MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 5. Techniques for microbiological analyses

Lesson 29
RAPID ENUMERATION OF HYGIENE AND SAFETY INDICATOR ORGANISMS

29.1 Introduction

Food spoilage is an enormous economic problem worldwide. Through microbial activity alone, approximately one-fourth of the world’s food supply is lost. Milk is a highly nutritious food that serves as an excellent growth medium for a wide range of microorganisms. The microbiological quality of milk and dairy products is influenced by the initial flora of raw milk, the processing conditions, and post-heat treatment contamination. Undesirable microbes that can cause spoilage of dairy products include Gram-negative psychrotrophs, coliforms, lactic acid bacteria, yeasts, and molds. In addition, various bacteria of public health concern such as Salmonella spp., Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, pathogenic strains of Escherichia coli and enterotoxigenic strains of Staphylococcus aureus may also be found in milk and dairy products. For this reason, increased emphasis should be placed on the microbiological examination of milk and dairy foods. Microbiological analyses are critical for the assessment of quality and safety, conformation with standards and specifications, and regulatory compliance.

Although bacteria are the first type of microorganisms that come to mind when discussing microbial food safety, they are by no means the only pathogenic food borne microorganisms. Mycotoxin producing moulds, human enteric viruses, protozoan parasites and marine biotoxins are also of importance. However, since foods are only screened for bacteria routinely, in this article we will focus on the techniques used to
detect bacterial contamination in food product by traditional methods.

Three basic principles for isolation and identification have been described for the detection of microorganisms are:

1. Direct plating on (selective) media
2. Direct (selective) enrichment
3. Pre-enrichment, followed by a selective enrichment

Depending on the number of cells expected in a sample and/or the standards described, one or more of these procedures may be used for the detection or enumeration of bacteria. Often an enrichment procedure is required for the detection of pathogens, whereas, due to higher expected numbers, direct plating is possible for the detection of indicator and spoilage organisms. The success of the basic protocols depends on: 1) the number and the state of the micro-organisms in the sample; 2) the selectivity of the media (a balance between inhibition of competitors and inhibition of the target organism); 3) conditions of incubation (time, temperature, presence of oxygen) and 4) the selectivity of the isolation medium (the ease of distinguishing the target organism from competitive micro flora). Since 1970s there has been considerable improvement in traditional methods: new apparatus for homogenizing samples (stomacher, pulsifier), spiral platers, (automated) colony counters and the TEMPO-method, based on the automated most probable number (MPN) techniques used in traditional microbiology for counting low numbers of micro-organisms. Another improvement on the traditional method is the development of chromogenic isolation and enumeration media, which make it easy to distinguish the target organisms.

29.2 Rapid Methods

Since traditional enumeration procedures often require rather long incubation times, there is a need for rapid methods to detect food borne pathogens, indicator and spoilage organisms. In most food legislation, microbiological criteria are stated for food borne pathogens, but to a lesser extent for indicator organisms. The laboratory needs to choose whether to use traditional or rapid methods. However, due to the very low numbers of some food borne pathogens present in a product (e.g. Salmonella in milk powder or Cronobacter in infant formula), time-consuming enrichment procedures are necessary (the time varying from 1-2 days, depending on the type of enrichment). Many rapid methods, mainly immunological and/or DNA-based, are commercially available for the detection of food borne pathogens. However, traditional methods are still first choice for the enumeration of indicator and spoilage organisms.

29.3 Detection and Enumeration of Microorganisms

There are several methods for detection and enumeration of microorganisms in food. The method that is used depends on the purpose of the testing.

