CHAPTER 3CHAPTER 3

MICROBIOLOGICAL METHODS MICROBIOLOGICAL METHODS ANALYSIS IN FOODANALYSIS IN FOOD

BCS 3214 FOOD BCS 3214 FOOD MICROBIOLOGYMICROBIOLOGY

• CULTURE, MICROSCOPIC AND SAMPLING METHOD

• PHYSICAL, CHEMICAL, MOLECULAR & IMMUNOLOGICAL METHODS

• BIO-ELECTRONIC MOLECULAR METHODS• BIOASSAYS AND RELATED METHODS

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WHY WE NEED TO EXAMINE FOOD?

2. To know the level of sanitation used during handling.

3. Assess the stability/ shelf life of food under normal storage condition.

1. Help one assess the safety of food consumers

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4. To know the whether the foods/ ingredients meet the normal standard/ guidelines/ specification

6. The examination of raw ingredients also provide information about heat processing parameters that would be necessary to meet the microbiological standard.

5. Help to determine the source/ load/ type of microorganisms in foods.

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Criteria for Choice of Methods

1. Accuracy2. Sensitivity or threshold3. Reliability4. Precision: repeatability and reproducibility5. Easy of implementation6. Equipment/operating cost7. Speed8. Official

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Traditional or conventional methods??

Traditional or conventional Standardized methods e.g. ISO methods THE GOLDEN STANDARDS!!!• Developed historically• International consensus Large volumes of materials Time consuming Laborious

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Conventional Methods: Enumeration Colony count method• Serial dilution• Inoculation on growth media• Spread plates• Pour plates• Counting colonies on agar plates

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Conventional Methods: Detection

Pathogens! (low numbers, sub-lethally injured, heterogenious distribution in foods)

•Resuscitation phase = pre-enrichment•Enrichment phase•Isolation of the pathogen•Identification/confirmation

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Rapid methods

Based on “Golden standard”: a “rapid method” can be defined as any method or system that reduces the time taken to obtain a microbiological test result. (Feng, 1996; Fung, 1994)

Save time – produce results in 24 h or lessSame (or greater) sensitivity, accuracy, andSavings of space and materialsReduction in human errors and labor cost

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Rapid method:

1.Culture Methods - Spiral Plater, Petrifilm®, Hydrophobic grid plates, Redigel®2.Physical Methods – Microcalorimetry, Spectroscopy, Fluorescence microscopy, Flow cytometry, Laser scanning.3.Chemical / Biochemical Methods - Substrate Used, Radiometry, Lux Gene Luminescence, Ice Nucleation Assay4.Molecular Methods – Serotyping, Phage Typing, Probe Based Technology, Polymerase Chain Reaction (PCR) Based Technology, Multilocus Enzyme Electrophoresis, Restriction Enzyme analysis of plasmid or genomic, Ribotyping

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Rapid method:

5.Immunological Methods- Flourescent antibody, ELISA, Latex, Lateral flow, Immunomagnetic Seperation, Hemagglutination, Biosensor, Bioluminescence, Imminofluorence6.Bioassay an Related Methods - Whole-animal assay, Cell culture systems, Other assay.7.Combination Methods.

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Types of Microbiologica

l Methods

2. Microscopic

7. Molecular7. Molecular

3. Physical3. Physical

8. Immunological8. Immunological

9. Bioassays9. Bioassays

6. Biochemical6. Biochemical 4. Chemical4. Chemical

1. Culture

Basic to food microbiology – examine food for the presence, types, numbers/ their products (e.g:toxin).

For basic methods for “total numbers”:a) Standard plate count (SPC) – for viable cellsb) The most propable numbers (MPN) methods - as

statistical determination of viable cellsc) Dye reduction techniques – to estimate number of

viable cells that posses reducing capacities.d) Direct microscopic counts (DMC) – for both viable &

non-viable.

