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Microbiological Services

 Food Microbiology

Microbiological quality of food and food product has become a major public health concern with the increasing number of reported and potential outbreaks of food borne diseases. This concern is reflected through the development and implementation of relevant government actions and programs such as the Hazard Analysis Critical Control Point Program (HACCP). This program puts strong emphasis on the importance of screening for pathogens and other microbiological contaminants in food and dairy products, and manufacturing processes and facilities.

PBR offers microbiological testing services using the methods approved by regulatory agencies such as CFIA (Canadian Food Inspection Agency) of Health Canada and USDA-FSIS (United States Department of Agriculture-Food Safety and Inspection Service). PBR strives to apply the most advanced analytical methods or technologies such as PCR (Polymerase Chain Reaction), immunoassays, flow cytometery, rapid microbial identification system (VITEK 2), etc. that are high throughput, significantly reduce analytical time and cost, and provide reliable results. PBR can validate and apply newly developed rapid screening methods to assist clients in shipping their products to the market faster.

A list of selected microbiological analyses for the detection, enumeration and identification of pathogens including bacteria, molds and yeasts is given in the following table.

 Microbiological Testing of Food and Feed


 
Aerobic and anaerobic Plate Count Enterobacteriaceae
Yeast and Mold Count Klebsiella pneumoniae
E. coli Staphylococcus aureus
E.coli 0157:H7 Pseudomonas aeruginosa
E. coli – Non-0157 Shiga toxin-producing (STEC) Shigella spp.
Salmonella species Yersinia enterocolitica
Listeria species Lactic Acid Bacteria
Listeria monocytogenes Total Coliforms/Fecal Coliforms
Bacillus cereus Mesophilic Aerobic Sporeformers
Campylobacter jejuni Mesophilic Anaerobic Sporeformers
Clostridium perfringens Other species of interest on request
 

 HACCP Services

The Hazard Analysis Critical Control Point (HACCP) Program is a new approach for monitoring the safety of food and food products, and the suitability and sterility of manufacturing processes and facilities. Regulatory agencies in both Canada and the United States continue revising and improving regulations concerning the quality and safety of food by incorporating stringent regulatory programs or standards such as HACCP, Good Manufacturing Practice (GMP) and ISO 9000.

PBR offers HACCP implementation programs and comprehensive microbiological services using standard testing protocols and applying new rapid screening methods to restaurants, food manufacturers, retailers, dairy, meatpacking and beverage plants and other food sector industries.

 Sterility and Sanitation Audits

Sterility audit is an integral part of GMP and HACCP and is routinely carried out to maintain a required level of sanitary conditions and sterile environment in a manufacturing facility or plant. Evaluation of microbial contamination of equipment, indoor air, critical areas, counter tops and working surfaces in the plant is essential to ensure standard quality and safety of both raw ingredients and finished food products. Specific microbial tests for sterility and sanitation audit are listed in the following table.

 Specific Tests for Sterility and Sanitation Audits


 
Total Coliform/Fecal Coliform Staphylococcus aureus
E. coli/E. coli 0157:H7 Clostridium perfringens
Fecal Streptococcus Group Pseudomonas aeruginosa
Salmonella spp Listeria monocytogenes
Fungal Count (Yeast/Mold) Mesophilic Aerobic Sporeformers
Heterotrophic Plate Count (Bacteria) Mesophilic Anaerobic Sporeformers
 

 Shelf-Life Studies

Shelf life is an important property of food products that provides a scientific basis for the setting of their minimum durability indication. More specifically, as defined by the UK Institute of Food Science and Technology, shelf-life represents the period of time during which the food product will remain, safe; retain its desired sensory, chemical, physical, microbiological and functional characteristics. Shelf-life is important to food manufacturers and processors as well as consumers.

PBR offers microbiological shelf-life testing with all applicable tests including challenge testing. In addition, PBR works closely with clients in planning the shelf-life studies of their products. Generally, the factors considered are: desired shelf-life durations; short (up to one week), medium (up to three weeks) or long (up to one year); safety considerations; storage conditions; and controlled conditions that mimic those the product is likely to encounter during storage.

PBR offers routine as well as, where applicable, accelerated shelf life testing (ASLT) for long shelf-life products. Some selected tests are listed in the table below.

 Specific Tests for Shelf Life Studies


 
Total Coliform Listeria monocytogenes
E. coli Staphylococcus aureus
Lactic Acid Bacteria (LAB) Bacillus cereus
Heterotrophic Plate Count (Bacteria) Fungal Count (Yeast/Mold)
Mesophilic Aerobic Sporeformers Mesophilic Anaerobic Sporeformers
 

 Water Microbiology

PBR offers complete analytical services for microbiological quality assessment and characterization of potable/drinking water for human consumption, health risk assessment, mortgage approvals, and treatment plant effectiveness. PBR also provides testing services for wastewater, effluent, sewage, sludge and surface and recreational waters for the assessment of microbiological contamination and potential environmental and public health impacts. Specific microbiological tests for these matrices are listed in the following Table.

