Water testing- Heterotrophic bacteria, coliforms and E. coli- Why and how to test

Water Quality

Water QualityWater is used in a variety of different industries as well as products within various industries, including Nutraceutical and Dietary Supplement, Pharmaceutical, cosmetics, toiletry industries.  Water can be used as a product ingredient, for example, to create the capsules that contain the supplement.  In the manufacture of the capsules many companies use their own water to create and encapsulate their products.   Water is also used for the cleaning of certain equipment and contact surfaces.

According to USP 1231, although there are no absolute microbial standards for water (other than water intended to be sterile), the CGMP regulations require the establishment of appropriate specifications. The specification must take into account the intended use of the water; i.e., water used to formulate a product should contain no organisms capable of growing in the product. Action or alert limits should be established based upon validation data and must be set low enough to signal significant changes from normal operating conditions.

Control of the microbiological quality of water is important for many of its uses. All packaged forms of water are required to be sterile because some of their intended uses require this for health and safety reasons. The needed microbial specification for a given bulk water depends upon its use. Some applications may require even more careful microbial control to avoid the proliferation of microorganisms ubiquitous to water during the purification, storage, and distribution.

To ensure adherence to certain minimal microbiological quality standards, water used in the production of drug substances or as source or feed water for the preparation of the various types of purified waters must meet the requirements of the National Primary Drinking Water Regulations (NPDWR) (40 CFR 141) issued by the U.S. Environmental Protection Agency (EPA) or the drinking water regulations of the European Union or Japan, or the WHO drinking water guidelines. Microbiological requirements of drinking water ensure the absence of coliforms, which, if determined to be of fecal origin, may indicate the potential presence of other potentially pathogenic microorganisms and viruses of fecal origin. Meeting these microbiological requirements does not rule out the presence of other microorganisms, which could be considered undesirable if found in a drug substance or formulated product.

USP<1115> deals with bioburden of non-sterile drug substances and products, and the chapter states that the biggest manufacturing risk is water as an ingredient.  Process water is the single most important risk factor contributing to the contamination of nonsterile products.  The purified waters that are used in manufacturing are deionized and do not contain chlorine that helps control microbial growth.  Purified water is capable of supporting growth of gram negative rod shaped bacteria and many different molds.

Water TestingThe FDA also covers a wide range of different types of water that can be used for pharmaceutical uses and describes different sources for water contamination.  The FDA even states that microbial contamination of oral liquids and topical drug products are a significant problem that is usually caused by contaminated water.  Due to the potential health risks involved with the use of contaminated water, particular attention should be paid to the deionized (DI) water systems, especially at smaller manufacturers.

Chlorinated water may be appropriate for early stage cleaning and sanitization activities, but the uses are risky and should only be used on a case by case basis.  Microbial enumeration is an integral component of a water monitoring system to assess the microbial quality of the water.  Some systems use both high-nutrient (PCA) and low-nutrient (R2A) media to allow the isolation of both heterotrophic organisms and slower growing oligotrophic bacteria.

Water testing is also important when dealing with well water, tap water and even bottled water.  The EPA uses coliform as an indicator of possible fecal contamination.  Coliforms naturally found in the environment, and are usually non-pathogenic, but their presence may indicate fecal coliforms.

The Rapid Automated BioLumix System

BioLumix SystemBioLumix automated; all-in-one microbial testing system is an ideal system for in plant water testing.  The system is fast, simple and cost-effective.  A novel optical system sensing color and fluorescence in ready-to-use vials provides faster results, labor savings, automation, and connectivity. The BioLumix system is capable of testing water for heterotrophic bacteria, total aerobic bacteria, E. coli, coliforms, fecal coliforms and yeast and molds. Using the BioLumix system will quickly determine the microbial quality of the water.

Heterotrophic Vial: This vial can detect organisms requiring low-nutrient media (similar to (R2A) to allow the isolation of both heterotrophic organisms and slower growing oligotrophic bacteria. In a study, over 50 samples of multiple different water types were tested by the BioLumix method and the plate count method side-by-side.  The BioLumix vials were directly inoculated with 0.1 mL of the water sample, or a 1.0 mL of a 1:100 dilution, and a few samples were inoculated with heterotrophic bacteria.  The samples were monitored in the BioLumix instrument for 35 hours.  The results showed that the BioLumix system was roughly 13 hours faster than the plate count method using Stand Methods Agar.  These particular samples were tested at specified levels <10 cfu/ml and <100cfu/ml, but the BioLumix method can detect organisms at levels of <1 cfu/ml of water.

Bottled water for human consumption also needs to be tested for coliforms, which are indicators of possible contamination. The FDA requires either MPN or membrane filtration to check 100 ml of water for any contamination. The MPN method which requires at least nine tubes to perform the test and up to 96 hours of testing; while BioLumix can do the same analysis using just one vial in less than quarter of the time.  The filter method can also be applied using the BioLumix system by filtering the 100ml onto a membrane filter and placing the filter directly into the vial.

What are the advantages of the BioLumix system?

The system serves, as a platform to perform all required assays- using the BioLumix system will allow the users to test for coliforms, heterotrophic bacteria, E. coli and Yeast/Mold. The system can be used for water testing as well as for testing raw materials, in process and finished products.

Saving time- The BioLumix system can save time when testing water for Heterotrophic bacteria instead of taking three days using traditional plates, the BioLumix system will give the same results in 35 hours.

Economical cost of assays: Instead of running an MPN assay, which will require up to 5 days of testing as well as 9 tubes of LTB and up to 9 tubes of EC Media to wait for confirmation of a positive fecal coliform, the BioLumix system requires less than 24 hours and a single vial.

