By Cynthia Martindale (Applied Rapid Microbiology Specialists, Ltd.), Scott Hooper (Merck), Miriam Guest (AstraZeneca), Hans-Joachin Anders (Novartis), Ulrich Georg Zuber (Roche), and Mike Russ (Genentech)

This the second article in a two-part series on conducting microorganism challenge studies using online water bioburden analyzers (OWBAs) in a laboratory setting. Part 1 discussed two different experimental approaches that have been successfully used for microorganism challenges. This second article explores the pitfalls to avoid and best practices to follow during execution of microorganism challenge tests.

The pitfalls and best practices were derived from various OWBA end users who are members of the OWBA Workgroup. The workgroup is composed of many companies within the pharmaceutical/biopharmaceutical industry who share a goal to accelerate OWBA implementation and routine use.

Pitfalls To Avoid In OWBA Microorganism Challenge Testing

Extended Use Of Microorganism Preparation

Unless it is specifically part of the experimental design (e.g., use of a prepared starved culture), limit the use of a fresh microorganism preparation to a few hours at most. Microorganisms become stressed in water, resulting in potential changes within the culture (e.g., viability, metabolism, clustering/clumping, and size). The changes in the culture dynamics are often difficult to measure and could result in confounding data.

Adding Particles To The Test

The successful execution of a microorganism challenge relies on a combination of particle control techniques and good aseptic practices. While microbiologists easily recognize the need to employ aseptic practices to provide for reliable data, it may not be clear why particulate control is just as important. Particle control is necessary since an OWBA detects microorganisms by measuring the cell as an appropriately sized particle that emits an appropriate fluorescent spectrum. Particles are ubiquitous in the environment, and some of these particles could also be fluorescent. Therefore, rinsing materials with “particle free” water is one technique used to remove extraneous particles from the test environment (e.g., materials, consumables, instruments, etc.). This level of particulate control is aimed at ensuring particulates in the sample can be detected and differentiated from those in the environment.

It is important to emphasize that materials that are “sterile” are not necessarily particulate free. Further, many sanitizing agents and detergents themselves are full of fluorescent particles. While sterile and sanitized materials are necessary in any microbiological test, care should be taken to ensure that the sterile/sanitized items do not significantly contribute to the level of fluorescent particles present in the test.

Using A Small Injection Rate On The Syringe Pump (Applicable To Online Testing)

When creating the injection scheme for online testing, consider the injection rates. Too small of an injection rate may lead to issues with precision between counts of the same injection rate, whereas too large of an injection may quickly consume the contents of the syringe and limit the number of concentrations that can be tested. Further, a period of adjustment may be necessary when changes to the injection rate are made, as every change in the flow rate (e.g., small pressure changes) leads to a temporal, non-stable value change in counts reported by the OWBA.

Expecting Linear Data From Experiments Using Discrete Sample Testing

It can be extremely difficult to consistently and predictably control the level of extraneous particulates in laboratory challenge testing. In experiments where discrete sample testing is used, it is the inconsistency in sample preparation that often leads to non-linear data sets.

Expecting A Single “Universal” Correlation Factor Between The OWBA And Conventional Plate Count Methods

A one-to-one correlation of the counts from an OWBA and the colony forming unit (CFU) from a conventional plate count method should not be expected, as the scientific basis of microorganism detection differs greatly between the two methods.

  • The conventional plate count method is a “growth based” method. One or more individual cells grow to a level that is visible and create a CFU. The formation of a CFU is dependent upon the media used, the growth conditions, and the viability of the cells. As a result, not all microorganisms present are recovered as a CFU on the conventional plate count method.
  • The OWBA relies on the intrinsic fluorescence of cellular components such as NADH and riboflavin. As water flows through the OWBA, a laser is used to excite these metabolites. The resulting intrinsic fluorescence emission spectra of these particles is used to determine whether the particle is a microorganism.

The differences in the two methodologies lead to differences in detection of microorganisms. An example of this would be the detection of microorganisms that are considered viable but not culturable (VBNC). A VBNC cannot, by definition, be cultured; however, the OWBA may be able to detect the cell due to inherent fluorescence.

Conversely, some microorganisms may have lower detection on the OWBA as compared to the conventional plate count due to the size and fluorescence signals. Where a microorganism’s size and/or fluorescent signatures fall outside the OWBA’s predefined set of detection thresholds, the cell(s) may not be reported as a count.

High Particulate Levels In Certain Water Types

A comparison of water types may be a desirable outcome of laboratory studies to assess if the instruments can detect the difference between “clean” and “dirty” water; however, when evaluating different types of water, consider the number of particulates that may be introduced. Water with the appearance of adequate control (e.g., pretreatment water) may be monitored by traditional methods and demonstrate a gold level of control with low bioburden; however, the water is not routinely monitored for particulates. The presence of non-microbial particulate contamination may blind the instrument, and, in these circumstances, the results obtained from the OWBA may appear “cleaner” than the higher-grade purified water or water for injection.

