Special Report: New Tools Join Old In Microbiological Research

Microbiological Research Author: RICKI LEWIS AND HOLLY AHERN, p.24 Microbiology has evolved from ancient art to savvy science. Yesterday's painstaking tasks of culturing and identifying microorganisms have been joined by rapid detection strategies and intricate genetic manipulations. Microbiologists may still streak specimens on agar plates and wait overnight for bacterial colonies to grow, as was done two centuries ago, but today they may also use spectrophotometers or chromatographs. Gas chromatograph MIDI Inc. 115 Barksdale Professional Center Newark, Del. 19711 Phone: (302) 737-4297 Product: Microbial identification system--Hewlett-Packard gas chromatograph, computer, software package: $46,000 Nutrient utilization screen Biolog Inc. 3447 Investment Blvd., Suite 2 Hayward, Calif. 94545 Phone: (415) 785-2564 Products: GN MicroPlate: $4-$6/tray MicroStation computer and reader: $11,080 MicroLog 1 software database (key in): $595 MicroLog 2 software database (reactions entered directly into microplate reader): $1,995 RFLP fingerprinting BIO-RAD Laboratories Inc. 1414 Harbour Way South Richmond, Calif. 94804 Phone: (800) 4-BIORAD Product: CHEF-DR II System (to DNA fingerprint cells): $5,380 Bioluminescence Analytical Luminescence Laboratories 11760 Sorrento Valley Rd., Suite E San Diego, Calif. 92121 Phone: (800) 854-7050 Products: ATP detection kit; includes discs treated with Firelight and Extra-light (promotes bacterial ATP release): 36 tests, $63 Camlight 500 luminometer: $575 Carbon dioxide detection Becton Dickinson Diagnostic Instrument Systems 7 Loveton Circle Sparks, Md. 21152 Phone: (800) 638-8656 Products: BACTEC NR-660 (60-vial capacity) with incubator shaker: $55,000 BACTEC NR-730 (30-vial capacity): $33,000 Immunoassays 3M Microbiology Products 3M Center St. Paul, Minn. 55144-1000 Phone: (800) 328-6553 Products: Petrifilm Aerobic Count plates: 100 plates, $60; 1,000 plates, $500 Petrifilm Coliform Count plates: 50 plates, $32.50; 1,000 plates, $540 Petrifilm E. coli Count plates: 50 plates, $62.50; 500 plates, $475 DNA Probes GEN-PROBE Inc. 9880 Campus Point Dr. San Diego, Calif. 92121 Phone: (619) 546-8000 Products: AccuProbe kits against various pathogens: 20 tests, $67-$210 Luminometer for analyzing AccuProbe assays: $3,495 "The field is both microbiology and analytical chemistry," says Herman Finkbeiner, manager of biological sciences at General Electric Co.'s research and development center in Schenectady, N.Y. "You can't put a finger on where one starts and one stops." However, as Annette Piette, senior technical specialist at Becton Dickinson Diagnostic Instrument Systems of Sparks, Md., explains, "As far as instrumentation goes, microbiology is pretty much the poor stepchild of the laboratory. In major chemistry and hematology labs, they just plop a specimen into a cuvette and push a button. In microbiology, more of the thought process is apparent on the part of the technologist." While bacteria remain a valued source of useful natural products--via the "old" biotechnology of fermentation--they have also served as model systems for deciphering the details of molecular biology. This, in turn, has allowed the development of recombinant DNA methodologies--the "new" biotechnology. The nature of infectious disease was for the most part a mystery to ancient cultures. Even with the arrival of the Black Death in 1348, which made the concept of infectious disease tragically obvious to all, the microbial culprits went undiscovered for several hundred more years. Not until the late 17th century did a major breakthrough occur--when Anton van Leeuwenhoek, a Dutch janitor, clothmaker, and amateur microscopist, peered under a crude lens system at tartar scraped from his teeth, and described "wee animalcules." By the late 19th century, the connection between microbes and infectious disease was established through the work of such scientists as Louis Pasteur, who put to rest the concept of spontaneous generation, and German physician Robert Koch, who linked a bacillus with anthrax and demonstrated the disease's infectivity. Today, while the bulk of microbiological research remains focused on human health-related investigations, researchers are studying microorganisms posing a range of impact well beyond the cause and spread of disease. Pseudomonas aeruginosa, the bacterium used to help clean up the Exxon Valdez oil spill in Prince William Sound, Alaska, in 1989, is an example of an industrially useful, naturally occurring microbe. And the ability to transfer Escherichia coli plasmids into other bacterial genera is