CULTURE, MICROSCOPIC & SAMPLING METHODS

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Standard plate count (SPC)

oMost widely used method for determining number of viable cells or colony-forming units (cfu).oPortions of food samples are blended/ homogenized, serial diluted, plated & incubated. Visible colonies are counted by Qubec or electronic counter.

Qubec counter Electronic counter.

oSPC often determined by pour plating & surface plating.

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Surface platingoPrepoured & hardened agar plates with dry surface emploed. Diluted specimen planted on the surface, aid with bend glass rod (‘hockey stick”), 0.1 mL inoculum per plate distributeover the entire surface. oAdvantages:

a)Determining the numbers of heat sensitive psychrotrophs in a food product – because org. do not come in contact with melted agar.

b)Colonial features of a colony are importan to its presumptive identification.

c)For most selective media

d)Strict aerobic favored by surface plating but microaerophilic tend to grow slower.oDisadvantages:

a)Problems of spreader (esp. when agar surface is not adequately dry prior to plating).

b)Crowding of colonies – make enumeration difficult.

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Homogenizing of food samplesoMechanical blenders vs. Stomacher?oStomacher is generally preferred over blending for the following reasons:

a)The need to clean & store blender container is obviated.

b)Heat buildup does not occur during normal operational time (usually 2 min).

c)Homogenates can be stored in Stomacher bags in a freezer for further use.

d)The noise level is not unpleasant as that of mechanical blenders.

e)Homogenization for dye reduction

tests – Stomacher does not cause

extensive disruption of meat tissue.

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Spiral plateroMechanical device, distributes liquid inoculum on the surface of a rotating plate containing poured & hardened agar medium.oEnumeration – use a special counting grid.oAn official Association of Official Analytical Chemists (AOAC) method.

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o Advantages:a) Less agar useb) Fewer plates, dilution blanks & pipette required.c) 3-4 more sample per hr can be prepared & examined.d) Little training required.

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Membrane filtersoPore size will retain bacteria (generally 0.45 μm) but allow water/ diluent to pass.oMethod: collect bacteria upon filtering, membrane is placed on agar plate, incubate (required temp. & time) & enumerate.oSuit for sample that contain low number of bacteria.oImproved – add fluorescent dye & epifluorescence microscopes to enumerate.

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Microscope colony countsoInvolve the counting of microcolonies that develop in agar layered over microscope slides, followed by incubation, drying & staining & microcolonies count with the aid of a microscope.oE.g: 2 ml melted agar mixed with 2 ml warmed milk & 0.1 ml inoculated agar is spread over a 4 cm2 area. Stain with thionin blue. View with microscope.

Agar dropletsoFood homogenate is diluted in tubes of melted agar.Counting procedure in which the a dilution of sample is placed (at increasing dilutions) into three tubes of melted agar. Small droplets of the agar are placed on the bottom of a Petri dish, incubated, and examined under magnification.

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Dry film & related methods

Petrifilm oConsist of two plastic film attached together on one side and coated with culture media. oProved by AOACoE.g: 1 ml diluent placed between two film & spread over the nutrient area by pressing. Following incubation, microcolonies appear red on non selective film bacause of the presence on tetrazolium dye.

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Redigel® / Easygel® oEasygel® (previously available as Redigel from 3M) replaces agar in microbiological media.oIt comes as a sterile 2-part test unit consisting of a bottle of liquid medium and a petri dish that is pretreated with a special formulation. oHere’s how it works: When the bottle of liquid medium is poured in to the pretreated petri dish, ions diffuse from the pretreatment layer into the liquid causing it to gel. Complete gelling takes around 40 minutes.oFor enumeration of psychrotopic organisms because there is no exposure to hot molten agar.

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Roll tubeoScrew-capped tubes or bottle is use. Predetermined amount of melted & inoculated agar are added to the tube. Agar is made to solidify as a thin layer. Followed incubation, colonies are count by rotating the vessel. oExcellent method for enumerating fastidious anaerobes.