Recommended Best Practices In OWBA Microorganism Challenge Testing

Microorganism Selection And Preparation

The selection of the challenge microorganism panel should encompass a variety of relevant microorganisms. While there is no comprehensive list of microorganisms, the firm may consider use of microorganisms of the following variety:

  • Culture collection strains (e.g., ATCC, NBRC, etc.)
  • Strains recovered from the firm’s water system (production water and/or source water)
  • Variety of water relevant Gram types and arrangements (e.g., Gram negative rods)
  • Stressed or starved preparations1,2
  • Mixed cultures3

The following sources may be used to develop an appropriate challenge panel:

  • Compendia1,2,3,4,5,6,7,8,9
  • PDA Technical Report Number 333
  • Firm’s historical water data – water in-house isolates

It is recommended to keep the microbial challenge panel relevant to those microorganisms that may be present in a water loop. Therefore, it would be appropriate to omit testing of non-relevant microorganisms such as fungi, spirochetes, mycoplasma, etc.

Microorganism Inoculum Preparations Should Be Created In Water

The OWBA instruments are designed to differentiate the presence of microorganisms from water in closed-loop pure water systems. Components found in common microbiological diluents (e.g., saline) are not present in these closed-loop systems and contain artifacts that may artificially increase the count reported by the OWBA.

Lyophilized cultures such as BioMerieux’s BioBalls or Microbiologics EZ-Accu Shot should not be used for direct preparation of the water sample.10 Ready-to-use culture preparations such as these contain a significant particulate load, and these particulates may also be fluorescent, leading to false positive results.

Sanitization Of The OWBA Prior To And Post Microorganism Testing

Prior to conducting microorganism testing, sanitize the OWBA and its components (e.g., inoculum feed line, sample needles/tubing etc.) following the vendor recommendations and avoid any hold time of the instrument after the sanitization is complete. Sanitization prior to testing is aimed at ensuring that only the target test microorganism is entering the OWBA flow path and being analyzed. After the test session is complete, an additional round of sanitization is recommended to ensure that the microorganism is removed from the OWBA instrument.

Control Particle Generation During Preparation And Testing

The unintended addition of particles to the microorganism preparation may falsely increase the count on the OWBA. Therefore, during microbial challenge testing, it is important to use not only aseptic technique, but to also exercise particulate control. It is recommended to control and understand the addition of extraneous particles during the microorganism preparation using the following techniques:

  • Rinse materials using copious amounts of “particle free” water or purified water that has been passed through a filter of ≤0.22 µm pore size.
  • Ensure that the materials used in the test have acceptably low counts on the OWBA. In some cases, materials such as bottles can be sonicated or filled with pure water (as described above) and soaked overnight to achieve low OWBA counts.
  • Corning Gosselin, polyethylene terephthalate glycol (PETG), or, in some cases, glass bottles have resulted in low particulate counts and have been used successfully in OWBA microbial challenge studies, whereas polypropylene bottles have proven problematic.

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OWBAs can readily enumerate microorganisms in water with essentially real-time detection. Their increased sensitivity and differing method of detection of microbes as compared to classical growth-based methods means that OWBA studies require additional care to avoid introducing artifacts into the tests. These lessons-learned from prior studies are provided as practical guidance for OWBA users to create and conduct their own controlled studies of water using online bioburden analyzers. The key is that OWBAs require nearly as much focus on particle science as they do on microbiology. Through this awareness and through exercising care in sample preparation and experimental design, OWBAs can be used to successfully enumerate microorganisms in aqueous samples.


  1. General Information Chapter G8 Water: Quality Control of Water for Pharmaceutical Use, The Japanese Pharmacopoeia, Seventeenth Edition, 2016, page 2547.
  2. General Information Chapter Rapid Microbial Methods, The Japanese Pharmacopoeia, Seventeenth Edition, 2016, page 2505.
  3. Technical Report No. 33: Evaluation, Validation and Implementation of Alternative and Rapid Microbial Methods; Parenteral Drug Association: 2013.
  4. General Chapter <1223> Validation of Alternative Microbiological Methods, USP41/NF36, U.S. Pharmacopeia 2018.
  5. General Chapter 5.1.6 Alternative Methods for control of Microbiological Quality. European Pharmacopoeia 9.4, European Directorate for the Quality of Medicines and Healthcare (EDQM), Strasbourg, Cedex, France: 2017.
  6. Chapter 2.6.12 Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests, European Pharmacopoeia 9.4, European Directorate for the Quality of Medicines and Healthcare (EDQM), Strasbourg, Cedex, France: 2017.
  7. Chapter 2.6.1 Sterility, European Pharmacopoeia 9.4, European Directorate for the Quality of Medicines and Healthcare (EDQM), Strasbourg, Cedex, France: 2017.
  8. General Chapter <61> Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests, USP41/NF36, U.S. Pharmacopeia 2018.
  9. General Chapter <71> Sterility Tests, USP41/NF36, U.S. Pharmacopeia 2018.
  10. Technical Bulletin LI021: Lyophilized Pellet Use in Testing LIF-based RMMs; Azbil BioVigilant, April 1, 2014.

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