Thursday, December 26, 2013

Understanding Environmental Monitoring in the Pharmaceutical Industry



Many microbiologists believe that the most critical sources of contamination in the pharmaceutical and nutraceutical industries are raw materials and water. While these are important, effective quality assurance practices also need to include environmental monitoring. Many microorganisms exist in the atmosphere and on almost all surfaces. When left unattended, they can be a serious cause of infection and contaminate sensitive manufacturing operations. Besides the fact that proper control of the environmental conditions where manufacturing takes place is an FDA requirement, manufacturers also take environmental monitoring seriously because it is a crucial step in safeguarding and ensuring high-quality products. 

Wednesday, December 18, 2013

Importance of an Efficient Quality Assurance System in Microbiology Testing



In the nutraceuticals, pharmaceuticals, and cosmetics industries, quality means conformance to standards set by both the manufacturer and the industry. These standards must not only reflect a strong emphasis on the desired features of a product but also on its safety and efficacy.

To maintain quality, quality control (QC) procedures are employed. These procedures are in place to identify any action that does not meet the specified standards for quality. As a tool, QC is said to be corrective in nature. Its goal is to find and eliminate where problems in quality originate from. An example of QC in a typical micro laboratory is the regular monitoring of reagents and test samples.

Wednesday, December 11, 2013

Water Testing: Rapid Detection Methods for Heterotrophic Bacteria


Bacterial growth in water distribution systems is a commonly used indication of potential health issues. Gaining an accurate assessment of the heterotrophic bacteria count is crucial for determining how effective water treatment systems are working. Unfortunately traditional testing methods for heterotrophic bacteria are often inaccurate and slow. Below you can discover some of the primary limitations of heterotrophic bacteria testing methods and why advanced rapid detection systems have been developed.

Wednesday, December 4, 2013

The Pharmaceutical Industry's Use of Rapid Microbiology Testing



Pharmaceutical products such as supplements and prescribed drugs are capsuled in either a hard or soft shell. Just like the granules they contain, these shells are brought to the laboratory for testing. For the longest time, microbiologists have used the plate count methodology to determine a capsule's bacterial, yeast, and mold counts. But while this method is effective, it poses several difficulties. For instance, it is labor-intensive. From the preparation of the agar up to the counting of colonies, it can take five days to finish one test. Aside from that, this method is also prone to errors. Enter simplified, rapid, automated microbiology testing.

Monday, November 25, 2013

Microbiology Instruments Appropriate for Enzyme Testing



Enzymes are intricate, stable, globular proteins that serve as catalysts in chemical reactions. These reagents reduce the activation energy in order to increase the rate of the reaction at the same temperature. Their stability prevents them from being consumed like other reagents in the reaction. While they can accelerate chemical reactions by more than 1,000 times, the effect of enzymes can either be enhanced by promoters, or reduced by inhibitors. Most industrial enzymes are obtained from microorganisms like fungi and bacteria, though they can also be obtained from animal and plant sources, unless they are activated.

Monday, November 18, 2013

Microbiology Quality Assurance: Rapid Yeast and Mold Detection



Mold and yeast are agents which can be responsible for contamination as well as spoilage in most products. A lot of these agents can develop something known as mycotoxins which can infuse itself into the product. If this is done in large quantities, it can cause poisoning. According to the American Society for Microbiology the presence of yeast can affect the product salability and quality, which can lead to significant financial loss for the food supplier. This is why rapid yeast and mold testing is important so that your organization will remain efficient and hygienic. Microbiology quality assurance can help you detect yeast, mold and other agents that may contaminate your products.

Monday, November 11, 2013

Quality Control in the Food and Beverage Industry: The Significance of Coliform Testing



Complying with U.S Food and Drugs Administration (FDA) and Environmental Protection Agency (EPA) laws and regulations related to safe food handling and water sanitation is important for all manufacturers of food and beverage products and water bottling facilities. This is particularly essential in meat, poultry and daily industries where food items are usually at a high risk of contamination by bacteria such as salmonella, E. coli and etc.

Monday, November 4, 2013

Understanding Quality Assurance in the Food Industry: Microbiological Testing of Yogurt



While yogurt is generally viewed as a health food there are specific dangers for the consumers and the manufacturers. The most important of these is the presence of harmful microorganisms. The main investigative procedures to determine the health of yogurt is testing for yeast, mold and the presence of coliform. Traditional testing methods tend to be slow and labor intensive. This makes them unsuitable for products with a relatively short life such as yogurt. This has led to an interest in more rapid methods of determine the safety of yogurt before it is delivered to store shelves.

