Microbiological examination of food and water

Prepared bySamira fattah

Assis. Lec.College of health sciences-HMU

Lecture 11

Why we need to examine food and water?

1. to know the level of sanitation used during

handling.

2. to know whether the food / ingredient meet the normal standard/ guidines.

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

4. To determine the source / load/ type of microorganisms in foods and water.

Criteria for choice of methods

1. Accuracy2. Sensitivity3. Easy of implementation4. Equipment/ operating cost5. speed

Common Test Methods• Although a wide range of technologies are used for the

identification and verification of microorganisms, but among these technologies, three types of method are commercially popular.

These are:• Culture Media,• Immunoassay• Polymerase chain reaction.

Culture Media

• Traditionally, cultural techniques have been the tests of choice for both ready-to-eat foods and fresh produce.

• However, today immunoassay and PCR methods are more accepted than cultural methods, because recent developments of newer testing methods and validation studies have demonstrated that cultural methods aren't suitable for all food groups.

• The traditional way of detecting and identifying bacteria from food, or other samples, is based on:

- Culturing- Enumeration- isolation of presumptive colonies for further

identification analysis.

• If necessary, the food sample must be homogenized, concentrated, and/or

pre-enriched prior to culturing.

• Bacterial cells can become injured or viable but nonculturable (VNC) due to the sublethal stressors, such as:

- Heat- cold- acid- osmotic shock during the food processing steps.

• These bacterial cells still pose a threat in the food industry and therefore, methods to improve the detection levels of these injured cells have been developed.

The pre-enrichment of the bacteria in a food sample can be performed by a:

- non-selective - or selective broth culture - or by the selective agar overlay

technique to resuscitate the injured cells.

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

• The pre-treated food sample can then be plated on:- non-selective- selective - differential media

• Non-selective media or standard methods agar, such as the aerobic plate count, can be used to detect and count the amount of bacteria in the sample.

• Selective medium contains a compound, such as an antibiotic, bacteriocin, a growth nutrient, which selectively inhibits the growth of specific microorganisms.

• Eg: PEMBA medium ( polymyxin pyruvate egg yolk mannitol bromothymol blue agar ) for the isolation and enumeration of Bacillus cereus in foods. Its able to detect small numbers of Bacillus cereus cells and spores in the presence of large numbers of other food contaminants.

PEMBA

• The third type is a differential medium which contains an indicator, such a chromogenic or fluorogenic substrate, which differentiates bacteria by various chemical reactions carried out during growth.

• By incorporating fluorogenic or chromogenic enzyme substrates into a selective media, identification of microorganisms can be done directly without further subculturing or biochemical tests.

• These culture media are based on bacteria producing specific and exact enzymes for substrates.

• As the enzyme then acts with the substrate( fluorogenic or chromogenic) the bacterial growth will fluoresce or change color, respectively.

A fluorogenic medium used to detect and enumerate E. coli and coliforms.

Chromogenic Media for Salmonella detection from food

samples

• Even though the culture methods are time consuming (Time to attain results can range from twelve hours to more than a week) and laborious.

• the isolation and purification of microorganisms allows for further subtyping analysis and for storage in culture collections.

• The more conventional methods for further subtyping of bacteria include the study of the phenotypic characteristics of the microorganisms.

• These phenotypic methods include:- Biotyping- serotyping,- phage typing .

• In biotyping some aspects of bacteria are investigated such as:

- the biochemical growth requirements.

- environmental conditions (pH, temperature, antibiotic resistance).

- physiological (colony and cell morphology, cell wall

composition by microscopy and membrane composition such as by fatty acid analysis)

• while serological and phage typing concentrate more on the surface structure differences of bacteria.

• Phages are not only useful in subtyping bacteria, but also in detecting pathogens directly from foods .

• However, these phenotypic typing methods are limited since microorganisms are capable of suddenly altering their phenotypic characteristics due to environmental changes or genetic mutations.

• Therefore, identification by genotypic characteristics has been developed to avoid these problems that can occur with phenotypic methods.

Immunoassay• Immunoassays are based on the specific bindings between

antigens and antibodies.

• Historically, they were used mainly in clinical diagnostics, but are becoming popular in rapid detection of microorganisms from food samples.

• Several types of immunoassays have been used in food sample analysis, including:

- enzyme linked immunosorbent assay (ELISA)- immunodiffusion tests- immunofluorescent microscopy- immunomagnetic separation- immunoprecipitation.

• Currently, a couple of commercial immunoassay-based detection systems are available to detect toxins or microbes in the food system, for example:

- the ImmunocardSTAT! E. coli O157:H7detection kit.

• Because of its accuracy and automation capability, most commercial immunoassay kits for food testing are based on ELISA.

• ELISA usually takes 1 h for toxin and 20-36 hours for cells .

• However, ELISA still has its limitations. For instance, without enrichment, the sensitivity of ELISA may not be enough to detect the microorganisms at very low level.

• Therefore, the time required for sample analysis including the pre-enrichment and/or enrichment procedures could be much longer than ELISA itself.

Polymerase Chain Reaction (PCR)• Polymerase chain reaction is the amplification of a nucleic acid target

sequence.

• The targeted sequences can be a specific gene or repetitive areas in the sequence .

• For foodborne bacterial pathogens, commonly targeted DNA areas are :

- virulence factors- Toxins- cellular metabolites- multicopy ribosomal RNA.

• The PCR-based techniques have also been developed for screening of genetically modified organisms and their derived materials in foods .

• Post- PCR detection methods vary from gel electrophoresis and usage of specific nucleic acid probes. In some cases, probes simplify the detection of the PCR product, in the similar way as gel electrophoresis.

• some PCR use DNA primers and fluorescent probes specific to the target organism in the same reaction mix.

•Detection of a fluorescent signal for the targeted amplified sequences indicates the presence of the pathogen in the sample being tested.