Rapid methods and automation in microbiology is a dynamic area in applied microbiology, dealing with the study of improved methods in the isolation, early detection, characterization and enumeration of microorganisms and their products in clinical, pharmaceutical, food, industrial and environmental samples. In the past 25 years, this field has emerged into an important sub-division of the general field of applied microbiology. It is gaining momentum nationally and internationally as an area of research and application to monitor the numbers, kinds and metabolites of microorganisms related to food spoilage, food preservation, food fermentation, food safety and food-borne pathogens.
Medical microbiologists started to be involved with rapid methods around the mid-1960s and started to accelerate in the 1970s, continuing developments into the 80s, 90s and up to the present day. Other disciplines, such as Pharmaceutical, Environmental, Industrial and Food Microbiology were lagging about ten years behind. Many symposia and conferences were held nationally and internationally to discuss the developments in this important applied microbiology topics.
Advances in Viable Cell Counts and Sample Preparation
The number of living organisms in the product, on the surface of manufacturing environment and the air of processing plants is very important for the pharmaceutical industry. Colony Forming Units (CFU) are the standard way to express the microbial loads. In the past 25 years, several ingenious systems in “massaging” or “pulsifying” the solid or liquid samples were developed so that the samples are homogeneously distributed in a disposable bag after one to two minutes of operation. Typically 1 ml (after dilutions) is placed into melted agar to encourage microorganisms to grow into discrete colonies for counting (CFU/ml). These colonies can be isolated and further identified as pathogenic or non-pathogenic organisms. In the past 25 years, convenient systems, such as nutrient housed in films, mechanical instrument to spread a sample over the surface of a preformed agar plate, trapping microorganisms on a bacteriological membrane and looking for growth of target microorganisms on selective and non-selective culture media, etc., greatly help to reduce labor time in performing viable cell count so important in pharmaceutical industry. Sterility testing can be done in these systems as well. The three or five tube Most Probable Number (MPN) system has been in use for more than 100 years. In 2007, a completely mechanized, automated and hands-off 16 tube MPN system had been developed for ease of operation of this tedious yet powerful viable cell count procedure. It was used in Public Health, Pharmaceutical and Food laboratories around the world.
Air Samples and Surface Samples
Pharmaceutical industry also needs to ascertain the air quality as well as surfaces for product manufacturing, storage and transportation. An active air sampling instrument can “suck” a known volume of air and deposit it on an agar surface (impaction) or trap it in liquid (impingement) to obtain CFU/ meter^3 or ml. A variety of swabs, tapes, sponges and other contact agar methods have been developed to obtain surface count of pharmaceutical manufacturing environment.
Aerobic, Anaerobic and “Real” Time Viable Cell Counts
By use of the correct gaseous environment or suitable reducing compounds, one can obtain aerobic, anaerobic, facultative anaerobic microbial counts of products. Typically, microbial counts were obtained in 24 to 48 hours. Several methods have been developed and tested in recent year that can provide “real” time viable cell counts, such as the use of “Vital” stains (Acridine Orange) to report living cells under the microscope to count fluorescing viable cells or measure ATP of micro-colonies trapped in a special membranes. These real time tests can give viable cell counts in about one to four hours. A simple double tube system using appropriate agar and incubation con-ditions has been developed that can provide a Clostridium perfringens count for water testing in ca. six hours.
Advances in Miniaturization and Diagnostic Kits
Identification of normal flora, spoilage organisms, clinical and food-borne pathogens, starter cultures, etc., in many specimens is an important part of microbiology. In the past 25 years, many miniaturized diagnostic kits have been developed and widely used to conveniently introduce the pure cultures into the system and obtain reliable identification in as short as two to four hours. Some systems can handle several or even hundreds of isolates at the same time. These diagnostic kits no doubt saved many lives by rapidly, accurately and conveniently identifying pathogens so that treatments can be made correctly and rapidly. There are no less than 20 miniaturized systems on the market to identify pathogens ranging from enterics (Salmonella, Shigella, Proteus, Enterobacter, etc.) to non-fermentors, anaerobes, gram positives and even yeasts and molds.