References:

http://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072925.htm -Water for Pharmaceutical Use

http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm064948.htm  Enumeration of Escherichia coli and the Coliform Bacteria

USP <1115> Bioburden Control of Nonsterile Drug Substances and Products

USP <1231> Water Treatment Systems For Industrial & Commercial Use.

Why is it important to test Pet Food for microbiology?

Pet Food microbiologyThe pet food industry is nearly a $22.2 Billion dollar a year industry and projected to almost double by the year 2017.  Nearly 101 million homes have at least one pet in the household, and there are nearly 170 million cats and dogs owned in the United States alone.  New trends in pet food are emerging as consumers want to give their pets the freshest food possible and make sure that it is healthier for them as well.  In the past few years there have been multiple outbreaks related to pet food affecting the health of both pets and humans.  Most people associate Salmonella as a bacterium linked to food borne illness in people food, but in recent years there have been quite a few outbreaks of Salmonella in pet food that has also affected humans.  The most concerning aspect is that it primarily caused illness in small children.

Several recalls of pet food due to Salmonella happened in the recent past as shown in the examples that follows. On February 5, 2014 – Pro-Pet LLC, has initiated a voluntary recall of a limited number of Dry Dog and Cat Foods for possible Salmonella contamination. A single field test indicated products manufactured during a two-day period, on a single production line might have the potential for Salmonella contamination1.  On January 25, 2014 – PMI Nutrition, LLC (PMI), has initiated a voluntary recall of its 20 lb. bags of Red Flannel® Cat Formula cat food for possible Salmonella contamination2. On November 4, 2013 – Bailey’s Choice LLC, had recalled its 5 oz. packages of chicken treats because they have the potential to be contaminated with Salmonella, an organism which can cause serious and sometimes fatal infections in young children, frail or elderly people, and others with weakened immune systems3.

The CDC also added an information page on keeping people and pet healthy and safe from Salmonella4.  There was also a pet food recall based on an aflatoxin contamination.  The Center for Disease Control (CDC) categorizes aflatoxin as a naturally occurring fungal toxin that contaminates maize and other types of crops during production, harvest storage or processing5.  The aflatoxin outbreak was linked to the death of over a hundred pets.  In the past year Kroger stores recalled a wide variety of pet foods due to a possible contamination caused by aflatoxin4.

Microbiology Testing of Pet Foods

Why test for indicator organisms? It is more effective to test for indicator organisms rather than to test for pathogens such as Salmonella.  Indicator organisms are used to measure potential fecal contamination of environmental samples. The presence of coliform bacteria, such as E. coli, is a common indicator of fecal contamination. Indicator organisms are typically used to demonstrate the potential presence or absence of groups of pathogens. The use of indicators is attractive because it reduces the complexity and cost of analyzing. Indicator bacteria are selected for the following reasons:

1) They are initially abundant in the matrix to be assayed.

2) A relatively rapid, accurate, and cost effective analytical method for enumerating the indicator exists or can be readily developed.

3) A reasonably strong correlation exists between the presence/absence of the indicator and a particular pathogen or group of pathogens. The strength of the correlation will determine the effectiveness and accuracy of the indicator as a measure of pathogen occurrence.

4) Indicator organisms can be used to pet food manufacturing to cleanliness and sanitary issues within the facility.

Assays Performed on pet foods: in pet food, testing is conducted for Enterobacteriaceae or fecal coliform as indicator of fecal contamination and yeast and mold as indicators for general quality and aflaxoins.

What are the advantages of the BioLumix system?

The system serves, as a platform to perform all required assays- using the BioLumix system will allow the pet food manufacturers to test their products not only for Salmonella and yeast/molds, but also for indicator organisms such as coliforms, fecal coliforms, Enterobacteriaceae and more.

Saving time- The BioLumix system can save time when testing pet food products for Yeast and Mold, instead of taking five days using traditional plates, the BioLumix system will give the same results in under 48 hours.  This can help the manufacturers to avoid a potential aflatoxin contamination by knowing if their product contains any amount of mold.

Economical cost of assays: Instead of running an MPN assay, which will require up to 5 days of testing as well as 9 tubes of LTB and up to 9 tubes of EC Media to wait for confirmation of a positive fecal coliform, the BioLumix system requires less than 24 hours and a single vial.  Finally, the last confirmation step is to streak the positive EC Media to L-EMB agar plates; the BioLumix system instead requires one test vial and 1ml of the sample in order to detect a level as low at <10 cfu/gram, and can give results in under 24 hours.  Similarly, the Enterobacteriaceae test in BioLumix requires one vial instead of multiple MPN tubes required by the European method.

Screening Products: BioLumix Rapid Microbiology Testing can also be helpful in screening products to determine what the next steps are.  Some manufacturers sample the product from the line and test for total aerobic count. If the level is below a certain number, then the product can be sent out to the market, if it is above the specification level then it has to go through a special sterilization procedure which costs more money as well as a delay in the product reaching the customer.

BioLumix Pet Food Study

BioLumix originally conducted a study of different store bought pet foods, ranging from dry dog food samples to wet (oil based) samples.  All samples matched the results for Yeast/Mold, Enterobacteriaceae, Total Aerobic Count, E. coli and fecal coliforms when comparing between the BioLumix System and traditional plating methods.  The products were processed and tested using FDA-BAM methods7.

Total Aerobic Count: There was 100% agreement between the two methods for all samples tested. Fourteen samples were below the specified level by both methods.  One sample was above the specified level by both methods.  One sample was inoculated to show the ability of the system to detect positive samples.

Yeast and Mold Count: There was 100% agreement between the two methods. Fifteen samples were below the specified level by both methods and two samples were above the specified level by both methods. One samples was inoculated with yeast or mold to show the ability of the system to detect positive samples.