Monday, October 28, 2013

Rapid Microbiological Testing: Importance of E. coli Testing


Escherichia coli, which is abbreviated as E. coli, refers to the rod-shaped bacterium that occur naturally in the lower intestines of endotherms, or warm blooded organisms, including human beings, as well as reptiles and fish. While most of these gram-negative organisms are harmless, some of the serotypes can result in serious food poisoning due to food contamination, which is a common reason for product recalls. There are five strains of E. coli that cause diseases. Some of these diseases include hemolytic uremia, copious bloody discharge, unique diarrhea, and Enterohemorrhagic (EHEC) class.

Monday, October 21, 2013

Quality Assurance: Microbial Testing of Cosmetics



The number of people using cosmetics continues to grow, and the quantities consumed keep increasing every year. As the production increases, the industry is faced with great concern regarding the microbial contamination of personal care and cosmetic products. The implications of microorganism contamination include massive product waste and financial losses for the manufacturer, not to mention consumer injury or infection. While there are very few cases of the latter happening, the occurrence of such episodes can drastically impact the reputation of a brand.

Monday, October 14, 2013

Nutraceutical Rapid Microbiology Detection Systems


Consumers want quality assurance and the FDA is backing them with their cGMP guidelines. Many manufacturing companies are choosing to build in-house programs or signing up with contract labs to beat their existing microbiology quality assurance practices. As a manufacturer, you should always be prepared to test your raw materials and products for unacceptable levels of certain microorganisms.

Monday, October 7, 2013

Preservative Efficacy Testing and Suitability Testing for Cosmetic and Toiletry Industries



The cosmetic and toiletry industries employ unique microbiology quality assurance systems that are specifically designed to detect the presence of microbes in cosmetic and personal care products, and deliver accurate and timely results. These microbiology systems perform a variety of typical assays including checking for unpleasant organisms, microbial content like molds and yeast, preservative efficacy testing, and suitability testing.

Thursday, September 26, 2013

The Pharmaceutical Industry and Its Dependence on Microbiology



Put simply, microbiology entails the study of microorganisms that one can't see with the naked eye. These are from bacteria, fungi, protozoa and such similar organisms. There is an intricate association between microbes to specific diseases. This has kept scientists at the front on research concerning them for centuries. The role of microbiology on advances within the healthcare industry, and especially the pharmaceutical industry, has led to great discoveries ranging from medicines and vaccines to devices.

Thursday, September 19, 2013

Microbiology Quality Assurance and Rapid Microbiological Methods



When it comes to microbiology quality assurance, this is the process whereby the quality of laboratory reports can be guaranteed. Quality control is the part of quality assurance that is mainly concerned with controlling errors when tests are being performed as well as in the verification of the test results. Materials, equipment and procedures have to be controlled adequately. Culture material has to undergo testing for sterility as well as performance. Every laboratory should have Standard Operating Procedures (SOPs). Quality assurances should be incorporated at every stage of a microbiological procedure, which can be done by ensuring that the laboratory is well lit, dust free and air-conditioned. 

Thursday, September 12, 2013

Understanding Microbiological Quality Control and Assurance Solutions



In the ever-changing pharmaceutical industry, competition is rife when it comes to the speedy development of products. Microbiological testing is essential to ensure safe products are made without any environmental hazards and at a reduced cost. This has led to an increased use of rapid detection kits that cut costs and time in detection of pathogens in any given sample. 

Wednesday, September 4, 2013

Regulations and Requirements for Pharmaceutical Microbiology Labs


In microbiology, quality assurance stands for the measures put in place to ensure quality control, as well as quality improvement. Quality control stands for the constant observation of work practices, paraphernalia and chemical substances so as to spot and correct defects. Once there are proper quality control measures in place, then quality improvement comes into focus as a way of making existing systems more efficient.

Tuesday, May 28, 2013

Everything is New at SupplySide Marketplace 2012!

New venue…new location…Stop by and see what’s new at BioLumix – booth #542 at the SupplySide Marketplace, Javits Center New York City on May 9th & 10th. The BioLumixsystem is the most advanced microbiological testing system of its kind. A demo of theBioLumix system will be available during the tradeshow.

Do you need Validated, Simplified Rapid Microbiology Testing? 

