Patent Application
20050026135
The growth of microorganisms in order to detect, enumerate and identify livable cells—bacteria, fungi, actinomycetes—is one of the most widely used methods in microbiology. The growth occurs on either solid or liquid artificial or natural nutrient media. Hundreds of different media for total count and growing of groups or species of microorganisms are known currently. It usually takes from several hours to several days to form well visible colonies or suspension of cells. Improvement of visibility (detectability) of colonies could shorten the time between inoculation and detection of the colony.
There are several different methods, instruments and devices employed to enhance colony visibility. Thus the addition of special, non toxic for cells substances (some artificial chromogenic or fluorogenic substrates) to solid nutrient media could change color of the colonies or make them fluorescent and improve colonies visibility on early stages. Toxic artificial substrates (example: Tetrazolium salts) or other substances could be used on late stage of colony or suspension of cell growth to colorize cells and make them more clearly visible.
Detection and enumeration of the colonies are done visually or with magnifying devices. Visual detection and enumeration using magnifying glass requires relatively big colonies; from hundreds of microns to millimeters in diameter. Microscopy helps to find micro colonies with tens of microns in diameter. These colonies contain at least several hundreds of cells and need at least 5-10 hours of incubation to form colony of this size.
Thus, modern microbiology utilizes three approaches to shorten time of needed growth and improve visibility of colonies; first—employ optimal growth nutrient media, second—addition of chemical matter in nutrient media or on a colony, or into suspension of cells, to change colonies optical characteristics, and third—employ optical instruments or devices.
There are no methods utilizing the shape of the colony during it growth in order to enhance its optical density (light absorbance). Changing of colony shape from regular semi-sphere with large volume and large amount of cell to thin cylinder shape with small volume and small amount of cells could strongly reduce the time between inoculation and colony counting. Smaller amounts of cells need shorter time for their production. The usage of chemicals producing color or fluorescence and optical instruments and devices together with detection of cylindrical colonies could improve visibility and reduce the time of analysis.
Reduction of the time between inoculation and detection is very important for early decision in quality and process control in food and biotechnological industry, medical microbiology and epidemiology, air, water and surfaces control of indoor and outdoor environment, and scientific research.
The invention is based on growing of micro colonies in thin and long micro channels, instead of regular growth on flat surface of solid nutrient media, or flat surface of filter placed on nutrient media, or growth in relatively big volume of liquid nutrient media.
The shape of regular micro colony is usually semi-sphere. The thickness (height) of micro colonies is crucial to make it visible using microscope because thick (high) colony has larger light absorbance—most important optical characteristic of visibility. Long and thin micro colony have the same light absorbance as regular semi-sphere colony of the same height—h (see
The growth of a cylindrical micro colony could be done with a help of grid that has large amount of very small and long channels. The diameter of this channel needs to be very small, only in 4-20 times larger than the size of investigated cells. Good example for these purposes could be MCGP—Micro Channel Glass Plate. MCGP contain thousands of extremely small precisely itched long channels. Regular MCGP has a diameter of each channel 10 microns, length 500 microns, and the amount of channels is 700,000 per cm2. Other grids or MCGP could be useful also.
Calculations below show obvious advantage in shortening of time of growth in micro channels in comparison with flat surface.
The regular shape of colonies growing on flat surface of solid nutrient media is, usually near to semi-sphere. The volume of semi-sphere is Vss=¶·h2·(R−h/3), where Vss—volume of semi-sphere, R—radius of sphere and h—part of radius—height of semi-sphere.
The volume of cylinder (cylindrical colony) is Vcc==¶·R2·h, where R—radius of cylinder, h—height of cylinder.
Micro colony with height (h) 10 μk and R=20 μk has volume:
Vss=3.14·102·(20−10/3)=5234 μk3
Cylindrical colony with the same height (h=10 μk) and R=2.5 μk has volume:
Vcc=3.14·2.52·10=196 μk3
Thus, the volume of a cylindrical colony is smaller than volume of semi-spherical micro colony with the same height in 27 times, and both have the same light absorbance.
