Public health microbiology for medical laboratory technologists

Laboratory method for detecting microbes and their products in food

1. Chapter 5
Laboratory methods for detecting
microbes & their products in food
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2. •Ensuring the microbiological safety and quality
of food requires various laboratory techniques
that can detect pathogenic bacteria, viruses,
fungi, parasites, and their toxins or metabolic
products.
•The methods employed vary from traditional
culture techniques to modern molecular and
immunological approaches.
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3. Importance of Detecting Microbes in Food
Food safety: Prevent foodborne illnesses.
Food spoilage prevention: Extend shelf life.
Regulatory compliance: Meet standards for
safe food distribution.
Quality control: Maintain food quality and
consistency.
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4. I. Microscopic Methods
•Microscopic examination involves direct observation of food
samples for the presence of microbes.
1. Direct Microscopic Count (DMC)

Principle: The DMC consists of making smears of food specimens or
cultures onto a microscope slide, staining with an appropriate dye, and
viewing and counting cells with the aid of a microscope (oil immersion
objective).

Strengths: simple and rapid visualization of microorganisms.

Limitations: Cannot differentiate between viable and non-viable
cells, some cells do not take the stain well and may not be
counted, labor-intensive, and requires trained personnel.
•There are three main types of direct microscopic count
methods:
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5. DMC…cont’ed
A. Breed Count:
•It is developed by R.S. Breed (Breed count). The sample is
spread (about 0.0 1 ml) over 1 cm
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of a microscope slide.
•Milk smear is dried and stained with Newman Lampert
stain; methylene blue milk smear stain.
•This stain fixes the smear, dissolves fat globules and stains
bacteria with methylene blue.
•Slide is then observed under several oil immersion
microscopic fields.
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6. DMC…cont’ed
B. Slide Method Using INT:
•A slide method to detect and enumerate viable cells have
been developed.
 
The method was found to be effective for pure cultures of
bacteria and yeasts.
Note: (INT) p-iodophenyl-3-p-nitrophenyl-5-phenyl
tetrazolium chloride
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7. DMC…cont’ed
C. Howard Mold Counts:
•This method involves the detection of fungi
especially molds.
•The method requires the use of a special chamber
(slide) designed to enumerate mold mycelia.
•By this method we can identify almost all molds
which are responsible for the spoilage of fruits and
vegetables.
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8. 2. Fluorescence Microscopy
•Principle: Fluorescent dyes or tagged antibodies
specific to microbes are used to label microorganisms,
allowing for visualization under a fluorescence
microscope.
•Strengths: High specificity and can detect specific
microorganisms.
•Limitations: Expensive and requires skilled personnel.
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9. II. Culture-Based Methods
Culture characteristics
•A wide variety of media are available for the growth of
microorganisms in the laboratory.
•To support microbial growth, a medium must provide an
energy source, as well as sources of carbon, nitrogen, sulfur,
phosphorus, and any organic growth factors the organism is
unable to synthesize.
•Most of these media, which are available from commercial
sources, have premixed components and require only the
addition of water and then sterilization.
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10. Culture-Based Methods…cont’ed
a. Plate Counting

Principle: Plate counting is used to estimate the number of
viable bacteria in a food sample. The sample is serially diluted
and spread on agar plates. After incubation, colonies are counted.

Types:
o
Total Plate Count (TPC): Measures total bacterial count.
o
Selective Plate Count: Uses selective media to target specific microbes
(e.g., XLD agar for Salmonella).

Strengths: Inexpensive, simple, and provides an estimate of
viable organisms.

Limitations: Time-consuming (24-72 hours) and may
underestimate non-culturable organisms.
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11. b. Most Probable Number (MPN)

Purpose: Quantitative estimation of microbes in low microbial
load samples (e.g., water or liquid food).

Principle: Involves serial dilution and incubation of the sample
in broth tubes. Based on the number of positive tubes, the most
probable number of organisms is estimated statistically.

Advantages: Suitable for estimating low levels of microbes.

Limitations: Less precise than direct plating, labor-intensive.
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12. c. Enrichment Cultures

Principle: Used when pathogens are present in very
low numbers. Samples are incubated in selective broth
to increase the number of target microbes before plating
on selective agar.

Strengths: Increases sensitivity for low levels of
contamination.

Limitations: Time-consuming and may require
multiple steps.
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13. III. Molecular Methods
a. Polymerase Chain Reaction (PCR)

Principle: PCR amplifies specific DNA or RNA sequences from
microorganisms present in the food sample. It is often used for
detecting pathogens such as Listeria monocytogenes, Salmonella,
and E. coli.

Types:
o
Conventional PCR: Detects specific gene sequences.
o
Real-time PCR (qPCR): Monitors amplification in real-time and
quantifies the microbial load.

Strengths: High sensitivity, specificity, and rapid results (within
hours).

Limitations: Expensive, requires specialized equipment, and only
detects known organisms.
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14. b. DNA Microarrays

Principle: DNA microarrays detect thousands of specific DNA
sequences simultaneously. Microbial DNA or RNA is
hybridized to an array containing probes for different microbial
genes.

Strengths: High rate detection and identification of multiple
microbes in a single assay.

Limitations: Complex, expensive, and requires expertise in
bioinformatics.
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15. c. Next-Generation Sequencing (NGS)

Principle: NGS provides a comprehensive analysis of the entire
microbial community (microbiome) in food. It sequences all
DNA present, allowing identification of both culturable and
non-culturable organisms.

Strengths: High sensitivity, can identify unknown and non-
culturable microbes.