Advances in Immunological Testings
Antigen and antibody reaction has been used for decades for detecting and characterizing microorganisms and their components in medical and diagnostic microbiology. This is the basis for serotyping bacteria such as Salmonella, Escherichia coli O157:H7, Listeria monocytogenes, etc. Both polyclonal antibodies and monoclonal antibodies have been used extensively in applied food microbiology. The most popular format is the “Sandwiched” ELISA test. Recently, some companies have completely automated the entire ELISA procedure and can complete an assay from 45 minutes to two hours after overnight incubation of the sample with suspect target organisms. Lateral Flow Technology (similar to pregnancy test with three detection areas on a small unit) offers a simple and rapid test for target pathogens (e.g. E. coli O157) after overnight incubation of food or allergens (e.g. wheat gluten). The entire procedure takes only about ten minutes with very little training necessary. A truly innovative development in applied microbiology is the immuno-magnetic separation (IMS) system. Very homogenized, paramagnetic beads have been developed, which can be coated with a variety of molecules, such as antibodies, antigens, DNA, etc., to capture target cells, such as E. coli O157, Listeria , Cryptosporidium ,Giardia, etc. These beads can then be immobilized, captured and concentrated by a magnet stationed outside a test tube. After clean-up, the beads with the captured target molecules or organisms can be plated on agar for cultivation or used in ELISA, Polymerase Chain Reaction (PCR), microarray technologies, biochips, etc., for detection of target organisms. Currently, many diagnostic systems (ELISA test, PCR, etc.) are combining an IMS step to reduce incubation time and increase sensitivity of the entire protocol.
Advances in Instrumentation and Biomass Measurements
Instruments can be used to automatically monitor changes (such as ATP levels, specific enzymes, pH, electrical impedance, conductance, capacitance, turbidity, color, heat, radioactive carbon dioxide, etc.) of a population (pathogens or non-pathogens), growth kinetic and dynamics in a liquid and semi-solid sample. It is important to note that, for the information to be useful, these parameters must be related to viable cell count of the same sample series. In general, the larger the number of viable cells in the sample, the shorter the detection time of these systems. A scatter gram is then plotted and used for further comparison of unknown samples. The assumption is that, as the number of microorganisms increases in the sample, these physical, biophysical and biochemical events will also increase accordingly. When a sample has 5 log or 6 log organisms/ml, detection time can be achieved in about four hours. Some instruments can handle hundreds of samples at the same time.
Advances in Genetic Testings
Phenotypic expressions of cells are subject to growth conditions such as temperature, pH, nutrient availability, oxidation-reduction potentials, etc. Genotypic characteristics of a cell are far more stable. Hybridization of DNA and RNA by known probes has been used for more than 30 years. More recently, Polymerase Chain Reaction (PCR) is now an accepted method to detect viruses, bacteria and even yeast and molds by amplification of the target DNA and detecting the target PCR products. By use of reverse transcriptase, target RNA can also be amplified and detected. After a DNA (double stranded) molecule is denatured by heat (e.g. 95C), proper primers will anneal to target sequences of the single stranded DNA of the target organism, for example Salmonella at a lower temperature (e.g. 37C). A polymerase (TAQ) will extend the primer at a higher temperature (e.g. 70C) and complete the addition of complement bases to the single-stranded, denatured DNA. After one thermal cycle, one piece of DNA will become two pieces. After 21 and 31 cycles one piece will become 1 million and 1 billion copies, respectively. At the beginning, PCR products are detected by gel electrophoresis. Ingenious ways exist to detect either the occurrence of the PCR procedure by fluorescent probes or special dyes, or by actually reporting the presence of the PCR products by molecular beacon. Since these methods generate fluorescence, the PCR reaction can be monitored over time and provide “real” time PCR results. Some systems can monitor four different targets in the same sample (multiplexing). These methods are now standardized and easy to use and interpret.
To further characterize closely-related organisms, detail analysis of the DNA molecule can be made by obtaining the patterns of DNA of specific organisms by pulse field gel electrophoresis (DNA finger-printing) or by “riboprinting” of the ribosomal genes in the specific DNA fragment. Since different bacteria exhibit different patterns (e.g. Salmonella versus E. coli) and even the same species can exhibit different patterns (e.g. Listeria monocytogenes has 49 distinct patterns), these information can be used to compare closely related organisms for accurate identification of target pathogens (such as comparing different patterns of E. coli O157:H7 isolated from different sources in an outbreak) for epidemiological investigations.
