From the earliest days of microscopy, scientists have been describing those parameters that indicate viability of microorganisms. In the late 1600’s, van Leeuwenhoek indicated that cells were viable based upon the motility of the cells. Much of his research was based upon his studies using human semen. The specimens that had a tail and were moving were alive, while the non-moving specimens were designated as dead [1]. Other scientists have equated cellular viability with the ability for the microorganism to reproduce; that is if it grows it is viable.
Today, scientists recognize that microorganisms have a wide range of states of being including terms like: active, dead, dormant, inhibited, injured, living, moribund, quiescent, resting, starved, sublethally damaged, viable but not culturable (VNBC), and vital [1]. In pharmaceutical environments, we often add the concept of the stressed microorganism. There are actually articles published that describe the correct way to define and use all of these terms [6]. The more we have learned about cells, we have found that the concept of cell viability is complex and can be more difficult to describe. Breeuwer and Abee [1] have defined viability as the ability of cells to be capable of performing all of the cell functions necessary for survival, i.e., the continuing existence of the species.
The concept of viable but not culturable (VBNC) cells refers to those cells that have metabolic activity but do not grow under the test conditions utilized. In a pharmaceutical laboratory, this might mean using the nutrient agars specified at the temperatures and times specified. Oliver [8] indicates that a large number of the microorganisms present in the environment exist in this VBNC state including Gram positive and Gram negative microbes as well as pathogenic and non-pathogenic microbes. The results obtained from traditional test methods may not accurately represent the number of microorganisms actually present in the environment. The problem inherent with the VBNC’s is that they maintain the ability to be virulent. As such, under the right conditions this VBNC can reproduce and cause disease.
In recent years, there has been an emphasis on the development and use of rapid microbiological methods (RMMs), also known as alternative microbiological methods. Growth-based microbiological methods take time for the cells to replicate sufficiently to be seen by the naked eye, as you need approximately 106-107 cells/mL. As such, scientists have looked at other methods of enumerating cells to reduce the time required. Some methods still employ cell growth, while others are based upon using a method to determine the number of viable cells present, eliminating the need for cell growth. The emphasis on viability methods has come from the concerns about VNBC’s.
There are a wide variety of methods available to assess cellular viability. Some of the available methods are included in Table 1.
Breeuwer and Abee [1] indicate that the most basic determinants of viable cells include: an intact and functioning (barrier properties) cytoplasmic membrane; DNA transcription and RNA translation (replication of the DNA); generation of energy to meet requirements for cellular metabolism, synthesis of proteins, nucleic acids, polysaccharides and other cellular components; and growth and replication. Unfortunately, there is no one single definition of cellular viability.
Even describing cell death can be difficult. Joux and LeBaron [5] define a viable cell a one that is capable of fission to produce similarly viable progeny under specified culture conditions. Basically, they push for a definition that includes cell division that can be evidenced to ensure that a cell is viable. Usingthe Joux and LeBaron 5] definition, one can culture a cell to determine if it is viable. The culture can be examined macroscopically or microscopically to determine viability. This definition limits the use of physiological probes to determine viability since they do not currently assess for cell division but rather for the presence of chemicals or components present in viable cells. Cells demonstrating metabolic activity would be classified active. Conversely, cells that do not show metabolic activity, but do have intact cellular membranes, would be deemed inactive. The so-called inactive cells may be dying. Those cells with membranes that are damaged would be considered dead cells. Inactive cells with intact membranes are considered intact. Detection methods can be used on dead cells until such time as cellular lysis occurs. In the event that lysis occurs, without destroying the cell membranes and with loss of the the nucleoid, the cells are called ghost cells [5].
Joux and LeBaron [5] also provided information on a variety of physiological probes and taxonomic probes that can be used to assess viability. Their information is summarized in Table 2.
Another type of viability marker used in some test methods includes NADH and Riboflavin. Both of these metabolites have been shown to be present in all living cells. They have been shown to be redox cofactors in microbial metabolism [4] and can be used to measure intracellular redox [7]. Additionally,flavoproteins have been found in the cell as a component of all microorganisms [3].
