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.

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