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Most probable numbers (MPN)

Serial dilution.Three serial dilution in 3x3 or 3x5 tubes.Number of positive tubes for each dilution.MPN table for calculation of numbers.Method of choice for determining fecal coliform densities.

Disadvantages:oLarge vol. of glassware is required (esp. for five tube method).oLack opportunity to observe the colony morphology.oLack precision.

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Dye reduction

Prepared supernatants of foods are added to standard solution of either dye for reduction from blue to white for methylene blue and from slate blue to pink or white for resazurin.Problems:-Food (raw meat) inherent reductive substances/ contain reductive enzymes – not applicable.-Not all organisms reduce the dyes equally.

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Direct microscopic count

Simplest form. Make smear of food specimens onto a microscope slide, stainning with appropiate dye, view & count with the aid of microscope (oil immersion objective). Advantages:

- rapid & simple- cell morphology can be assessed

oDisadvantages:-fatiguing to analyst-both viable & nonviable cells are enumerated-food particles not always distinguishable for microorganism-microbial cells are not uniformly from distributed relative to single cells & clumps.-some colony do not take the stain well & may not be counted.28

MICROBIOLOGICAL EXAMINATION OF SURFACES

The need to maintain contact surface or utensils in a hygienic state is of obvious importance. A few common methods for surface assessment:1)Swab/swab-rinse methods

-oldest & most widely used method-not only in food & dairy industries, but also in hospitals &

restaurants 2)Contact plate

-Environmental sampling (for disinfection control) is usually carried out using RODAC plate or HYcheck

contact slide (Difco).29

Figure 1: RODAC plate containing TSA agar is ensure to have perfect contact with the tested surface.

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3) agar syringe/ ‘agar sausage’ methods- The cut surface of the agar sausage is pressed lightly against the

surface of the equipment which has to be checked. In this way a stamp of the bacteria on the surface of the equipment is transferred to the sterile surface of the agar sausage.

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3) Direct surface - melted agar poured onto surface/ utensils. Upon hardening, agar mold is placed in a petri dish & incubated.

5) Sticky film- pressing sticky film /tape against surface to be examined & pressed the exposed side on an agar plate.

5) Swab/ agar slant- Sampling with cotton swabs that are transferred directly to slants7) Ultrasonic devices

- examine small size & removal / placed inside a container immersed in diluent. Ultrasonic apparatus generate effects, release microorganism into the diluent.

8) Spray gun- spray washing solution onto area of surface. Plating of the washing solution.- air pressure is necessary.

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Types of Microbiologica

l Methods

2. Microscopic2. Microscopic

7. Molecular

3. Physical

8. Immunological

9. Bioassays

6. Biochemical 4. Chemical

1. Culture1. Culture

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PHYSICAL, CHEMICAL, MOLECULAR & IMMUNOLOGICAL METHODS, BIO-

ELECTRONIC MOLECULAR METHODS, BIOASSAYS AND RELATED METHODS

Physical method

The basic of these techniques is the direct of microorganisms in the water samples, without any prior enrichment. The techniques usually consist of the labeling of the organism or their components, followed by the detection of the labels.

1.Microcalorimetry•Calorimetry is the science of heat. •Study of small heat changes: enthalpy change involved in the breakdown of growth substrates (cell catabolic activities).•Two type: 1) batch & 2) flow•batch - E.g: identification & characterization of foodborne organism, characterized commercial yeast.•Flow – E.g: study spoilage canned food, differentiate between Enterobacteriaceae, detect present of S. aureus.

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VITEK identificationEach identification card comprises 30 wells which contain biochemical substrates in a dehydrated form. No additional reagents are needed, thus eliminating any risk of omission or error. VITEK identification covers over 300 species encountered clinically, and in the industrial field.

