Abstract
Technological development in microbiological instrumentation has been progressing at an accelerated rate and with a level of sophistication difficult to predict just a few decades ago. However, today’s microbiological quality control laboratory still uses fundamental tools that were initially developed centuries ago. Why are instruments such as inoculation loops, broth tubes and Erlenmeyer flasks, agar plates, incubators, autoclaves, and microscopes still so popular, while many new detection and enumeration devices applicable in alternative and rapid microbiological methods struggle with adoption?
This article aims to pinpoint the main attributes and design characteristics of older instruments and understand why – centuries after their models were introduced – they remain key components of modern laboratories. Today’s traditional instruments will also be compared to the new wave of alternative or rapid microbiology systems in terms of functionality and sensitivity. By arming readers with this information, the author hopes that modern rapid microbiological instrumentation will further develop, leading to increased adoption by the pharmaceutical industry.
Microbiology in the Quality Control Laboratory
The monitoring of microbial contamination is one of the essential procedures in the manufacturing of pharmaceuticals, as well as in the cosmetics, food, and beverages industries. Microbiological test methods can be grouped into three broad categories based on their function. They are (1) Detection of the presence or absence of microorganisms in a test sample; (2) Enumeration of microorganisms present in a sample; and (3) Identification of microorganisms either present in a test sample or from a pure culture isolated from a test sample [1].
Each of the traditional and rapid (new, alternative) microbiological methods (RMMs) covers one or more of the above mentioned test methods. For the purpose of this paper, discussions will focus on methods dedicated to detection and enumeration of microbial contaminants. Due to challenges with current technologies, the pharmaceutical industry has not yet achieved broad acceptability of RMMs and opportunities for growth are addressed here.
A Brief Historical Perspective
Recently, a number of publications have introduced and summarized a plethora of new rapid microbiological methods [2-3]. Table 1 lists a selection of the most fundamental tools and instruments used in microbiology and the approximate date they were invented [4-7].
Table 1.
The Traditional Microbiological Instruments
As evidenced in Table 1, most of the listed instruments were developed during the second half of the nineteenth century. All these tools were designed with a specific application in mind. They covered specific needs. Their design and the concepts behind them were simple by our technological level but their applicability was, and still is, straightforward.
Applicability of the Traditional Instruments in the Detection and Enumeration of Microorganisms
Each of these instruments, from microscopes, autoclaves, and incubators to simple tools such as loops, culture tubes, pipettes, plates and more, has its own role in completing the required quality control microbiological testing and related assays in today’s laboratories [8-9]. One or more of these instruments are still employed in each of the most broadly applied detection and enumeration methods used in the microbiological laboratories, such as: (1) Most-Probable- Number (MPN) method; (2) Turbidimetric method; (3) Plate Count; (4) Membrane Filtration; and (5) Direct Count (Microscopic) [10].
How Sensitive is a Traditional Enumeration Method?
Sensitivity, Analytical Sensitivity, Functional Sensitivity, Limit of Detection (LoD), and Limit of Quantification (LoQ) are terms used to describe the smallest concentration of an analyte (or in microbiology: the smallest number of microbial cells) that can be reliably detected and/or measured by an analytical procedure [11]. In microbiology, particularly in quality control for the pharmaceutical industry, the determination of these parameters is very important in measurements of microbial samples at low count. Defining these parameters is relevant to understand the test capability and limitations. In analytical chemistry, the determination of Limit of Blank (LOB), LoD and LoQ are well described in the guideline EP17 of the Clinical and Laboratory Standards Institute (CLSI) [12]. In microbiology QC, there also is guidance on the determination of these parameters [13].
Some of these traditional detection and enumeration methods are very sensitive, particularly the methods listed in the previous section as Most-Probable-Number (1), Plate Count (3) and Membrane Filtration (4). Assuming that sufficient replicates are run, microbial cells are not clustered together and are randomly distributed, the liquid or solid growth medium and incubation temperature are appropriate for the microorganisms under study, and the incubation period is long enough to allow even a single viable cell to grow to a visible colony or a turbidity visible to the naked eye, these traditional methods potentially have a limit of detection down to a single cell.