Enterobacteriaceae: There was 100% agreement between the two methods. Thirteen samples were below the specified level by both methods and two samples were above the specified level by both methods.

E. coli: Fifteen products were tested for E. coli at a level of Absence in 10 grams.  There was 100% agreement between the two methods. Fifteen samples were below the specified level by both methods. One sample was inoculated with E. coli and were detected as containing E. coli by both methods

Salmonella: Ten products were tested for Salmonella at a level of Absence in 25 grams.  There was 100% agreement between the two methods. Ten samples were below the specified level by both methods after a confirmation step. One sample was inoculated with Salmonella and was detected as containing Salmonella.

BioLumix has also conducted a study using fresh pet food, which is an emerging product in the marketplace.  The study yielded similar results as the initial BioLumix study, except Lactic Acid Bacteria was also tested.

The BioLumix System showed a high correlation between the instrument results and the BAM methodology.  It simplified the microbiological testing, offers a significant reduction in time to obtain results and reduces hands-on labor due to its automation and simplicity of use.  The time to results for bacteria was hours rather than days while yeast and mold required only 48 hours instead of 5 days.

  1. http://www.fda.gov/Safety/Recalls/ucm384876.htm
  2. http://www.fda.gov/Safety/Recalls/ucm374043.htm
  3. http://www.cdc.gov/features/salmonelladrypetfood/
  4. http://www.cdc.gov/features/salmonelladrypetfood/
  5. http://www.cdc.gov/nceh/hsb/aflatoxin/
  6. http://www.prnewswire.com/news-releases/kroger-recalls-pet-foods-due-to-possible-health-risk-112125284.htm
  7. http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm2006949.htm

International Dairy Show 2013

International Dairy Show 2013
November 3–6, 2013, McCormick Place • Chicago, Illinois

International Dairy Show 2013The International Dairy Show is back in Chicago and is bigger, and better than ever. Dairy, food and beverage processors will see technology and innovations in formulation, processing, packaging, distribution, food safety, and more. Operations, research, and sales and marketing teams will attend to learn trends, cost-cutting innovations, and ways to deliver more value to your customers. We at BioLumix wanted to take this opportunity to invite you to our exhibit (booth #452) and show you what’s new at BioLumix. We continue to innovate, growing our assay repertoire and capabilities.
BioLumix has developed a simple rapid microbiological method for the detection of various groups of bacteria, yeasts and molds. The system can make the microbiology testing simpler, faster and automated, saving you time, labor and money. BioLumix has a comprehensive range of microbiological tests, including specific vials for Coliforms and Pseudomonas. The system is designed to accelerate product release in a simplified automated approach. Complete coliform test results are obtained within 12 hours with one vial substituting for nine or more MPN (Most Probable Number) test tubes. Yeast and mold results are obtained in 48 hours rather than 5-7 days using plate methods. Many products can be introduced directly into the BioLumix test vial without the need of a dilution step. These include yogurts and sour cream, as well as milks.
Dairy product Testing for Yeast and Mold and Coliform
The yogurt can be directly added (Figure 1) into BioLumix vials to measure growth of Coliforms or Yeast and Molds. Specialized high pH BioLumix vials can also be used for yogurt samples. When a low pH yogurt sample is added to the BioLumix high pH Coliform vial (CC), the pH conditions become near neutral. This ensures the yogurt manufacturer to be able to correctly test for coliforms using a direct (without dilution) sample of product. As much as 1 gram of product can be directly tested in each BioLumix test vial.

A clear advantage of the BioLumix vial is that it can be used on the same work-day to yield data about the status of any contamination in dairy samples and thus enable the manufacturer to make a decision on the acceptability of the lot of product for sale and consumption.

Rapid Detection of Pseudomonads in Dairy Products as an Indicator of Product Shelf-Life The data presented in this study suggested that the BioLumix Pseudomonas vials are capable of early detection of Pseudomonads in dairy products and in process water. The system offered a reduction in time to results as compared to the plate methodology and eliminates any product interference. It allows for rapid assessment of any post processing associated problems.

The BioLumix Advantage
• Vial design prevents product interference
• Automation and connectivity allows faster product release
• Real-time communication for immediate action
• Expedited results: most results in 12-18 hours; Yeast and mold assay in 48 hours
• Automated data archiving and audit trail
• Streamlined testing increases laboratory efficiencies
• Paperless laboratory: centralized test data automatically stored and protected
• Barcode capability for automated sample entry
• Environmental testing made easy

Free Product Trial
Give us your most difficult samples and we will test them for free. We will provide you with a detailed report which includes a side-by-side comparison to your current manual methodology. The data generated is strictly confidential and is only used to show the high correlation of results should your company decide to purchase.

We look forward to working with you and earning another satisfied customer!

Rapid Microbiology Testing of Dairy Products using the BioLumix Instrument

Background

Testing Dairy Products using Rapid Microbiology with the BioLumix InstrumentCommercial Dairy Products need to be tested for microbial contaminants.  Dairy products include: cultured dairy products (kefir, cultured buttermilk, sour cream, and yogurt), cheeses (soft cheese products, semi-hard, hard, and extra hard cheeses), processed cheeses, butters, butter creams, dried milks, and ice creams.

Manufacturers require a simple, cost effective and rapid microbiological method to assess samples for the presence of specific organisms as an indication of the sanitary conditions.  The most common dairy microbiology tests include: Aerobic plate count, Yeast and Molds, Coliforms, and could also include some indicator of cold spoilage such as Pseudomonads.  Rapid and early testing for microorganisms can reduce product quarantine time, allows for faster response to contamination, decreases inventory holding and cold warehouse costs, and aids in prediction of product shelf-life for manufacturers.

