The use of rapid microbiological methods (RMMs) is quickly gaining favor in the pharmaceutical, biotech, personal care, and cosmetic manufacturing industries. Combining this BioPhorum article and our experience assisting labs in a wide variety of industries, we have compiled this step-by-step guide and related resources to help you.
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Quantitative Method Detection
Flow Cytometry-based detection is typically used for the measurement of cells. Flow cytometry is conducted by focusing cells into a narrow stream, or “single file”, in a sheath fluid. Photomultipliers detect light scattering and fluorescence, which gives information about the cell’s size and morphology. Solid phase cytometry is similar, except the sample is first filtered through a membrane. Microbes that remain on the filter are labeled with a fluophore, which are then scanned by the laser to detect microorganisms but also fluorescent particles.
Fluorescence Assisted Enumeration
Fluorescence- assisted enumeration is an imaging-based technology performed through visual counting of fluorescently labeled colonies on a membrane. Similar to solid phase cytometry, microorganisms captured on a filter are directly labeled with a non-fluorescent substrate which is cleaved by living microorganisms and taken into the cell. Only living cells will fluoresce, but these technologies still require a user to perform enumeration. These systems are routinely able to detect microcolonies, comprised of multiple cells or colony forming units, and may have difficulty detecting individual cells.
Automated Colony Counters: Autofluorescence Assisted enumeration
Autofluorescence based colony counters are imaging-based technologies that utilize naturally occurring fluorescence when colonies of microorganisms are exposed to light of a specific wavelength. In these systems, plated samples are exposed to a narrow wavelength of this high powered light, their momentary autofluorescence is captured by a charged couple device (CCD) as data. An image is rendered from this data. The resulting images are either manually reviewed to determine if contamination is present in the form of glowing colonies, or an algorithm is employed to identify colonies from the background via signal to noise ratios. One drawback to these instruments is that the repeated exposure to this high powered light generates heat in the sample being imaged, which may impact recovery over time.
Automated colony counter/ non-fluorescence assisted enumeration
Non-fluorescence assisted enumeration, or automated colony-counting is image-based enumeration which does not utilize fluorescent dyes or stains. Some technologies utilize algorithm-based image analysis to predict colonies; while others also require manual enumeration. Since these instruments do not utilize any fluorescence to discern colonies at very early stages in their growth, or to discern them from the background, it can be difficult for the imaging system to discern individual cells against microcolonies.
Qualitative Method Detection
ATP bioluminescence utilizes a naturally occurring reaction which occurs in the presence of adenosine triphosphate (ATP), when the luciferase enzyme converts luciferin into oxyluciferin. As ATP is present in all living bacterial, fungal, and yeast cells, and that this reaction only occurs when ATP is present, allows for the detection of microbiological contamination in a sample. The light produced via this reaction is automatically detected by Celsis luminometers. This reaction is also the same reaction which gives fireflies their famous glow.
CO2 Monitoring is the qualitative measurement of the increasing of CO2 levels as an indicator of microbial growth inside a closed vial. Changing CO2 levels inside the closed vial will initiate a change in the pH of the vial, which changes the color of an indicator on the vial containing the sample. An instrument measures the change in this indicator, resulting in a decrease in pH levels, to report whether a contamination is present.
PCR-based Selective Detection
In PCR-based Selective Detection, bacterial are detected by targeting specific conserved regions of the bacterial or fungi/yeast genomes. The 16S rRNA coding region for bacteria and 18s sRNA coding region for fungi/yeasts are amplified using PCR. The amplified fragments are then detected to indicate the presence of a contamination. This technology is often employed specifically for mycoplasma, as these are not recovered through traditional methods for bacteria, yeasts, and molds. This assay can be very sensitive; however, it requires a considerable amount of manual preparation with very tight experimental protocol parameters.
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