The Single Microbial Cell Detection Paradigm
Traditional methods have a limit of detection still unmatched by many of the new rapid technologies. In order to improve their sensitivity level, some rapid methods use an enrichment step of the potential microbial contaminant previous to the assay. If enrichment is required to allow growth of the potential contaminant to reach the LOD of the assay, the time consumed in doing this step may compromise the benefit of using a “rapid” method. In addition, enumeration is also affected as the test will produce a qualitative (presence or absence) result.
There is a significant amount of guidance on the validation of rapid and alternative microbiological methods [14-16]. As discussed previously, and in more detail in the chapter 1223 of the USP [15], the LOD is the lowest number of microorganisms in a sample that can be detected under the stated experimental conditions. The limit of detection discussed in the guidance refers to the number of organisms present in the original sample before any dilution or incubation steps; it does not refer to the number of organisms present at the point of assay. Regarding quantification, the LOQ is the lowest number of microorganisms that can be accurately counted, and as with the LOD, this parameter also refers to the number of organisms present in the original sample. Also, as stated in chapter 1223, the LOQ should not be a number greater than that of the traditional method. In addition, for validation purpose, the alternate method need only demonstrate that it is at least as sensitive as the traditional method to similar lower limits (USP1223). One of the methods referenced in [15] to demonstrate equivalence between the two quantitative methods (traditional vs. RMM) is through the use of the Most Probable Number technique that was mentioned in a previous section.
This discussion raises the following questions: Are all of the rapid enumeration systems capable of meeting these requirements? How do they compare in terms of sensitivity to the traditional methods?
Despite encouragement by regulators and professional associations’ conferences to move forward with RMMs [17-20], pharmaceutical microbiologists have yet to fully accept these methods. Poor acceptance may be due to the fact that the sensitivity of some technologies is below the levels obtained with traditional methods. Systems that are based on very sophisticated and sensitive technologies, such as nucleic acid amplification (e.g., quantitative polymerase chain reaction or qPCR), still aim to achieve the sensitivity reached by traditional membrane filtration/plate count based methods. The sensitivity attained using qPCR remains at least 1 log lower than the traditional method due to the Instrument Detection Limit (IDL), that takes into account not only the limitations of the instrument detection system, but also some limitations of the PCR reaction in terms of purity of the DNA polymerase, inhibitors, and background level.
In addition, many new technologies require sample preparation protocol in order to achieve a degree of purity of the target under analysis compatible with the assay and eliminate potential inhibitors of the reaction and/or detection parameters. Each added step due to sample prep contributes to losses of the initial microbial count and opportunities for error. All of these factors will naturally increase the measured detection limit. This detection limit that combines the limitations intrinsic to the assay with those errors or limitations originated during the sample preparation is called the Method Detection Limit (MDL). Some rapid methods when used in tests without any pre-enrichment have a 2-log lower sensitivity than the reference method. This is a challenge when working with enumeration methods at the lowest ends of detection. However it is encouraging to see that many of the new RMM technologies are addressing these challenges. A qPCR based microchip combining high selectivity antibody based recognition (sorting), an electric field mediated capture by dielectrophoresis (DEP), and a highly specific real time qPCR process with a very low LOD and short overall process time have been recently described [21-22]. This platform presents an LOD of about 10 microbial cells inside the microchip and has the possibility to operate with a continuous flow.
Lessons on design and applicability of the traditional microbiological instruments provides ideas that may stimulate developers of new technologies to seek further refinements and achieve similar or better sensitivity than the old tools and methods.
Inspiring Designs
The previous review of historical microbiology tools was intended to act as inspiration to influence and perhaps guide the further development of new technologies for the twenty-first century.
What are the characteristics of those simple designs that could serve as guidelines or checkpoints in the development of the new molecular microbiology based systems of today? Not surprisingly, most traditional instruments mentioned comply with the Principles for a Good Design (see Table 2) elaborated by the famous and contemporary German designer Dieter Rams [23].