1. Direct Enumeration: Using direct microscopic counts (DMC), Coulter counter etc. allows a rapid estimation of all viable and nonviable cells. Identification through staining and observation of morphology also possible with DMC.
2. Viable Enumeration: The use of standard plate counts, most probable number (MPN), membrane filtration, plate loop methods, spiral plating etc., allows the estimation of only viable cells. As with direct enumeration, these methods can be used in the food industry to enumerate fermentation, spoilage, pathogenic, and indicator organisms.
3. Metabolic Activity Measurement: An estimation of metabolic activity of the total cell population is possible using resazurin, methylene blue dye reduction, acid production, electrical impedance etc. The level of bacterial activity can be used to assess the keeping quality and freshness of milk. Toxin levels can also be measured, indicating the presence of toxin producing pathogens.
4. Cellular Constituents Measurement: Using the luciferase test to measure ATP is one example of the rapid and sensitive tests available that will indicate the presence of even one pathogenic bacterial cell.
5. DNA/ RNA probes: A probe is a nucleic acid sequence typical of the organism of interest, used to detect homologous DNA or RNA sequences in the target organism. RNA as target sequence has an advantage in having 104 copies per cell versus DNA which has only one or two copies per cell. Nucleic acid fragments for testing are prepared using restriction endo-nucleases.
6. Fluorescent antibody method: Fluorescent antibody (FA) reagent reacts with test antigen. Resultant antigen-antibody complex emits fluorescence and is detected using a fluorescence microscope. AOAC-approved method for detecting Salmonella yields results within 52 hours.
7. Immunodiffusion: Gel diffusion methods are widely used for detecting and quantifying bacterial toxins and enterotoxins, for example, S. aureus enterotoxin and C. botulinum toxins. Most widely used test is the Crowle modification of the Ouchterlony slide.
8. Immunomagnetic separation (IMS): The Dynal system uses Dynabeads, polystyrene beads coated with specific antibodies. The antibodies combine with the target organism in the test sample and the bead bacterial complexes are separated using a magnetic particle concentrator. Incorporation of the IMS step greatly reduces the isolation time for the target organism. IMS can be used for isolation of E. coli O157:H7, Listeria, and Salmonella.

29.4 Direct Enumeration using Microscopic Technique

29.4.1 Breed-smear or DMC

The direct microscopic count (DMC) also known as Breed-smear is the method based on the microbiological examination of the milk sample. It is one the first method to be used for counting bacteria in milk. The basis of this method is that the representative sample of milk the average number of bacteria are counted to detect the quality of milk by comparing with the standard counts. The procedure involves preparing milk smear with breeds pipette on a grease free slide in a specified area with specific amount of milk is 0.01 ml milk in 1 cm2 area. It is stained with Newman's stain after fixing the organisms and then washed. The numbers of organisms are counted in different fields.

The microscopic factor is the ratio of area of the smear to the area of the microscopic field and calculated as

MF = 100 * 100/ πr2

Where r is the radius of the field
Counts = Average count x M.F. x Dilution factor

Advantages

This method is one of the most popular and most rapid one to be used in dairy microbiology.

Disadvantage

• This method involve the failure to stain,
• Irregular distribution of bacteria.
• This method include the use of a very small volume which leads to increased errors
• Large microscopic factor which limits the sensitivity to about 5,00,000 bacteria/ ml
• Operator fatigue resulting from prolonged use of the microscope.

29.4.2 Electronic counting and fluorescence labelling

Fluorescent stains have been used to count bacteria in wet or dried preparation of milk. Bacteria often stain better with fluorochromes than with conventional stains.

29.4.2.1 Bactoscan

The basis of Bactoscan is the use of fluorescent microscopy. The somatic cells and the casein micelles are dissolved chemically. Later bacteria are separated by continuous centrifugation. Bacteria thus obtained are incubated with proteases to remove the residual protein and then stained with acridine orange. The treated sample is applied in a thin film on the surface of rotary disc and passed under a microscope objective the fluorescent impulses in a microscope are converted into electrical impulses and recorded. The instrument has capacity to analyze 68 samples per hour with analysis time of 7 minutes. The method correlates well with the SPC with correlation of up to 0.88. The Bactoscan can be used to rapidly detect the milk of poor bacteriological quality and could be used to grade milks at the 100,000 bacteria per ml.

29.4.2.2 Flow cytometry

This method is new to microbiological slides. In this method, the microbial suspension must first be subjected to an enzymatic treatment for the purpose of disrupting aggregates. The cells are treated with flurochromes which bind to nucleic acid, protein etc. To get the stoichiometric binding to the cell, the fluorochromes are used at saturation levels. The fluorescent marked suspended cells are focused into a narrow flow path delimited by a liquid sheath which enters the detection system at high speed. The detection system consists of a fine laser brush which spans the liquid stream. If a cell passes through this brush the bound fluorochrome produce detectable and measurable fluorescent emission. This method is capable of examining at least 1,000 cells/ sec and is therefore very rapid.

