The detection and enumeration of microorganisms in food are an essential
part of any quality control or food safety plan. Traditional methods of detecting foodborne pathogenic bacteria are often time-consuming because of the need for growth
in culture media, followed by isolation, biochemical and/or serological identifi cation,
and in some cases, subspecifi c characterization. Advances in technology have made
detection and identifi cation faster, more sensitive, more specifi c, and more convenient than traditional assays. These new methods include for the most part antibodyand DNA-based tests, and modifi cations of conventional tests made to speed up
analysis and reduce handling.
3. Introduction
o The detection and enumeration of pathogens in food and on surfaces that
come into contact with food are an important component of any
integrated program to ensure the safety of foods throughout the food
supply chain.
o Traditional methods of detecting foodborne pathogenic bacteria are often
time-consuming because of the need for growth in culture media,
followed by isolation, biochemical and/or serological identification, and
in some cases, sub-specific characterization.
o Advances in technology have made detection and identification faster,
more sensitive, more specific, and more convenient than traditional
assays.
4. Importance
It is important to determine the safety and quality of food.
Rapid detection methods are important, particularly in food
industry, as they are able to detect the presence of pathogens
in raw and processed foods immediately.
Rapid methods are also sensitive enough to detect pathogens
that present in low numbers in the food.
6. 1. Culture, Microscopic and Sampling methods
The examination of foods for the presence, types and numbers of microorganisms and
their products is basic to food microbiology. In spite of the importance of this, none of
the methods in common use permits the determination of exact numbers of
microorganisms in a food product.
The four basic methods employed for “total” numbers are as follow:
a. Standard plate counts (SPC) or Aerobic plate counts (APC) for viable cells or colony
forming units.
b. The most probable numbers (MPN) method as a statistical determination of the viable
cells.
c. Direct microscopic counts (DMC) for both viable and non-viable cells.
d. Dye reduction techniques to estimate numbers of viable cells that possess reducing
capacities.
7. Conventional Standard Plate Count
By the conventional SPC method:
Portions of food samples are blended or homogenized
Serially diluted in an appropriate diluents
Plated in or onto a suitable agar medium
Incubated at an appropriate temperatures for a given time
After that all visible colonies are counted by use of a Quebec or electronic
counter
This SPC is far the most widely used method for determining the numbers of
viable cells or colony-forming-units (cfu) in a food products.
8.
9. When total viable counts are reported for a product, the counts/numbers
should be viewed as a function of at least some of the following factors:
Sampling methods employed
Nature of the food biota
Nature of the food material
The pre-examination history of the food product
Nutritional adequacy of the plating medium employed
Incubation temperature and time used
pH, water activity and oxidation-reduction potential of the plating medium
Type of diluents used
Relative number of organisms in the food
Existence of other competing or antagonistic organisms
10. The spiral plater
The spiral plater is a mechanical device that distributes the liquid inoculums
on the surface of a rotating plate containing a suitable poured and hardened
agar medium.
The dispensing arm moves from the near centre of the plate toward the
outside, depositing the sample in an Archimedes spiral.
The attached special syringes dispense a continuously decreasing volume of
a sample so that a concentration range of up to 10,000:1 is effected on a
single plate.
Following incubation at an appropriate temperature, colony development
reveals a higher density of deposited cells near the centre of the plate, with
progressively fewer toward the edge.
12. Cont…
Advantages:
• Less agar is used
• Fewer plates, dilution banks and pipettes are required
• Three to four times more samples per hour can be examined
Disadvantage:
• Food particles may cause the blocking in the dispensing stylus.
13. Membrane filters
• Membranes with a pore size that will retain bacteria but allow water or
diluents to pass are used.
• Following the collection of bacteria upon filtering a given volume, the
membrane is placed on an agar plate or an absorbent pad saturated with the
culture medium of choice and incubated appropriately.
• Following growth, the colonies are enumerated
Cellulose filters were among the earliest used, however,
polycarbonate Nucleopore filters offer the advantage of retaining
all bacteria on the filter.
14.
15. Direct Microscopic Count (DMC)
In this method:
The organisms are collected on the membranes are viewed and
counted microscopically following appropriate staining,
washing and treatment of the membrane to render it
transparent.
These methods are especially suited for samples that contain
low number of bacteria.
16.