VITEK susceptibility testingThe susceptibility test cards include 30 or 45 wells containing dehydrated antibiotics. Specific tests for detecting resistance mechanisms are systematically included in the susceptibility test cards 

VITEK automates all the steps needed to perform identification and susceptibility tests, using VITEK cards. It comprises a filler/sealer, an incubator/reader, a computer and a printer.

The filler/sealer enables inoculation of cards within a few minutes. The incubator/reader simultaneously incubates and reads the cards, and has a capacity of between 32 and 480 cards depending on the model.

The computer with its VITEK software continuously monitors the operations under way, stores values, processes and interprets results.

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2. Flow cytometry

• Measure components (cells) & the properties of individual cells in liquid suspension.

• Detector by means of a flow channel. • A wide range of fluorophores can be used as labels in flow

cytometry - detecting and measuring the amount of fluorescent dye on particles, and basically consists of one or more lasers for supplying excitation energy, and a series of filters and detectors for measuring the resultant fluorescent emissions.

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Chemical/ biochemical method

Some of the fluorogenic & chromogenic substrate employed in culture media in food microbiology.

1.Thermostable nuclease•Presence of S. aureus in significant number in food can be determined by examining food for the presence of thermostable nuclease (DNAse).•Number of cell (bacteria) increased, amount of extractable thermostable nuclease is increase.

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2. Limulus Lysate for Endotoxins• Gram –ve bacteria are characterized by their production of

endotoxin, which consist of lipoloplysaccharides (LPS) layer (outer membrane) of the cell envelope & is composed of lipid A.

• LPS is pyrogenic & responsible for some of the symptoms that accompany infections caused by gram –ve bacteria.

• The Limulus amoebocyte lystate (LAL) test employs a lysate protein obtained from the blood (actually haemolymph) cells (amoebocyte) of the horseshoe crab (Limulus polyphemous). The lysate protein is the most sensitive substance known for endotoxins.

• E.g: suitable for rapid evaluation for hygienic quality of milk relative to detection of coliform before & after pasteurization.

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3. Adenosine Triphosphate Measurement• Adenosine Triphosphate (ATP) is the primary source of energy in all

living organisms. Its disappears within 2 hrs after cell death & the amount per cell is generally constant.

• One of the simplest way to measure ATP is by the use of the firefly luciferin-luciferase system.

• In presence of ATP, luciferase emits light, which is measured with a liquid scintillation spectrometer or a luminator. Amount of light produced by firefly luciferase is directly proportional to the amount of ATP added.

• Major problem in food – to remove the nonmicrobila ATP• E.g: microbial load on chicken carcasses, food handling surface

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4. Radiometry • Based on incorporation of a 14C-labeled metabolite in a growth so then

when the organisms utilize this metabolite, 14CO2 is released & measured by the use of a radioactivity counter.

• E.g: detect organisms in frozen orange juice concentrate.

5. Fluorogenic and Chromogenic Substrates• Methods based on the application of chromogenic and

fluorogenic substrates enable specific and rapid detection of a variety of bacterial enzymatic activities.

• Some fluorogenic and chromogenic substrates applied are:5. 4-Methylumbelliferyl beta-D-glucuronide (MUG)6. 4-Methylumbelliferyl beta-D-galactosidase(MUGal)7. Ortho-Nitrophenyl-ß-D-Galactopyranoside (ONPG)8. L-alanine-p-nitroamilide (LAPN)9. 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (BCIG/ X-Gluc/ X-GlcA))10. 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Gal) is also used for

which the cleavage product is a blue precipitate.11. Indoxyl-β-D-glucuronide (IBDG)

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• These substances are employed in various ways in plating media, MPN broths & for membrane filtration methods.

• E.g: ONPG –specific for coliforms – produce yellow color that can be countitated at 420nm – Colilert & ColiQuik systems.

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6. Lux Gene Luminescence • Luminescence in marine bacteria such as Vibrio ficheri & V. harveyei is

controlled by genes, & the capacity to produce luminescence can be transferred to other organisms by effecting transfer of some of these genes (lux genes – luciferase).

