Microbiologists' Tools Reflect The Diversity Of The Discipline

NUMEROUS APPLICATIONS: Bio-Tek Instruments' FL600 automated fluorescence microplate reader tells the researcher how many living cells are in a bacterial culture. In the natural world, microorganisms wear many hats. They are agents of disease, producers of proteins, remediators of rubbage, and brewers of beer. The field of microbiology is just as diverse. A microbiologist may search for ways to inhibit bacterial growth or for methods to make bacteria grow faster. Scientists may coax bacteria to mass-produce proteins or manipulate them to be more adept at performing desired tasks, such as degrading toxic compounds. Clearly, microbiologists need tools to take full advantage of the diversity and utility of microorganisms. Additionally, many life sciences rely on the microbes themselves as tools. SEMIAUTOMATIC: With the Touch-O-Matic Bunsen burner, the researcher controls ignition via an on-off platform. Using culture techniques, microorganisms can be grown in populations as small as a colony or as large as hundreds of liters. "Microbiology is a science that requires a considerable amount of manual effort," says William Crandall, a research associate in the department of biology at the University of North Carolina in Chapel Hill. "Even as the practice of microbiology becomes more automated, in some labs the only pieces of equipment you'll find are Bunsen burners and incubators." Bunsen burners are connected directly to a gas supply. When ignited, they product a hot flame that incinerates microbes to sterilize instruments. Standard Bunsen burners can be purchased from supply companies such as Fisher Scientific Inc. of Pittsburgh; VWR Scientific Products in West Chester, Pa.; and Albany, N.Y.-based Krackeler Scientific Inc. The Touch-O-Matic Bunsen Burner-an updated, semiautomatic version of the old classic-is available from Bellco Glass Inc. of Vineland, N.J., as well as from Fisher Scientific and VWR. It has a low-burning pilot light that ignites the burner only as needed. Ignition is controlled by touching an on-off platform or with an optional foot-operated accessory that maintains the flame until the foot is removed. Automation has also made its mark on incubators. For example, Lab-Line Instruments Inc. in Melrose Park, Ill., manufactures several incubators designed to provide optimal growth temperatures for bacteria. The Ambi-Hi-Lo Models 3550DT, 3550DT-1, 3554-40, 3554-41, 3554-42, and 3554-43 incubators from Lab-Line are microprocessor-controlled, with a seven-day timer that allows two temperatures to be set and switched automatically. For labs that need to culture anaerobic organisms, Lab-Line's microprocessor-controlled Programmed Anaerobic Controlled Environment (P.A.C.E.) chamber provides appropriate growth conditions. The microprocessor regulates two sequential purges with nitrogen and one with mixed gas, which maintains the anaerobic conditions inside of the chamber even when samples or supplies are moved in or out. Fisher Scientific, also a distributor of Lab-Line products, sells a variety of small-capacity incubators and larger environmental chambers. Fisher's Isotemp incubator is a standard-capacity, refrigerated unit used in labs performing biochemical oxygen demand (BOD) studies on sewage or waste flow. BOD samples can be stored overnight at cooler temperatures (3ºC to 4ºC ), and later incubated at the standard 0ºC required for BOD testing. Electrical outlets inside of the chamber allow investigators to use auxiliary equipment, such as shakers or recording thermometers, in the incubator. An increasing number of researchers are using bacteria to mass-produce proteins with commercial value. However, to coax bacteria to grow in high-density populations, they must be provided with conditions akin to a bacterial smorgasbord-rich food served at the right temperature. Difco Laboratories in Detroit and Becton Dickinson Microbiology Systems (a supplier of BBL products), based in Cockeysville, Md., both supply a broad selection of microbiological culture media for research and clinical lab applications. Massive quantities of bacteria are required to produce large quantities of a particular gene product. Cultures larger than a few hundred milliliters can be prepared in large flasks, in bioreactors known as fermenters, or in much larger vessels used primarily in industrial laboratories. To promote optimal growth, bacteria in flasks must also be agitated to distribute nutrients and to keep the temperature in the culture uniform. New Brunswick Scientific Co. in Edison, N.J., manufactures advanced biological shakers, as well as fermenters and bioreactors for research and industrial-scale applications. New Brunswick's Innova