 Microbiological Analysis of Water and Wastewate


 
Total Coliforms/Fecal Coliforms Staphylococcus aureus
E. coli and  E.coli 0157:H7 Clostridium perfringens
Fecal Streptococcus Group Pseudomonas aeruginosa
Salmonella spp. Pseudomonas cepacia
Klebsiella spp. Listeria species
Legionella spp. Listeria  monocytogenes
Legionella pneumophila Enterococcus group
Actinomycetes Nocardia
Bacillus cereus Yeast and Mold Count/identification
Heterotrophic Plate Count (Bacteria) Fecal Streptococcus Group
Mycobacterium Giardia and Cyrptosporidium
Candida albicans Enteric Viruses
Algae Other species of interest on request
 

 Indoor Air Quality Monitoring

Airborne mold, bacteria and other microorganism are known to cause asthma, hypersensitivity, allergic reactions, toxic effects and other disease manifestation in humans. The monitoring of the indoor air and environment for the presence and level of microbial contamination is therefore of paramount significance particularly for evaluating potential adverse health effects.

Comprehensive microbiological monitoring of indoor environment begins with a walkthrough survey for assessing the nature and extent of the problem and developing a monitoring strategy. This is followed by collection of indoor air samples; surface sampling; and  bulk sampling of  household dust, wood, carpets, and any materials suspected to have mold contamination.  In addition, water samples from drip pans, humidifiers and air conditioners may also be collected as part of the surveillance strategy.

The main objectives of monitoring indoor environment and air quality include determination of the presence and level of mold contamination, estimation of predominant molds, identification at genus and species level, and determination of contamination source for undertaking remedial actions.

The testing of the collected samples is performed  either by direct examination of field samples for the presence of mold spores or by performing viable counts using culturing techniques. The choice between direct examination and culture-based analysis depends upon the nature of the sample and the mandatory turnaround time.  Direct examination techniques such as spore trap analysis provide relatively quicker turnaround time.

PBR offers genus or species level identification of predominant mold and bacterial pathogens  along with comments regarding their environmental significance and potential adverse health effects.

PBR provides interpretation of test results based on the guidelines and exposure standards (limits) of relevant national and international organizations. These include: Health Canada, American Conference of Government Industrial Hygienists (ACGIH), United States Environmental Protection Agency (EPA), European Union (EU) and World Health Organization (WHO). A list of specific tests is given in the table below.

 Specific Tests for Indoor Air Quality Monitoring


 
Heterotrophic Plate Count, (Bacteria)    Aspergillus/Penicillium Group
Staphylococcus aureus Stachybotrys chartarum
Pseudomonas aeruginosa Bacteria and Mold Identification
Legionella species Microbial Biomass Determination
Legionella pneumophila Pet Antigen Detection
Bacterial Endotoxin Dust Mites Detection
Fungal (Mold/Yeast) Count Other pathogens of concern on request
 

 Equipment Rental and Sampling Supplies

PBR provides rental equipment, sampling materials, supplies, and offers advice for proper tests and procedures if clients are qualified to do the sampling. However, it is recommended that clients involve PBR’s qualified staff in designing the sampling scheme and protocol to ensure the relevance of the laboratory results to the problem under investigation.

 Soil Microbiology

PBR offers complete analytical services for microbiological quality assessment and characterization of agricultural and contaminated soils based on the tests listed in the following table. These tests and parameters are useful in determining the level and source of microbiological contamination as applicable to environmental impact assessment, soil bioremediation, and soil fertility evaluation.

 Specific Tests for Soil Microbiology


 
Microbial Biomass Determination Bacillus cereus
Heterotrophic Plate Count (Bacteria) Salmonella spp.
Nitrogen Cycle Bacteria Total Coliform/Fecal Coliform
Fecal Streptococcus Group Fungal (Yeast/Mold) Count
E. coli Any other  microbes on request
 

 Environmental Microbiology

PBR offers specialized testing for the presence and level of microorganisms that are of significance in diverse environmental and industrial processes and systems, including cooling waters. These involve microorganisms that are responsible for biological corrosion, biofouling, biofilm (slime) formation and other health hazards.

Corrosion and clogging of iron and steel pipes can occur due to the growth of microorganisms such as iron related bacteria (IRB’s) and sulfate reducing bacteria (SRB’s).