29.4.2.3 Direct epifluorescent filter technique (DEFT)

The Direct Epifluorescent Filter Technique is rapid method for counting bacteria in milk which uses membrane filtration and epifluorescent microscopy. Membranes can be made from nitrocellulose, cellulose acetate esters, nylon, polyvinyl chloride and polyesters. Membrane filters are used in modified conventional methods for a variety of purposes:

• To concentrate target organisms from a large volume to improve detection limit
• To remove growth inhibitors
• To transfer organisms between growth media without physical injury through resuspension

DEFT is a direct method used for enumeration of microbes based on binding properties of flurochrome acridine orange. In this food samples are pretreated with detergents and proteolytic enzymes, filtered on to a polycarbonate membrane stained with acridine orange and examined under fluorescent microscope. The number of viable cells is determined based on the count of orange cells on the filter and can be performed in 10 min. Before filtration the milk (2 ml) is treated with proteolytic enzyme (trypsin) and a non-ionic detergent (Triton X-100) for 10 minutes at 50°C. This treatment enables lysis of somatic cells and dispersion of fat globules so as to enable the treated milk to be filtered through a 0.6 µm pore size polycarbonate membrane filter. Bacteria remain intact and are retained on the surface of the membrane. Then the membrane along with bacteria is stained with the acridine orange a fluorechrome, mixed with Tinopal AN (fluorescent brighter), and then counted by means of an epifluorescent (incident fluorescent) microscope.

Acridine orange can be used to differentiate deoxyribonucleic acid (DNA) from ribonecelic acid (RNA) by green as opposed to orange fluorescent. In the DEFT actively growing bacteria because of higher RNA content fluoresce orange. The bacteria can easily be distinguished from the small amounts of fluorescent debris present on the filter. The DEFT count is rapid, taking less than 25 minutes to complete, inexpensive and is suitable for milks containing 6000-10 million bacteria/ ml. The DEFT count correlates well (r = 0.91) with the SPC.

29.5 Viable Enumeration

It is done by using standard plate counts, most probable number (MPN), membrane filtration, plate loop methods, spiral plating etc.,

29.5.1 Plate loop method

The standard plate count is time-consuming and requires a considerable amount of equipment-pipettes and dilution bottles. This procedure requires the use of standardized loops (0.01 and 0.001) for making serial dilutions instead of pipettes and dilution bottles. (1). It consists briefly of: (1) a 0.01 and a 0.001 ml calibrated loop, Luer-Lok hypodermic needle, Cornwall continuous-pipetting outfit: Becton-Dickerson & Co. No. 1251 (consisting of a metal pipetting holder, a Cornwall Luer-Lok syringe and a filling outfit), 2 ml capacity, adjusted to deliver 1.0 ml. An approximate 30° bend has been made about 3-4 mm from the loop.

The PLC method employs 0.01 and 0.001 ml loops. The PLC counts using the 0.01 ml loop was determined on a 1:10 dilution of the same sample, thus the 0.01 and 0.001 both represented a 1:1000 dilution. Incubation was at 30°C.

29.5.2 Spiral plate

Spiral plater spreads continuously the sample using stylus (dispenser) moving away from centre to periphery of the petri-plate based on Archimedes principle and creating the dilution by means of difference caused in the distance of the deposition on the petri-plate containing the medium for a fixed volume of the sample delivered per unit time. Counts should be calculated, where possible, using dilutions giving 20 or more colonies on the plate.

29.5.3 Droplet technique

Standard Plate Count Agar (SPCA) is prepared in 9 ml amounts in screw capped test tubes. For each viable count, three test tubes of medium are to be melted and cooled to 45°C in a water bath. The bottom of a petridish should be marked with the sample number, and three equally spaced parallel lines on the outer surface.

One ml of the product suspension is to be pipetted out into the first test tube of cooled molten agar and well mixed. With a sterile dropping pipette delivering, 5 x 0.096 ml droplets were formed in the Petri dish along one of the marked parallel lines. Three drops (0.096 ml) were then added to the second test tube of agar. The contents of the test tube are thoroughly mixed using a fresh capillary pipette by repeatedly filling and emptying, and a second row of 5 droplets should be deposited along the second parallel line. A third dilution (0.096 ml in 9 ml) was made in the remaining test tube of cooled agar and droplets made in the same way above the third line in the Petri dish. The Petri dish then contained one total viable count (Fig. 29.1).

Fig. 29.1 Droplets in petridishes after incubation

A second count was placed in the lid of the same dish in exactly the same way. In some instances, droplets are overlaid with 1 or 2 drops of sterile agar to prevent growth of surface colonies. The dishes are to be counted after 24 and 48 hours at specified incubation temperatures.