17. Direct Epifluorescent Filter Technique (DEFT)
• This technique is viewed as an improved modification of the
basic method.
• Employs fluorescent dye and fluorescent microscopy.
• DEFT has been employed successfully to estimate numbers of
microorganisms on meat, poultry and on food contact surfaces.
18. Cont..
A diluted food homogenate is filtered through a 5-µm nylon filter
Filtrate is collected and treated with 2 ml of Triton X-100 and 0.5 ml of
trypsin
Reagents are used to lyse the somatic cells and to prevent clogging of filters
After incubation, the treated filtrate is passed through a 0.6 µm Nucleopore
polycarbonate membrane
Filter is stained with acridine orange
Stained cells are enumerated by epifluorescent microscopy
Number of cells per gram is calculated by multiplying the average number per
field by the microscope factor
Results can be obtained in 25-30 minutes and the numbers as low as around
6,000 cfu/g can be obtained from meats and milk products.
19.
20. Microcolony- DEFT
DEFT allows for the direct microscopic determination of cells whereas
microcolony-DEFT is variation that allows one to determine viable
cells only.
Food homogenates are filtered through DEFT membranes
Placed on the surface of an appropriate culture media
Incubated for microcolony development
3-hour incubation can be used for Gram-negative bacteria
6-hour incubation is used for Gram-positive bacteria
Developed microcolonies viewed with microscope
For coliforms, pseudomonas and staphylococci, as few as 103/g could be
detected within 8 hours.
21. Hydrophobic Grid Membrane Filters (HGMF)
o The method employs a specially constructed
filter that consists of 1600 wax grids on a single
membrane filter that restricts growth and colony
size of individual grids.
o On one filter, from 10-9 x 104 cells can be
enumerated by an MPN procedure and
enumeration can be automated.
o Method can detect as few as 10 cells per gram
and results can achieved in 24 hours or so.
o It can be used to enumerate all cfus or specific
groups such as indicator organisms, fungi,
salmonella and yeast and molds.
For use:
1 ml of 1:10 homogenate is filtered
through a filter membrane
Place membranes on suitable agar
medium
Incubate overnight to allow colonies
to develop
Grids that contain colonies are
enumerated and the MPN is
calculated
22. Microscopic colony count
1.
Spreading 0.1 ml of milk-agar
mixture over 4-cm2 area on a
glass slide
Incubation, drying and staining
Microcolonies developed with the
aid of a microscope
2.
2 ml of melted agar are mixed
with 2 ml of warmed milk
After mixing, 0.1 ml of inoculated
agar is spread over a 4-cm2
area
Staining with thionin blue
Slide is viewed with the 16-mm
objective of a wide-field
microscopic
This method involve the counting of micro-colonies that develop in agar
layered over microscopic slides
23. Agar droplets method
The food homogenate is diluted in tubes of melted agar (45°C)
For each food sample, three tubes of agar are used
First tube being inoculated with 1 ml of food homogenate
After mixing, a sterile capillary pipette (delivering 0.033 ml/drop) is used to transfer a line of 5 x 0.1 ml
droplets to the bottom of an empty petri dish
With the same capillary pipette, three drops (0.1 ml) from the first 9 ml tube are transferred to the second
tube
After mixing, another line of 5 X 0.1 ml droplets is placed next to the first
This step is repeated for third tube
Petri plates containing agar droplets are incubated for 24 hrs
Colonies are enumerated with the aid of a 10x viewer
Method is three time faster and 24 hours incubation gave counts equal to those obtained after 48 hours
by the conventional plate count. Dilution blanks are not required and only one Petri dish per sample is
needed.
24.
25. Dry film and related methods
• A rehydrated dry film method consisting of two plastic films attached together
on one side and coated with culture medium ingredients and a cold-water-
soluble jelling agent.
• The method can be used with non-selective ingredients to make aerobic plate
counts (APC) and with selective ingredients, certain specific groups can be
detected.
For use:
1 ml of diluent is placed between the two films
Spread over the nutrient area by pressing with a special flat-surface device
Incubation
Micro-colonies appear red on the non-selective film because of the presence of a
tetrazolium dye in the nutrient phase
26.
27. Cont…
Redigel plating
o It is a plating medium that does
not use agar as a solidifying
agent .
o It is employed by inoculating pre-
sterilized ingredients with food
homogenates by mixing and
holding to allow for solidification
which occurs in about 30 minutes.
o It is attractive for enumerating
psychrotrophic organisms.