Biofouling is the undesirable accumulation of microorganisms, plants or algae on surfaces and structures that are in contact with water for a period of time. The three most important agents involved in biofouling are IRB’s, SRB’s, and Heterotrophic Aerobic Bacteria (HAB’s).

Biofilm is an aggregate of diverse microorganisms in which cells adhere to each other on a surface. These adherent cells are generally embedded within a self-produced matrix of extracellular polymeric substances, which are also referred to as slime. Biofilms may form on living or non-living surfaces and can be prevalent in natural, hospital, industrial and wastewater settings.

Presence of Legionella species in cooling towers, air conditioners, humidifiers and drip pans is a major health concern.

PBR can provide microbial consortia capable of degrading harmful contaminants in the treatment of industrial effluents and contaminated soils. PBR also provides testing for hydrocarbon utilizing bacteria and nitrogen cycle bacteria. Specific tests and parameters for all of the above matrices and processes are listed in the following Table.

 Specific Tests for Biofouling, Biofilm, Corrosion and Other Environmental Samples


Heterotrophic Aerobic Bacteria (HAB)  Iron Related Bacteria (IRB’s)
Slime Forming Bacteria and Fungi Microbial Biomass Determination
Pseudomonas aeruginosa biofilms Legionella spp.
Nitrifying/Denitrifying Bacteria Acidifying Bacteria
Hydrocarbon Utilizing Bacteria Wastewater Microbial Consortia
Sulfate Reducing Bacteria (SRB’s) Algae Enumeration and Identification

 Product Safety and Efficacy Evaluation

PBR provides specific testing services to determine and evaluate the anti-microbial activity and efficacy of various products, as well as to ascertain the level of bacterial and fungal contamination (product sterility). Most commonly tested products include probiotics, consumer and industrial products, antimicrobial agents, sanitizers and disinfectants.

Specific microbiological tests are performed using Standards and Protocols of the United States Pharmacopoeia (USP), Canadian General Standards Board (CGSB), Association of Official Analytical Chemists (AOAC) International, American Society of Testing Materials (ASTM) International and International Organization for Standardization (ISO).

 Bacterial and Fungal Identification

The genus and species level identification of microorganisms present in food, air, water, soil and other matrices is essential for the safety evaluation and assessment of potential health hazards, as well as for the required regulatory compliance and approval.    

PBR provides comprehensive microbiological services for the identification of non-pathogenic and pathogenic bacteria, mold and yeast in food, water, air, soil and other environmental samples. Generally, both bacteria and fungi are identified to the genus level, but species, strain and serotype levels identification is provided if and as required.

Basic microbiological techniques, microscopic examination, biochemical methods and specific automated systems are used for the genus and species level identification of bacteria, yeast and mold present and detected in diverse samples.
As an example, PBR uses the state-of-the art PCR technology and VITEK 2 automated system (from BioMerieux) that provide highly accurate and reproducible results for the identification of microorganism that are of clinical, hygienic and environmental significance. 

 Allergens and Toxin Testing Services

PBR offers comprehensive analytical services for the screening and detection of various allergens and specific toxins in foods, food products, natural health products and water using highly sensitive test kits and immunoassays. Testing is essentially conducted to prevent the occurrence of allergic reactions in sensitive individuals and to minimize environmental and public health concerns.

 Food Allergens

A food allergy is an adverse immune response to a food protein, and is distinct from other pharmaceutical and toxin-mediated reactions or food intolerance. Most allergic reactions could be prevented by simply avoiding potentially allergenic food or food products and carefully reading food labels.

People can be allergic to any food, but only a handful of foods are responsible for most (90%) allergic reactions. These common foods are known as the “Big Eight” and include milk, eggs, peanuts, tree nuts (such as walnuts, almonds, and hazelnuts), soy, wheat, fish and shellfish (such as shrimps, mussels, crabs).

In addition to food allergies, some people may react to certain other foods and their components such as Gluten.
PBR provides specialized analytical services for food allergens and gluten testing using the standard immunoassay kits.

 Cynobacterial Toxins

Cyanobacterial toxins are the naturally produced poisons stored in the cells of certain species of cyanobacteria (blue-green algae) and are released in the water when the cells rupture or die naturally or by a chemical disinfectant treatment. Cyanobacteria generally occur in warm, shallow, slow-moving or still waters, and their massive growth leads to the production of algal blooms, scums and mats in nutrient-enriched freshwater bodies like ponds, dugouts and lakes.

Two major groups of cyanobacterial toxins are called Microcystins and Nodularins. Microcystins are monocyclic heptapeptide liver toxins produced by species of cyanobacteria belonging to the genera Microcystis, Anabaena, Oscillatoria and Nostoc. Nodularins (monocyclic pentapeptides) are also liver toxins produced by the brackish water cyanobacterium Nodularia.