Series incubator shakers feature microprocessor controls to store set points, alarms, and running time. Several benchtop and larger-capacity floor models are available, including the Innova 4300 Series incubator shakers, in which temperature and gaseous atmosphere in cultures can be controlled. The Innova Model 4335 includes dual-temperature programming to automatically reduce and hold the temperature at a refrigerated level, and to shut off agitation at a predetermined time to limit microbial metabolic activity. Such automation can mean more sleep for some researchers, notes Suzy Kedzierski, product manager at New Brunswick Scientific. "Someone growing cells doesn't have to come in on a weekend or in the middle of the night just to change the growth conditions," she says. Lab-Line Instruments manufactures Environ shakers, which are available for benchtop use or as floor models. The Model 3597 is a refrigerated shaker with a temperature capacity down to 0ºC. It provides continuous or timed orbital shaking. This instrument can accommodate flasks up to the 6-liter size. For even larger bacterial cultures, researchers may perform a fermentation. The BioFlo 3000 from New Brunswick Scientific is a PC-compatible benchtop fermenter with a selectable two- or four-gas controller to optimize cell yield. Automatic data-logging as well as control over process conditions may be added with AFS-BioCommand, a Windows-based software package. Complete pilot- and production-scale systems that hold up to 15,000 liters are available in a range of standard or custom designs. Before proteins can be produced by bacteria on a large scale, genes must be cloned. Once cloned genes are inside of bacterial cells, the protein of interest will be produced along with the normal bacterial proteins. Bacterial cultures are easier to grow and maintain than are cultures of animal cells or animals themselves, and engineered proteins are often secreted into the culture supernatant, from which the protein can be purified. Stratagene Cloning Systems of La Jolla, Calif., offers several products designed to make cloning and expressing proteins from genes easier. The company carries different bacterial and viral cloning vectors, such as the pCR-Script plasmid vectors for cloning polymerase chain reaction (PCR) products in bacteria and the pET plasmid vector series. The pET expression systems from Stratagene allow researchers to achieve high levels of expression from cloned genes under the inducible control of a bacterial promoter. The pGEM-T and pGEM-T Easy Vector systems from Promega Corp. of Madison, Wis., may be used to clone PCR products. They are designed with a single thymidine "overhang" at each 3« end of the insertion site. The thymidine overhangs prevent the plasmid from re-forming into a circle after being cut with a restriction enzyme. The T overhang also provides a compatible overhang for PCR products generated by certain thermostable polymerases, such as Taq, which adds a single adenine to the 3' ends of amplified fragments. Promega also offers the pET Vector System for cloning and expressing recombinant proteins in E. coli. The ZipLox vector from Life Technologies Inc. in Gaithersburg, Md., is used to clone and express cDNAs produced from RNA isolated from cells. When the target gene is from an animal rather than bacterial source, the pFastBac plasmids from Life Technologies permit researchers to carry out the initial cloning steps in bacteria. Once the target gene is cloned, it can be "shuttled" to insect cells, where proteins are expressed in a more natural form. The Bac-to-Bac Baculovirus Expression system, also from Life Technologies, is an expression system designed for such applications. To express genes cloned in plasmids or viruses, the vector has to be "transformed" into the appropriate cells, which reproduce the cloned genes every time the cells divide. Since the average E. coli cell reproduces about every 20 minutes, billions of copies of genes cloned in this bacterial workhorse can be obtained in a day. While many researchers prefer to prepare their own "competent" cells (bacteria that readily take up foreign DNA), bacterial cells made competent by either chemical or electrochemical means can be purchased from Stratagene and Life Technologies. Stratagene's Epicurean Coli product line consists of various strains of E. coli that have been genetically manipulated to transform vectors with extremely high efficiency. E. coli strains made competent for transformation are also available at the levels of Max Efficiency, Library Efficiency, and Subcloning Efficiency from Life Technologies. Expressing cloned genes isn't the only use for bacteria. They can also be used in toxicology studies to determine the