Sim plating
o It is a culture method based on
the activity of several enzymes
common to many food born
organisms.
o The special plates have holes
or wells (in two sizes-84 and
198 incubation wells).
o It does not allow for the
characterization of colony
features.
28. Most Probable Number
o In this method, dilutions of food samples are prepared as for the SPC
o Three serial aliquots or dilutions are then planted into 9 or 15 tubes of
appropriate medium for the three or five-tube method, respectively
o Numbers of organisms in original sample are determined by use of standard
MPN tables
o The method is statistical in nature and MPN results are generally higher than
SPC results.
Advantages include:
Relatively simple
Specific groups of organisms can be determined by use of appropriate
selective and differential media
It is method of choice for determining fecal coliform densities
Disadvantages:
Large volume of glassware required
Lack of opportunity to see the morphology of the colonized organism
Lack of precision
29. Presence of acids and carbon dioxide indicates the positive MPN test
30.
31. Dye Reduction
Two dyes are commonly employed in this procedure to estimate the number of
viable organisms in suitable products:
Methylene blue
Resazurin
To conduct a dye-reduction test, properly prepared supernatants of foods are
added to standard solutions of dye for reduction:
From blue to white for methylene blue
From slate blue to pink or white for resazurin
The time for dye reduction to occur is inversely proportional to the number of
organisms in the sample.
Advantages includes that they are simple, rapid, inexpensive and only viable cells
actively reduce the dyes.
Disadvantages are that not all organisms reduce the dyes equally and they are not
applicable to food organisms that contain reductive enzymes unless special steps
are employed.
32. Microbiological examination of surfaces
The most commonly used methods for surface assessment in food
operations are presented below:
1. Swab/swab-rinse method
2. Contact plate
3. Agar Syringe/Agar Sausage method
Other surface methods are:
Direct surface
Sticky film
Swab/Agar slant
Ultrasonic devices
Spray Gun
33. Swab/Swab-Rinse methods
o It is the oldest and most widely used method
o Not only in the food and dairy industries but also in hospitals and restaurants
Two types of swabs can be used:
Cotton swab – when used, the organisms must be dislodged from the fibers
Calcium alginates – when used, the organisms are released in the diluent upon
dissolution of the alginates by sodium hexametaphosphate
o The organisms in the diluent are enumerated by a suitable method such as
SPC but any of the culture media may be used to test specifically for given
groups of organisms.
In an innovation in the swab-rinse method presented by Koller:-
1.5 ml of fluid is added to a flat surface, swabbed for 15 seconds over a 3-cm2
area and volumes of 0.1-0.5 ml collected in microliter pipette.
The fluid may be surface or pour plated using plate count or selective media.
34. Contact plate
o The replicate organisms direct agar contact (RODAC) method employs special
Petri plates which was poured with 15.5-16.5 ml of an appropriate plating
medium, resulting in a raising agar surface.
o When the plate is inverted, the hardened agar makes direct contact with the
surface.
o When surfaces are examined that have been cleaned with certain detergents, it
is necessary to include a neutralizer in the medium.
Drawback :-
The covering of the agar surface by spreading colonies and its ineffectiveness
for heavily contaminated surfaces.
These can be minimized by using plates with dried agar surfaces and by using
selective media.
35. Agar Syringe/Agar Sausage Method
o By this method, 100 ml syringe is modified by removing the
needle end to create a hollow cylinder that is filled with
agar.
o A layer of agar is pushed beyond the end of the barrel by
means of the plunger and pressed against the surface to be
examined.
o The exposed layer is cut off and placed in a Petri dish,
followed by incubation and colony enumeration.
37. Other Surface Methods
Direct Surface:- In this method, melted agar is poured onto the surface or utensils to be
assessed. Upon hardening, the agar mold is placed in a Petri dish and incubated.
Successfully used to determine the survival of Closteridium sporogenes ensospores on
stainless steel surfaces.
Direct Surface:- In this method, melted agar is poured onto the surface or utensils to be
assessed. Upon hardening, the agar mold is placed in a Petri dish and incubated.
Successfully used to determine the survival of Closteridium sporogenes ensospores on
stainless steel surfaces.
