Advances In Miniaturization And Diagnostic Kits

Identification of microorganisms constituting normal flora, spoilage organisms, foodborne pathogens, starter cultures, etc., in food microbiology is an important part of microbiological manipulations. Conventional methods, dating back more than 100 years, utilize large volumes of medium (10 ml or more) to test for a particular characteristic of a bacterium (e.g., lactose broth for lactose fermentation by Escherichia coli). Inoculating a test culture into these individual tubes one at a time is also very cumbersome. According to Hartman [10], over the years many microbiologists have devised vessels and smaller tubes to reduce the volumes used for these tests. This author has systematically developed many miniaturized methods to reduce the volume of reagents and media (from 5 to 10 ml down to about 0.2 ml) for microbiological testing in a convenient microtiter plate which has 96 wells arranged in an 8 x 12 format. The basic components of the miniaturized system are the commercially sterilized microtiter plates for housing the test cultures, a multiple inoculation device, and containers to house solid media (large Petri dishes) and liquid media (in another series of microtiter plates with 0.2 ml of liquid per well). The procedure involves placing liquid cultures (pure cultures) to be studied into sterile wells of a microtiter plate (~0.2 ml for each well) to form a master plate. Each microtiter plate can hold up to 96 different cultures, 48 duplicate cultures, or various combinations as desired. The cultures are then transferred using a sterile multipoint inoculator (96 pins protruding from a template) to solid or liquid media. Sterilization of the inoculator is accomplished by alcohol flaming. Each transfer represents 96 separate inoculations in the conventional method. After incubation at an appropriate temperature, the growth of cultures on solid media or liquid media can be observed and recorded, and the data can be analyzed. These methods are ideal for studying large numbers of isolates or for research involving challenging large numbers of microbes against a host of test compounds. Using this miniaturized system the author has characterized thousands of bacterial cultures isolated from meat and other foods, studied the effects of organic dyes against bacteria and yeasts, and performed challenge studies of various compounds against microbes with excellent results.

Other scientists also have miniaturized many systems and developed them into diagnostic kits in the late 1960s and early 1970s. Diagnostics systems such as API, Enterotube, Minitek, Crystal ID, MicrolD, RapID, Biolog, and VITEK systems are currently available. Most of these systems were first developed for identification of enterics (salmonella, shigella, proteus, enterobacter, etc.). Later, many of these companies expanded the capacity of their diagnostic systems to identify nonfermentors, anaerobes, Gram-positive organisms, and even yeast and molds. Originally, an analyst needed to read the color reaction of each well in the diagnostic kit and then use a manual identification code to "key" out the organisms. Recently, diagnostic companies have developed automatic readers interfaced with a computer to provide rapid and accurate identification of the unknown cultures.

The most successful and sophisticated miniaturized automated identification system is the VITEK system (bioMerieux, Hazelwood, MO) which utilizes a plastic card containing 30 tiny wells in each of which there is a different reagent. The unknown pure culture in a liquid form is "pressurized" into the wells in a vacuum chamber, and then the cards are placed in an incubator for a period of time ranging from 4 to 12 hours. The instrument periodically scans each card and compared the color changes or gas production of each tiny well with the database of known cultures. VITEK can identify a typical Escherichia coli culture in 2 to 4 hours. Each VITEK unit can automatically scan 120 cards or more simultaneously. There are a few thousand VITEK units currently in use in the world, and the database is especially good for clinical isolates.

Biolog system (Hayward, CA) is also a miniaturized system using the microtiter format for growth and reaction information. Pure cultures are first isolated on agar and then suspended in a liquid to the appropriate density (~6log cell/ml). The culture is then dispensed into a microtiter plate containing different carbon sources in 95 wells and one nutrient control well.

The plate with the pure cultures is then incubated overnight, after which the microtiter plate is removed, and the color pattern of the wells with carbon utilization is observed and compared with profiles of typical patterns of microbes using computer software to obtain identification. This system is very ambitious and tries to identify more than 1400 genera and species of environmental, food, and medical isolates from major groups of Gram-positive, Gram-negative, and other organisms. There is no question that miniaturization of microbiological methods has saved much material and operational time and has provided needed efficiency and convenience in diagnostic microbiology. The systems developed by the author and others can be used in many research and developmental laboratories for studying large numbers of cultures. These miniaturized systems and diagnostic kits can be used efficiently in identifying isolates from fruits and vegetables.

The conventional viable cell count method and the MPN (3- or 5-tube MPN) procedure have been used extensively for water and food testing for almost 100 years. The conventional methods are too cumbersome, time consuming, and utilize too many tubes, plates, and media. More than 30 years ago, Fung and Kraft [11] miniaturized the viable cell count procedure by diluting the samples in the microtiter plate using 0.025 ml size calibrated loops in 1:10 dilution series. One can simultaneously dilute 12 samples to 8 series of 1:10 dilutions in a matter of minutes. After dilution, the samples can be transported by a calibrated pipette and spot plating 0.025 ml on agar; one conventional agar plate can accommodate 4 to 8 spots. After incubation, colonies in the spots can be counted, and the number of viable cells in the original sample can be calculated since all the dilution factors are known. The accepted range of colonies to be counted in one spot is 10 to 100. The conventional agar plate standard is from 25 to 250 colonies per plate. This procedure actually went through an AOAC International collaborative study with satisfactory results [12]. However, the method has not received much attention and is waiting to be "rediscovered" in the future.

In a similar vein, Fung and Kraft [13] also miniaturized the MPN method in the microtiter plate by diluting a sample in a 3-tube miniaturized series. In one microtiter plate one can dilute 4 samples, each in triplicate (3-tube MPN), to 8 series of 1:10 dilution. After incubation, the turbidity of the wells is recorded, and a modified 3-tube MPN table can be used to calculate the MPN of the original sample. This procedure has recently received renewed interests in the scientific community.

Walser [14] in Switzerland reported the use of an automated system for microtiter plate assay to perform classic MPN of drinking water. He used a pipetting robot equipped with sterile pipetting tips for automatic dilution of the samples. After incubation, the robot placed the plate in a microtiter plate reader and obtained MPN results with the use of a computer. The system can cope with low or high bacterial load from 0 to 20,000 colonies per milliliter. This system takes out the tediousness and personnel influences on routine microbiological work and can be applied to determine MPN of fecal organisms in water as well as other microorganisms of interest in food microbiology.

Irwin et al. [15] in the U.S. also worked on a similar system using a modified Gauss-Newton algorithm and a 96-well micro-technique for calculating MPN using Microsoft EXCEL spreadsheets. These improvements are possible today compared with the original work of the author in 1969 because: (1) automated instruments are now available in many laboratories to dispense liquid into the microtiter plate and automated dilution instruments are also available to facilitate rapid and aseptic dilutions of samples; (2) automated readers of microtiter wells are now commonplace to read efficiently turbidity, color, and fluorescence of the liquid in the wells for calculation of MPN; and (3) elegant mathematic models, computer interpretations and analysis, and printout of data are now available which the author could not have envisioned back in 1969.

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