While all of these indicators of viability are available, the question is what do they mean? Each marker has been developed to look for a specific indication of viability, e.g., enzyme activity, cellular function, or intact cell membranes. Many scientists assume that each marker is definitive, meaning that the instance the cell dies the marker knows the cell is no longer viable. The inherent problem is that when there is no clear cut definition of viability or even cell death, it is unlikely that a viability marker that checks for one specific indication of viability will clearly tell you whether the cell is or is not viable.
Consider an analogy to the human body. When a human body “dies”, the entire body does not die at the same time. It is possible for the brain to be “dead” and still have many other organs in the body working for some period of time. Individuals in respiratory arrest, where their breathing has stopped, still have other organs functioning. The microbial cell has similar characteristics. You might use a viability marker to indicate that the DNA is intact and find this to indicate “viability” for some period of time after the cell has actually dies. Other markers like riboflavin or NADH may continue to show viability for a period of time after the cell dies. This complicates the issue of viability.
In most pharmaceutical applications, the presence of a viable cell is a worst case condition, meaning that it is more significant than having a dead cell present. As such, if a system detected a dead cell as viable it does not pose a higher risk to the product than a true viable cell being present. Detecting a “false” viable cell is more of a business risk, than a regulatory risk since you would have to treat the data as if viable cells are present. There is a concern among some that this higher count due to “false” viable cells could negatively impact your business. But how do you know if you really have viable cells present? Some of the commercial systems have more than one specific test performed to determine viability. These tests are also called discrimination parameters. One of the systems available uses about 20 different parameters to decide whether a cell is or is not viable. Other systems available use as few as one specific test to determine viability.
As an informed consumer, you need to understand the advantages and disadvantages of the viability markers used in your system. Increasing the number of viability markers or discrimination parameters used can reduce your risk of incorrectly identifying a cell as viable. Until such time as there are clear definitions of what constitutes a viable cell and a dead cell, these types of issues are likely to remain.
References
1. Breeuwer, P. Abee, T. (2000) “Review: Assessment of Viability of Microorganisms Employing Fluorescence Techniques.” International Journal of Food&Microbiology 55:193-200.
2. Breeuwer, P. Drocourt, J-L.,Rombouts, F.M., Abee, T. (May, 1994) “Energy- Dependent, Carrier-Mediated Extrusion of Carboxyfluorescein from Saccharomyces cerevisiae Allows Rapid Assessment of Cell Viability by Flow Cytometry.” Applied and Environmental Microbiology 60(5): 1467-1472
3. Chance, B. (1996) “Spectrophotometric and kinetic studies of flavoproteins in tissues, cell suspensions, mitochondria and their fragments.” In Slater, EC, editors. Flavins and flavoproteins. New York: Elsevier; p.496–510.
4. Davis, B.D., Bulbecco, R., Eisen, H.N., and Ginsberg, H.S., (1980) Microbiology—Including Immunology and Molecular Genetics, Harper & Row. p. 33.
5. Joux, F. and Lebaron, P. (2000) “Use of Fluorescent Probes to Assess Physiological Functions of Bacteria at Single-Cell Level.” Microbes and Infection 2: 1525-1535.
6. Kell, D.B., Kaprelyants, A.S., Weichart, D.H., Harwood, C.R., Barer, M.R. (1998) “Viability and Activity in Readily Culturable Bacteria: A review and Discussion of the Practical Issues.” Antonie van Leeuwenhoek 73: 169-187.
7. Li, J.K., Asali, E.C., Humphrey, A.E. and Horvath, J.J. (1991) “Monitoring cell concentration & activity by multiple excitation fluorometry.” Biotechnology Prog. 7: 21-27.
8. Oliver, J.D. (February 2005) “The Viable but Nonculturable State in Bacteria.” The Journal of Microbiology 43(special issue S): 93-100.
Jeanne Moldenhauer is Vice President of Excellent Pharma Consulting. She has over twenty-five years experience in the pharmaceutical and biotechnology industries. She is the chair of the PDA’s Microbiology and Environmental Monitoring Interest Group. She also sits on the Scientific Advisory Board and the Technical Book Advisory Boards. Jeanne is the author/editor of many books include: Environmental Monitoring Volumes 1-4 Steam Sterilization, Systems Based Inspections, Preparation for a FDA Inspection: Review of Warning Letters, Biological Indicators (with M. Gomez)and has authored chapters in many other books. She has an extensive listing of journal articles and presentations as well. She is a certified quality engineer and a certified quality manager. Readers may email the author directly at: [email protected]