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.

Detection of psuedomonads in dairy and water samples using a quantatative one-step testing protocol in just one day

By Roger Brideau*
*Presented in part at the International Association of Food Preservation
‘IAFP’ Conference in Charlotte NC USA

dairy microbiology detection of psuedomonads Introduction- Pseudomonad organisms are a major cause of bacterial spoilage of pasteurized milk and dairy products due to post process contamination.  Early detection of Pseudomonad’s can be a predictor of product shelf-life as they are the predominant psychotropic bacteria present.  BioLumix has developed a rapid method for the detection of Pseudomonad’s in dairy products and the method is also applicable to their detection in process water.

Purpose- To evaluate the ability of the BioLumix system to detect Pseudomonad’s in dairy products, determine the speed to results, sensitivity, selectivity and ability to predict shelf-life.

Methods- the BioLumix system is an optical system that detects growth of Pseudomonad’s using a CO2 sensor in selective growth media.  The BioLumix system was directly compared to the plate count methodology for milk samples stored at refrigerated temperatures and held overnight at room temperatures (enriched).  Testing of water was also accomplished in side by side studies to show the capability of the BioLumix system for quantitation of Pseudomonads.

Results:  Growth of Pseudomonad’s in the BioLumix vial
Table of quantitation of Pseudomonads
A growth comparison was made for detection of each Pseudomonad in the BioLumix system using PSE-B vials and on CFC (Pseudomonas agar) spread plates.  Table 1 summarizes the growth of freshly diluted samples of organisms that were enriched in TSB during the prior 18-24 hrs.  The PSE-B vials are selective, as shown by not allowing growth of unrelated gram positive and gram negative bacteria, yeast or mold.  Four different species of Pseudomonad’s grew in the PSE-B vial and on CFC plates.

Milk Sample Testing
Commercial milk samples were tested upon arrival in the laboratory.  Five of twenty were positive for the presence of Pseudomonad’s using both PSE-B vials and CFC spread agar plates.  After storage for 3-7 days, twelve of twenty samples were positive for Pseudomonad’s including after enrichment at RT for 16-18 hrs.  Thus, refrigerated milk samples have varying incidence of Pseudomonad flora.

Dairy Microbiology Calibration dataMilk Calibration Curve
Organisms from milk samples that grew in PSE-B vials and on CFC plates were used to generate the Calibration Curve shown in Figure 1. These data suggest that low numbers (~10) of Pseudomonad’s should detect within 24 hrs in the PSE-B vial. The Calibration Curve can be embedded into the BioLumix software on the instrument and used to generate a read-out of cfu per gram of milk.  This enables quantitation of the milk sample for the presence of Pseudomonad’s before 24 hr; a distinct advantage over plate methodology taking 48-72 hours.

Dairy Microbiology detection time distributionDistribution of Data
In the dairy settings the goal is to separate a “good” sample that has a potential to maintain quality over a product’s shelf-life from a “bad: sample that will have a shorter shelf life. Criteria for separation between a “good” and “bad” product based upon the Pseudomonad’s numbers can be established. If one selects a count of 1,000 cfu/ml as the separation point: the Histogram shown in Figure 4 indicated at 12.5 hrs all samples with higher counts (in red) detected, while all the samples below 1,000 cfu/ml did not.

Results: Testing of Process Water for the presence of Pseudomonads
Eight different types of process water samples were found to be free of Pseudomonad’s after testing using PSE-B vials and CFC spread plates (data not shown).  Clean process water samples were then inoculated with individual isolates of Pseudomonad’s were used to generate a calibration curves for water, similar to the milk calibration curve. Pseudomonad growth in inoculated process water was measured using PSE-B vials and PA spread plates and was used to generate the Calibration Curve. Detecting vials were confirmed to contain Pseudomonas by the Oxydase test.

Summary
The data presented show equivalency between the BioLumix PSE-B vial and CFC (Pseudomonas agar) plates for the detection of Pseudomonad’s found in commercial milk samples and in inoculated process water samples.  PSE-B vials detected as little as 1-3 organisms (data not shown).
The number of organisms in commercial milk was found to increase over time at refrigerated temperatures and this agreed with a previously published report (Burdova et al 2002) showing the affect of storage temperature on milk shelf-life.
The BioLumix assay is completed in 18 hours and offers an advantage over spread plate methods for time to results and ease of calculation of cfu per gram of milk or water.  A single vial is all that is needed and thus both time and material costs are reduced.  Calibration Curves were easily generated for both milk and water sampling and can be used to generate a cfu/ml of sample in less than 1 day to yield an estimate of cfu/gram.

REFERENCE:  Burdova, O. et al (2002).  Bulletin Vet Med. Poland. 46:325-329. Hygiene of Pasteurized Milk Depending on Psychrotrophic Microorganisms.