expanding the repertoire of recombinant DNA-derived microbial products. Transferring foreign genes to bacteria can accomplish in one step what years ago took several cycles of mutation and selection of useful traits. Within the field of medicine, of course, microbiology is blooming. According to a Food and Drug Administration report, "Drug Utilization in the United States" (1989), systemic anti-infectives, including many microbe-derived antibiotics, constitute the second most prescribed class of drugs, out of 83 classes, accounting for 13 percent of all prescriptions annually. Microbial products have other interesting biomedical properties and applications beyond antibiotics. For example, a top-selling cholesterol-lowering drug, mevinolin, is derived from a microbe, and inhibits a rate-limiting enzyme in the liver's cholesterol synthetic pathway. Other microbial biochemicals intervene in receptor-ligand binding, or affect immune system molecules, such as the interferons and interleukins, implicated in cancers and inflammatory conditions. Although many microbiology tools have withstood the test of time, things move along much faster now that these methods are being combined with newer techniques. Michael Sinclair, president of Microcheck Inc., a microbial testing company based in Northfield, Vt., recalls, "I've been doing bacterial identification since grad school, in the early 1970s, by biochemical methods. I would search for antibiotic properties in Sphagnum moss, doing test after test, 25 to 30 tests. Now I can do what once took weeks in two days." G.E.'s Finkbeiner agrees: "People complain that it now takes 25 minutes to do an analysis. If they could just do it in 10 minutes, it would be better. Not long ago, it would have taken a week!" A look around Finkbeiner's laboratory vividly reveals the stepped-up timetable of modern microbiology. The G.E. biology group works on bioremediation--the use of microbes to degrade toxins. Nine analytical stations, including combinations of gas chromatographs, mass spectrometers, and high-performance liquid chromatographs (HPLC), enable the workers to identify 15 contaminants of a chemical plant's waste water, presented in parts-per-million amounts, in a half-hour; or to distinguish among 80 types of polychlorinated biphenyls, totaling 10 to 25 ppm. A gas chromatograph separates molecules on the basis of boiling point and polarity, and it is often combined with a mass spectrometer, which measures the weights of molecules and their breakdown products. An HPLC separates molecules on the basis of size or polarity. "We can literally do dozens of analyses every night," says Finkbeiner. "Every component is separated and quantified--a totally impossible task as recently as 10 years ago. The GC [gas chromatograph] and HPLC have completely revolutionized our ability to separate, isolate, and quantify components in any metabolic study." Gas chromatography is used to identify microbes by determining their characteristic ratios of fatty acids. A sample containing an unidentified microbe can be assigned a "similarity index" to known organisms. Myron Sasser of the University of Delaware, Newark, has compiled a database of fatty acid profiles. "We can now identify about 1,200 species and subspecies, and the list is growing rapidly," he says. "We can test for aerobes, anaerobes, yeast, and actinomycetes." His company, MIDI Inc., also in Newark, markets the "microbial identification system," a package consisting of a gas chromatograph, a computer, and software. But identifying microorganisms with a gas chromatograph is not as simple as plugging in the bug and getting an answer. Here is where a sound background in classical techniques pays off. In what Microcheck's Sinclair describes as "database oddities," two unrelated organisms may reveal strikingly similar fatty acid profiles. Distinguishing between the two then depends upon a traditional differential staining test, such as the Gram stain, which classifies according to cell wall morphology. Sinclair has spun off his dual expertise in microbiology and gas chromatography into his business, Microcheck. His diverse clientele cannot afford to purchase equipment or do not have the time or experience to make the most of it. "We do lots of confirmation testing, as well as isolating and speciating bacteria from raw materials," he says. "We have dealt with cheese makers who have too many gas pockets in their products, bacteria in Antarctica that survive 5 C temperatures, and a Drosophila lab with three types of bacteria contaminating the media--and all sorts of dead flies." Flow cytometry is another fairly complex