Swab/Agar Slant:- The method involves sampling with cotton swabs that are transferred
directly to slants and incubate.
Swab/Agar Slant:- The method involves sampling with cotton swabs that are transferred
directly to slants and incubate.
Sticky Film:- The method consists of pressing sticky film or tape against the surface to be
examined and pressing the exposed side on an agar plate and has been employed
successfully on meat surfaces.
Sticky Film:- The method consists of pressing sticky film or tape against the surface to be
examined and pressing the exposed side on an agar plate and has been employed
successfully on meat surfaces.
Ultrasonic Devices:- These devices are used to assess the microbiological contamination of
surfaces, but the surfaces to be examined must be small in size and removable so that they
can be placed inside a container immersed in diluent. Energy generated makes the release of
microorganisms into the diluent.
Ultrasonic Devices:- These devices are used to assess the microbiological contamination of
surfaces, but the surfaces to be examined must be small in size and removable so that they
can be placed inside a container immersed in diluent. Energy generated makes the release of
microorganisms into the diluent.
Spray Gun:- It is based on the impingement of a spray of washing solution against a
circumscribed area of surface and the subsequent plating of the washing solution. It was
shown to be much more effective than the swab method in removing bacteria from meat
surfaces.
Spray Gun:- It is based on the impingement of a spray of washing solution against a
circumscribed area of surface and the subsequent plating of the washing solution. It was
shown to be much more effective than the swab method in removing bacteria from meat
surfaces.
38.
39. CHEMICAL, BIOLOGICAL AND
PHYSICAL METHODS
These are the methods based on metabolic activity of the
microorganisms on given substrate, measurements of growth response
and measurements of some parts of cells including nucleic acid or
combinations of these.
40. 2. Chemical methods
The chemical methods used primarily to detect, enumerate or
identify food born organisms or their products are:
1. Thermostable nuclease (for Staphylococcus aureus)
2. Limulus amoebocyte lysate (LAL) assay (for Gram negative
bacteria)
3. ATP assay (for live cells)
4. Radiometry
5. Fluorogenic/chromogenic substrats (to identify/differentiate
microbial species or strains)
41. Thermostable Nuclease
o The presence of S. aureus in significant numbers in a food can be determined by
examining the food for the presence of thermostable nuclease (DNase).
o This is possible because of the high correlation between the production of
coagulase and thermostable nuclease by S. aureus, especially enterotoxin
producers.
o It has been found to be as good as coagulase in testing for enterotoxigenic strains.
Among the advantages of testing for heat-stable nuclease as an indicator of S.
aureus growth and activity are the following:
Because of its heat-stable nature, the enzymes will persist even if the bacterial cells
are destroyed by heat, chemical or bacteriophage or if they are induced to l-forms
Can be detected faster than enterotoxin
The nuclease appears to be produced by enterotoxigenic cells before enterotoxin
appear
Nuclease is detectable in un-concentrated cultures of food specimens whereas
enterotoxin detection requires concentrated samples
It is stable to heat as are the enterotoxins
42.
43. Limulus Lysate for Endotoxins
• The Limulus amoebocyte lysate (LAL)
test employs a lysate protein obtained
from the blood (actually hemolymph)
cells (ameobocytes) of the horseshoe
crab (Limulus polyphemous).
• The lysate protein is the most
sensitive substance known for
endotoxins and the presence of
endotoxins causes gel formation of
the lysate material.
• LAL test is a good, rapid indicator of
the total numbers of Gram-negative
bacteria.
• The method has been applied
successfully to monitor milk and milk
products, microbial quality of raw fish
and cooked turkey rolls.
44. Adenosine Triphosphate Measurement
• Simplest way to measure ATP is by use of the firefly luciferin-luciferase system.
• In the presence of ATP, luciferase emits light which is measured with a luminometer.
• The amount of light produced by firefly luciferase is directly proportional to the ATP
added.
• It has been adapted for the determination of microbial load on chicken carcasses as well
as pork and beef.
• It is widely used as rapid and on the spot method for monitoring food handling surfaces
by swabbing designated areas and reading the relative light unit (RLU) from a
luminometer.
45. Radiometry
The radiometric detection of microorganisms is based on the incorporation of a
C-labeled metabolite in a growth medium so that when the organisms utilize this
metabolite, CO2 is released and measured by use of a radioactivity counter.