Pseudomonads and Their Rapid Detection

Description of Pseudomonad Organisms

Pseudomonas bacteria (Pseudomonads) encompass gram negative, motile, non-fermenting rods. This genus is ubiquitous in nature and these organisms can impact a number of environments and patient populations. The Pseudomonads may be found in soil, on plant material, in water, and can be isolated from various tissues and body fluids from mammals. In human health, some of these organisms, primarily Pseudomonas aeruginosa, can be an opportunist pathogen and cause serious health problems. If allowed to reach unsafe levels, this organism may cause several health problems including skin rash and other skin infections, ear infection, urinary tract infection, and in rare instances, pneumonia. Other Pseudomonads, for example, P. stutzeri can be isolated from wounds but are generally not associated with human disease. Many Pseudomonads found in the soil can damage plant materials by causing spoilage.

Who tests for Pseudomonas and why?

Water Testing: Pseudomonas aeruginosa is a bacterium commonly found in purified water systems. Pseudomonas grows in water. It thrives at warm temperatures, which is why it is so often associated with spas. It can also grow in purified water systems.

Pharmaceutical and Cosmetic Products: Analysis of FDA product recall data for 134 non-sterile pharmaceutical products from 1998 to September 2006 demonstrated that 48% of recalls were due to contamination by either Burkholderia cepacia, or Pseudomonas spp (Jimenez L. 2007). In cosmetic products, P. aeruginosa was recovered from contaminated mascara material and was identified as the agent responsible for corneal ulcers in the 1970s (Ortho 2009). Pseudomonads can survive and grow in DI water—Contaminated DI water may be the source of microbial contamination if it is used for the final rinse of equipment that has been cleaned and sanitized, and it may be the source of contamination for finished products in these industries.

Dairy and Food: The predominant microorganisms limiting the shelf life of processed fluid milk at 4°C are Pseudomonas spp. these species are able to grow to high numbers during refrigerated storage. Pseudomonas species accounted for79% of the psychotropic isolates that spoiled pasteurized milk (Dogan and Boor 2003). Important characteristics of Pseudomonads include their abilities to grow at low temperatures (3–7?C) and to hydrolyze and use large molecules of proteins and lipids for growth.

Biolumix Offers Two Options for Detecting Pseudomonads

For certain industries it is important to detect Pseudomonas aeruginosa, while for others it is important to detect all Pseudomonas spp, including the closely related Burkholderia cepacia. As a result BioLumix offers two different types of vials: the PSE vial for the detection of P. aeruginosa; and the PSB vial for the detection of all strains of Pseudomonas and for B. cepacia.

Detection of Pseudomonas aeruginosa (PSE Vial)

For the Pharma (OTC), Cosmetic and Nutraceutical Industries the primary cause for concern is the absence or presence of Pseudomonas aeruginosa. P. aeruginosa is common and is able to become an opportunistic pathogen in people and may cause severe disease (Hugh and Gilardi 1974). The ability to detect P. aeruginosa is critical in the non sterile Pharmaceutical products, Cosmetic and Nutraceutical Industries to ensure the product material is safe. BioLumix offers a highly selective media in the form of a test vial (PSE) that primarily only allows for the growth of Pseudomonas aeruginosa organisms. Confirmation of the presence of this organism is accomplished using the simple Oxidase reaction on vial contents. The test sample is merely enriched in TSB (Tryptic Soy Broth) per USP instructions and then tested directly in the BioLumix PSE vial. Other common Pseudomonads and closely related organisms, including B. cepacia and P putida, as examples, are excluded from growth due to the use of antibiotic supplements in the BioLumix PSE vial. P. aeruginosa is typically more antibiotic resistant than other Pseudomonas organisms (Blazevic, DL et al 1973). Figure 1 illustrates the growth curve of Pseudomonas aeruginosa ATCC 9027 in the BioLumix PSE vial.

KEY:Dark Blue Curve- P. aeruginosa Green Curve- Negative Control

Detection of other Pseudomonads (PSB Vial)

For many industries including the dairy industry and manufacturers using water, there is a need to test for all Pseudomonads as they impact these industries economically. Other Pseudomonads may include P. fluorescens, P. putida, and P. stutzeri. Burkholderia cepacia, can also be detected using the BioLumix PSE-B vial. Specific to the use of water in manufacturing: Pseudomonas bacteria can be found naturally in the ground and within drinking water sources such as aquifers. Contamination of either dairy products or water systems by Pseudomonads is something to avoid and early detection of goods using a rapid microbiological detection system such as the BioLumix Instrument System, would offer an advantage to the manufacturer. Figure 2 illustrates the growth of many types of Pseudomonads and Burkholderia cepacia in the BioLumix PSE-B vial.