mutagenicity of chemical compounds. The implication here is that substances that cause mutations in the genomes of bacteria may eventually cause cancer in humans. In vitro studies that rely on cultured mammalian or bacterial cells are replacing animal tests that were used by the cosmetics and pharmaceutical industries to access the toxicity of their products. The Ames test, developed by microbiologist Bruce Ames, was the first of these so-called in vitro tests for mutagenicity. SELECTED SUPPLIERS OF PRODUCTS FOR MICROBIOLOGISTS: Becton Dickinson Microbiology Systemlass Inc. Bioscience International Bio-Tek Instruments Inc. Difco Laboratories Fisher Scientific Inc. Gen-Probe Inc. Krackeler Scientific Inc. Lab-Line Instruments Inc. Life Technologies Inc. Molecular Probes Inc. New Brunswick Scientific Co. Promega Corp. Stratagene Cloning Systems VWR Scientific Products Xenometrix Inc. NEW VERSION OF A CLASSIC: Xenometrix’s Ames II Assay detects base-specific mutation events in Salmonella. Xenometrix Inc. in Boulder, Colo., manufactures and sells an integrated system of cell-based assays that test the genetic response of bacterial and human cells when exposed to a compound. The company's bacterial and mammalian cell Stress Gene Assay systems allow investigators to determine the specific molecular mechanisms that underlie the activity of a chemical compound. The bacterial assays include the Pro-Tox (C) and Pro-Tox (D) kits. The mammalian assays include CAT-Tox (L) and CAT-Tox (D), which contain human liver and human colon cells, respectively. These Stress Gene Assays are engineered with stress-inducible genes that respond to DNA damage and other cellular stresses. Each assay creates a histogram-or "fingerprint"-for a given compound. Researchers can compare the fingerprint obtained from a test compound with those generated by previously tested compounds in the XenoMatrix database. The Ames II Assay from Xenometrix improves upon the original Ames assay. It utilizes six specially constructed strains of the bacterium Salmonella typhimurium, each with a different mutation in the histidine operon (the genes the bacteria use to synthesize the amino acid histidine). If a test compound is mutagenic, one or more of the histidine mutants may revert back to the wild-type, a phenotype that can be detected using a colorimetric system. In the Ames II High Through Put Assay, the six Salmonella strains are combined into a single rapid screening mixture, and the test is performed in a 96-well microtiter plate format. The Ames II is also available in a manual and automated mutational spectra format. Other microbe-based genotoxicity assays from Xenometrix that employ the same principles as the Ames II Assay include the Yeast DEL Assay and the E. coli Trp Assay. Numerous products-including several from Rockville, Md.-based Bioscience International-are available to standardize microbiological practices. The company's Whitley Automatic Spiral Plater (WASP) system is an automated, microprocessor-controlled instrument that replaces the standard inoculating loops and Bunsen burners. The WASP system deposits a sample from a liquid bacterial culture on a rotating agar plate, then spreads the sample uniformly over the surface to produce isolated colonies of the bacteria after the plate is incubated. LUMINOMETER: Gen-Probe’s Leader 400 can be used with the company’s Amplified MTD Test, which labels probes with a chemiluminescent dye. Bioscience also offers the Protos Colony Counter to automate the tedious task of counting colonies, plaques, or cells. Protos will count objects of just about any shape, provided there is a detectable contrast between the objects and the background. This means it can be used to count colonies on agar plates, viral plaques, and fluorescent or stained cells on membrane filters or in agarose gels. Protos software permits researchers to store results in memory or to send them to external PCs for further analysis. "Manually counting bacterial plates can take several minutes per plate, depending on colony density," explains William Richman, regional sales manager for Bioscience International. "The Protos instrument counts plates in approximately one second. The plate counter also standardizes the counting procedure." For many applications, researchers need to know how many living cells are in a bacterial culture. The FL600-an automated fluorescence microplate reader developed by Bio-Tek Instruments Inc. of Winooski, Vt.-can provide this information when used with the appropriate fluorescent probes. The LIVE/DEAD BacLight system, a kit produced by Eugene, Ore.