Colonies were counted at 10x magnification using a bench lens, or stereomicroscope, or a specially designed projection viewer. The mean counts for up to 5 droplets are to be taken, depending on the number of colonies. For example, if the count exceeded l00/ droplet, only two are counted. Up to 200 colonies/ droplet could be conveniently counted (Fig. 29.2).

By using 9 ml quantities of agar and regarding the transferred volume as 0.1 ml, it was, in fact, 0.096 ml - the error introduced through the pipetted volume was insignificant. Possibility of error and the need for judgment in using dropping pipettes have been eliminated by a specially designed diluter/ dispenser.

Fig. 29.2 A droplet photographed from the ground glass screen of the viewer

29.5.4 Hydrophobic grid membrane filter

It is a direct method for counting viable bacteria. The principle involved in this is that it uses a membrane filter imprinted with hydrophobic material in a grid pattern, the hydrophobic lines divide the filter surface into compartments of equal and known size and act as a barrier to the spread of colonies.
Square occupied by colonies are counted and converted to MPN estimate of the organisms by application of the formula.

Where,

N = Total number of Squares
Log = Natural logarithm
P = Number of positive squares.

In this method bacteria are retained on the membrane filters after the filtration of the sample. The protocol of the method involves the placing of filter on a pad soaked in nutrient medium and counting the colonies formed after 1-3 days of incubation. The variation of this method is available in form of Hydrophobic Grid Membrane Filter (HGMF) given by Sharpe and Michand in 1974. The processing time is reduced as the dilution series is not required. Also efficiency is better as compared to conventional filters. This method has a square grid pattern printed in hydrophobic material likewise. On a conventional membrane filter dividing filter in 2000-4000 compartments depending on size of the grid. It functions as MPN technique and size variations and lateral species of colonies is prevented. Automated counting of colonies is done. The HGMF method has been used in the enumeration of coliforms in a variety of foods. The long incubation time is a disadvantage with this method.

29.5.5 Petrifilm

Other rapid method kits speed up standard microbiological methods by using special substrates, enzymes, or other apparatus. For example, a Petri film plate count card contains prepared media. One just adds one’s sample at the appropriate dilution and incubates it. One can then count the bacteria present in the sample. It is disposable and eliminates the need to make agar plates.

The 3M™ Petrifilm™ Plate Reader (Fig. 29.3) provides consistent, automated reading and recording of results of 3M™ Petrifilm™ Plates (Aerobic, Coliform, E. coli/Coliform Count and Select E. coli) in 4 seconds, thereby increasing productivity, reducing costs and eliminating variation between lab techs. The included software displays results and colour images of the plate on the computer screen, marking the colonies for easy verification.

Fig. 29.3 3M™ petrifilm™ plate reader

29.6 Enzyme/Substrate Based Detection Method

With a positive MUG test kit a special chemical reaction alerts the microbiologist that the organism one is looking for is present. One type of MUG test kit is called a Coli complete test. The discs are impregnated with two chemicals that react in the presence of coliforms and E. coli. One inoculates the tube, adds one of the discs, and incubates it. If a blue colour develops, one has a presumptive positive for coliforms. One then shines an ultraviolet light on the tube. If the tube fluoresces, one has a presumptive positive for E. coli (Fig. 29.4).

Fig 29.4 Enzymatic detection of E. coli and E. coli O157:H7

29.7 Metabolic Activity Measurement

An estimation of metabolic activity of the total cell population is possible using resazurin, methylene blue dye reduction, acid production, electrical impedance etc. The level of bacterial activity can be used to assess the keeping quality and freshness of milk. Toxin levels can also be measured, indicating the presence of toxin producing pathogens.

29.7.1 Dye reduction tests

The basis of the dye reduction tests is the ability to produce enzyme like dehydrogenases, which can transfer hydrogen from a substrate to a redox dye which undergoes change in color. The rate of reduction of color depends upon the enzyme activity which is taken as index of number of organisms present in milk. The reduction time is inversely related to the bacterial count of the sample. These tests are used for non-refrigerated, bulked raw milk.

29.7.2 Electrical methods

This method is based on the principle that the growth of micro organisms results in changes in the composition of the culture medium as nutrients are converted into metabolic end products. The complex uncharged molecules like carbohydrates or lipids are converted to simpler charged molecules like lactic acid and acetic acid. Charged proteins are poly peptides are converted to amino acids, bicarbonates etc. This overall conversion leads to the increase in conductance and capacitance of the medium in which they grow thus decreasing the Impedence.

