RMM Tutorial: Growth-based Technologies
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Rapid methods that employ the use of growth-based platforms are, for the most part, decreasing the time at which we can detect actively growing microorganisms. Many currently used growth-based systems continue to use conventional liquid or agar media. As a result, the same types of applications that traditional methods are used for can also be applied to growth-based RMMs. Examples include bioburden testing, Microbial Limits, environmental monitoring, sterility testing, and the identification or presence/absence of microorganisms. Examples of the types of core technology principles that are currently used are based on impedance microbiology, the detection of carbon dioxide (CO2), the utilisation of biochemical and carbohydrate substrates, the use of digital imaging and auto-fluorescence for the rapid detection and counting of micro-colonies, fluorescent staining and enumeration of micro-colonies by laser excitation, and the use of selective media for the rapid detection of specific microorganisms.
Microbial growth results in the breakdown of larger, relatively uncharged molecules into smaller, highly charged molecules (e.g., proteins into amino acids, fats into fatty acids and polysaccharides or sugars into lactic acid). RMMs that utilize impedance employ sample holders containing incubation wells in which two electrodes are located at the bottom of each well. These systems can detect changes in measurable electrical threshold during microbial growth; for example, by monitoring the movement of ions between electrodes (conductance), or the storage of charge at the electrode surface (capacitance). Liquid media is added to each well, in addition to the sample under test, and the holder is incubated at an appropriate temperature. If microbial growth occurs, changes in impedance are detected faster than observing turbidity in the media by an operator. Usually, the levels required for detection are ~100,000 colony forming units (CFU) for bacteria and ~10,000 CFU for yeast and mould. Impedance is primarily used for the rapid detection of growth (presence/absence) and for the estimation of viable cell concentration. An example of using impedance for a semi-quantitative screening tool is during preservative effectiveness testing in support of formulation development.
The Detection of Carbon Dioxide
Microorganisms, when grown in liquid culture, produce carbon dioxide (CO2) and other metabolites. In a closed container, the amount of CO2 produced may be monitored and used as a measure of organism viability. A number of RMMs that utilize the detection of CO2 have been employed in hospital clinical labs for many years, and are used for the rapid detection of microbial contamination in blood and urine samples. Now, this type of technology is being used by the pharmaceutical industry for rapid sterility testing and other assays where the presence or absence of microbial growth is determined. Test samples are added to media bottles that contain a liquid emulsion or silicone sensor. During microbial growth, CO2 in the medium diffuses into the sensor. Hydrogen ions will then interact with the sensor resulting in a decrease in pH, and the sensor will turn color (e.g., from gray to yellow). The rate at which CO2 is detected depends on the initial concentration of microorganisms; for example, a higher initial concentration will provide a faster detection response. However, the sensitivity level required to produce this response (i.e., a color change in the sensor) is not known. Currently, a number of companies have received FDA-approval to use CO2 detection RMMs for rapid sterility testing of tissue and cell-based products. For one company, the 14-day sterility test was reduced to just three days.
Detection of Changes in Head Space Pressure
As organisms grow in a liquid medium, they will consume oxygen and produce CO2 as a result of microbial respiration. If the microorganisms are respiring in a closed container, changes in head space pressures will result. Rapid methods employing sensors (electronic transducers) can detect both positive and negative pressure changes. general microbiological media may be used for the detection of a wide range of microorganisms; however, if a selective medium is used, the detection of specific or target microorganisms is possible.
Utilization of Biochemical and Carbohydrate Substrates
There are a variety of RMMs that employ a microorganism’s ability to utilise biochemical and carbohydrate substrates as sole carbon or energy sources for the rapid and automated identification of microorganisms. A suspension of a pure culture (usually from an isolated colony on an agar plate) is inoculated onto test cards or strips. Each card or strip is composed of incubation wells, and individual wells contain a single substrate in dehydrated form. The inoculated cards or strips are incubated, and if the organism under test utilizes any of the substrates for cellular metabolism and growth, the turbidity, color and/or fluorescence in the well will change. One system incorporates a dye in each carbohydrate test well, and if growth occurs, the well will turn purple. The resulting data (normally in the form of positive and negative responses) are compared with an internal database or reference library and a microbial identification is provided.
Digital Imaging and Auto-fluorescence of Micro-colonies
During microbial growth, cells will fluoresce in the yellow-green spectral region when illuminated with blue light. Cellular auto-fluorescence in this spectral region is a property of all microbial cells due to the presence of ubiquitous fluorescent biomolecules including flavins, riboflavins, and flavoproteins. One RMM technology utilizes this phenomenon to enumerate micro-colonies developing on an agar plate in approximately one-half the time required to visualize colonies using the naked eye. Samples are filtered and the membrane is placed onto an agar cassette and incubated. During incubation, a laser excites micro-colonies to autofluoresce, which are automatically enumerated by a CCD imaging system. Particles that do not grow in size over time are ignored (via software algorithms), and because this technique is non-destructive, the agar medium can continue to incubate to obtain larger colonies that can subsequently be used for microbial identification. Companies are using this type of RMM for the rapid enumeration of microorganisms during bioburden, water, environmental monitoring, in-process, finished product and sterility testing.
Fluorescent Staining and Laser Excitation of Micro-colonies
A recently introduced RMM utilises fluorescent staining and laser excitation for the rapid enumeration of micro-colonies. The technology is applicable for all filterable samples, including water, in-process and finished product. A test sample is filtered and the membrane is placed onto an agar cassette. Following an appropriate incubation period, the membrane is stained with a non-fluorescent substrate. Microorganisms on the filter will take up the substrate, which is then enzymatically cleaved, liberating free fluorochrome in the microorganism cytoplasm. As the fluorochrome accumulates inside the cells, the signal is amplified. Following incubation, the membrane is placed into a reader and exposed to the excitation wavelength of the fluorescent stain. Fluorescent micro-colonies can then be counted in the instrument window or on a computer via a camera. This RMM is also non-destructive, as the membrane can be placed back onto the agar cassette and re-incubated to allow larger colonies to form that can then be used for microbial identification.
Use of Selective Media for the detection of Specific Microorganisms
Another RMM targets the presence of specific microorganisms by monitoring changes in color or fluorescence in selective media and also detects total aerobes and yeast or mould by monitoring the generation of CO2. The system utilizes disposable two-zone vials that contain an incubation zone (top of vial) for the sample and microorganism, and a reading zone (bottom of vial). The two-zones eliminate masking of the optical pathway by the product and by microbial turbidity. The time to detection depends on the initial concentration of organisms in the product sample, and the threshold for bacteria is 100,000 cells/mL and 10,000 cells/mL for yeast and mould. Current test vials allow for an estimation of total aerobic count, and the detection of yeast and mould, coliforms, E. coli, lactic acid bacteria, Enterobacteriaceae, Salmonella, Pseudomonas, and Staphylococcus.
Growth-based rapid microbiological methods provide opportunities for enhanced detection, enumeration and identification of microorganisms. Applications for these types of RMMs include, but are not limited to, raw material and component testing, in-process and pre-sterilization/filtration bioburden, fermentation and cell culture monitoring, purified/process water testing, environmental monitoring, Microbial Limits, antimicrobial effectiveness testing and sterility testing.