Troublesome spoilage microorganisms include aerobic Psychrotrophic bacteria, yeasts, molds, heterofermentative lactobacilli, and spore-forming bacteria (Ledenbach and Marshall 2009).  Psychrotrophic bacteria can produce large amounts of extracellular hydrolytic enzymes, and the extent of recontamination of pasteurized fluid milk products with these bacteria is a major determinant of the milk products shelf life. Fungal spoilage of dairy foods is manifested by the presence of a wide variety of metabolic by-products, causing off-odors and flavors, in addition to visible changes in either color or texture and may also cause gassing.

BioLumix has developed a simple rapid microbiological method for the detection of various groups of bacteria, yeasts and molds.  The system can make the microbiology testing simpler, faster and automated, saving manufacturers time, labor and money. BioLumix has a comprehensive range of microbiological tests, including specific vials for Coliforms and Pseudomonas.  The system is designed to accelerate product release in a simplified automated approach.  Complete coliform test results are obtained within 12 hours with one vial substituting for nine or more MPN (Most Probable Number) test tubes.  Yeast and mold results are obtained in 48 hours rather than 5-7 days using plate methods.  Many products can be introduced directly into the BioLumix test vial without the need of a dilution step.  These include yogurts and sour cream, as well as milks.

BioLumix Applications

Yogurt Testing

Yogurt is a dairy product, which is made by blending fermented milk with various ingredients that provide flavor and color.  Manufacturers have responded to the growth in the yogurt market by introducing many different types of yogurt including low fat and no-fat, creamy, bio-yogurt, organic, baby, and frozen.  BioLumix has developed assay methods for measuring contaminating microorganisms in dairy products.

The yogurt can be directly added (Figure 1) into BioLumix vials to measure growth of Coliforms or Yeast and Molds.  Specialized high pH BioLumix vials can also be used for yogurt samples.  When a low pH yogurt sample is added to the BioLumix high pH Coliform vial (CC), the pH conditions become near neutral.  This ensures the yogurt manufacturer to be able to correctly test for coliforms using a direct (without dilution) sample of product.  As much as 1 gram of product can be directly tested in each BioLumix test vial.

Coliform organisms generally include four key groups of enteric bacteria.  They comprise of the following species: Escherichia, Citrobacter, Klebsiella and Enterobacter.  Each of these organisms can grow in the BioLumix high pH CC vial.  Figure 2 illustrates the growth of a coliform (Citrobacter) in the BioLumix CC vial (green) while the Dark Blue curve shows a clean sample.

A clear advantage of the BioLumix vial is that it can be used on the same work-day to yield data about the status of any contamination in dairy samples and thus enable the manufacturer to make a decision on the acceptability of the lot of product for sale and consumption.

Fermented Dairy Products

Yeast and Mold Testing: An example of the growth of a mold in cream cheese is shown below. Figure 3 illustrates the clean test result, using BioLumix Yeast & Mold vials, for the cream cheese product as shown in the Green curve.  The Dark Blue curve illustrates the growth curve in BioLumix YM vials when cream cheese was inoculated with the mold organism Geotrichum candidum.

Coliforms Testing:Similarly, testing of sour cream showed the lack of Coliforms in the product as illustrated in the Dark Blue curve in Figure 4.  The sour cream product sample was found to have other flora as shown by growth in the BioLumix TAC vial which detected total aerobic bacterial counts.  Thus, the BioLumix high pH CC vial was used to show selectivity for the presence or absence of Coliform organisms.

Rapid Detection of Pseudomonas in Dairy Products as an Indicator of Product Shelf-Life
Pseudomonad organisms are a major cause of bacterial spoilage of pasteurized milk and dairy products due to post process contamination.  Early detection of Pseudomonas in can be a predictor of product shelf-life as it is the predominant psychotropic bacteria present.  BioLumix has developed a rapid method for the detection of Pseudomonads in dairy products; the method is also applicable to detection of Pseudomonads in process water.

The BioLumix system was directly compared to the plate count methodology for milk samples stored at refrigerated temperatures and held overnight at room temperatures and to detected Pseudomonads in process water and other dairy products.

Commercial milk products were used to measure the presence of Pseudomonads during refrigerated and elevated temperature storage.  Pseudomonads were present at varying levels in dairy samples and were detected within 16-24 hours using BioLumix vials.  All process water samples tested were free of Pseudomonads by both methods.  Process water samples inoculated with different Pseudomonads strains were found to detect in the BioLumix system.  The vial were selective enough not allowing for growth of unrelated gram positive and gram negative bacteria, mold or yeast.  The combination of the Pseudomonas vial with overnight pre-incubation could serve as an indicator of shelf-life of products.

The data in this study suggests that the BioLumix Pseudomonas vials are capable of early detection of Pseudomonads in dairy products and in processing water. The system offers a reduction in time to results as compared to the plate methodology and eliminates any product interference.  It allows for rapid assessment of problems associated post processing problems.

BioLumix has developed a vial specific for testing of Pseudomonads that are known to commonly contaminate milk and also survive pasteurization, albeit in low numbers. These Pseudomonads may include P aeruginosa, P fluorescens, P putida, and P stutzeri.  Each of these species of pseudomonas was found to grow readily in the BioLumix PSE-B vials.