For organisms that utilize glucose, C-glucose is usually employed.
For those that cannot utilize this compound, others such as C-formate or C-
glutamate are used.
Application:
Capped 15 ml serum vials are used
Add 12-36 ml of medium containing labeled metabolite
Vials are made aerobic or anaerobic by sparging with
appropriate gases and then inoculated
Incubation
Headspace is tested periodically for the presence of
CO2
The time required to
detect the labeled CO2 is
inversely proportional to
the number of organisms in
a product.
The Bactec is a
commercially available
detection system.
46. Fluorogenic and Chromogenic Substrates
Some of the fluorogenic and chromogenic substrates employed
in culture media in food microbiology are :
4-methylumbelliferyl-β-D-glucuronide (MUG)
4-methylumbelliferyl-β-D-galactoside (MUGal)
4-methylumbelliferyl phosphate (MUP)
O-nitrophenyl- β-D-galactopyranoside (ONPG)
L-alanine –p-nitroanilide (LAPN)
5-bromo-4-chloro-3-indolyl- β-D-glucuronic acid (X-GlcA)
5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside (X-Gal)
Indoxyl- β-D-glucuronide (IBDG)
47. Cont..
These substrates are employed in various ways in plating media, MPN broths and for
membrane filtration methods.
MUG is the most widely used of the fluorogenic substrates and it is hydrolyzed by β-
D-glucuronidase (GUD) to release the fluorescent 4-methylumbelliferyl moiety which
is detected with long-wave ultraviolet light.
The value of MUG is that E.coli is the primary producer of GUD and this substrate
has found wide use as a deifferential agent in media and methods used for this
organisms.
A few Salmonella and Shigella are also GUD positive.
48. 3. Immunological methods
Serotyping:
o Serotyping is most widely applied to Gram-negative bacteria Enteric bacterial
pathogens such as Salmonella and Escherichia.
o Among Gram-positives, serotyping is important for the genus Listeria.
o The gist of a typical serotyping scheme is the use of specific antibodies
(antiserum) to identify homologous antigens.
This typing scheme results in the recognition of three antigenic sites:
Somatic antigen (O)
Capsular antigen (K)
Flagellar antigen (H)
49. Fluorescent antibody (FA):
o This technique has had extensive use in both clinical and food microbiology.
o An antibody to a given antigen is made fluorescent by coupling it to a
fluorescent compound and when the antibody reacts with its antigen, the
antigen-antibody complex emits fluorescence and can be detected by use of
fluorescence microscope.
The fluorescent markers used are :
Rhodamine B
Fluorescein isocyanate
Fluorescein isothiocyanate
FA technique can be carried out by two basic methods:
Direct method – Antigen coated by specific antibody with fluorescent label
and it detects the presence of antigen.
Indirect method – antigen coated by homologous antibody which is in turn
coated by antibody to the homologous antibody bearing the fluorescent label
and it detects the presence of homologous antibody.
50.
51. Enrichment Serology (ES):
• The use of ES is a more rapid method for recovering salmonella from foods
than the conventional culture method (CCM).
It is carried out in four steps:
Pre-enrichment in a non-selective medium for 18 hours
Selective enrichment in selenite-cystine or tetrathionate broth for 24 hours
Elective enrichment in M broth for either 6-8 hrs or 24 hrs
Agglutination with polyvalent H antisera at 50°C for 1 hour
Results can be obtained in 50 hours (depending on elective enrichment time
used) compared to 96-120 hrs by CCM.
A modified ES methods has been proposed involving a 6-hr pre-enrichment
thus making it possible to obtain results in 32 hrs.
The Oxoid Salmonella rapid test (OSRT) is a variation of ES.
52. Salmonella 1-2 Test:
This method is similar to ES and OSRT.
It employs the use of a semisolid phase.
The test is conducted in a specially designed plastic device that has two
chambers, one for selective broth and other for non-selective motility
medium.
It addition to selective ingredient, also contains the amino-L-serine
which is elective for salmonella.
Application:
Inoculation of selective medium chamber
Incubation
Motile salmonella moves into the non-selective medium chamber
When motile organisms enter the antibody area, an immuno-band develops,
indicating antigen-antibody reaction
Following non-selective enrichment, results can be obtained in 8-14 hours
53. Radioimmunoassay:
This technique consists of adding a radioactive label to an antigen,
allowing the labeled antigen to react with its specific antibody and
measuring the antigen that combined with the antibody by the use of a
counter to measure radioactivity.