The state-of-the-art BioLumix system helps streamline microbial testing, and allows for easy compliance with cGMP, with over 200 instruments performing assays daily. All of BioLumix customers’ audits by FDA and NSF passed their inspection while using the BioLumix System.
Dealing with difficult to read plates or manual data transfer is a thing of the past. With automated monitoring of ready-to-use assay vials and automated data processing and archiving (paperless), the microbiologist’s job isa lot easier, with the same accurate results inless than half the time.

What is the BioLumix System? 

Test vials areincubated and the assay are monitored every 6 minutes. Each Instrument is capable of simultaneously running 32 test vials at one temperature. The modular structure of the BioLumix System enables flexible growth as needed; up to 1,024 samples can be tested simultaneously with up to 32 instruments connected to a single PC. There is random access to all instrument vial positions at any time. Therefore,no batching is required.

The Biolumix System is designed to accelerate product release with a simplified, automated approach.This yields fast, accurate results while reducing costs. No more waiting days for completed assays. The BioLumix System brings automated microbiological testing in house with most assay results achieved over night, and an Automated Certificate of Analysis within 48 hours for all USP assays. It is simple enough to be operated by no-microbiologists, providing accurate data of raw materials, environmental samples, and finished products.
What would you like to save? Time, money, labor? BioLumix does it all. The BioLumix system is both simple and cost-effective, with its streamlined testing design and single platform testing. The system is designed to accelerate product release using a modified simple methodology. It saves time to results,labor for microbiological testing and money due to its cost effectiveness. In many cases, return oninvestment is takes less than 6 months.

What would you like to test? Tablets, capsules, oils, liquids and powders are easily analyzed andresults are obtained quickly and with real time communication. The simple sample preparation allowstesting for raw materials, in-process product, finished goods, processing water and environmenta ltesting. All of which have been Validated by us, according to USP methodology, so you don’t have to. Infact, we are so excited about our system that we ask your company to send us samples and we will testthem for free so you can see the results!
What Assays would you like to test?

•••••••••
Total Aerobic Microbial Count Total Combined Mold and Yeast Count Enterobacterial Count (Bile-Tolerant Gram Negative Bacteria) ColiformE. coli Pseudomonas aeruginosa Staphylococcus aureus Salmonella Probiotic

Would you like No Product Interference?
The vial design is separated into two zones: the incubation zone where the sample is present, and the detection zone where the readings take place.Therefore, the system prevents any product Interference.

The BioLumix Advantage is:
•••••••••••••
Simplified testing procedures – can be operated by non microbiologists Ready to us vials-shorter sample preparationVial design prevents product interference Real-time communication for immediate action Early warning of contaminated samples An automated Certificate of Analysis within 48 hours including all organisms required by USP Validation package against USP methodology including IQ, OQ and PQ Allows for cost effective internalization of microbiological assays 21 CFR part 11 compliant with audit trail and login/ log out Centralized test data automatically stored and protected Cost reduction of microbiological assays Automation and connectivity – faster product release Allows internalization of microbiological testing
Come See Us at Booth #542!

BioLumix Microbial Limit Vial (MC)

Introduction:

The Microbial Limit vial is used to test primarily Personal Care, Cosmetic and over the counter Pharmaceutical (OTC) products for microbial content (contamination). Each of these types of products may have preservatives in their composition and the Microbial Limit vial helps to neutralize the inhibition of microbial growth that many preservatives provide. Neutralization of the preservative allows for a proper evaluation of whetheror not the product has contaminants. Often the contaminating bacteria in the product while in the presence of the preservative remain “injured” and unable to replicate. Thelack of replication might be interpreted as the lack of contamination.

How It Works


The Microbial Limit vial’s sensor detects production of CO2 by microorganisms, based upon the principle that CO2 is a universal metabolite produced by all microorganisms. The disposable vial contains a transparent solid sensor located at the bottom which changes its optical properties whenever CO2 diffuses into it. Only gases can penetrate the sensor; blocking liquids, microorganisms, and particulate matter. Consequently, the optical readings are not masked by the sample. CO2 generated by bacterial metabolism in the liquid medium diffuses into the sensor and interacts with an indicator reagent to provide an indication of the presence of the carbon dioxide.