Summary

The BioLumix System is designed to accelerate product release with a simplified, automated approach.  This yields fast, accurate, real-time results while reducing costs and eliminates the time required for the assays to be completed.  The system offers real-time results of contaminated samples saving hours, possibly days.  The Coliform or Enterobacteriaceae results can be available within 12 hours; Yeast and Mold results within 48 hours.  Thus the BioLumix system allows for rapid detection of Coliforms and Yeast and mold.  For low coliform numbers, one vial can substitute for 9+ MPN tubes.  BioLumix will streamline and simplify the microbiological procedures, save labor and create a paperless laboratory, while generating results that correlate well with plate count methodology.

Reference
Ledenbach L. H. and R.T. Marshall. 2009 In “Compendium of the Microbiological Spoilage of Foods and Beverages” . Chapter Microbiological Spoilage of Dairy Products.

The Significance of Coliform Testing

Complying with FDA and EPA rules regarding food and water sanitation is essential for every manufacturer of food and bottler of water and other beverages. This is especially significant for the dairy and meat industries, where food products are exceedingly susceptible to contamination by bacteria like E. coli, salmonella and many others. Due to the numerous possibilities of contamination, coliform testing is a priority. It should be performed regularly to indicate problems in the production and packaging processes.

Coliforms as Safety Indicators

Testing for the presence of coliforms is the standard way to indicate the possible additional presence of more harmful pathogens in food, beverages and water. While some of these “indicator bacteria” are frequently present in our everyday lives to a certain extent, they, themselves usually only cause issue when they reach elevated numbers, such as with E. coli bacteria. It is present in most warm-blooded animals including people, and exists harmlessly in most cases. When numbers elevate, or certain strains are introduced however, this is when illness is more likely to occur.

What is more important about the presence of E. coli is that it indicates the likelihood of the presence of other, more harmful organisms, which is why it is considered to be an indicator organism. Numerous other coliforms behave in the same way, so they have become useful in pinpointing where there are issues in food and water supplies. Essentially, if the indicator organisms are present, there should be strong suspicion that some other, more harmful pathogens that live in the same environments as these indicators are present as well.

Quality Control with Coliform Testing

The importance of regular coliform testing for food producers and processors should be a fairly obvious one, although there is much more that can be learned through this testing. Depending on which type of pathogen is found, and at what point in the production or packaging process it is found, steps can then be taken to remedy the problem quickly, which protects both the manufacturer and the consumer. Manufacturers can refer to their suppliers and packagers to locate the source of either poor handling or contamination, and take the necessary steps to remove the risk of pathogens quickly, which reduces waste and the need for more massive, widespread product recalls.

Quick and Convenient Results with BioLumix

Given how significant coliform testing is, and the effect that it can have on food and beverage production, and the substantial losses that can occur with contamination, having answers as soon as possible is essential. This is where the biolumix system shines above the rest, since we have developed a highly accurate testing method that provides those answers in as little as two days, as opposed to the 7 to 8 days that most other types of coliform testing require. In terms of efficiency, our system offers the fastest means for manufacturers, bottlers and packagers to find problems with raw ingredients or within their processing techniques, and isolate them before production has continued for upwards of a week, creating huge production losses.

The debate: Coliforms, Fecal coliforms, and Enterobacteriaceae as Indicator Organisms

Which one Should you Use?

What are Indicator Organisms and why use them?

Indicator organisms are organisms used as a sign of quality or hygienic status in food, Dietary supplements, water, or the environment. The initial goal in finding a safety indicator was to find a group of bacteria that could indicate the presence of fecal material and serve as a surrogate for Salmonella, but was easier and simpler to detect. Such a group may signify the potential presence of pathogens, a lapse in sanitation as required in good manufacturing practices (GMPs), or a process failure.

The longest used indicator organism was the coliform group that was recommended for use in the early 1900s for water testing. Fecal coliforms and E. coli followed as more specific indicators of potential presence of pathogens. The Pasteurized Milk Ordinance includes a requirement of coliform testing of pasteurized for milk and milk products.

Many different types of safety indicators have been proposed for use in particular applications. A thorough review of the indicator organisms is given in Tortorello (2003).

Definitions

Coliforms

Coliforms are gram negative, oxidase negative, non spore-forming, aerobic or facultative anaerobic rod shaped bacteria. The coliform group is not a distinct valid taxonomic group, but is defined functionally as organisms that ferment lactose with both gas and acid production at 35°C. The coliform members include Citrobacter, Enterobacter, Escherichia, and Klebsiella. Some also add to the group Serratia and Hafnia to the coliform group. Many of these bacteria are found naturally in the intestines of humans and animals, and some are even found naturally in soil and water. However, of the 1% of coliforms found naturally in the human gut, E. coli represents the majority and is found exclusively in the intestines of humans and animals. It is important to note that many of the coliforms can be found also in plants and the environment, thus, a positive coliform test does not necessarily indicate fecal contamination.

Enterobacteriaceae

The family Enterobacteriaceae encompasses approximately 20 genera, including E. coli and all members of the coliform group; in addition it includes foodborne pathogens Salmonella, Shigella, and Yersinia. The family was originally proposed as an indicator alternative to the coliform group because testing for the entire family would be more inclusive for the pathogenic bacteria. The Enterobacteriaceae may be superior to coliforms as indicators of sanitation GMPs because they have collectively greater resistance to the environment than the coliforms. This group is more widely used as indicators in Europe than in the United States. The determining factor separating coliforms from Enterobacteriaceae is the ability of coliform to ferment lactose, while the Enterobacteriaceae family ferments glucose.

Fecal Coliform

These organisms are a subset of the total coliform group. The fecal coliforms have the same properties as the coliform group, except that the fermentation is able to proceed at 44.5°–45.5°C. They are considered a better indicator of fecal contamination than the coliform group.