procedure that may soon occupy an expanded niche in the microbiology lab. The flow cytometer sends a pressurized stream of saline containing labeled particles through a pipe with a vibrating nozzle, only a few microns across at its end. Drops leaving the nozzle are so small that they contain, at most, one particle or cell. A detection system such as a laser distinguishes between the types of particles and deflects them into collection containers (The Scientist, Dec. 10, 1990, page 29). Light-scattering patterns or voltage change can indicate particle (cell) size, and techniques using fluorescent dyes can reveal DNA, RNA, and protein content; surface antigens; intracellular enzyme activity and pH; and membrane potential. Together, these parameters can paint a portrait of a particular microorganism. Size can distinguish a bacterium from a eukaryotic cell, and a membrane potential indicates a living particle. Even DNA base composition can be estimated, providing clues. For example, Staphylococcus aureus characteristically has 31 percent G-C base pairs, whereas E. coli has 50 percent. Gas chromatography and flow cytometry represent the high-tech end of the spectrum of bacterial detection systems. Several simpler, yet ingenious, tools are also available. Each type of kit targets a particular biochemical sign of life. Many detection systems highlight the biochemical reactions of metabolism, which produce pyruvic or lactic acid, carbon dioxide, and ATP. Becton Dickinson's popular BACTEC series, a now-standard piece of equipment in the microbiology lab first marketed in 1971, spots bacteria in blood by measuring the carbon dioxide the bacteria give off during metabolism. Older BACTEC models used radioactively labeled 14C; newer models use nonradioactive labeling. A steady stream of ATP is a sure sign of life. Bioluminescence-based devices exploit the role of ATP in the conversion of the complex carboxylic acid luciferin to light--the same chemical process that makes a firefly glow. San Diego-based Analytical Luminescence Laboratories markets Firelight, a reagent that is a mixture of luciferin and the enzyme needed for its conversion to light, luciferase. If a sample glows, it has ATP--a microorganism is present. As far as bacterial detection goes, what could be simpler than a tray of wells that literally spells out a microbe's identity? Biolog Inc. of Hayward, Calif., markets the GN MicroPlate and accompanying MicroLog software, based on nutrient utilization. GN MicroPlate consists of 96 wells in a culture tray, each of which has a low concentration of complex medium, a high concentration of a particular nutrient, and tetrazolium violet, a dye that yields a violet color if a microbe utilizes the nutrient in the well. A particular microbe has a characteristic design formed by the wells that turn purple. The pattern for E. coli strain K12 spells out an "E," and the Salmonella typhimurium strain LT2 wells spell "S." The kit can identify 434 different bacteria. Many bacterial detection kits are based on the specificity provided by proteins and nucleic acids. Antibodies selectively bind to the surfaces of particular microorganisms, allowing them to be detected. 3M Microbiology Products of St. Paul, Minn., for example, offers immunoassays for the bacteria most commonly responsible for food poisoning. DNA probes highlight microbial genes, providing rapid and sensitive diagnostic tools to replace standard culturing methods. GEN-PROBE Inc., based in San Diego, offers DNA probes for such common pathogens as streptococcus A and B, staphyloccus, Haemophilus influenzae, and the organisms behind several sexually transmitted diseases. Restriction fragment-length polymorphism (RFLP) mapping, the basis of DNA fingerprints and genetic markers, can also help to identify bacteria. Restriction enzymes cut a bacterium's DNA into a set of species-specific sizes reflecting the DNA sequence. Bio-Rad Laboratories Inc. of Richmond, Calif., markets CHEF-DR II, a pulsed field gel electrophoresis apparatus (The Scientist, July 23, 1990, page 25) that separates such cut DNA. It is used, for example, to distinguish strains of Lactococcus lactis important in cheesemaking. As microbiology continues to evolve, advances in biotechnology and molecular biology will further contribute to its development. But as in the past, microbiology--"the poor stepchild of the lab"--will add as much to these sciences as it draws from them. Ricki Lewis and Holly Ahern teach biology at the State University of New York, Albany.

Special Report: New Tools Join Old In Microbiological Research

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