• When added to specific host bacteria, lux gene –bearing phages enter & multiply, & thus cause host cells to luminesce by increased production of more lux genes.

• E.g: MPN method; detection wide range of bacteria in food – initial no. is low, need sample enrichments.

7. Ice Nucleation Assay• Similar to Lux Gene Luminescence.• A number of genera of Gram –ve plant inhibiting bacteria carry a gene

(ina) that encode the synthesis of a protein that act as an ice nucleator.

• Formation of ice crystal at temperature around -9°C, by coupling a fluorescent freeze indicator dye, a green color indicate freezing & thus indicate the presence of Salmonella while orange indicates no freezing

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Molecular method

i.Serotypingii.Phage typingiii.Nuclei acid probesiv.Polymerase chain reactionv.Multilocus enzyme electrophoresisvi.Restriction enzyme analysisvii.Ribotyping

SerotypingoWidely applied to Gram –ve pathogens, Salmonella, EscherichiaoAmong Gram +ve, Listeria

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o Serotype or serovar refers to distinct variations within a subspecies of bacteria or viruses. These microorganisms, viruses, or cells are classified together based on their cell surface antigens.

o Determining serotypes, the process of serotyping, can be based on a variety of factors, including virulence, lipopolysaccharides (LPS) in Gram-negative bacteria, presence of an exotoxin (such as pertussis toxin in Bordetella pertussis), plasmids, phages, genetic profile (such as determined by polymerase chain reaction), or other characteristics which differentiate two members of the same species, allowing the epidemiologic classification of organisms to the sub-species level.

o A group of serovars with common antigens is called a serogroup

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Bacteriophage

oPhage typing is a method used for detecting single strains of bacteria. It is used to trace the source of outbreaks of infections. The viruses that infect bacteria are called bacteriophages ("phages" for short) and some of these can only infect a single strain of bacteria. These phages are used to identify different strains of bacteria within a single species.oA culture of the strain is grown in the agar and dried. A grid is drawn on the base of the petri dish to mark out different regions. Inoculation of each square of the grid is done by a different phage. The phage drops are allowed to dry and are incubated: The susceptible phage regions will show a circular clearing where the bacteria have been lysed, and this is used in differentiation.

Phage typing is the identification of bacterial species and strains by determining their susceptibility to various phages

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Nucleic Acid (DNA) probes

oNucleic acid hybridization is a technique in which single-stranded nucleic acids are allowed to interact so that complexes called hybrids are formed by molecules with similar complimentary sequences. This means that single-stranded nucleic acid molecules are made into double-stranded nucleic acid molecules.

Southern blotting and DNA probes, including DNA chips, are examples of hybridization techniques.

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DNA Amplification (Polymerase Chain Reaction)

oThe polymerase chain reaction is a procedure that can amplify a specific segment of DNA without the use of host cells.oPolymerase Chain Reactions have three cycles, producing more and more copies of specific parts of DNA throughout each cycle; it starts with the denaturing of DNA strands by adding heat. After it is heated a cooling or annealing of the separated strands of DNA begins, which cools the DNA and allows primers to form hydrogen bonds with the ends of the original DNA strands. DNA polymerase will then add nucleotides creating base-pairs to the 3' ends of the primers that were created. o The DNA polymerase needed for

this process is a unique polymerase is a heat-stable polymerase that is taken from cells of bacteria first discovered in Yellow Stone National Park living in some of the hot springs located there and are not found in the original cell.

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Ribotyping

oRibotyping is a method that can identify and classify bacteria based upon differences in rRNA. It generates a highly reproducible and precise fingerprint that can be used to classify bacteria from the genus through and beyond the species level. oDNA is extracted from a colony of bacteria and then restricted into discrete-sized fragments. The DNA is then transferred to a membrane and probed with a region of the rRNA operon to reveal the pattern of rRNA genes. The pattern is recorded, digitized and stored in a database. It is variations that exist among bacteria in both the position and intensity of rRNA bands that can be used for their classification and identification.

oWhy Ribotype?Contamination source bacteria in finished products can originate from any one of the ingredients, processing personnel or the environment. Ribotyping allows the establishment of unequivocal relationships between bacterial isolates recovered from any of these sources and the finished product.

