an antibiotic,
bacteriocin, a growth nutrient, which selectively inhibits the
growth of specific microorganisms.
• Eg: PEMBA medium ( polymyxin pyruvate egg yolk mannitol
bromothymol blue agar ) for the isolation and enumeration of
Bacillus cereus in foods. Its able to detect small numbers of
Bacillus cereus cells and spores in the presence of large
numbers of other food contaminants.

13. • The third type is a differential medium which contains an indicator,
such a chromogenic or fluorogenic substrate, which
differentiates bacteria by various chemical reactions carried out
during growth.
• By incorporating fluorogenic or chromogenic enzyme substrates
into a selective media, identification of microorganisms can be
done directly without further subculturing or biochemical tests.
• These culture media are based on bacteria producing specific and
exact enzymes for substrates.
• As the enzyme then acts with the substrate( fluorogenic or
chromogenic) the bacterial growth will fluoresce or change color,
respectively.

14. A fluorogenic medium used to detect and
enumerate E. coli and coliforms.
Chromogenic Media
for Salmonella detection from food
samples

15. • Even though the culture methods are time consuming (Time to
attain results can range from twelve hours to more than a week)
and laborious.
• the isolation and purification of microorganisms allows for further
subtyping analysis and for storage in culture collections.
• The more conventional methods for further subtyping of bacteria
include the study of the phenotypic characteristics of the
microorganisms.
• These phenotypic methods include:
- Biotyping
- serotyping,
- phage typing .

16. • In biotyping some aspects of bacteria are investigated such
as:
- the biochemical growth requirements.
- environmental conditions (pH, temperature, antibiotic
resistance).
- physiological (colony and cell morphology, cell wall
composition by microscopy and membrane composition such
as by fatty acid analysis)

17. • while serological and phage typing concentrate more on
the surface structure differences of bacteria.
• Phages are not only useful in subtyping bacteria, but also in
detecting pathogens directly from foods .
• However, these phenotypic typing methods are limited
since microorganisms are capable of suddenly altering
their phenotypic characteristics due to environmental
changes or genetic mutations.
• Therefore, identification by genotypic characteristics has
been developed to avoid these problems that can occur
with phenotypic methods.

18. Immunoassay
• Immunoassays are based on the specific bindings between
antigens and antibodies.
• Historically, they were used mainly in clinical diagnostics, but
are becoming popular in rapid detection of microorganisms
from food samples.

19. • Several types of immunoassays have been used in food
sample analysis, including:
- enzyme linked immunosorbent assay (ELISA)
- immunodiffusion tests
- immunofluorescent microscopy
- immunomagnetic separation
- immunoprecipitation.

20. • Currently, a couple of commercial immunoassay-based
detection systems are available to detect toxins or microbes
in the food system, for example:
- the ImmunocardSTAT! E. coli O157:H7detection kit.

21. • Because of its accuracy and automation capability, most
commercial immunoassay kits for food testing are based on
ELISA.
• ELISA usually takes 1 h for toxin and 20-36 hours for cells .
• However, ELISA still has its limitations. For instance, without
enrichment, the sensitivity of ELISA may not be enough to
detect the microorganisms at very low level.
• Therefore, the time required for sample analysis including the
pre-enrichment and/or enrichment procedures could be
much longer than ELISA itself.

22. Polymerase Chain Reaction (PCR)
• Polymerase chain reaction is the amplification of a nucleic
acid target sequence.
• The targeted sequences can be a specific gene or repetitive
areas in the sequence .
• For foodborne bacterial pathogens, commonly targeted DNA
areas are :
- virulence factors
- Toxins
- cellular metabolites
- multicopy ribosomal RNA.

23. • The PCR-based techniques have also been developed for
screening of genetically modified organisms and their
derived materials in foods .
• Post- PCR detection methods vary from gel electrophoresis
and usage of specific nucleic acid probes. In some cases,
probes simplify the detection of the PCR product, in the
similar way as gel electrophoresis.

24. • some PCR use DNA primers and
fluorescent probes specific to the
target organism in the same
reaction mix.
•Detection of a fluorescent signal
for the targeted amplified
sequences indicates the presence
of the pathogen in the sample
being tested.