Limitations: Expensive, complex data analysis, and longer
processing time.
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16. IV. Immunological Methods
a. Enzyme-Linked Immunosorbent Assay (ELISA)

Principle: ELISA detects specific antigens or toxins produced
by microbes using antibodies linked to an enzyme. The reaction
produces a measurable color change.

Types:
o
Direct ELISA: Detects antigens directly.
o
Indirect ELISA: Detects antibodies in response to infection.

Strengths: High specificity and sensitivity, rapid results.

Limitations: Requires specific antibodies, and cross-reactivity
may occur.
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17. b. Immunofluorescence

Principle: Antibodies tagged with fluorescent dyes
bind to specific microbial antigens, which are then
visualized using fluorescence microscopy.

Strengths: High specificity for pathogen detection.

Limitations: Requires specialized equipment and
reagents.
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18. c. Lateral Flow Immunoassays

Principle: Portable devices using antibodies to detect
specific antigens or toxins in food. Examples include
rapid tests for Listeria, Salmonella, and toxins like
aflatoxin.

Strengths: Simple, rapid, and suitable for field testing.

Limitations: Limited sensitivity compared to lab-based
methods.
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19. V. Chemical Methods for Microbial Metabolite Detection
•Microbiologists distinguish many bacteria that are similar
in microscopic appearances and growth characteristics on the
basis of differences in their ability to utilize or produce certain
chemicals as well as the isolate’s ability to grow or
survive in the presence of certain inhibitors (e.g., salts, toxins,
and antibiotics).
•Biochemical tests include procedures that determine an organism’s ability
to
Produce different enzymes
Utilize various substrates, such as specific amino acids, citrate, and gelatin
ferment various carbohydrates
produce waste products, such as hydrogen sulfide (H2S)
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20. V. Chemical Methods … cont’ed
a. High-Performance Liquid Chromatography (HPLC)

Principle: HPLC separates and quantifies microbial toxins or
metabolic products in food (e.g., mycotoxins, histamines).

Strengths: High sensitivity and accuracy for detecting
microbial toxins.

Limitations: Expensive and requires skilled personnel.
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21. Chemical Methods … cont’ed
b. Gas Chromatography-Mass Spectrometry (GC-MS)

Principle: GC-MS separates volatile microbial metabolites,
followed by identification using mass spectrometry. It is useful
for detecting compounds such as biogenic amines, toxins, and
volatile spoilage compounds.

Strengths: High sensitivity and specificity.

Limitations: Complex, expensive, and requires technical
expertise.
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22. Chemical Methods … cont’ed
c. Catalase Test
•Catalase acts as a catalyst in the breakdown of
hydrogen peroxide to oxygen and water.
•An organism is tested for catalase production by
bringing it into contact with hydrogen peroxide.
•Bubbles of oxygen are released if the organism is a
catalase producer.
•The culture should not be more than 24 hours old.
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23. Chemical Methods … cont’ed
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Catalase tube test
Right: Shows a positive test.
Left: Shows a negative test.

24. Chemical Methods … cont’ed
d. Oxidase test
•The oxidase test is used to assist in the identification of
Pseudomonas, Vibrio, Brucella, and Pasteurella species, all
of which produce the enzyme cytochrome oxidase
•Testing for the presence of oxidase can be performed by
rubbing bacterial colonies onto filter paper impregnated
with the tetramethyl-p-phenylenediamine dihydrochloride
(TPD)
•A positive reaction is indicated by the development of a
purple color.
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25. Chemical Methods … cont’ed
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26. Chemical Methods … cont’ed
e. Urease test
•This test is used to determine an organism’s ability to
produce the enzyme urease, which hydrolyzes urea.
•Hydrolysis of urea produces ammonia and CO2.
•The formation of ammonia alkalinizes the medium, and
the pH shift is detected by the color change of phenol
red from light orange at pH 6.8 to magenta (pink) at pH
8.1.
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27. Chemical Methods … cont’ed
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28. Chemical Methods … cont’ed
f. Citrate utilization
•An agar medium that contains sodium citrate as the
sole carbon source may be used to determine ability to
use citrate.
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29. Chemical Methods … cont’ed
g. Indole reaction tests
•It test the ability of the organism to produce indole, a
benzopyrrole, from tryptophan.
•Indole is detected by the formation of a red dye after
addition of a benzaldehyde (Kovac’s reagent) reagent.
•Positive test is indicated by a pink ring.
–Positive indole test – pink ring
–Negative indole test - yellow ring
•Indole positive – E.coli
•Indole negative – Salmonella
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30. Chemical Methods … cont’ed
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31. VI. Rapid Detection Kits

Principle: These kits provide simplified versions of lab-based
assays for detecting specific microbes or toxins. They often
combine molecular or immunological methods.

Examples: Rapid tests for E. coli O157, Salmonella, and
Listeria.

Strengths: Simple, fast (results within minutes to hours), and
user-friendly.

Limitations: Limited sensitivity and specificity compared to
laboratory methods.
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32. VII. Biosensor-Based Methods
a. Optical Biosensors

Principle: These sensors detect changes in optical properties
(e.g., fluorescence, absorbance) when target microbes or toxins
interact with specific probes (e.g., antibodies or nucleic acids).

Strengths: Highly sensitive, fast detection.

Limitations: High cost and limited to specific targets.
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33. b. Electrochemical Biosensors

Principle: These devices measure electrical changes when target
molecules interact with specific probes (e.g., antibodies,
enzymes).

Strengths: Portable, rapid, and suitable for on-site testing.

Limitations: Limited sensitivity for complex food matrices.
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