E. coli

E. coli is present in all mammalian feces at high concentrations; it does not multiply appreciably, but can survive in water for weeks, and so it is useful as an indicator of fecal pollution of drinking water systems. E. coli meets all the criteria used for the definition of both total coliforms and fecal coliforms. In addition, the organism can be distinguished from other fecal coliforms by the lack of urease and the presence of B-glucuronidase enzymes.

When to test and how to test

There are some regulations in various industries that require testing of one or several of these organisms. In some industries, product manufacturers use these indicators to assure that there is no lapse in sanitation or process failure.

Coliform

This test is required in the dairy industry, bottled water and drinking water. Many producers in the food industry also utilize the coliform test, especially in the USA. The BioLumix Coliform vial (CC-C) is useful for water testing, for food, and in some cases also in dietary supplements. The coliform vial contains lactose as the sole carbon source and selective ingredient such as bile salts. The assay is simple to perform – just add the appropriate amount of the liquefied sample to the ready-to-use vial and run it in the instrument at 35°C for 16-18 hours.

Enterobacteriaceae

USP(ENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS) recommends the testing of Enterobacterial Count (Bile-Tolerant Gram-Negative Bacteria) in dietary supplements. Most European producers prefer to use this test instead of the coliform test for foods. The BioLumix Enterobacterial vial (ENT-C) is very similar to the Coliform vial, except the medium contains glucose in addition to lactose. It is mainly used for dietary supplement industry.

Fecal coliforms

The fecal coliform test is used instead of the coliform test in industries where it is considered to be more directly associated with fecal contamination from warm-blooded vertebrates than are other members of the coliforms, such as in seafood, nuts, etc. The BioLumix coliform vial is used for this application however is incubated at an elevated temperature of 44.0°C.

E. coli

E. coli testing is required in drinking water systems. Also, USPsuggests the testing of Dietary supplement for the absence of E. coli in 10 grams of product. In the meat industry there are regulations relating to the presence of generic E. coli on carcasses.

The BioLumix E. coli vial (EC) contains a highly selective medium and includes MUG that is the most commonly used fluorogenic substrate for the detection of E. coli. It detects the activity of glucuronidase by E. coli. For tests with a specified level of 10 cfu/g of product, the liquefied sample can be added directly into the EC vial and the vial is run in the instrument at 35°C for 18-20 hours. For tests with a specified level of absent in 10 grams, the samples are diluted 1:10 in TSB for 18-24 hours followed by the addition of 0.1 mL of sample into the EC vial.

BioLumix has them all!

The BioLumix system is ideal for testing of any of the indicator organisms. With the BioLumix automated microbiological system, users have control over their testing, leading to accurate results and accelerated product release while reducing costs. The BioLumix Advantage:

  • All assays can be performed on one Automated Microbial testing system
  • Fast, automated results of all assays
  • Can be operated by non-Microbiologist
  • Complies with FDA cGMP Regulations
  • System is validated during system installation
  • Designed to accelerate product release
  • Fully automated data archiving
References:
  • Ockerman, C. “Rapid Microbiological Testing of E. coli with the BioLumix Vial” (2012). BioLumix Blog.
  • Tortorello, M. “Indicator Orgnanisms for Safety and Quality – Uses and Methods for Detection: Minireview.” Journal of AOAC International 86 (2003): 1208-1217.
  • United State PharmacopeiaENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS (2005). The National Formulatory . Rockville MD.
  • United State PharmacopeiaMICROBIOLOGICAL ATTRIBUTES OF NONSTERILE NUTRITIONAL AND DIETARY SUPPLEMENTS. (2005). The National Formulatory . Rockville MD.

Rapid Coliform, Yeast and Mold Testing of Yogurt

Testing of Yogurt and Other Dairy Products

Yogurt is a dairy product, which is made by blending fermented milk with various ingredients that provide flavor and color.  Manufacturers have responded to the growth in the yogurt market by introducing many different types of yogurt including low fat and no-fat, creamy, bio-yogurt, organic, baby, and frozen.  The popularity of yogurt has increased due to its perceived health benefit resulting in significant increase in consumption (Chandan et. al 2006) as shown in Figure.

Recently Reuters reported that Dannon, the world’s largest yogurt maker, announced that it expects annual double digit percentage dairy sales growth in the United States over the long term.

Microbiological Testing of Yogurt

Undesirable microorganisms constitute the primary hazard to safety, quality, and wholesomeness of milk and dairy foods.   The primary assays performed in yogurt are yeast and mold (requiring up to 7 days for results) and the coliform assay that is used as an indication of appropriate processing. Traditional methods are slow, tedious, labor intensive, and often not suitable for assessing the quality and shelf-life of perishable dairy products. 

 The emphasis on the programs based on HACCP (Hazard Analysis and Critical Control Points) for total quality management in the dairy industry and increased demand for microbiological surveillance of products, process, and environment have led to increased interest in rapid methods and automation in microbiology.

BioLumix technology can make the microbiological testing simpler, faster, and automated.  Saving time, labor and money. 

The BioLumix test method for detection of coliform and yeast & molds involves a direct addition of the sample into ready to use vials and automated monitoring of the samples in the instrument.  The coliform assay is completed in 12 hours, while most contaminated samples are flagged within a typical shift.  The yeast & molds assay is completed in 48 hours as compared to the 5 days required for the standard assay.

The ready–to-use vials come with media that is pre-adjusted for pH, such that after the addition of the sample the appropriate pH for the growth of the microorganisms is attained.  BioLumix has developed such a media that is custom made with a higher pH to accommodate the low pH of yogurt while still maintaining the capability to test as much as 1.0 gram of product directly in the vials.    