Applications:

The Microbial Limit vial is used to test primarily Personal Care, Cosmetic and over the counter Pharmaceutical (OTC) products for microbial content (contamination). Each of these types of products may have preservatives in their composition and the Microbial Limit vial helps to neutralize the inhibition of microbial growth that many preservatives provide. Neutralization of the preservative allows for a proper evaluation of whether or not the product has contaminants. Often the contaminating bacteria in the product while in the presence of the preservative remain “injured” and unable to replicate. The lack of replication might be interpreted as the lack of contamination.
The first step of the assay is to perform a 1:10 dilution of the product in neutralizing broth such as D/E (Dey/Engley) broth, Letheen Broth, or TAT (Tryptone-Azolectin-Tween) Broth. There after 1.0-0.1 ml of the sample is added to the Microbial Limit vial. The Microbial Limit vial contains the neutralizers that inhibit many common preservatives and this neutralization event helps the customer to correctly measure the presence of contaminating organisms.

Examples of Growth Curves Using the Microbial Limit Vial:


In the curves shown below in the Figure, there is an example of both a positive curve and a negative curve. The bacterium used was Pseudomonas aeruginosa.
The BioLumix Microbial Limit vial was specifically designed to be used in complying with USP. Due to the fact the Microbial Limit vial has both Lecithin and Tween in its media composition helps allow for neutralization of the preservative in the sample to be maintained during the assay for viable organisms. Thus, this vial is useful to the customer that has already determined the amount of neutralizing buffer and its content of neutralizer to be used when the product sample is first prepared in diluent. Together the use of the correct neutralizer and the use of the BioLumix Microbial Limit vial helps ensure an accurate assay for the replicating organisms.
Table 1 summarizes the types of Products that customers test in the BioLumix Microbial Limit vial to measure the presence of organisms.


Summary:


The versatility of the BioLumix Microbial Limit vial includes the ability to support growth of most aerobic bacteria, many yeast and some mold organisms. In most cases YMC vial is used for the detection of yeast and molds. The BioLumix Microbial Limit vial can be used for determination of microbial content (contamination), for use in suitability studies that test whether a product can support growth of microorganisms, and in Preservative Efficacy Studies (PET analysis) that is used for cosmetic products. The BioLumix Microbial Limit vial can also be used by customers whose products include Dietary Supplements and Nutraceutical products for which preservatives (natural or chemical) are also added. Supplement products with natural preservatives also need to be neutralized and tested for their ability to support microbial growth.

Tuesday, April 30, 2013

Lactic Acid Bacteria Testing

Background

Lactic acid bacteria (LAB) is a group of Gram-positive, generally non sporulating, non-respiring rod or cocci. A common metabolic characteristic is their ability to produce lactic acid as a major metabolic end product of carbohydrate fermentation and their increased tolerance to grow at a lower pH range. This allows the LAB to partially outcompete other bacteria in natural fermentation, since they can withstand the increased acidity caused by the lactic acid production. All LAB grow anaerobically, but unlike most anaerobes, they grow in the presence of O2 as “aerotolerant anaerobes”. Because they obtain energy only from the metabolism of sugars, lactic acid bacteria are restricted to environments in which sugars are present.

LAB can spoil products or produce health benefits


A few LAB are pathogenic for animals, most notably some members of the genus Streptococcus. In humans, Streptococcus pyogenes is a major cause of disease (strep throat, pneumonia, and other pyogenic infections, scarlet fever and other toxemias), Streptococcus pneumoniae causes lobar pneumonia, otitis media and meningitis; some viridans and nonhemolytic oral streptococci play a role in dental caries and may be an insidious cause of endocarditis.

LAB can cause spoilage of a variety of foods including ready-to-eat meats, fish, vegetables, salad dressing, mayonnaise, and wine. There is no legal requirement to test for LAB in foods; however, quality-focused food manufacturers around the world choose to monitor lactic acid bacteria in their products and environments to ensure customer satisfaction of their product.

LAB are best known for their role in the preparation of fermented dairy products, pickling of vegetables, baking, wining making, curing fish, meats and sausages. For dairy manufacturing it involves a microbial process by which lactose is converted to lactic acid.
In yogurt, manufacture depends on a symbiotic relationship between two bacteria; Streptococcus thermophilus and Lactobacillus bulgaricus, where both species of bacteria help each other grow. The LAB created in this product can help supplement the normal healthy flora in the human intestine.
LAB are the most common microbes that are used as probiotics. The two primary probiotic bacteria used are members of the Lactobacillus and Bifidobacterium genera. Lactobacillus acidophilus is the most commonly used LAB. It ferments sugars into lactic acid, and can grow readily at lower pH values and has an optimum growth at 37C. Strains of this bacterium are used many in dairy products.

