USP Sample Preparation Comparison

By Jason Kircos

Many companies use in house laboratories and perform using USP methodology. In these cases, when it comes to preparing their own media and reagents, the lab needs to pay special attention to USP because the quality of the results depends on the quality of the culture media prepared. This includes accurate weighing of dehydrated components, the use of high quality (USP purified) water, completely dissolving the dehydrated media or individual ingredients, and the need to control the heating of the media to avoid damaging heat-labile components of the media. The quality control of each batch of the media is a critical concern. However, a major issue with microbiological testing is the hidden costs in labor and inventory hold time.

The BioLumix system simplifies the testing due to the pre-made vials that do not require a technician to run quality control on the vial.

Sample preparation for the USP to BioLumix is very similar, so the transition for most laboratories is very simple. The benefit that is gained is the time it takes to get results and the ease of processing samples.

One example for sample processing is Yeast and Mold, which shows the advantage of the BioLumix system. Both the USP and BioLumix sample preparations start with a 10 gram sample into 90ml of either Butterfields Buffer or Fluid Soybean-Casein Digest Medium (TSB). The sample is then tested for pH to make sure it is the optimal range (6.0-8.0). From this point the BioLumix system shows how much easier and efficient an automated system is over the traditional methodology.

For the BioLumix system, the sample is then added to the vial, the information is added to the system, the sample starts running and 48 hours later, the data is archived and completed.

Now, compare that to the traditional USP methodology. First is the need to melt the agar used to pour the plates, and the time it takes to allow the media to temper down to an appropriate temperature that does not damage any possible organisms in the product. Then you must take the sample and pipette it onto the plate, and then pour the tempered media on the plate. Next, comes swirling the media and sample to evenly distribute it throughout the plate. Finally, then use must allow it to solidify and then place in the incubator for 5-7 days. Once the incubation is complete, read the plate and record the results.

The BioLumix system gives the result in 48 hours and automatically tells the customer if it is above or below the specification level. While the traditional USP methodology needs to wait 5-7 days before results are complete.

Many other assays that use the BioLumix system reduce the time to get results much quicker than USP. For example, total aerobic count which instead of 2-3 days using traditional USP methodology, is less than 24 hours on the BioLumix system.  E. coli testing takes three days using USP methodology but only two days using the BioLumix system.

The BioLumix System is a system that fits the needs of the supplement industry in terms of speed, working with all product matrices, and automatically documenting the results in real-time, thereby reducing the labor requirement for microbiological testing.

The BioLumix system was extensively validated for dietary products and compared to the methodology described in USP <2021> and <2022>.  The validation methodology followed USP <1223> “Validation of Alternative Microbiological Methods”.  This included performance criteria such as: side-by-side comparison to the USP methodology, accuracy and precision, specificity, limits of detection and quantification, along with ruggedness and robustness, reproducibility of the results, false positivity rate and false negative rate.

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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

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 Microbiological Testing of E. coli with the BioLumix Vial

Escherichia coli are Gram negative rod-shaped organisms found naturally in the lower intestines of warm blooded organisms. Most serotypes of this organism are relatively harmless, making up a small percentage of bacterial colonization in the gut. These serotypes prevent the establishment of pathogenic strains.

E. coli is one of the most common bacteria found in the gut of animals. This includes humans. There are other animal species that contain this organism in the gut including reptiles and fish. E. coli colonizes the gut and can cause infection in the urinary tract and brain stem (meningitis) as well as intestinal diseases referred to as gastroenteritis. There are five classes of E. coli that produce disease. The most serious disease is the Enterohemorrhagic (EHEC) class. These organisms can cause diarrhea distinct from some others (including Shigella) in that there is copious bloody discharge and no fever. The life threatening situation is its toxic effects on the kidneys (hemolytic uremia).

Why Test for the Presence of E. coli?

