Doing it Through Rapid Testing, Automation, and "Chemistry" Instruments

Microbiology—the study of microscopic organisms, mostly single-celled— is one of the deepest, broadest scientific disciplines. Microbiology is an essential component of human and veterinary medicine, pharmaceuticals production and safety, wastewater treatment and bioremediation, food fermentation and quality, agriculture and plant biology, environmental and workplace testing, and at least a dozen areas of pure research.

Several subsets exist within each category, and each business segment is characterized by unique workflows and, to a lesser degree, equipment, instrumentation, and consumables.

Cell culture and microbiology labs differ in several important aspects. In cell culture, the cells themselves or the molecules they produce are usually the “product.” In microbiology, the “answer” is a test to determine the presence, viability, or quantity of some organism.

But two important overlaps exist, in the form of cell-based assays for cultured cells and fermentation cultures in microbiology.

Agar Media Dishes / Microsart @media Sartorius / www.sartorius.com	Both cell culture and microbiology labs use microplates. Plates are used in cell culture for screening before amplification within flasks and bioreactors. Microbiology does not generally scale up beyond what is necessary to conduct an assay, whether the format be a microplate, petri dish, or instrument. Cell culture labs follow good laboratory practices and good manufacturing practices (for therapeutic cells and cell-derived proteins), while microbiology labs operate under a myriad of laws and standards from the EPA, OSHA, American Society for Microbiology, College of American Pathologists, and various industry groups and the FDA’s food regulations.   Yet operationally the two labs are quite similar. Alex Esmon, PhD, global product manager at Thermo Fisher Scientific (Asheville, NC), notes that both keep an eye on costs and readily acquire equipment that offers advantages to users, workflows, and obtaining consistent results. Both labs use centrifuges, microscopes, clean benches and hoods, rockers, rotators, and mixers. “A microbiology lab looks a lot like other labs, with some equipment differences,” Esmon explains. “For example, they typically ask different questions of their organisms of choice than a cell culture lab does.” Culture media are equally critical for microbiology and cell culture labs, as both industries seek quality, trustworthy basal media that support the expansion of cells and microorganisms. Unless they are following set protocols, cell culture labs often tweak media to encourage growth and protein productivity in recombinant cells. Industrial microbiology tends to use off-the-shelf media optimized for their specific organism; media optimization occurs only in media development and general research labs. “Media tends to be more standardized in microbiology, particularly in clinical labs,” Esmon explains. “But in research they’re probably asking the same types of questions as in cell culture labs, where media are much more variable.”
96-Well Microplates with 3D Epoxy Surface Greiner Bio-One / www.greinerbioone.com
Air-cooled Benchtop Centrifuge / Allegra X-5 Beckman Coulter / www.beckmancoulter.com

Speed to result is important in any analytical industry, but it is particularly critical in microbiology. “Labs are always looking for a faster result,” comments George Tice, R&D director for molecular diagnostics at DuPont Nutrition & Health (Wilmington, DE). “In the food industry, products are kept on hold until they have a result. If you’re dealing with fresh ground meat, time is money.” Here, sampling variability adds a dimension that is rarely seen elsewhere. “Some assays are designed to detect one CFU (colony-forming unit) or viable cell per sample. That sample can be anywhere from twenty-five to 375 grams, but you must still detect that one CFU,” Tice adds.

Vendors of media, instrumentation, and equipment are assiduously pursuing workflow improvements in sample preparation, sample processing, workflow integration, and making organism testing more user-friendly. Medical and food labs, moreover, are looking for assays that provide information on multiple pathogens through a single test.

These improvements will take time. Some are in their earliest stages, while others have already hit the market. The vibrancy and growth in microbiology almost ensure that they will be achieved.

Rapid microbiology

Slow acceptance, strong take-up

Rapid microbiology methods are taking the world of microorganism testing by storm, with applications in medical diagnostics, foods, and agriculture leading the way. PCR is now routine for field testing and, in hospitals, to manage life-threatening infections. In foods, both farms and factories have adopted PCR, particularly in rapid or portable formats.

Because of the diversity of microbiology assays, defining “rapid” is not straightforward. Some rapid tests take a few minutes to run but require more or less traditional culturing. Other methods take a few hours. Michael J. Miller, PhD, president of Microbiology Consultants (Lutz, FL), describes rapid methods in relative terms: “Any method that provides identification, quantitation, or enumeration in significantly less time than classical methods, often with greater sensitivity.”

Miller’s website, microbiologyconsultants.com, is an educational resource used by thousands of microbiologists worldwide.

Some growth-based rapid methods home in on surrogates of colony-forming units, such as metabolites or cellular targets such as ATP (a measure of viability); others employ fluorescence, Raman, or light scattering to detect microcolonies long before humans are capable of seeing them. Still other methods use stains or fluorescent tags that prefer one organism over others. These techniques are often used without enriching (culturing) the sample. Detection occurs through flow cytometry or solid-phase cytometry.