Solid-phase radioimmunoassay (RIA):
Principle – The ability of antibody-coated phase to bind specifically with
radioactive tracer antigens.
It employs solid materials or surfaces onto which a monolayer of antibody
molecules binds electrostatically.
The solid materials used are:
Polypropylene
Polystyrene
Bromacetylcellulose
54.
55. ELISA (Enzyme-linked immunosorbent assay):
ELISA or Enzyme immunoassay (EIA), is an immunological method
similar to RIA but employing an enzyme coupled to either an antigen or
an antibody rather than a radioactive isotope.
This technique is widely used to detect and quantitate organisms and
their products in foods.
Synonymous with ELISA are:
Enzyme-multiplied immunoassay technique (EMIT)
Enzyme-linked antibody technique (ELAT)
Variations of this basic ELISA consists of:
Sandwich ELISA – The antigen is required to have at least two
binding sites.
Double sandwich ELISA – It employs a third antibody.
56. Cont..
ELISA is performed with a:
Solid-phase (polystyrene) coated with antigen and incubate with
antiserum
Following incubation and washing, an enzyme labeled preparation of
immunoglobulin is added
After gentle washing, the enzyme remaining in the tube or microtiter well
is assayed to determine the amount of specific antibodies in the initial
serum
A commonly used enzyme is horseradish peroxidase and its presence is
measured by the addition of peroxidase substrate.
The amount of enzyme present is ascertained by the colorimetric
determination of enzyme substrate.
57.
58. Gel diffusion:
Gel diffusion methods have been widely used for the detection and
quantitation of bacterial toxins and enterotoxins.
The four most widely used methods are:
Single-diffusion tube
Microslide double diffusion
Micro-Ouchterlony slide
Electroimmunodiffusion
They have been employed to measure enterotoxins of
Staphylococcus and C. perfringens and the toxins of C. botlinum.
Recent studies suggest that results can be obtained in <8 hours when
slides are incubated at 45 °C.
59.
60.
61.
62.
63. Immunomagnetic separation:
This method employs paramagnetic beads (about 2-3 µm in size) that
are surface activated and can be coated with antibody by incubating in
the refrigerator for varying periods of time up to 24 hours.
The unabsorbed antibody is removed by washing.
When properly treated, the coated beads are added to the food
slurry that contains the homologous antigens (toxins or whole cells
in the case of Gram-negative bacteria), thoroughly mixed and
allowed to incubate from a few minutes to several hours to allow
for reaction of antigen with antibody coated beads.
This method may be used for a number of other organisms including
viruses and protozoa.
64.
65. Hemagglutination:
Whereas gel diffusion methods generally require at least 24 hrs for results,
two comparable serologic methods yield results in 2-4 hrs:-
o Hemagglutination-inhibition (HI) – specific antibody is kept constant
and enterotoxin (antigen) is diluted out. Following incubation for about
20 minutes, treated sheep red blood cells (SRBCs) are added.
o Reverse-passive hemagglutination (RPH) – antitoxin globulin in RPH
is attached directly to SRBCs and used to detect toxins.
Hemagglutination (HA) occurs only when antibody is not bound to
antigen. When diluted toxin preparation are added, the test is read
for HA after incubation for 2 hrs.
No HA occurs if no toxin or enterotoxin is present.
68. Nucleic acid (DNA) probe:
o A DNA probe consists of the DNA sequence from an organism of interest that can be
used to detect homologous DNA or RNA sequences.
o In effect, the probe DNA must hybridize with that of the strain to be sought.
o Ideally, the probe contains sequences that code for a specific product.
o The probe DNA must be labeled in some way in order to assess whether hybridization
has occurred.
o Chromosomal DNA is often the source of target nucleic acid, but it contains only one
copy per cell in most cases.
o Multiple targets are provided by mRNA, rRNA and plasmid DNA, thus making for a
more sensitive detection system.
o Synthetic oligonucleotide probes may be constructed of 20-50 bases and under proper
conditions, hybridization times of 30-60 minutes are possible.
69.
70. Polymerase chain reaction (PCR):
This method is fast becoming the most widely used of all molecular genetic
methods for detecting and identifying bacteria and viruses in food. Its
increasing use is due to its high sensitivity, specificity, its availability in a
number of formats and the commercial availability of PCR-based methods in
kit-like formats.