These electrical changes in microbial cultures form the basis of detecting micro organisms and their metabolic effects. The impedence is defined as the resistant to flow of alternating current through a conducting material. A model with resistor and capacitor in series is formed completing the electrical circuit by placing the electrodes in the microbiological media. The following formula gives the total impedence in this model.

Z² = R² + 1

(28fc)²

Where

Z-----> = <---- Impedance, R = ----> resistance
C ---> = <---- Capacitor and f = --->frequency of alternating current

The impedance changes are detected by passing a small alternating current through media and comparing impedence of the inoculated medium with that of uninoculated one.

29.7.3 Pyruvate test

Pyruvate is the key intermediate of the metabolism as the lipid, polysaccharides and protein degradation leads to pyruvate formation. Thus estimating the pyruvate content can give the amount of bacterial contamination. It is suitable because of its pooling nature and high solubility. It is not affected by pasteurization and UHT.

The principle behind this method is an enzymatic reaction. In this presence of reduced form of co-enzyme NADPH₂, Pyruvate is completely converted to lactate by enzyme lactate dehydrogenase and NADH₂ is oxidized to NAD, Decrease in NADH₂ is proportional to amount of pyruvate at 340 nm. Pyruvate count of 2.25 ppm is correlated with 3 x 105 cells/ml and 11.0 ppm with 4 x 107 cells/ml.

29.7.4 Limulus lysate test

This test can be used rapidly and specifically to determine the cumulative content of Gram negative bacteria in foods. The G-ve bacteria produce a lipopolysaccharide (LPS) (endotoxin) which is a high molecular weight complex. This LPS is released into the surrounding medium after the death and lysis of cells, although some viable cells may also release. The horse shoe crab called amoebocyte, whose cytoplasm is packed with granules. In the presence of lipopolysaccharide the limulus blood clots.

This test is specific for LPS and very sensitive as little as 10^-12 g lipopolysaccharide per ml can be detected, occasionally even 10^-15 g/ ml. A single G-ve organism contains approximately 10^-14 g. Lipopolysaccharide, because of the extreme sensitivity of the test all utensils must be absolutely free from lipopolysaccharide (LPS).
In this test, a ten-fold dilution series of the sample is prepared and equal volume of limulus lysate is mixed in a test tube.

1. The tube is then incubated at 37°C for 4 h.
2. Before being inverted and read, if the mixture remains unchanged and runs down the wall of the tube then that dilution of the sample does not contain lipolpolysaccharide
3. If a firm opaque gel is formed which sticks to the bottom of the tube that dilution of the sample contains lipopolysaccharide
4. Generally visual reading of 10-fold dilution will give sufficient information about the level of polysaccharide present in the sample. The accuracy of the sample can be increased by using a 2-fold dilution series.

As the lipopolysaccharide is heat resistant, the limulus test has been used to assess the cumulative contamination of G-ve bacteria in dairy products. An estimate of the bacteriological quality of the raw milk is possible by applying the limulus test to UHT milk.

29.8 Cellular Constituents Measurement

29.8.1 Bioluminescence

The ATP is considered currency of energy for cell. The function and significance of this in the metabolism of living cells suggests that its assay should be an excellent monitor of biological activity in the sample. The firefly luciferase reaction, where light is produced by an enzymatic reaction, is frequently used as an assay as it is specific for ATP.

Following reactions occur here

The light is recorded by photometer and is proportional to the concentration of ATP. There are two factors which adversely affect the estimation of bacterial numbers by the measurement of ATP - first, non-bacterial ATP and second quenching of the emitted light. Many biological materials such as Urine, meat and milk contain non-bacterial ATP which must be destroyed before an accurate estimate of bacterial ATP can be made. In milk the somatic cells are lysed and then incubated with an enzyme. Apyrase to destroy the ATP released. Following this treatment, the bacteria are chemically disrupted and the released ATP measured. Failure to destroy the non-bacterial ATP will give a high estimate of bacterial ATP, and hence bacterial numbers. ATP has been used as an indicator of bacterial numbers in food. The bioluminescent method and SPC hold a 93% correlation for range of 10⁴ - 10⁷ cells/ml. These techniques are suitable for detecting raw milk of poor quality.

