Problem: Counting the total number of live microorganisms (TVC/TVO) on a Petri dish is one of the main laboratory procedures. It has been used worldwide in microbiological, medical, food, biotechnological, and environmental laboratories ever since the Petri dish was invented more than 120 years ago. Each cell in the sample produces a colony on the surface of solid nutrient agar. It takes at least 24 hours of growth to produce a clearly visible colony on solid nutrient agar. Using microscopy at the early stage of colony forming is impossible because the majority of microcolonies are colorless and/or transparent. The result of microbiological analysis after 24-48 or more hours does not satisfactorily meet the modern medical and industrial practice. It is one of the most critical modern microbiological diagnostics problems. Reducing the time of TVO/TVC analysis to 8 hours (one work shift) or even less in a simple and cost-effective way will have a significant impact in medicine and industry.
Solution: Micro-colonies start appearing on solid nutrient agar within several tens of minutes of inoculation. Thus E. coli having a duplicating time of 20 minutes will contain around 500 cells in the micro-colony after three hours of growth, 4,000 after four hours, more than 250,000 after six hours, and more than 2 million after seven hours. Micro-colonies containing 0.5-2.0 million cells are practically invisible because they are small and pellucid. Nevertheless they become clearly visible and countable if cells can be stained. Invented RTVC membranes contain vital chromogenic dyes, polymers and biochemical enhancer immobilized on a highly smooth (much less surface roughness) cellulosic hydrophilic membrane. They are designed to stain micro-colonies to an intense dark blue color and enable them to be clearly visible. The method contains the following steps: grow sample on appropriate nutrient agar 1/4-1/3 of regular growth time; place RTVC membrane over agar with micro-colonies for 5-10 minutes for bacteria or 15-20 minutes for yeasts. Chromogens slowly released from membrane penetrate cell walls and are transformed by enzymes to a dark blue insoluble precipitate. The precipitate is collected inside each cell but not in the surrounding space. Thus micro-colonies obtain an intense blue color and become clearly visible at the early stage of colony forming. Stained micro-colonies can be enumerated either on the membrane or from the bottom of the Petri dish if the membrane is not peeled. Method works with any kind of nutrient agar and any bacteria or yeasts.
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