This technique is applicable more to the identification of food born organisms
than to their enumeration.
A general outline of PCR test is as follow:
Denaturation – at 95 °C
Annealing – at 55-60 °C
Extension – at 72 °C
When the starting material is RNA (e.g. RNA virus), it is converted to dsDNA
by use of reverse transcriptase (RT-PCR).
When this process is repeated, the two strands become four, four becomes eight
and so on each additional cycle, resulting in the several million copies of the
original if enough cycles are run.
71.
72. Cont..
Among commercially available kits are the following:
o BAX system – Screening of E. coli (Qualicon, Dupont Corp.)
o Probelia (Sanofi Diagnostic Pasture)
o Foodproof (Biotecon Diagnostics)
Other PCR-based methods are:
1. Multiplex PCR
2. RT-PCR
3. Molecular beacon PCR
4. PCR-DGGE
73. Lux Gene Luminescence:
o Luminescence in marine bacteria such as Vibrio fisheri and V. harveyi is
controlled by genes and the capacity to produce luminescence can be
transferred to other organisms by effecting the transfer of some genes.
o The primary genes for luciferase are lux A and lux B.
o In the food microbiology application of lux phages, one starts with
bacteriophages that are specific for the bacterium of interest and thus takes
advantage of the highly specific relationship that exits between the phages and
their hosts.
If Y. enterocolitica is the bacterium of interest:
One selects the phage that will in infect the widest range of strains
To this phage, lux genes are inserted by recombination methods, which accounts
to about 2kb of DNA
lux-gene bearing phages enter and multiply
Cause the host cells to luminesce by the increased production of more lux genes.
74.
75. Ice Nucleation Assay:
The bacterial ice nucleation diagnostic (BIND) test developed by
scientists at the DNA Plant Technology Corporation, was developed for
the detection of salmonella.
The ina gene from Pseudomonas syringae is cloned into genetically
engineered bacteriophages that are specific for salmonella.
If salmonellae are present, the phage infect and lead to the synthesis of
ice nucleation protein as part of the outer cell membrane.
This is evidenced by the formation of ice crystals at a temperature
around -9 °C.
By coupling a fluorescent freeze indicator dye, a green color indicates
freezing thus the presence of salmonellae while an orange color
indicates no freezing.
With salmonellae phage P22, as few as 25 cells per gram can be detected
within 24 hrs.
76.
77. Fingerprinting methods
It includes the following:
Phage typing
Amplified-fragment length polymorphism
Multilocous enzyme electrophoresis
Restriction enzyme analysis
Random amplification of polymorphic DNA
Pulsed field gel electrophoresis
Restriction fragment length polymorphism
Ribotyping
Microarrays
78. 5. Physical methods
Biosensors: (Father of Biosensor – Professor Leland C Clark Jnr)
A device containing a biological sensing element connected to a transducer.
Enzyme-substrate or antigen-antibody reactions may be the components of a
biosensor.
Biosensors that have been demonstrated to be of value for food-born
microorganisms are:
1. Accoustical Biosensors (Piezoelectric Crystals) – With the quartz coated with
an antibody, the target analyte is the homologous antigen which, when it binds
to the antibody, changes the mass with a resulting decrease in frequency.
2. Fiber optics – A fiber optic biosensor uses electronic or optical transduction to
monitor a biological reaction and reports it as an optical signal.
3. Impedance – When microorganisms grow in cultured media, they metabolize
substrates of low conductivity into products of higher conductivity and thereby
decreases the impedance of the media.
79.
80. Microcalorimetry:-
This is the study of small heat changes, measurement of the enthalpy change
involved in the breakdown of growth substrates. The heat production that is
measured is closely related to the cell’s catabolic activities. There are two
types of calorimeters: batch and flow.
Flow cytometry:-
It is the science of measuring components (Cells) and the properties of
individual cells in liquid suspension. In essence, suspended cells, one by
one are brought to a detector by means of a flow channel.
BioSys Instrument:-
The BioSys-32 instrument makes automatic and computer-analyzed changes in
the color of reaction vials as organisms grow in specified culture media that
contain a substrate that changes color in proportion to increase in number of
cells. It was used to detect Salmonella and Listeria.