E. coli is often used as indicator organisms to test the effectiveness of effluent disinfection in a wastewater treatment plant, on animal products as well as in nutraceutical and pharmaceutical products. While these organisms are generally harmless, they do live under the same conditions that human pathogens live. Since we cannot test for every pathogen, we test for easily detectable indicator organisms. The assumption is that if we kill the indicator organisms then we most likely kill the pathogens during effluent disinfection. E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Current Methodology

The current methodology can take anywhere from 3-7 days and includes multiple broths, agars,
transfers and temperatures. Different methodologies are utilized by the various industries. Below
are some examples:

Nutraceutical and Dietary supplements: The protocol described in USP <2022> requires
absence of the organisms in 10 grams of product. A 1:10 dilution of product is made into TSB or
another enrichment medium. This mixture is incubated for 24 to 48 hours at 30°C to 35°C, and
then 1.0 mL is transferred to 10 mL of MacConkey broth. This mixture is incubated for 24 to
48 hours at 42°C to 44°C. A loop from the MacConkey broth is transferred to MacConkey agar
and the plate is incubated for 18 to 24 hours at 30°C to 35°C. If typical colonies appear, these
colonies are then transferred to Levine Eosin Methylene Blue agar and incubated 24 to 48 hours
at 30°C to 35°C. If none of the colonies exhibit green metallic sheen under reflected light or if
none of the colonies exhibit a blue-black appearance under transmitted light, the sample meets
the requirement for the absence of Escherichia coli. Because results can sometimes be misread
due to interpretation of plates, identification may be run on the sample(s) adding another 2-3
days of testing.

Pharmaceutical: The protocol described in USP <62> again requires the absence of E. coli in
10 grams of product. A 1:10 dilution is made and incubated for 18 to 24 hours at 30°C to 35°C.
From this mixture, 1.0 mL is transferred to 100 mL of MacConkey broth and incubated 24 to 48
hours at 42°C to 44°C. A loop is transferred to MacConkey agar and this plate is incubated 18 to
72 hours at 30°C to 35°C. Growth of typical colonies indicates the presence of E. coli which is
confirmed with an identification test.

Food Testing: The food industry follows Bacteriological Analytical Manual (BAM). In most
cases, the Most Probable Number (MPN) method is utilized. This is labor intensive, multi-
step assay consists of presumptive (in LST tubes), confirmed (in BGLB tubes), and completed
phases (in EC tubes). In the assay, serial dilutions of a sample are inoculated into broth media.
Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2
phases of the assay are performed and then uses the combinations of positive results to consult a
statistical table. From this table, the analyst is able to estimate the number of organisms present.
The 3-tube MPN test is used for testing most foods. The 5-tube MPN is used for water, shellfish
and shellfish harvest water testing, and there is also a 10-tube MPN method that is used to test
bottled water or samples that are not expected to be highly contaminated. Positive EC tubes
must be transferred to L-EMB agar and if typical colonies are observed, they must be further
identified.

The BioLumix E. coli Vial

The E. coli vial, or EC vial, is a membrane vial that is monitor by the fluorescent
signal in the instrument. The membrane separates the incubation zone from the
reading zone. If E. coli is present, it utilizes MUG (4-Methylumbelliferyl-3-D-
Glucuronide) through an enzymatic reaction to create fluorescence.
A 1:10 dilution is made in TSB or a similar growth medium and this mixture is
incubated for 18-24 hours at 35°C. From the TSB dilution, 0.1 mL is added to an
EC vial containing the MUG supplement and then tested in the BioLumix instrument. The EC
assay runs for 18 hours in the instrument. An
example of E. coli curves is shown in the graph. If
detection occurs, a confirmation test is performed.
From the time the sample is prepared to the time
the confirmation is complete, the EC test takes
only 2 days, saving valuable time.

The BioLumix assay is much simpler to perform, requiring less labor and disposables as any to
the standard method. It is faster (completed in 30 hours), accurate, automated, and it eliminates
any product interference due to the two zone vial.

References:

USP <2021> MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY
SUPPLEMENTS
USP <2022> MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED
MICROORGANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS
USP <62> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TESTSFOR SPECIFIED MICROORGANISMS
Peter Feng, Stephen D. Weagant, Michael A. Grant, 2002. Bacteriological AnalyticalManual BAM). Chapter 4: Enumeration of Escherichia coli and the Coliform Bacteria

Confirmation Testing of Presumptive Positive Assays Using the BioLumix System

In Microbiology, the initial test result using selective or differential media is called Presumptive Test. Most presumptive tests require confirmation. Confirmation can be accomplished using specific reagents and materials. However, due to the critical importance of testing for pathogens and/or objectionable organisms as contaminants; it may be necessary to perform identification of any organisms isolated from samples. Identification measures microorganisms to the species level.