Coliform Assay:

The curves obtained by the addition of 1.0 gram of yogurt to a high pH vial (pH 8.2) of coliform are shown:

The Dark Blue curve shows the 1.0 gram sample.  All product interference has been eliminated as depicted by the flat part of the curve.  The Green Curve illustrates a yogurt sample inoculated with a coliform.




In North America, BioLumix has tested yogurt samples manufactured by a number of companies.  The Table below includes a series of products from one such company Yoplait, Ontario, Canada.  These samples include low fat and regular varieties. 

Lemon Cream Pie Light Fat Free
Key Lime Pie Original 99% Fat Free
Strawberry Shortcake Light Fat Free
Raspberry Mousse Whips
Strawberry Thick & Creamy Low Fat
Key Lime Pie Original
Delights Parfait Chocolate Raspberry

All un-inoculated products resulted in flat curves as seen in the Figure.  Product interference was not found.

All products tested were shown to be clean (<1 coliform/ gram).  All inoculated products detected in the system in less than 8 hours.

Yeast and Mold Assay:
Similar results were obtained for yeast and molds. The curves show the results obtained with a variety of yogurt products.

Fieldberry Stirred Yogurt Dark blue
Raspberry Stirred Yogurt Green
Raspberry Stirred Yogurt-inoc CAD L- blue
Yoplait Mingo Red
Yoplait Mingo-inoc ASP Purple
Tubes- Raspberry Yellow
Tubes- Grapes Tan
Irresistible Creamy yogurt Vanilla Dark green

The inoculated samples (light blue- inoculated with Candida albicans; purple inoculated with Aspergillus niger) detected in less than 24 hours.  All the un-inoculated samples were clean and did not detect in the vials.  A total of 50 different yogurts were tested using this method and the data showed that all the samples that contained either yeast or mold detected in the system and none of the clean samples detected.  The plate count results agreed 100% with the BioLumix vial results.

Conclusion:

The BioLumix system is designed to accelerate product release with a simplified, automated approach. This yields fast, accurate, real-time results while reducing costs. The BioLumix offers real-time results of contaminated samples saving hours, possibly days. Completed Coliform or Enterobacteriaceae results are obtained within 12 hours; Yeast and Mold results are obtained within 48 hours.

References:

R.C. Chandan, C.H. White, A. Kilara, and Y.H. Hui. 2006. in Manufacturing Yogurt and Fermented Milks. Blackwell Publishing.

Pet Food and Microbiology

Why is it important to test Pet Food for microbiology?

In the past few years there have been multiple outbreaks related to pet food affecting the health of both pets and humans.  Most people associate Salmonella as a bacterium linked to food borne illness in people food, but in recent years there have been quite a few outbreaks of Salmonella in pet food that has also affected humans.  The most concerning aspect is that it primarily caused illness in small children.  It was not believed to be caused by the children eating the dog food, but having interactions with the dog.  After it was discovered that the outbreak was caused by tainted dog food, over 23,000 tons of pet food was recalled, and when the outbreak continued, the plant that produced the tainted dog food was closed down.  There was also a pet food recall based on an aflatoxin contamination.  The Center for Disease Control (CDC) categorizes aflatoxin as a naturally occurring fungal toxin that contaminates maize and other types of crops during production, harvest storage or processing1.  The aflatoxin outbreak was linked to the death of over a hundred pets.  In the past year Kroger stores recalled a wide variety of pet foods due to a possible contamination caused by aflatoxin2.

However, it is more effective to test for indicator organisms rather than to test for pathogens such as Salmonella.  Indicator organisms are used to measure potential fecal contamination of environmental samples. The presence of coliform bacteria, such as E. coli, is a common indicator of fecal contamination. Indicator organisms are typically used to demonstrate the potential presence or absence of groups of pathogens. The use of indicators is attractive because it reduces the complexity and cost of analyzing

Indicator microbes are generally selected for the following reasons:

1) They are initially abundant in the matrix to be assayed.

2) A relatively rapid, accurate, and cost effective analytical method for enumerating the indicator exists or can be readily developed.

3) A reasonably strong correlation exists between the presence/absence of the indicator and a particular pathogen or group of pathogens. The strength of the correlation will determine the effectiveness and accuracy of the indicator as a measure of pathogen occurrence.

In pet food testing is conducted for Enterobacteriaceae or fecal coliform as indicator of fecal contamination and yeast and mold as indicators for general quality and aflaxoins.

What are the advantages of the BioLumix system?

Using the BioLumix system will allow the customers to test their products not only for Salmonella and yeast/molds, but also for indicator organisms such as coliforms, fecal coliforms, Enterobacteriaceae and more.  Indicator organisms can be used to pet food manufacturing to cleanliness and sanitary issues within the facility.  Also the presence of the organisms can affect appearance, taste and texture of the pet food.  The BioLumix system can save time when testing pet food products for Yeast and Mold, instead of taking five days using traditional plates, the BioLumix system will give the same results in under 48 hours.  This can help the manufacturers to avoid a potential aflatoxin contamination by knowing if their product contains any amount of mold.  Detecting fecal coliforms is even faster and saves even more money using the BioLumix system.  Instead of running an MPN assay which will require up to 5 days of testing as well as 9 tubes of LTB and up to 9 tubes of EC Media to wait for confirmation of a positive fecal coliform.  Finally, the last confirmation step is to streak the positive EC Media to L-EMB agar plates; the BioLumix system instead requires one test vial and 1ml of the sample in order to detect a level as low at <10 cfu/gram, and can give results in under 24 hours.  Similarly, the Enterobacteriaceae test in BioLumix requires one vial instead of multiple MPN tubes required by the European method.