The occurrence of these cyanobacterial toxins in water bodies is a well-recognized public health hazard. These toxins have been linked to incidences of human illnesses including allergies, skin irritation, vomiting, diarrhea, fever, headache, and liver damage and cancer. The major human exposure routes include the consumption of contaminated drinking water, recreational use of polluted lakes and rivers and the consumption of certain algal health food products. Livestock, wildlife and fish exposure to microcystins is through the consumption of algal cells from contaminated water

Microcystins are extremely stable in water because of their chemical structure and can survive in both warm and cold water. They can withstand radical changes in water chemistry, including extreme pH.  About 60 different kinds (congeners) of microcystin have been found; however, Microcystin-LR is the most commonly found cyanobacterial toxin.

In Canada, the Federal-Provincial-Territorial Subcommittee on Drinking Water has recommended a maximum acceptable concentration for total microcystins in drinking water as 1.5 ppb (ug/L) or 0.0015 mg/L microcystin-LR equivalent. In USA, the FDA has established monitoring guidelines for blue-green algae based foods at 1ppm (ug/g) microcystin equivalent.

Microcystins and nodularins have been found to be potent inhibitors of protein phosphatase types 1 and 2A. Accordingly, a colorimetric protein phosphatase inhibition (PPI) assay has been described for the detection of microcystins and nodularins. PBR employs the PPI and specific immunoassays for rapid screening and quantitative determination of microcystins and nodularins in food and water.

 Mycotoxins

Mycotoxins are secondary metabolites produced by a variety of molds on several agricultural commodities under specific environmental conditions. It has been estimated that about 25% of the grain produced each year worldwide is contaminated with mycotoxins, which are capable of causing diseases (mycotoxicoses) through dietary, respiratory and dermal exposure routes.

Since mycotoxins generally resist decomposition or digestion, they can persist in the food chain in both meat and dairy products for a long time. They are generally not destroyed by either heating or freezing.

Based on the recognition that most fungal toxins occur in groups of chemically related metabolites, approximately 400 compounds are now designated as mycotoxins. Of these, about a dozen groups regularly receive attention because of their threat to human and animal health. In Canada, the mycotoxins that are of major significance and concern are the aflatoxins, trichothecenes (predominantly deoxynivalenol,DON, also called vomitoxin; diacetoxyscirpenol, DAS; nivalenol, NIV; T-2 toxin and HT-2 toxin), zearalenone (ZEN), fumonisins (mainly FB1), and ochratoxins (predominantly ochratoxin A, OA).

Aflatoxins (AF), a most potent human carcinogen, are commonly encountered in food and feed products imported in Canada from warmer tropical and subtropical regions and countries. The four major aflatoxins are called B1, B2, G1, and G2. Of these, aflatoxin B1 is considered the most potent natural Carcinogen, and is usually the major aflatoxin produced by toxigenic fungi.

Regulations and guidelines for recommended tolerance levels for several mycotoxins including aflatoxins have been established in Canada and USA.

PBR provides specialized analytical services for the screening and testing of mycotoxins in food, feed, dairy products, cereal grains, nuts, corn and other crops using highly sensitive immunoassays.

 Endotoxins

Endotoxins are heat-stable toxins associated with the outer membranes of certain gram-negative bacteria such as species of Salmonella, Shigella, Pseudomonas, Neisseria, Bordetella, Brucella, Vibrio and E.coli. They are not secreted by cells in the surrounding medium but are mostly released when the cells are disrupted or destroyed. Endotoxins, also called lipopolysaccharides (LPS), are a combination of lipid (Lipid A) and polysaccharide side chains.

The biological activity of endotoxins is mainly associated with the LPS, toxicity with Lipid A, and immunogenicity with the polysaccharide components. Boiling for 30 minutes does not destroy endotoxins. However, certain powerful oxidizing agents such as peroxides, superoxides and hypochlorite have been reported to neutralize endotoxins.

Exposure to large quantities of endotoxins produce hemorrhagic shock and severe diarrhea, while smaller amounts may cause fever, altered resistance to bacterial infection, leucopenia, leukocytosis, and numerous other health effects. Therefore, it is essential to remove all traces of endotoxins from drugs, injectabales, pharmaceuticals, and other biological products to prevent illnesses and adverse health effects in humans.

PBR uses a very sensitive Limulus Ameobocyte Lysate (LAL) assay, based on the Gel Clot Method, for detecting the presence of endotoxins in diverse samples. Most of the endotoxins testing are performed on Reverse Osmosis (RO) water (frequently used in dialysis), serum, drug vials, hospital specimens, pharmaceuticals products and a variety of environmental samples.

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