The Basics

Many in the industry are now using viability-based technologies which can differentiate living cells from dead cells, and can even target specific types of microorganisms using nucleic acid, enzymatic or monoclonal antibody probes. In many cases, direct labeling of individual cells with viability stains or fluorescent markers followed by laser excitation has been demonstrated with no requirement for cellular growth. And because microbial growth is not required, many organisms that would have a difficult time growing in or on conventional medium, such as spores, stressed and physically-injured cells, fastidious organisms, and viable, but non-culturable isolates, can now be easily and quickly detected and/or enumerated. Since viability labeling of microorganisms can occur within hours or even minutes, near real-time detection and quantitative results may be attained.

Flow Cytometry

Flow cytometry represents one of the viability-based RMM technologies currently available. In flow cytometry, individual particles are counted as they pass through a laser beam in a very narrow flow cell. To be used in microbiological applications, organisms are pre-labeled with a viability marker or probe, and the labeled cells are injected into the flow cell. Individual cells pass single file, through a focused laser beam, and are enumerated as the cells fluoresce and light scatter signals are detected. Usually, low levels of test sample volumes are utilized, such as 1 mL or less, because it takes time for this volume to pass through the narrow flow cell. The process of labeling and evaluating viable cells in these types of RMMs can be accomplished in as little as 4 minutes, and some systems operate automatically with little or no operator intervention or manipulations. Flow cytometry offers a relatively large range of detection operation (10-106 cells/mL) and has been demonstrated to enumerate bacteria, spirochetes, mycoplasmas, parasite cysts, yeasts, and mold.

Although flow cytometry offers relatively fast and hands-off capabilities, there is one limitation: the limit of quantification (LOQ). Flow cytometry systems usually have a LOQ of approximately 10-50 cells; therefore, it may be difficult qualifying this technology for the detection of single cells, as what would be required during sterility testing (i.e., an enrichment step would probably be required). However, flow cytometry also offers additional capabilities that other technologies do not possess. For example, flow cytometry can make use of fluorescent dyes and microbe-specific probes, such as antibodies, rRNA or peptide nucleic acid (PNA), making the simultaneous enumeration and detection of target organisms possible. Test methods applicable to the use of flow cytometry include bioburden testing of purified water, liquids and non-filterable material, such as creams and lotions. Other uses include the screening of raw materials, in-process samples and finished product for microbial contamination. In the latter application, samples can be enriched (i.e., incubated in conventional microbiological media), in order to encourage the growth of very low levels of microorganisms (e.g., less than 10 cells).

Solid Phase Cytometry

Another viability-based technology, called solid-phase cytometry, uses a similar staining and laser excitation method as does flow cytometry; however, the microorganisms are first captured onto a solid surface and subsequently labeled with a viability stain, instead of labeling the cells first and then passing the labeled cells through a flow cell.

Test samples are initially passed through a 0.4 µm membrane filter, and then the membrane is exposed to a non-fluorescent substrate. Microorganisms that are retained on the filter will take up the substrate, and within the cytoplasm of metabolically active cells, the substrate is enzymatically cleaved by an esterase. This enzymatic cleavage results in the release of a fluorochrome, which can be excited when exposed to a laser of an appropriate wavelength. If the retained organisms have an intact cell membrane, the fluorescent label is concentrated within the cell and this signal is detected by the system as the membrane is scanned by the laser. Photo-multiplier tubes detect emitted fluorescent light from the cells, and software algorithms and discrimination processes will determine if the fluorescent signals originate from labeled viable microorganisms or from an auto-fluorescent particle.

The use of this type of RMM allows for the evaluation of a large volume of filterable materials, with single cell detection as early as 90 minutes, making solid phase cytometry an excellent candidate for testing purified water and finished product for sterility. However, the advantage of single cell quantification in large volumes of test sample comes with one limitation; the samples must be filterable.

Test methods applicable to the use of solid phase cytometry include bioburden testing, environmental monitoring, sterility testing and the detection of bacterial contaminants in mammalian cell cultures. Solid phase cytometry can also use antibodies and nucleic acid probes to detect and quantify specific microorganisms within a few hours. For example, municipal water authorities have used this RMM to rapidly detect and quantify E. coli and Cryptosporidium in their test samples.

Summary

Viability-based rapid microbiological methods take us to the next level of detection and enumeration of microorganisms. Applications for these types of RMMs are fairly broad, ranging from raw material to in-process to finished product release testing. The end-user is only limited by their imagination and the compatibility of the test samples with the intended technology platform.