Advances in Biosensor, Microchips and Biochips
Biosensor is an exciting field in applied microbiology. The basic idea is simple but the actual operation is quite complex and involves much instrumentation. Basically, a biosensor is a molecule or a group of molecules of biological origin attached to a signal recognition material. When an analyte comes in contact with the biosensor, the interaction will initiate a recognition signal which can be reported in an instrument. Many types of biosensors have been developed. Sometimes, whole cells can be used as biosensors. Analytes detected include toxins, specific pathogens, carbohydrates, insecticides and herbicides, ATP, antibiotics, etc. The recognition sig-nals used include electrochemical (e.g. potentiometry, voltage changes, conductance and impedance, light addressable, etc.), optical (such as UV, bioluminescence, chemiluminescence, fluorescence, laser scattering, reflection and refraction of light , surface phasmon resonance, polarized light, etc.) and miscellaneous transducers (such as piezoelectric crystals, thermistor, acoustic waves, quartz crystal, etc.).
Recently, much attention has been directed to “biochips”and “microchips” developments to detect a great variety of molecules, including foodborne pathogens. Due to the advancement in miniaturization technology, as many as 50,000 individual spots (e.g. DNA microarrays), with each spot containing millions of copies of a specific DNA probes can be immobilized on a specialized microscope slide. Fluorescent labeled targets can be hybridized to these spots and be detected. Biochips can also be designed to detect all kinds of foodborne pathogens by imprinting a variety of antibodies or DNA molecules against specific pathogens on the chip for the simultaneous detection of pathogens, such as Salmonella, Listeria, Escherichia coli, Staphylococcus aureus, etc. The market value is estimated to be as high as $5 billion at this moment. This technology is especially important in the rapidly developing field of proteomics and genomics which require massive amount of data to generate valuable information.
The potential of biochips and microarrays for microbial detection and identification in pharmaceutical and related samples is great but, at this moment, much more research is needed to make this technology a reality in applied microbiology.
Testing Trends and Predictions
There is no question that many microbiological tests are being conducted nationally and internationally in pharmaceutical and food products, environmental samples, medical specimens and water samples. The most popular tests are total viable cell count, coliform/E. coli count and yeast and mold counts. Alarge number of tests are also performed on pathogens such as Salmonella, Listeria and Listeria monocytogenes, E. coli O157:H7, Staphylococcus aureus, Campylobacter and other organisms. According to reliable sources, in 1998, the worldwide microbiological tests were estimated to be 755 million tests with a market value of $1 billion. The projection is that, in 2008, the number of tests will be about 1.5 billion tests with a market value of $5 billion. In 2007, about one third of the tests are being performed in North America (USA and Canada), another third in Europe and the last third in the rest of the world. This author predicts that, in twenty years, the rest of the world will perform 50 percent of the tests, with North America and Europe performing 25 percent each. This is due to rapid economic developments and food and health safety concerns of the world in the years to come.
Prediction of the Future
The following are the ten predictions made by the author in 1995. Many predictions have been correct in 2007. (+) is a good prediction. (?) is an uncertain prediction.
1. Viable cell counts will still be used in the next 25 years. (+)
2. Real-time monitoring of hygiene will be in place. (+).
3. PCR, ribotyping and genetic tests will become reality in food laboratories. (+)
4. ELISA and immunological tests will be completely automated and widely used. (+)
5. Dipstick technology will provide rapid answers. (+)
6. Biosensors will be in place for Hazard Analysis Critical Control Point programs. (?)
7. Biochips, microchips and microarrays will greatly advance in the field. (+)
8. Effective separation and concentration of target cells will assist rapid identification. (+)
9. A microbiological alert system will be in food and pharmaceutical packages. (?)
10. Consumers will have rapid alert kits for pathogens at home. (?)
In conclusion, it is safe to say that the field of rapid method and automation in microbiology will continue to grow in numbers. The kinds of tests to be done in the future will rise due to the increase concern on food safety and public health. The future looks very bright for the field of rapid methods and automation in microbiology. The potential is great and many exciting developments will certainly unfold in the near and far future in pharmaceutical areas.
References
1. Fung, D. Y. C. 2002. Rapid Methods and Automation in Microbiology. Comprehensive Reviews in Food Science and Food Safety. Inaugural Issue Vol 1, Issue 1. Pp 3 to 22. (www.ift.com>