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Immunological method

i.Fluorescent Antibodyii.ELISAiii.Latexiv.Lateral flowv.Immunomagnetic seperationvi.Hemaglutinationvii.Biosensorviii.Biolumonacenceix.Immunofluorence

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Fluorescent Antibody•Direct fluorescent antibody (DFA or dFA) (also known as "Direct immunofluorescence") is a laboratory test that uses antibodies tagged with fluorescent dye that can be used to detect the presence of microorganisms.

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ELISA

ELISA techniques use antibodies linked to an enzyme, such as horseradish peroxidase or alkaline phosphatase.Antigen – antibody reactions are detected by enzyme activity. An antibody linked to the indicator enzyme is added to the test well and is bound in the well if the antigen it is specific for is present. To determine whether or not the enzyme-linked antibody is bound in the well substrate for the enzyme is added. If the enzyme linked antibody is present the substrate is converted to a product that causes a color change.

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o ELISA kits are available for both clinical diagnostics and home use. These tests are used for everything from screening blood for anti-HIV antibodies to home pregnancy tests.

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Lateral flow

A simple device intended to detect the presence (or absence) of a target analyte in sample (matrix). Lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with an antibody or antigen. Depending upon the analytes present in the sample the coloured reagent can become bound at the test line or zone.

Handheld PDA based lateral flow strip reader- Developed by Detekt Biomedical 

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Detection of Listeria monocytogenes in foods with Singlepath® L’mono

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Principle Lateral Flow Tests

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VTEC:BHICAYE BrothSMAC

ListeriaBuffered LEB (BLEB)Demi Fraser Broth

UVM BrothPALCAM AgarOXFORD AgarChromocult Listeria Agar

Legionella:CYE Agar BaseBCYE SupplGVPC Suppl

Campylobacter:Bolton BrothCCDA Agar

Bacillus cereus:M.Y.P. AgarCGY BrothBHI

NEWNEW

E.Coli O157:mEC BrothmTSB Broth suppl.CT-SMAC suppl.

Salmonella:Rappaport-Vassiliadis BrothSelenite Cyctine BrothSalmosyrt BrothXLD AgarRambach Agar

NEWNEW

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Bioassay & related method

1.Whole animal assayMouse lethalityRabbit & mouse diarrheaKitten (cat) testRabbit & guinea pig skin test2.Cell culture systemsHuman mucosal cellHuman fetal intestineGuinea pig intestinal cellsHeLa cells - a cell type in an immortal cell line used in scientific research. It is the oldest and most commonly used human cell lines. The line was derived from cervical cancer cells taken from Henrietta Lacks, a patient who eventually died of her cancer on October 4, 1951. The cell line was found to be remarkably durable and prolific as illustrated by its contamination of many other cell lines used in researchY-1 adrenal cell assay3.Other assay.

Standard Microbiological Methods• Government– FDA’s Bacteriological Analytical Manualhttp://www.cfsan.fda.gov/~ebam/bam-toc.html– FSIS’s Laboratory Guidebookhttp://www.fsis.usda.gov/OPHS/microlab/mlgbook.htm

Standard Methods• Scientific or Commodity Association– AOACI’s Official Methods of Analysis (AOACInternational)– APHA’s Standard Methods for Examination ofDairy Products (American Public HealthAssociation)– APHA’s Recommended Methods for theMicrobiological Examination of Foods

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Standard Methods• International– Codex Alimentarius’s Volume 13 (1999)Methods of Analysis and Sampling– ISO’s General methods of tests and analysis forfood products (67.050)– IUPAC

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