Current Methodology:

Currently the methodology to detect Lactic Acid Bacteria is one of the most time consuming and higher resource requiring media. To properly grow LAB in traditional methods most standard require the use of MRS Agar, APT, or Universal Beer Agar. Each sample tested using this method requires two plates of each media for a total of four plates. The plates also require an anaerobic chamber with a gas pack. The LAB then can require up to 7 days at 30C or 35C to grow. The plates have to be examined for growth and confirm the morphology of the colonies via gram staining. Sometimes further biochemical testing or identification techniques are required to complete the test.

BioLumix Methodology:


BioLumix has developed a Lactic Acid Bacteria vial that helps detect LAB in products. The LAB vial detects the presence of LAB using a modified version of MRS broth. The curves show four of the most common LAB (L. lactic, B. longum, L. acidophilus, and L. rhamnosus).

BioLumix has tested a variety of products using the LAB vial, such as mayonnaise, lunch meats, salad dressings, tartar sauce, yogurt, and enzymes. All products were tested both uninoculated and inoculated, and some products already contain LAB, such as yogurt (which in this case contained LABs such as L. Bulgaricus, S. Thermophillus, L. Acidophilus, Bifidus, and L. Casei). The BioLumix system can detect the LAB without interference from the products, which can be more difficult on traditional plating methods.


Advantages: The results of all these assays are available within 35-48 hours for most LAB and 72 hours for very slow growing LAB. The vial also can detect results in as early as 6 hours for some cultures. This is saves a substantial amount of time over the traditional seven days. Due to the depth of the broth in the vial no gas packs of unique environment is required. 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.

Thursday, March 14, 2013

Who Needs Environmental Monitoring and Process Water Testing?

Introduction 

Controlling manufacturing environmental conditions is not only a regulatory requirement but also part of protecting and producing a quality product. Environmental monitoring (EM) of manufacturing facilities provides assurance that the environment is both adequately controlled and in compliance. There is substantial evidence establishing a direct relationship between the level of environmental control and the final quality of the product. 

EM serves a critical role in product safety by ensuring that the environment in maintained properly. Swabs are often used for sampling irregular or hard-to-reach surfaces and critical surfaces where contact plates are not practical. In addition, cleaning hold-time studies are often performed using swabs. In general, the purpose of a Microbial EM Program is to: provide crucial information on the quality of the work environment during manufacturing; prevent future microbial contamination by detecting and reacting to adverse trends; prevent the release of a potentially contaminated batch if the appropriate standards are not fulfilled; prevent the risk of contamination of the product; ensure there are environmental controls in the production areas; and provide a profile of the microbial cleanliness of the manufacturing environment. 

Current Methodology 

Most EM is done by plate counting of colonies which is both simple and inexpensive. However, plate counting methods are slow requiring two to seven days to complete, thereby causing a delay in the detection of contamination, which can increase product loss, plant downtime and result in expensive clean up. The delay in obtaining results impacts reaction to contamination issues and can make investigations very difficult. For example, the rooms in question typically have been cleaned numerous times, so re- sampling results are almost always meaningless and determining the root cause of the contamination is difficult. Since real-time response is not possible, batches are jeopardized. The plate count methodology is also labor intensive and requires manual data entry and documentation. Such documentation is prone to human errors and compliance issues. 

Available RMM Methods 

Methods are available to measure total particles in the air, including Total Organic Carbon (TOC), and ATP (Adenosine Tri-phosphate). These methods are very fast to perform but do not correlate well with total bacterial count or any specific group of organisms and do not measure viable organisms (Carrick et. al. 2001 and Easter 2010). Therefore, these results do not measure viable organisms in the environment or on production lines. The standard plating methodologies can take several days. Rapid microbiological methods (RMM) can provide rapid and efficient solutions over traditional plating methodologies. Therefore, both manufacturers and regulators are motivated to develop initiatives and help in the implementation of rapid testing methods (FDA 2004). 

On June 8th the conference on Contamination Control ( http://www.pdamidwest.org/) the data from the validation of BioLumix growth based system for EM and water testing will be presented. The BioLumix Optical System is based upon the detection of microorganisms due to color or fluorescence changes caused by the growth and metabolic activity of microorganism in the test vials. 