BioLumix Pet Food Study

BioLumix recently conducted a study of different store bought pet foods, ranging from dry dog food samples to wet (oil based) samples.  All samples matched the results for Yeast/Mold, Enterobacteriaceae, Total Aerobic Count, E. coli and fecal coliforms when comparing between the BioLumix System and traditional plating methods.  Since there are no specifications by AAFO or FDA for indicator organisms in pet foods the levels tested for Enterobacteriaceae were based on European standards for pet food.   The products were processed and tested using FDA-BAM methods3.  Only one sample came up positive for fecal coliforms, results of the BioLumix vial matched the MPN results.  Two of the samples had counts >10 cfu/g for Enterobacteriaceae and were the only ones that were above the specification level of cfu’s of the manufacturer. 

The BioLumix system detects optical changes in the test vial, presenting results of the assays as soon as detections occur with no need for involvement of an operator or a microbiologist, providing significant savings on laboratory labor. Any out-of-spec samples are flagged in red, demanding attention. The greater the contamination level, the faster the result, ensuring a rapid warning of poor-quality raw materials, finished products or any equipment line issues.

The BioLumix System showed a high correlation between the instrument results and the BAM methodology.  It simplified the microbiological testing, offers a significant reduction in time to obtain results and reduces hands-on labor due to its automation and simplicity of use.  The time to results for bacteria was hours rather than days while yeast and mold required only 48 hours instead of 5 days. 

  1.  http://www.cdc.gov/nceh/hsb/aflatoxin/
  2. http://www.prnewswire.com/news-releases/kroger-recalls-pet-foods-due-to-possible-health-risk-112125284.html
  3. http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

Rapid Detection of Coliforms in Yogurt

What are coliforms?


Coliforms are a group of bacteria commonly found in the environment, including soil, surface water, vegetation and the intestinal tracts of warm-blooded animals. Most coliforms do not cause disease, but a small percentage can cause illness in people, especially young children, the elderly, and those with weakened immune systems. Coliforms are rod-shaped Gram-negative non-spore forming organisms. They can ferment lactose with the production of acid and gas when incubated at 35-37°C. Coliform bacteria are not a traditional taxonomic group, like Salmonella, Escherichia coli, or Listeria. Instead, the coliform bacteria are a collection of strains in the Enterobacteriaceae family. E. coli, Enterobacter spp., Klebsiella spp., and Citrobacter spp. are the most common coliforms.

How do coliforms get into milk?

Coliform bacteria are normally shed in the feces of healthy livestock, including dairy cattle. Thus, poor herd hygiene, contaminated water, unsanitary milking practices, and improperly washed and maintained equipment can all lead to elevated coliform counts in raw milk at the dairy farm. The milking of cows with wet and manure-soiled udders and inadequately cleaned milking equipment are the most common ways for coliform bacteria to enter milk on-farm.

Coliforms in milk: What does it mean?

The coliform bacteria count is used as an index of the level if sanitation and/or water quality employed in the handling and processing of milk products. In dairy products, the process of pasteurization easily kills coliform bacteria. Therefore, the finding of coliforms in pasteurized products indicates some level of contamination has occurred after pasteurization during product manufacturing or packaging. Hence, coliforms are used as a general indicator of sanitary conditions in dairy production and processing environments.

Traditional methods for testing Coliforms


Traditionally the agar plate count method using VRBA (Violet Red Bile Agar) and the MPN methods are being used.  The plate count method takes 24 hours to perform and can use 1.0 ml of 1:10 dilution and as a result has a sensitivity of <10 cfu/gram.

When higher sensitivity is required the MPN (Most Probable Number) method can be used.  Multiple tubes (typically 3) are inoculated for each dilution.  The method involves the inoculation of at least 3 decimal dilutions (a total of 9 tubes) of LST (Lauryl Tyrptose Broth).  Any LST tube that shows growth and gas production is transferred to BGLB (Brilliant green lactose bile), the MPN is calculated based upon the positive BGLB tubes.  This assay is very labor intensive and takes 3-5 days to complete.

Rapid High volume Automated Method for the Detection of Coliforms

Direct inoculation of Product and Speed to Results


In a recent study 25 different types of yogurts were tested by the BioLumix method and the plate count method.  The BioLumix ready to use vials were directly inoculated with 1.0 ml of various yogurts and monitored in the BioLumix system for 12 hours.  Figure 1 shows the curves obtained.  Key:  Dark blue – Grape yogurt, Green – Grape yogurt inoculated with Citrobacter, Lt Blue – Raspberry yogurt, and Red – Raspberry yogurt inoculated with E. coli.

 All products tested using the automated BioLumix assay for coliforms yielded results in a considerably shorter test time (typically 10-12 h) than the conventional Violet Red Bile Agar (VRBA) method (24 h to 72 h with confirmation). The BioLumix method can detect <1 cfu/gram of product, being 10 times more sensitive that the plate count method.  The BioLumix coliform medium had better selectivity in detecting coliforms, eliminating the need for the confirmation step.

Labor Savings

The BioLumix System offers a significant reduction in time to obtain results while reducing hands-on labor due to its automation and simplicity of use.  The procedure involves 2 simple steps:

  1. Add 1.0 ml of product directly to the ready to use vial
  2. Add the sample information to the computer

The system automatically will run the system and generate the desired reports. Therefore, there is no sample preparation required no media preparation or counting of plates.  Due to its simplicity the assays can be performed by less skilled personnel.

Automation

The BioLumix system is a fully automated system offering automated data achieving, and automated reporting.  It offers the ability to operate the laboratory as a paperless operation. Due to its connectivity through the Intranet it allows for automated transfer of product release information to the warehouse.

Free Product Trial

Interested in a free product trial?  Contact BioLumix today!  P.734-984-3100.