Initial Testing

For testing of any sample for the presence of microorganisms it is critical to perform a measure of total organism counts (viable organisms). BioLumix provides testing for both Total Aerobic Counts (Bacteria) and for Total Yeast and Mold Counts (Fungi). The BioLumix system in this regard mimics testing for both bacteria and fungi using USP or BAM plate methodology. In these initial tests for total counts there isn’t any discrimination of objectionable organisms from common organisms and common flora. Objectionable organisms for Nutraceutical Samples as an example may include E coli, Staphylococcus aureus, Salmonella or Pseudomonas aeruginosa.

Use of Selective Media

For most samples, it will be necessary to test for at least some objectionable organisms. In order to perform tests for each specific objectionable organism it is necessary to use selective media specifically designed to select for the target organisms. For example, for E coli testing, it is necessary to use selective media that contains both inhibitors that prevent the growth of non-E coli organisms and substrates that can utilized by E coli and not by most other microorganisms. BioLumix make use of such a media, referred to as the EC vial.

Confirmation Test

Unique confirmation tests that can be performed directly from the vials are described for the various objectionable organisms.

E. Coli- Indole Test

For samples that grow and detect in the BioLumix system, a series of Confirmation Tests can be utilized to begin the process of understanding whether the organism(s) are genuine E coli or not. For E coli testing a common initial confirmation test is the Indole Test using the Kovacs reagent. The Indole Test measures the presence of any indole in the growth media as a by-product of tryptophan metabolism by E. coli. Figure 1 depicts a negative (yellow ring) and positive Kovacs Reaction (Red ring) at the top of the media in the test vial.

Staphylococcus aureus – Coagulase Test

The BioLumix vial for testing for the presence of Staphylococcus aureus contains inhibitors of non-Staphylococcus organisms and substrates, such as mannitol as the sole carbon source used by S aureus. If growth is found in the BioLumix STA vial, the analyst can begin to confirm the presence or absence of S. aureus, directly from the vial, using the classic coagulase tests. The coagulase test that has been used for decades uses a known antisera specific for S. aureus epitopes. When S aureus is present, the antiserum reacts with the specific epitopes and forms a lattice of antibody-antigen, and the material coagulates within hours. Figure 2 illustrates the coagulase positive (upper tube) and negative (lower tube) reaction.

Pseudomonas aeruginosa- Oxidase Test

The BioLumix vial for testing for the presence or absence of Pseudomonas aeruginosa contains inhibitors such as Centrimide to prevent the growth of non-pseudomonads and substrates for use by P. aeruginosa. If growth is found in the BioLumix PSE vial, the analyst can begin to confirm the presence or absence of P. aeruginosa using the classic oxidase test. When P. aeruginosa is present, the oxidase test strip reacts with the centrifuged precipitate material (bacterium) and yields a rapid dark blue reaction. This reaction is based on the presence of certain cytochrome oxidase that are found intracellularly in the P. aeruginosa.Figure 3 illustrates the positive oxidase color test ( + ) from the negative reaction ( – ).

Salmonella- Immunoassay Strip

The BioLumix vial for testing for the presence of Salmonella contains inhibitors of non-salmonella bacteria and substrates utilized by Salmonella. If growth is found in the BioLumix SAL vial; the analyst can begin to confirm the presence of absence of Salmonella using commercially available test kits that typically make use of Immunological reactive endpoints. One such kit is shown in the cartoon (Figure 4) and depicts immuno-reactive bands on a test strip.

Identification

Any negative confirmation assay indicates that the target organism is absent and the result is negative. However, in the rare occasions that the vial shows growth and the confirmation assay is positive, it does not necessarily mean that an objectionable organism is present. In these situations further identification of the growing organism might be required. The growing organism could be isolated on selective or non-selective medium and identified by any appropriate identification system.