29.9 DNA/ RNA probes

Some of the rapid methods involve using antibodies, nucleic acids, or robotics to detect pathogens and toxins. Of these, the antibodies are most versatile and are used in various test kits. They take advantage of antibody-antigen interactions that are specific to a particular pathogen. A latex agglutination test works that way. If the reaction is positive, the latex beads cause the bacteria to clump.

The identification of bacteria by DNA probe hybridization methods is based on the presence or absence of particular genes. This is in contrast to most biochemical and immunological tests that are based on the detection of gene products such as antigens or chemical end products of a metabolic pathway.

The physical basis for gene probe tests stems from the structure of DNA molecules themselves. Usually, DNA is composed of two strands of nucleotide polymers wound around each other to form a double helix. These long nucleotide chains are held together by hydrogen bonds between specific pairs of nucleotides. Adenine (A) in one strand binds to thymine (T) in the complementary strand. Similarly, guanine (G) in one strand forms a hydrogen bond with cytosine (C) in the opposite strand. The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. Single-stranded molecules result and the DNA is considered denatured. When the pH or temperature is lowered, the hydrogen bonds are reestablished between the AT and GC pairs, reforming double-stranded DNA. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.

A gene probe is composed of nucleic acid molecules, most often double-stranded DNA. It consists of either an entire gene or a fragment of a gene with a known function. Alternatively, short pieces of single-stranded DNA can be synthesized, based on the nucleotide sequence of the known gene. These are commonly referred to as oligonucleotides. Both natural and synthetic oligonucleotides are used to detect complementary DNA or RNA targets in samples. Double-stranded DNA probes must be denatured before the hybridization reaction; oligonucleotide and RNA probes, which are single-stranded, do not need to be denatured. Target nucleic acids are denatured by high temperature or high pH, and then the labeled gene probe is added. If the target nucleic acid in the sample contains the same nucleotide sequence as that of the gene probe, the probe will form hydrogen bonds with the target. Thus the labeled probe becomes specifically associated with the target.

The unreacted, labeled probe is removed by washing the solid support, and the presence of probe-target complexes is signaled by the bound label. In addition to DNA, probes and/or their targets can be made of RNA. A number of commercially available gene probe kits use synthetic DNA probes specific for ribosomal RNA targets. DNA:RNA and RNA:RNA hybrids are somewhat more thermally stable than DNA:DNA duplexes, but RNA molecules are quite labile at alkaline pH.

29.10 Fluorescent Antibody Method

The use of fluorescently-labeled monoclonal antibodies, with detection by multi-parameter flow cytometry, was investigated for the rapid detection of salmonellas. Accurate detection of specific Salmonella serotypes was demonstrated down to levels of below 10⁴ cells ml^-1 (within 30 min) and 1 cell ml^-1 (after 6 h non-selective pre-enrichment). This level of sensitivity was attained even in the presence of high levels of other bacterial species that would otherwise have interfered with the results. With combinations of different antibodies, each with a unique fluorescent label, simultaneous analysis for two species was possible

29.11 Immunomagnetic Separation (IMS)

The isolation stage can be shortened by replacing a selective enrichment stage with non growth related procedures. IMS uses super-paramagnetic particles, which are coated with antibodies against the target organisms to selectively isolate the organisms from a mixed population. IMS is analogues to selective cultural enrichment, whereby the growth of other bacteria is suppressed while the pathogen of interest is allowed to grow. The separation process is consists of two fundamental steps, where the suspension containing target cells is mixed with immunomagnetic particles for incubation no longer than 60 min and finally, they are separated using an appropriate magnetic separator. In the second step, the magnetic complex is washed repeatedly to remove unwanted contaminants and the target cells with attached magnetic particles can be used for the further experiments. Polystyrene beads coated with iron oxide and antibodies are the most common magnetic carriers used for concentration and separation of selected microorganism from foods. The Immunomagnetic beads have been used for capture of E. coli O157:H7, Salmonella and Listeria. In recent years, applications of IMS coupled with PCR assays are showing very promising results for the detection of E. coli O157:H7, Salmonella enteritis and Listeria monocytogenes. The detection limit for IMS with PCR was 1 cfu/1-25 g of sample following enrichment for L. monocytogenes. The immune magnetic separation may be employed either directly or indirectly. However, in selective enrichment stage separation, chemical reagents are antibiotics are used to select pathogens,. Since reagents can be harsh and may cause cells stress are injury, LMS is a milder alternative to enrichment; also the elimination of selective enrichment step shortens analysis time. The major drawbacks of the IMS-based assays are the requirement of enrichment and a sample clean up step.