Table 2. Ten Principles of Good Design
Table 3 lists characteristics of what a good new microbiology product should have to be competitive according to the author. If any of the traditional instruments and tools previously discussed (e.g., microscope) are selected, it is evident that most, if not all of these instruments, share a good number of the characteristics listed in Table 3 for a solid microbiology instrument.
Table 3. Characteristics of a Good Microbiology Instrument or System
Finally, Table 4 summarizes the author’s view of what attributes an ideal RMM instrument should have.
Table 4. An Ideal Rapid Microbiology Instrument or System
The more checkpoints an RMM platform accumulates in Table 4, the more powerful the appeal and acceptability this platform will have for adoption and implementation replacing a traditional method. Note that the list of attributes refers to an “ideal” instrument. Therefore, it is not necessarily feasible with current knowledge levels and technologies. Readers may complete the comments and checks in Table 4 when either assessing a new RMM platform or those considering developing one.
Conclusion
The sensitivity limitation of some RMM systems may contribute to delayed acceptance on a broader basis, despite other obvious advantages of current RMMs over traditional methodologies [2, 17-20]. Many currently available rapid methods undoubtedly provide significant benefits to the user as they have advantages over traditional methods in a wide range of applications [24-27] and it is important to note that the traditional or compendial methods have their own set of limitations. It is well acknowledged that the microorganisms detected in the traditional methods are limited to those able to grow in the media and culture conditions used in these methods. This is why RMMs usually detect more and provide deeper information to the microbial monitoring test. Therefore, many current RMMs present clear advantages to the pharmaceutical industry and have the potential to become the “ideal” system.
My hope is that RMMs will overcome barriers to being accepted by a broader audience. Ideally they will become applicable to a wider range of microbiological tests, particularly in the pharmaceutical field where many test samples carry zero or very few counts of microbial contamination, and alert and action limits are quite low.
Acknowledgments
I am indebted to Ron Adkins, Gilberto Dalmaso, and Elizabeth Bennett for their critical reading of the manuscript.
Author Biography
Claudio Denoya, Ph.D., has extensive experience in developing rapid microbiological methods, both as a vendor and end-user. Previously, Dr. Denoya was a Research Fellow and Group Leader of the Microbiological Technology Assessment group at Pfizer Global R&D, and a co-chair of the Global Rapid Microbiology Methods (RMM) Steering Team. He has received numerous distinctions throughout his career and has authored more than 80 patents, book chapters, reviews and journal peer-reviewed articles. Dr. Denoya is a frequent microbiological speaker and has presented over 200 technical dissertations at national and international scientific meetings.
References
1. Madsen, R.E. (2001) “New Technologies for Microbiological Testing” In Microbiology in Pharmaceutical Manufacturing (R. Prince, Ed), p. 147-156, Parenteral Drug Assoc, Baltimore
2. Hussong, D. and Mello, R. (2006) Alternative Microbiology Methods and Pharmaceutical Quality Control. American Pharmaceutical Review. 9(1): p. 62-69.
3. Denoya, C.D. (2011). “Alternative Microbiological Methods and New Pharmaceutical Microbiology Curriculum”. In Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices (M. R. Saghee, T. Sandle and E. C. Tidswell, eds.), First Edition, Business Horizons Pharmaceutical Publishers, New Delhi, India.Chapter 20 pp. 491-518
4. Boorstin, D.J. (1983) “The Discoverers: A History of Man’s Search to Know his World and Himself”. Random House, NY
5. Hugo W.B. (1991). “A brief history of heat and chemical preservation and disinfection”. J. Appl. Bacteriol. 71 (1): 9–18.
6. Davis, B.D. (1990) “Evolution of Microbiology and of Microbes”. In: Microbiology (Ed. Bernard Davis et al). pages 3-20, J.B. Lippincott Co., Philadelphia.
7. Pommerville, J.C. (2013) “Microbiology: Then and Now”. In Fundamentals of Microbiology, pages 3-34, Jones & Bartlett Publishers, Burlington, MA
8. Cappuccino, J. G., and N. Sherman. (2008). Microbiology a laboratory manual, 8th ed. Pearson/Benjamin Cummings, San Francisco, CA.