A Novel Rapid Automated Method for Suitability Testing

Suitability Testing by USP Methodology

Suitability testing is performed in order to verify that the method utilized eliminates the effect of any
antimicrobial properties of the product. Therefore, the media diluent combination does not inhibit the
recovery and growth of microorganisms, if present in the sample. The goal of the suitability testing is to
establish the ability of the test to detect microorganisms in the presence of product.

The suitability described in USP <61> verifies the
validity of the testing method by showing the
recovery of microorganisms in presence of the
product. Total Aerobic Microbial Count and Total
combined Yeast and Mold can be carried out by
membrane filtration, pour plating or spread plate
method.

Suitability using USP <62> can use selective media to
detect various organisms such as: Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli,
bile-tolerant gram-negative bacteria, Clostridia,
Salmonella and Candida albicans. The samples are
first enriched by incubating in Trypticase Soy Broth
(TSB) or another appropriate neutralizing media, and then streaked onto selective agars for the
determination of presence of specified or the objectionable microorganisms.

The new USP <61> and USP <62> tests also provide harmonization to existing European Pharmacopeia
methods for testing non-sterile pharmaceutical products. Additional in order to verify the testing
conditions, a negative control is performed using the chosen diluent that shows no growth of
the microorganisms. While conducting the suitability testing precautions must be taken to avoid
contamination so they do not affect the microorganisms that are being tested. The procedure involves
the inoculation of the neutralized sample with low (not more than 100 cfu) and detecting the organisms
by the prescribed method.

Even though USP uses traditional microbiology methods, from the USP <61> and <62> states
that “Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopeial method has been demonstrated” and “any validated method,
including, Rapid Methods can be used”.

The New BioLumix Method

A study was recently conducted to show the utilization of BioLumix system (see figure below) in
suitability tests using a variety of products from both the pharmaceutical and cosmetic industries.

Sampling was conducted by taking ten gram of the product
and placing it into 90ml of M Letheen broth (or another
appropriate neutralizing broth) for a final dilution of 1/10.
An overnight culture of the target organism was diluted to
not exceed 100 cfu and the inoculum not exceeds 1% of the
volume of the diluted product. Then 1.0 mL of the
neutralized sample containing organism was placed into the
appropriate vial and a side by side comparison was done
with the appropriate USP method.

The products tested included Aloe, Hand Sanitizer, Lip Balm, Flavored Lip Balm, Medicated Lip Balm,
Breath Spray, Medicated ointment, and Sun Screen. A variety of different types of each product were
tested.

32 product samples were tested for suitability. Four bacteria (Staphylococcus aureus ATCC 6538;
Pseudomonas aeruginosa ATCC 9027; Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739; a
yeast (Candida albicans ATCC 10231), and a mold (Aspergillus niger ATCC 16404) were used to show the
effectiveness of the neutralization step. Thirty one
products were properly neutralized by the M Letheen
Broth as evidenced by detection time in the vials and
colonies on the plates. Only one product tested contained
a high level of ethanol, which required a 1:100 dilution in
M Letheen Broth to obtain neutralization. There was 100%
correlation between the two methods.

Typical data obtained by the system is shown in the figure:

The product was inoculated with ~ 100 cfu/g of three organisms: Staphylococcus aureus ATCC 6538 (light blue); Bacillus subtilis ATCC 6633 (Dark blue) and Escherichia coli ATCC 8739 (green). The Detection times obtained (shown as triangles on the curves) are comparable to data obtained without product.

What are the advantages of the BioLumix system?

Time Saving: The results are available much faster, for example, the results of the Yeast and Mold vialsoccurred in less than 48 hours, while the Aspergillus took nearly five days for countable colonies. All
products tested with bacteria using the automated BioLumix assay yielded results typically 10-13 hours,
instead of 48 hours for the plate count methodology. The advantage is that you can see results an
entire day early if the product is exhibiting any sort of inhibition. Data generation is slower using classic
microbiology so it can also slow down production improvements as well.
Labor Saving: The setup of the assay can be done much faster using the BioLumix system as opposed
to traditional plating methods, saving significant hands-on labor due to its automation and simplicity of
use. BioLumix can make the microbiological testing simpler, faster, and automated- saving significant
time and labor. It is paperless, increasing efficiency and saving on disposables, time and space, and best
of all the system is unaffected by product interference. The System is extremely easy to operate, with
its straightforward, streamlined testing design it offers accurate results leading to reduced material-
holding time for faster product release.

