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 Testing for Gram negative Bacteria- the BioLumix Approach

Gram Negative Bacteria- Cell Wall

Gram negative cell walls are more complex than Gram positive cell walls, both structurally and chemically. Structurally, a Gram negative cell wall contains two layers external to the cytoplasmic membrane. Immediately external to the cytoplasmic membrane is a thin peptidoglycan layer, which accounts for only 5% to 10% of the Gram negative cell wall by weight. External to the peptidoglycan layer is the outer membrane, which is unique to Gram negative bacteria. For Gram negative species, many of the lytic virulence factors such as collagenases, hyaluronidases, proteases, and beta-lactamase are located within the periplasmic space.

The outer layer of the Gram negative bacterial cell wall is made up of lipopolysaccharide and proteins. This outer layer protects a thin layer of peptidoglycan. For the Gram positive bacteria, the outer layer of the cell wall is the peptidoglycan layer and does not contain lipoproteins. Below the outer layer of lipopolysaccharide, there exist layers of periplasmic space and the plasma membrane.

The pathogenic capability of Gram-negative bacteria is often associated with their cell walls, especially, the lipopolysaccharide layer (also known as LPS or endotoxin layer). In humans, LPS can triggers an immune response characterized by cytokine production and immune system activation.

Gram Negative Bacteria- Who are they?

Important gram negative bacteria include: the Enterobacteriaceae family, Pseudomonads, Aeromonas, Plesiomonas, Xahothomonas, Burkholderia, Haemophilus.

The Enterobacteriaceae family contains a number of pathogens including E. coli, Salmonella, Shigella, Klebsiella pneumoniae, Pasteurella, and Yersinia. Pseudomonas aeruginosa is an opportunistic pathogen and can cause urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, etc. Pseudomonas aeruginosa is found in many natural and domestic environments including plants, soils and surface water, especially warm moist environments containing organic material or contaminated by human or animal waste.

Members of the genus Aeromonas and Plesiomonas are involved in human intestinal disease. The species Hemophilus influenzae is a cause of meningitis in children, while Pasteurella. multocida causes cholera in fowl. Burkholderia cepacia, an important pathogen of pulmonary infections in people with cystic fibrosis.

Current methodology

In USP <61-62> the test for Bile-tolerant Gram-negative Bacteria (BTGNB) replaces the “Test for Enterobacteriaceae and Certain Other Gram-Negative Bacteria”. In older USP protocols. USP <62> does require testing for absence of P. aeruginosa and Burkholderia cepacia.

The method usually involves a 1:10 dilution using Soybean–Casein Digest Broth (TSB) as the diluent, and incubation at 20 to 25 C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours). Thereafter the appropriate amount is sub-cultured on Violet Red Bile Glucose Agar (VRBGA) and incubates at 30 to 35 C for 18 to 24 hours. For the testing of lower numbers of BTGNB, the MPN method can be used. For P. aeruginosa the TSB is pre-incubated for at 30 to 35 C for 18 to 24 hours. Growth is sub-cultured onto a plate of Cetrimide Agar and incubate at 30 to 35 C for 18 to 72 hours

The BioLumix approach

The BioLumix system offers two approaches for the detection of gram negative bacteria:

  1. The Two Vial Approach: An approach similar to the USP procedure where two separate vials are used. One vial detects Bile-tolerant Gram-negative Bacteria and another vial detects P. aeruginosa.
  2. The Single vial approach: An approach where a single vial is used to detect all gram negative bacteria.

A 1:10 dilution of the product is made in TSB. For specified levels of >10 cfu/g, a 1.0 ml sample of this dilution is added to the ENT (Enterobacteriaceae) vial and to a PSE (Pseudomonas) vial. The system automatically monitors the vials and will determine in if the samples are clean or contaminated. When the specified level of absent in 1.0 gram or 10 grams is required, the TSB is pre-incubated overnight as an enrichment step, and then 0.1 ml of the sample is added to the vial.

Single Vial Approach

The GN (Gram Negative) vial has excellent inclusivity for gram negative organisms and does not detect most of the gram positive bacteria. After the first dilution in TSB, with or without pre-incubation depending on the specified level, 0.1 ml of the TSB is added to a single GN vial. This is a very simple and fast procedure to access the absence or presence of gram negative bacteria. This single vial can replace the two vials listed above with better coverage of gram negative bacteria.

ENT Vial: The Enterobacteriaceae vial monitors a change in color due to a pH shift as Enterobacteriaceae organisms ferment glucose in the presence of selective media. The vial medium selectivity is similar to VRBGA.

PSE Vials: The Pseudomonas vial uses the same CO2 sensor as the BioLumnix Total Aerobic Count vial, but with a selective medium. The vial medium selectivity is similar to Centrimide agar.

GN Vial: The hydrolysis of fluorogenic synthetic substrates by bacterial enzymes causes an increase in fluorescence. A fluorogenic synthetic enzyme substrate containing 4-methylumbelliferon, common to all gram negatives, is used in the presence of selective media that inhibits the growth of gram-positive bacteria. The GN vial detects the presence of all Enterobacteriaceae, Pseudomonas, Burkholderia and many other gram negative bacteria. The curves below show the growth of quadruplicate samples for P. aeruginosa (figure on the right: green, lt. blue, red, and purple) and for E. coli (figure on the left: green, lt. blue, red, and purple). A negative control (Bacillus species) is shown in each figure and is represented along the baseline in dark blue.

Advantages: The results of all these assays are available overnight (24 hours), the system is fully automated including achieving of data, data maintenance and report generation, it can be used to create a paperless laboratory. The system is unaffected by product interference, delivering accurate results with faster product release. These assays are simpler to perform than the standard methods saving time, labor, and money.

References

United States Pharmacopeia (2009) Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.

United States Pharmacopeia (2009) Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.

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

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.