How To Select the Right Rapid Microbial Method

Rapid methods of microbial analysis can eliminate days from your standard assay time and follow-up investigations while still delivering the critical information needed to maintain control of your production process. Determining which method is best for your lab can be complex, but we've prepared some materials to help guide you toward the most appropriate RMM solution for your needs.

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Validation and Implementation of a rapid microbiological method of analysis for the detection of bacteria, yeasts, and molds in a pharmaceutical or personal care industry quality control microbiology laboratory

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.

lab technician preparing a microbial sample

1. Define Your Needs

Start by identifying your challenges. Are you trying to protect the integrity of your data? What about the speed at which you obtain it? Microbiology quality control departments should identify the key drivers and requirements that are most important to their workflow and business. Requirements such as sample compatibility, vendor expertise, and ease of implementation can vary from organization to organization, and even within organizations, from site to site.
Lab technician using Celsis rapid microbial detection instruments for sterile and non-sterile products, including cell therapies, ATMPs, and other short shelf-life products

2. Consider Other RMM Applications

The second step is to identify the many available rapid microbiological methods and determine which are compatible with your goals and manufacturing processes. It was once widely believed that most rapid detection systems were solely used for a single purpose or were only compatible with certain sample types, but that is no longer the case. Celsis® ATP bioluminescence, for example, can be used on both sterile and non-sterile product testing. When paired with the Celsis Adapt™ Concentrator System, it can be used for testing cell-based samples, cellular therapies, and short shelf-life products.
Quality control microbiologist working in biological safety cabinet for validation of microbiological methods

3. Compare Rapid Detection Technologies

Next, determine which RMM products best align with your goals by comparing their strengths and weaknesses. Current rapid microbiology technology instruments can be either qualitative or quantitative and can further be categorized by the type of detection technology they employ.
  • Quantitative Method Detection

    Cytometry-Based 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 

    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 

    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. 

lab technician preparing microbial samples

4. Develop an RMM Business Case

Presenting your findings to key decision makers within your organization can be tricky. Our Financial Impact Assessment tool can help you build your case by outlining the rationale behind your selection and the associated risks, costs, and ROI. This ebook lays out the business case for RMMs with a conceptual road map, guidance, and insights obtained by an industry expert over 30 years. Finally, this webinar outlines some of the benefits using Celsis® methods, such as shorter product hold times and improved production efficiency.
lab technician pipetting sample into cuvettes for rapid microbiology

5. Plan Your RMM Validation Strategy

How rapid microbial methods are implemented into manufacturing procedures will vary by industry and site. Gather the information you need to successfully execute your chosen technology and establish contact with regulatory agencies, if possible. This will allow for a smoother transition to your testing efforts. In order for rapid microbiological methods to be approved for routine testing, they must first be validated. What was once a lengthy and complicated process is now simplified with our two validation support packages, Celsis® Complete and Celsis® Advantage.
Lab technicians working to implement a rapid microbial method into their global processes 

6. Implementation and Global Deployment

The final step is to engage the RMM practice globally to all production facilities within your organization. Although this may seem like a daunting task, we’ve seen successful implementation from industry leaders in both the sterile and non-sterile markets. Once you come to a decision, our team will be happy to help you determine the best path toward implementing a rapid microbiological method specific to your product, method, and laboratory.

Once you come to a decision, our team is happy to help you determine the best path forward to implementing a rapid microbiological method specific to your product, method, and laboratory.

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