High Correlation with USP: The BioLumix System showed a high correlation between the instrument
results and the USP methodology. The system is fully automated with automated data archiving,
data maintenance in databases, and automated report generation. Regulators encourage rapid
microbiology methods for improved process control and product release. The BioLumix system
is validated as being at least equivalent to the compendial method. Under general notices of the
USP states that “Automated procedures employing the same basic chemistry as those assay and
test procedures given in the monograph are recognized as being equivalent in their suitability for
determining compliance.”

References:

USP <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

USP <62>: MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TEST FOR SPECIFICIED
MICROORGANISMS

Rapid Microbiological Testing of Capsules, Softgels and Pills Encapsulation of Pharmaceuticals

Pharmaceutical products may be encapsulated in a relatively stable shell for oral consumption.  These shells are called capsules and can either be hard-shelled or soft-shelled.  Hard-shell capsules are commonly filled with dry powdered ingredients, pellets, or granules.  Soft-shell capsules (softgels) are made from gelatin and primarily contain oils or active ingredients that are dissolved or suspended in oil.  Both hard and soft-shell capsules may contain colorants, dyes, opaquing, dispersing, or hardening agents, and preservatives.  Tablets are solid doses of medicinal substances and may be soluble effervescent, chewable, molded, or compressed.

Traditional methods for testing

Plate count methodology as described in USP <61> is regularly used to test capsules and tablets.  Using this methodology, it takes two days for Aerobic Count results, and five days for Yeast and Mold results.  When testing for objectionable organisms, it may take several days using selective broths or agars to determine the absence or presence of these bacteria.   The colors of the shells and the products and the viscosity of the 1:10 dilution sometimes interfere with the reading of the plates.

Rapid Detection with the BioLumix System

The BioLumix System simplifies testing, expedites time to results, reduces the testing cost and accelerates product release while providing better control of microbial contamination.  The system can be used to automate microbial testing with a more cost effective and streamline manner, and reduces the error rates produced by paper-based activity recording and batch data entry.  The BioLumix system also helps automate microbiological quality control processes.

To validate the equivalency of the BioLumix system to USP <61> or USP <2021>, over 100 types of capsules, softgels, and tablets were tested with the BioLumix system and plate count methodology at various specified levels.  The products were tested for total aerobic microbial count (TAMC), Total yeast and mold count (TYMC), and the absence of E. coli, P. aeruginosa, S. aureus, and Salmonella (objectionable organisms) in 10 grams of product.  For each assay, a 1:10 dilution was created by adding 10 grams of product to 90 mL of TSB and further dilutions were performed depending on the desired specified level.  Some of the samples were inoculated with various bacteria.


Figure 1 shows the BioLumix curves obtained for the Total Aerobic assay.  The purple curve represents an inoculated colored capsule.  All the curves along the baseline represent different kind of capsules that were not contaminated.


Figure 2 shows the BioLumix curves for the Yeast and Mold assay.  The dark blue curve represents an inoculated product. All the curves along the baseline represent different kind of capsules that were not contaminated.

 

As seen in Figure 3, the BioLumix vial design is separated into two zones: the incubation zone where the sample is present and the detection zone where the reading takes place.  Therefore, the system prevents any product interference.

Figure 3 shows two vials that contained dissolved blue capsules.  The sample on the right contained bacteria while the sample on the left was clean.

Final results are seen in the BioLumix system roughly 25-40 hours faster than the plate count method in the Total Aerobic Count assay and 40-72 hours faster in the Yeast and Mold assay.

When testing for objectionable organisms, results may be obtained several days sooner than the plate count method.  Following the BioLumix protocol, 0.1 mL of the incubated sample in TSB is added to a selective vial and data is collected in the BioLumix instrument for 18-24 hours, depending on the assay.  If detection occurs, the sample may be verified with a confirmation assay.  This procedure eliminates hours or days waiting for plate results.