KEY: Dark Blue Curve- B. cepacia; Green Curve -P aeruginosa; Light Blue Curve – P. putida; and Red Curve– P. fluorescens growth

REFERENCES

Blazevic, DJ, Koecke, M.H., and Mastsen J.M. (1973). Incidence and identification of Pseudomonas fluorescens and Pseudomonas putida in the clinical laboratory. Applied Microbiology 25: (1)

Dogan, B. and Boor, K J. (2003). Genetic diversity and spoilage potentials among Pseudomonas ssp. isolated from fluid milk products and dairy processing plants. Appl. Microbiol.,69: 130-138.

Hugh, R. and Gilardi, G. (1974) In “Manual of Clinical Microbiology” Edited by Spaulding, Lennette, Spaulding and Truant. Chapter 23 Pseudomonas.

Jimenez L.(2007). Microbial diversity in pharmaceutical product recalls and environments. Review. PDA J Pharm Sci Technol. 2007 Sep-Oct;61(5):383-99.

Ortho D. (2009). Insight into Cosmetic Microbiology, Chapter 8 263-267

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.

REFERENCES:

Carrick, K, Barney M, Navarro, A. and Ryder D. (2001). The Comparison of Four Bioluminometers and Their Swab Kits for Instant Hygiene Monitoring and Detection of Microorganisms in the Brewery. J. Institute of Brewing 107, 32-37

Easter M. (2010) A comparison of commercial ATP hygiene monitoring systems. Next Generation Food issue 9, 2010

PDA Midwest Chapter- June 8, 2012 – All Day Contamination Control Event (http:// www.pdamidwest.org/)

United States Pharmacopeia XXI (1985) Chapter <1231> Water for Pharmaceutical Purposes. The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.

Rapid Detection of Heterotropic Bacteria in Water

What categorizes Heterotrophic organisms?

Heterotrophic organisms are categorized by the requirement of organic carbon sources to produce energy.  These organisms can include bacteria, yeasts and molds, and can be found throughout the environment in both natural and treated water (including tap water).  They occur normally and are unique in their ability to proliferate in the nutrient-poor environment of water systems at various temperatures. 

Why Test Water for Heterotrophs?


The concentration of Heterotrophs in treated water is an indicator of water treatment efficacy.  The presence of these microorganisms does not necessarily indicate concentrations high enough to cause disease.  However, the concentration of these microbes is important when testing for coliforms in water.  If Heterotrophic organisms are found at a concentration of approximately 500 colony forming units (cfu) per mL or higher, then the detection of coliforms may be compromised.  An increase of Heterotrophic counts may indicate the presence of coliforms in the water being tested.

Traditional methods for testing water

Heterotrophic Plate Count (HPC) is the procedure traditionally used to estimate the number of viable organisms in a sample of water.  Three different methods – (i) spread plate; (ii) pour plate; and (iii) membrane filtration – are utilized with different types of media.  Although these methods are widely accepted, there are some disadvantages.

  • In the pour plate technique, organisms are suspended in agar until the media cools.  During this time, damaged organisms are susceptible to heat shock from the higher temperature of the agar.  Also, because the colonies are surrounded by agar, the count of obligate anaerobes becomes compromised.
  • If an automated spreader is used in the spread plate method, heavy growth of colonies may make counting of colonies difficult, rendering counts non-applicable.
  • When filtering water, samples may contain suspended solids that cannot pass through the filter, interfering with accurate counts.

Due to different techniques and different types of media used, the counts achieved may vary significantly.  In addition to multiple protocols and media, temperature and incubation time vary.  Samples may be incubated anywhere from a few hours to 7 days or even a few weeks at temperatures ranging from 20°C to 40°C.  This makes processing water samples laborious with much data collection.

Rapid Detection with the BioLumix System 

BioLumix is the most advanced microbiological testing system of its kind. This automated, all-in-one microbial testing system is extremely easy to operate. The system is both simple and cost-effective, revolutionizing your current testing methodology. A novel optical system sensing color and fluorescence in ready-to-use vials provides faster results, labor savings, automation, and connectivity. The system has a large repertoire of assays that it can perform including: Total Aerobic Count, Yeast and Mold, Coliforms, Enterobacteriaceae, E. coli, Pseudomonas, Staphylococcus, and Salmonella.

Recently a new assay for the detection of Heterotrophic bacteria in water was developed for the BioLumix system.  The developed assay was validated by testing 50 samples of multiple types of water that 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 1.0 mL of a 1:100 dilution (depending on the desired specified level), and a few water samples were inoculated with heterotrophic bacteria.  These samples were monitored in the BioLumix instrument for 35 hours.  Figure 1 shows the curves obtained.

Final results were seen in the BioLumix system roughly 13 hours faster than the plate count method using Standard Methods Agar.  These particular samples were tested at specified levels <10 cfu/mL and <100 cfu/mL, but 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. 

  Caron Ockerman