9. Lammert, J. M. (2007). Techniques in microbiology. A student handbook. Pearson/Prentice Hall, Upper Saddle River, NJ.
10. Hammack T. (1998) FDA Bacteriological Analytical Manual, 8th Edition, Revision A, Silver Spring, MD
11. Armbruster, D.A. and T. Pry (2008) Limit of Blank, Limit od Detection and Limit of Quantitation. Clin Biochem Rev.; 29 (Suppl 1): S49–S52.
12. Guideline EP17A (2004) “Protocols for Determination of Limits of Detection and Limits of Quantitation”. Clinical and Laboratory Standards Institute (CLSI), Wayne, PA
13. United States Pharmacopeia (2012) Chapters <1225> Validation of Compendial Procedures. And <1227> Validation of Microbial Recovery from Pharmacopeial Articles. 35th revision. United States Pharmacopeial Convention, Rockville, MD.
14. PDA Technical Report No. 33 (2000) Evaluation, Validation and Implementation of New Microbiological Testing Methods. PDA Journal of Pharmaceutical Science and Technology. Volume 53(3) Supplement TR33.
15. United States Pharmacopeia (2012) Chapter <1223>, Validation of Alternative Microbiological Methods 35th revision. United States Pharmacopeial Convention, Rockville, MD.
16. European Pharmacopoeia (2011) Chapter 5.1.6. Alternative Methods for Control of Microbiological Quality. 7th ed. Strasbourg, FR: European Directorate for the Quality of Medicines.
17. Riley, B., A Regulators View of Rapid Microbiology Methods. (2011) European Pharmaceutical Review. 16(5): p. 3-5.
18. Denoya, C., J. Reyes, M. Ganatra and D. Eshete (2012) “Feasibility Studies on Rapid Sterility Testing Using a Bioluminescence-based Method” In Encyclopedia of Rapid Microbiological Methods (M. J. Miller, Ed) PDA, Bethesda, Maryland, and DHI Publishing, LLC, IL, USA Chapter 15.
19. Denoya, C. (2011) “PCR and Other Nucleic Acid amplification Techniques – Challenges and Opportunities for Their Application to Rapid Sterility Testing” In Rapid Sterility Testing (J. Moldenhauer, Ed.) PDA, Bethesda, Maryland, and DHI Publishing, LLC, IL, USA
20. Denoya, C.D. (2011). “Alternative Microbiological Methods and New Pharmaceutical Microbiology Curriculum”. In Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices (M. R. Saghee, T. Sandle and E. C. Tidswell, eds.), First Edition, Business Horizons Pharmaceutical Publishers, New Delhi, India.Chapter 20 pp. 491-518
21. F.R. Madiyar, L.U Syed, P. Arumugam, J. Li (2013) Electrical Capture and Detection of Microbes Using Dielectrophoresis at Nanoelectrode Arrays In, Advances in Applied Nanotechnology for Agriculture Chapter 6, pp 109–124, American Chem Society, Washington, DC
22. M. Nayak et al (2013) Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms. Scientific Reports (Nature) 3:3266 1-7
23. Dieter Rams (2012) San Francisco Museum of Modern Art (SFMOMA) exhibition “Less and More: The Design Ethos of Dieter Rams”.
24. John Duguid, Ed Balkovic, and Gary C. du Moulin. (2011) Rapid Microbiological Methods: Where Are They Now? American Pharmaceutical Review - Volume 14, Issue 7
25. Miller MJ, ed. (2012) Encyclopedia of Rapid Microbiological Methods. PDA, Bethesda, Maryland, and DHI Publishing, LLC, IL
26. Denoya C, Colgan S, du Moulin GC. (2006) Alternative Microbiological Methods in the Pharmaceutical Industry: The Need for a New Microbiology Curriculum. Am. Pharm. Rev.; 9:10-18.
27. Cundell AM. (2006) Opportunities for Rapid Microbial Methods. Eur. Pharm. Rev.; 1:64-70.