The BioLumix system is faster, less labor-intensive, and more sensitive than the plate count method.  With an automated certificate of analysis generated within 48 hours, the time-to-results is reduced and allows for quick release of products.

Caron Ockerman

References:

USP <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS

USP <2021> MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS

USP <2022> MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MICROORGANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS 

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

Microbiological Testing of Gelatin Capsules

Introduction

Two-piece capsules have been used for almost a century in the pharmaceutical field, and gelatin has been adopted as the main material of these capsules due to its excellent characteristic as a gelling agent. The gelatin dissolves under high concentration into water of a high temperature and quickly gels in room temperature. The thickness of the film made by the gelatin becomes uniform.

The gelatin capsules consist of gelatin, plasticizers and water. Modern day shells may, in addition, consist of preservatives, colors, opacifying agents, flavors, sugars, acids, enteric materials etc. A mixture derived from pork skin and bones is used in capsules.  Pork skin gelatin contributes plasticity while bone gelatin gives firmness.

One important reason for the exclusive use of gelatin for making hard and soft capsules is its solubility characteristics in stomach fluids. It absorbs cold water readily, though the rate of absorption depends upon moisture content of gelatin. Bloom Strength is an empirical gel strength measure which gives an indication of the firmness of gel. The plasticizers used are glycerin, sorbitol etc.  Preservatives, if included, are generally a mixture of methylparaben (4 parts) and propylparaben (1 part) to the extent of 0.2%. Flavors, if added, should not exceed 2% and are generally ethyl vanillin or essential oils. Sugar, if included, may be up to 5% to give the gelatin shell desirable chewable characteristics.

Microbiology Testing

Each incoming lot of capsules needs to be tested using USP <61> and USP<62>. The total aerobic bacterial count should not exceed 3,000 cfu per gram, the combined yeast and mold counts along with Bile-Tolerant Gram-Negative Bacteria should not exceed 300 cfu per gram.  Material must also meet the requirements of the tests for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.

Challenges of Current Methodology

Testing gelatin capsules for microbiology might result in several challenges.  The 1:10 dilution of the product has high viscosity and is sometimes difficult to pipette. Many capsules have vivid colors that interfere the reading of the plates. 

The current methods used in microbiology originated over 100 years ago.  There have been limited improvements in methods used for microbiological testing in the last decade. The current USP methodology is slow, requiring up to 5-7 days for product release, is manual, and in many cases is inaccurate.  Paper-based QC laboratory processes can be expensive, error-prone, time and labor-intensive.  

Rapid microbiological methods (RMM) offer a cost effective alternative to USP methodology.  With an RMM’s high degree of automation, significant reduction in time to results, faster product release, and improved process control, while providing enhanced accuracy, better repeatability, and total automation

BioLumix Alternative

The BioLumix System simplifies testing, expedites time to results, reduces the testing cost and accelerates product release while providing better control of microbial contamination.  The system can be used to automate microbial testing with a more cost effective and streamline manner. The system reduces the error rates produced by paper-based activity recording and batch data entry.  The BioLumix system also helps automate microbiological quality control processes.

The BioLumix technology is based on continuous monitoring of changes in color or fluorescence as a result of microbial metabolism in ready to use test vials. The results are presented as soon as detection occurs without any involvement of the operator. The fully automated system offers a paperless operation with increase efficiencies.

The key to the technology is the two-zone ready to use vials which eliminates any product interference.  Many types of gelatin capsules were tested in the BioLumix system for total aerobic bacterial count, combined yeast and mold counts, Bile-Tolerant Gram-Negative Bacteria, and for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.  As can be seen in the figure, there is no product interference even with capsules containing the most vibrant colors.  All assays yielded clear results that correlated well with the USP methodology.

Key: Dark Blue-clear capsule;Green- Brilliant Red Capsule; Light Blue-Brown capsule;Red- Teal capsule; Purple-Inoculated Brilliant Red Capsule.

The BioLumix system is validated against the USP methodology.  All assay results are complete in 48 hours with an automated Certificate of Analysis; resulting in faster product release.

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.