Droplet Digital PCR System / QX200 / Bio-Rad / www.bio-rad.com

Flow Cytometry System / Navios / Beckman Coulter / www.beckmancoulter.com

PCR Workstations / AC600 Series AirClean Systems / www.aircleansystems.com

Polymerase chain reaction (PCR) is the basis of a separate branch of microbiology that amplifies genes over the course of several hours, then detects signature genetic sequences. In addition to positive identification, PCR provides an estimation of cell count.

“Some techniques approach real-time detection and counting of viable microorganisms, which is a far cry from waiting days for colony-forming units,” Miller says.

While all classical and many rapid techniques depend on expanding organism populations through culturing, some organisms will not grow in culture due to stress or nonoptimized culture conditions. Rapid methods that do not rely on culturing are by definition more sensitive than other methods because they detect fewer organisms.

Infectious disease, pharmaceutical, and biopharmaceutical assays normally focus on viable organisms with the ability to reproduce inside a host. In injected drugs, even dead organisms and their fragments can cause severe allergic responses.

Infectious agent testing is big business. In its 2012 report, The World Market for Infectious Disease Diagnostics, Kalorama Information estimated this market at $14.5 billion in 2012. With predicted percent yearly growth, it will reach $18.3 billion in 2017. While traditional immunoassays and culture-based microorganism identification and antimicrobial sensitivity testing are holding their own, demand for rapid, specific, sensitive tests fuel this market segment’s growth.

It takes time

Rapid methods are not yet mainstream in all markets. Although the methods were introduced nearly simultaneously in clinical and pharmaceutical markets, in the early 1990s clinical labs adopted rapid methods, while their use in drug safety testing lagged.

Both industries require validation and qualification of assays and instrumentation, and both are highly regulated, but their cost structures differ significantly. Medical testing is a commodity business, while pharmaceutical businesses have been slow to adopt cost cutting. A large roadblock for therapeutics was a lack of regulations for adopting rapid methods. Absent such guidance, companies are loath to embrace new technologies.

But relief is on the way. For the past five years, Miller has chaired a Parenteral Drug Association task force on validating rapid methods for the drug industry. Technical Report 33, published in October 2013, is expected to serve as the pharm/biotech industries’ primary regulatory guidance on rapid testing.

As with classical microbiology, no rapid method does everything. All platforms favor certain samples or organisms. For example, flow cytometry works only with liquids, whereas samples must in addition pass through a filter membrane for solid-phase cytometry. Fluorescence-based methods are prone to interferences from naturally fluorescing components in the organisms or sample, whereas DNA interferences can thwart PCR analysis.

“You have to match the right technology with the analysis need,” Miller cautions. And time to result varies widely. “The term ‘rapid’ can mean anything from close to real time to overnight to increase the organism count.” Even PCR, which itself is an amplification method, requires a critical mass of organism DNA to initiate.

Automation

What it delivers, what it does not

Automation can streamline traditional microbiology workflows by providing consistency and increasing walkaway time. Many platforms exist that borrow ideas and components from cell culture. The BioLumix automated microbiology instrument from BioLumix (Ann Arbor, MI) is an example of a platform dedicated to microbiology.

A traditional assay for total count of yeast/mold, coliform, and salmonella from a food sample is lengthy and labor-intensive. After diluting liquid samples or liquefying and diluting non-liquids, technicians pour plates and cover them with liquid agar, followed by incubation for as long as five days. They then pull the plates and count the colonies, adjust for dilution, and collate the three results into a report.

With BioLumix, operators pipette samples into assayspecific vials containing a sensor and all necessary media and reagents. The instrument takes over, first culturing the organism, then conducting the assay, performing all calculations, and producing a report. Most bacterial assays take less than 24 hours; for molds the holdup is up to 48 hours.

BioLumix president Ruth Eden says no hard and fast rules dictate when a microbiology lab should automate. “A small company lacking a dedicated microbiology lab must send samples out. Automation allows labs to internalize testing, get results faster, and ultimately save money.” Eden adds that the BioLumix instrument does not require a degreed microbiologist for operation. Turning operation into a technician-level job, however, does not eliminate deciding on what to test and interpreting results.

The automation imperative is just as compelling at the high end, in labs that run many hundreds of samples per day. “Going paperless, having almost all operations automated, consistency of results, and cutting labor by half are the main drivers for high-throughput labs,” Eden says. But in the end, “different customers have different reasons to automate.”

A spectrum of solutions

“Automation means many things to many people,” explains Paul Held, PhD, manager of BioTek Instruments (Winooski, VT), “with gradations throughout the spectrum.” BioTek offers stand-alone products at the lower end that perform one or two specific tasks. Third-party automation companies often integrate BioTek components with additional instruments to provide full, end-toend automation.

Microplate Stacker / BioStack™ 4 BioTek / www.biotek.com

PCR Setup Workstation / PCR setup STARlet™ Hamilton Robotics / www.hamiltonrobotics.com

Industrial microbiology has many characteristics that favor automation. Repetitive, multiplicative tasks involving measurement, dilution, liquid dispensing, and incubation followed by manual manipulation or semi-manual instrumental analysis are what robots and liquid-handling systems do best. While some labs believe that automation cedes a degree of control over operations, the benefits of walk-away time, consistency, and results transferred directly into printable reports are often enough to tip the scales during decision making.