-based Molecular Probes Inc., contains a mixture of fluorescent stains for nucleic acids. Live bacteria with intact plasma membranes exclude the dyes, so the proportion of live bacteria in the sample can be directly related to the ratio of green fluorescence signals to red. Using fluorescence technology, the Live BacLight Bacterial Gram Stain Kit from Molecular Probes will distinguish between Gram-positive and Gram-negative bacteria. AUTOMATED MEDIA PREPARATION: New Brunswick Scientific’s AS-10 agar sterilizer and MP-1000 plate pourer and stacker Several other automated systems are dedicated to identifying bacteria in specimens. The ESP System from Difco Laboratories is a fully automated microbial testing system, often used for detecting bacteria in blood samples. The ESP system continuously monitors cultures for consumption of gases like oxygen, and for the production of gases such as carbon dioxide, hydrogen, or nitrogen. BBL Crystal Identification Systems from Becton Dickinson Microbiology Systems are miniaturized identification methods that employ conventional biochemical tests. The systems were designed to identify clinically significant aerobic Gram-negative bacteria, as well as other types of fermenting and nonfermenting Gram-negative bacilli of human origin. In this system, the Crystal device is inoculated with a broth culture of the unknown bacterium and incubated. The chemical reactions that occur in the microwells of the device are observed and recorded, generating a profile number that can be punched into a computer and identified from a database. Preparing culture media is one of the most time-consuming tasks in a lab. Bioscience International also sells media-preparation stations that automatically prepare and pour culture media into petri dishes. The AGARSTER MINI II (the media preparator) and ADD MINI-SS (the plate filler), for example, will prepare and pour up to 3 liters of media. The AGARSTER and ADD-SS have a capacity of up to 8 liters of media, which translates into approximately 320 petri dishes full of media. Automated media preparation instruments are also available from New Brunswick Scientific. The AS-10 sterilizer processes 1 liter to 10 liters of agar per hour; it can be programmed to mix, sterilize, and maintain nutrient broth at a preset temperature. The MP-1000 dish filler dispenses the processed media into petri dishes and then stacks the sterile plates into a carousel for cooling. DETECTS rRNA SEQUENCES: Gen-Probe’s Amplified MTD Test is designed for high sensitivity. Microbiology is moving into the realm of molecular biology as scientists struggle to find out what makes microbes tick. The study of tuberculosis is an example. Molecular investigations should help researchers elucidate the microbe's mechanisms for disease and resistance. Mycobacterium tuberculosis grows extremely slowly, so quick detection of the bacterium has always been a challenge. However, new probe-based assays available from Gen-Probe Inc. in San Diego allow researchers to identify the bug in clinical samples in less than a day. Gen-Probe's Amplified MTD Test detects ribosomal RNA (rRNA) sequences from M. tuberculosis. "Targeting rRNA results in a much more sensitive assay because rRNA is present in bacterial cells in many thousands of copies," explains Craig Hill, manager of scientific affairs for Gen-Probe. "Thus, this system is sensitive enough to detect as little as one copy of M. tuberculosis DNA or RNA in a clinical sample." The MTD test is based on an amplification technology known as transcription mediated amplification (TMA), which does not require PCR's characteristic rounds of temperature increases and decreases. Preparation and amplification of the target sequences can be performed in a single tube. To identify probe-bound sequences, a chemiluminescent detection system is used in conjunction with a patented technique called the hybridization protection assay (HPA). Because the probes are labeled with a chemiluminescent dye, the assay requires the use of a luminometer, such as Gen-Probe's Leader 400 luminometer or the fully automated Leader 450i. The entire MTD assay can be completely automated with Gen-Probe's MultiPROBE Liquid Handling System to process samples and reagents. Such automation seems to be a hallmark of many of the products available for microbiologists who use tools to study microbes or use microbes as tools. "Microbiology is really the only life science where we can study events on a molecular, cellular, and organismal level practically simultaneously," comments Ann Wilson, an independent microbiology consultant based in Madison, Wis. "The variety of the products on the market simply reflects the diversity of the discipline." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Microbiologists' Tools Reflect The Diversity Of The Discipline

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