The volume of one cell of Escherichia coli (E. coli) is near to 1 μk3. The speed of multiplying of E. coli is around 20 min at optimal temperature, on optimal media. One cell of E. coli can produce 8 cells in one hour, 64 in two hours, 512 in 3 hours, 4096 in 4 hours and 32768 in 5 hours. Thus, one micro colony contains 5234 cells could be formed in 4.2 hours. The cylindrical colony with the same height and light absorbance (196 cells) could be formed in 2.5 hours.
Therefore growth of micro colonies with cylindrical shape has significant advantage because of visualization of colony could be done at much earlier stages.
The visualization of microorganisms in micro channels filled by liquid nutrient media is much faster than in regular tubes or wells of immunological plate, or other known laboratory devices for microorganisms growth, because of very small volume of micro well and it long cylindrical shape. Thus, one cell in a cylindrical micro channel, with a length 500 μk and diameter 10 μk (V=40,000 μk3) correspond to concentration of 25 millions cells per ml (V=1012 μk3). 40 cells in a micro channel correspond to the concentration 10{circumflex over ( )}9 cells per ml—well visible concentration. One cell of E. coli can reach this concentration (concentration 40 cells per micro channel=10{circumflex over ( )}9 cells per ml) in 1.7 hours.
Experiments show that 10 layers of colorless small cells (for example E. coli) are enough to find visual differences between micro channels contain cells and empty micro channels using regular light microscope with even small magnification of ×100. Smaller diameter of the channel needs smaller amount of cells to create 10 layers of cells in the channel.
Table 1 represent amount of layers of E. coli that could be produced in micro channels of different diameters in different time.
Table 1 shows that 10 layers of cells will be reached in micro channel with diameter 2 μk in 1.5 hours; in 3 μk micro channel in 2 hours; in 4 μk micro channel in 2.3 hours; in 5 μk micro channel in 2.7 hours; in 7 μk micro channel in 2.9 hours and in 10 μk micro channel in 3.5 hours. Thus, the detection and enumeration of long cylindrical micro colonies according this invention could be done in 10-20 times faster than regular growth, detection and enumeration of CFU.
The channels containing micro colony look like dark dots. Addition of artificial chromo- or fluorogenic substrates to micro colonies could reduce time between inoculation and detection: color or fluorescence could make micro colonies much more visible on earlier stage.
This invention differs from other methods of detection of CFU, by using of plate containing hundreds of thousands of extremely small and long channels (micro channel plate). The combination of micro channel plate and filter allows trapping of cell on the filter surface and growing colonies inside channel. Those colonies will obtain high cylindrical shape. High cylindrical shape of colony has long optical way (high light absorbance) but smaller volume and amount of cells which drastically reduces time of analysis. This method could be realized with a simple device consisting of a plate with channels, filter to trap cells by filtration from air or liquid and frame consists from several parts.
This invention is based on the method and device for trapping cells from liquids or air, grow relatively short time on solid nutrient media or in liquid nutrient media and find dark (not colored), colored or fluorescent channels that looks like large round dots under regular or fluorescent microscope. The time of analysis could be reduced, and sensitivity could be enhanced by the usage of channels of smaller diameter and substances produced color or fluorescence. Physical factors like heating in order to coagulate proteins and increase light absorbance or addition of the substances produced gas bubbles like O2 produced from H2O2 by Catalase could be employed also.
Simple device for trapping cells in the channels by filtration shown on
Procedure for sampling, growth and enumeration of the colonies is following:
This method and device could be used with a broad range of different solid and liquid natural or artificial media. Micro channel plate without a filter could be used to find contamination on surfaces by spraying liquid nutrient media on surface and placing micro channel plate above, incubate needful time and read results under microscope.
This invention could be realized in many different optical or opto-electronic instruments and devices.