Many companies view automation simply through the lens of throughput. Few would argue that processing an additional two or three plates per day justifies the purchase of expensive robotic systems, while almost everyone agrees that automation can save time and money for labs that process 200 plates per day.

“At some point it is less costly to buy an instrument than to hire more employees,” Held notes. Instruments take up less room, do not receive a salary, and never ask for longer coffee breaks. And for labs somewhere in the middle of the throughput continuum, where number of replicates provides no conclusive justification, consistency and scheduling are often the deciding factors. “Instruments always do what they do the same way. An instrument may not be as good as the best technician, but you can count on the results.”

Consistency is particularly desirable in, for example, drug discovery labs where sub-microliter samples and dispensing are the norm.

Jeff Papi, director of microbiology at bioMérieux (Hazelwood, MO), defines automation as any instrumentor device-based strategy that takes a manual process and improves it in a way that reduces labor, speeds production, and ensures consistency and quality. “Ultimately, automation reduces total time from sample to decision, so products reach the market faster.”

Not everyone is enamored of robotics and automation, however. “You lose a little something, I believe, when you automate,” comments Christopher A. Catani, marketing director at Hardy Diagnostics (Santa Maria, CA). “Automation is wonderful, but when the machine breaks down, labs descend into crisis mode. And it’s not a matter of if but of when. You also lose some proficiency as a workforce. Sure, you gain consistency and can even cut costs, but it’s a trade-off.”

Automation is expected to affect how microbiology labs are built and operated. “Particularly, adopting alternative methods will make labs leaner,” explains Michael Miller. Instrument footprints are shrinking, but rapid techniques tend inherently to take up less space. “Labs may be able to reduce personnel or education and training requirements. In some instances portability and rapid testing enable moving traditional laboratory activities into manufacturing or out to the field.” Despite these long-term trends, classical microbiology is by no means extinct.

Microbiology labs will change, but the most significant impact will be on microbiologists who, over time, face the need to adopt new skills. Miller predicts that because many rapid platforms are based on chemistry, “microbiologists are going to have to pick up analytical chemistry skills. Or in the case of PCR, they’re going to have to learn genetics techniques. They’re going to have to learn a lot more to keep up with these systems.”

Miller warns that microbiology cannot continue to be relevant by losing its classical mind-set. “The data still must be understood and translated by classically trained microbiologists who still must understand what the relevance of data means to product safety or contamination.”

Two goals, one instrument

One of the most exciting developments in microbiology, which satisfies the desire for rapidity and automation, has been micro’s embrace of instrumentation from chemistry and biology, particularly mass spectrometry (MS). Two instrument types, MALDI-TOF and electrospray ionization (ESI) mass spectrometry, provide a “soft” ionization critical to preserving the integrity of signature large-molecule fragments from cell membranes or walls. Both techniques work with or without cultivation/ expansion or chromatographic fractionation to generate data through mass, affinity, or other parameters.

Triple Quadrupole Mass Spectrometer / LCMS-8050 / Shimadzu www.ssi.shimadzu.comAccording to Yen-Peng Ho, PhD, of Taiwan’s National Dong Hwa University, “Rapid and reliable identification of microorganisms without extensive manipulation is a major goal in environmental and clinical microbiology.” MS fits the bill, Ho continues, for its robustness, low cost per sample, rapidity, and suitability for high-throughput analysis. MALDI-TOF uses very low sample mass and requires little or no preparation, yet it can confirm the presence of microorganisms in minutes. Two general workflows are possible. One involves cultivation/expansion or cell enrichment followed by fractionation (gas chromatography, liquid chromatography, or capillary electrophoresis) and MS analysis. The other works without sample prep or achieves cell or sample enrichment through affinity, microfluidic interactions, or PCR. Among the leading approaches to separation/enrichment are magnetic microbeads coated with organism-specific antibodies or antibiotics that bind to test organisms.

“At its simplest, you can scrape a colony off a plate, pop it into the MALDI, and get the genus and species directly,” Miller says. “These same methods have existed in the world of analytical chemistry for years and are now finding application in microbiology.”

One does not normally think of acquiring a mass spectrometer as a cost-cutting measure, but that is how Catani describes MALDI-TOF. Catani claims that MS cuts assay costs from about $10 per test for some automated systems to about $1 per test. “And it takes seconds, not hours or days. Yes, you need a mass spec, and they’re quite expensive, but a busy lab can make its money back in less than a year.”

Another “cool trend,” Catani says, is the emergence of chromogenic media that provide superfast detection without the need to purchase an MS. Hardy, Sigma Aldrich, Becton-Dickinson, BioRad, and bioMérieux (among other companies) manufacture chromogenic media for microbiology.

As its name implies, chromogenic media uses color to identify specific pathogens, even in mixed-pathogen samples. The media are optimized for the test organism, both for nutrients and biochemical-colorimetric detection and reporting. Some media also contain antibiotics to inhibit the growth of competing organisms.