Study Design: 10×10 cm surface coupons made out of 5 various materials (Stainless Steel, Aluminum Alloy, High Density Polyethylene, Silicone Rubber, and Perspex, Plexiglas) were inoculated with different organisms (Bacillus spizizenii var subtilis ; Escherichia coli; Pseudomonas aeruginosa; Staphylococcus aureus; Citrobacter freundii; Candida albicans; and A. brasiliensis formerly Aspergillis niger). The coupons were then swabbed and testing using three assays: (i) Total aerobic count; (ii) Yeast and Molds and (iii) Gram Negative Bile Tolerant Bacteria. 

In total, 550 coupons were tested, 290 coupons were inoculated above the specified levels while 260 coupons had counts below the specified levels. There was very good correlation between the BioLumix results and the plate count results, with an overall agreement for samples above spec of 97.2%. None of the 260 un-inoculated coupons detected in the BioLumix system or had plate counts above the specified level. Consequently there was 100% agreement between the two methods. The overall agreement between the two methods was 98.5%. 

Total Aerobic Count: A total of 129 swabs were analyzed using the BioLumix TAC vial and the standard plate count method with TSA. All the swabs with count above specified level signaled as being above the specified level in the vials. Five marginal samples detected in the vials and had counts just below the specified level. The agreement between the methods was 96.1%. 

Yeast and Molds: A total of 85 coupons were analyzed using the BioLumix YM vial and the standard plate count method with SDA (Sabouraud Dextrose Agar W/ Chloramphenicol). All the swabs with count above specified level signaled as being above the specified level in the vials. A few coupons with count very close to the specified level (e. g. 50-80 cfu/swab for a specified level of < 50 cfu/swab) did detect in the vials. One coupon that had a count of 40 cfu/swab, while technically found to be below the specified level, was a very marginal result being so close to the specified level of 50 cfu/swab, did detect in the vial. The agreement between the two methods was 98.8%. 

Gram Negative Bile Tolerant Bacteria: A total of 75 coupons were analyzed using the BioLumix ENT vial and the standard plate count method with VRBGA (Violet Red Bile Glucose Agar). One swab with a marginal count of 310 cfu/swab did not detect in the vial. A few coupons with count very close to the specified level (e. g.300-400 cfu/swab for a specified level of < 300 cfu/swab) did detect in the vials. One coupon that had a count of 190 cfu/swab did detect in the vial. The agreement between the two methods was 97.3%. 

Conclusion: The BioLumix system was validated as an alternative to the plate count method for EM. The study involved a total of 550 surface coupons representing five diverse types of surface material. These five surfaces represent those encountered in manufacturing, including metal, plastics and rubber. Some of the coupons were inoculated with bacteria or yeast or mold. There was 100 % agreement between BioLumix assay and the plate count assay for the 260 coupons that were determined to be below the specified level by the plate count method. There was an overall agreement of 97.2 % between the two methods when swabs containing counts above the specified level were used. 

The advantages offered by the BioLumix system include: automation of results, great speed to results, paperless EM, direct detection of viable microorganisms, detection of multiple types of organisms, increased operation efficiency and consequently an improvement in product quality, reduction in costs, and both enhanced reporting and ability to track trends. 

Water Testing 

Water is widely used as a raw material, ingredient, and a solvent in the processing, formulation, and manufacture of pharmaceutical products, active pharmaceutical ingredients and intermediates. As such, all water purification systems must be monitored regularly to verify the quality of the water produced. Monitoring of water for microbiological quality may include testing for total heterotrophic plate count, coliforms/E. coli, or by checking for the presence of other organisms suspected to be present in a water sample. The relevant standards relating to pharmaceutical grade water are USP <1231> Water for Pharmaceutical purposes. 

The BioLumix system is capable of testing water for heterotrophic bacteria, coliforms, E. coli, and Pseudomonas. For levels of < 1 cfu/ml the water can be inserted directly into the vial. To test for levels such as < 1/100 ml the water is filtered and then the filter is added directly to the vial. 

Water study summary: Ninety- two water samples were analyzed with two specified levels (10 cfu/ ml and 100 cfu/ml). Sixty samples were below the specified level by both methods while 28 samples were above the specified level by both methods. Four samples were below the specified level by the BioLumix method, but above by the plate count method. All these samples had very low counts (1-3 colonies on the plate). There was 96.9% agreement between the two methods. 

BioLumix advantage for water testing: Final results were seen in the BioLumix system roughly 13 hours earlier than the plate count method using Standard Methods Agar. The BioLumix method can detect organisms at a level of < 1 cfu/mL of water. The BioLumix system is faster, less labor-intensive, and more sensitive than the plate count method.

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.