Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-10-15
2001-04-17
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S288100, C435S288700, C435S810000
Reexamination Certificate
active
06218176
ABSTRACT:
BACKGROUND OF INVENTION
This application relates to a device and a method for rapid hygiene testing by detecting ATP found in biomass on a test surface, such as a food preparation surface. From a sample taken from the surface, ATP is detected using a luciferase/luciferin bioluminescent reaction. The device and method of the present invention provides a quick, accurate determination of the cleanliness of surfaces.
It is important in many industries, such as food preparation, medicine, beverages, toiletries, and pharmaceuticals, to provide clean and sanitary surfaces. It is not enough to simply clean or sanitize a surface and assume it is free from microorganisms such as bacteria. Instead, a test must be performed to detect whether the surface is actually free of microorganisms. Thus, random areas of a surface, such as a food preparation surface, are tested for microorganisms to determine the general cleanliness of the surface.
One of the oldest methods to check for cleanliness involves culturing samples for bacteria. A test surface is chosen and wiped with a swab, and then the swab is smeared onto a culture medium. The medium is incubated and then checked for the presence of bacteria colonies grown in the medium. Over the years, various types of culture media have been developed along with numerous products based thereon. While the results of bacterial cultures are accurate, they are limited by the time that it takes to incubate the culture, usually in the order of days.
In response for a need to obtain results more quickly, other methods for detecting microorganisms were developed. Research soon focused on the detection of biomass on the test surface. Biomass includes living cells, dead cells, other biotic products such as blood, and food residue. It was discovered that biomass could be detected by detecting ATP, adenosine triphosphate, a chemical found in all living organisms.
The specific test for ATP involves the “firefly” reaction. The following is the reaction:
ATP
+
Luciferin
+
Luciferase
+
O
2
→
Mg
2
+
AMP
+
CO
2
+
2
Pi
+
Light
ATP, luciferin (D-luciferin cofactor), luciferase (enzyme) and oxygen are reacted in the presence of magnesium ion. Luciferin and luciferase are the same cofactor and enzyme present in fireflies that yields their namesake light. The products of the reaction are AMP (adenosine monophosphate), inorganic phosphate, carbon dioxide, and light (photons). The reaction, just as in fireflies, produces light. This light can be quantified and used to correlate to an amount of ATP. However, the amount of ATP does not necessarily relate directly to the number of microorganisms or bacterial cells or colonies. In fact, ATP may be from non-microbe biomass such as beef blood; thus the amount of ATP would not be related to microorganisms.
The lack of correlation may be due to the variation in ATP concentration within cells and the degradation of ATP in dead cells. ATP is found in all living cells, but the amount of ATP in cells can vary significantly. For example, prokaryotic cells have about one hundredth the amount of ATP as eukaryotic cells and different strains of bacteria will contain significantly different amounts of ATP. In addition, if a cell is growing or about to divide, it will contain more ATP than a dormant cell. Furthermore, cells that have just died contain ATP and even dead cells may contain ATP. In dead cells, any ATP present may degrade, often caused by a reaction between ATP and intracellular enzymes contained within the dead cells. All of these variables in ATP concentration mean that ATP testing is limited as a means to quantify the number of microorganisms or bacterial cells or colonies. However, ATP testing remains a method to qualitatively determine the presence of biomass, including microorganisms or bacteria.
Thus, the detection of ATP can be used to determine the presence of biomass, whether viable or nonviable. The ability to detect nonviable biomass is important, for example, in testing a surface for cleanliness because nonviable biomass (dead cells) such as food residue can provide a medium for living cells to grow.
Typically, the luciferase, luciferin, and magnesium ion are sold as a single combined reagent, not as individual reagents. The luciferase must be at the proper pH of 7.8 in order to be effective, usually achieved by employment of a buffer solution. If the proper pH is not mainined, the reaction will not work efficiently, and the results will be erroneous. However, luciferase is unstable while in solution, and will degrade, particularly at higher temperatures. Generally, at room temperature, the luciferase solution will remain effective for a period of hours whereas at near freezing temperatures, the luciferase solution will last for a period of days. In addition, luciferin in solution is light sensitive. Light causes the dissolved luciferin to degrade. Once the luciferin has degraded, no cofactor remains to unleash the bioluminescent reaction resulting in false negatives.
To prevent degradation, the luciferin and luciferase can be dried and protected from light. Methods for drying include, but are not limited to, freeze drying and lyophilization. The luciferase is more stable if kept out of solution. When ready to use, the dried luciferin and luciferase are dissolved in water containing an appropriate buffer to form an aqueous solution having the proper pH.
By mixing the luciferase/luciferin reagent with a sample taken from a test surface, extracellular ATP is immediately reacted and detected. However, intracellular ATP cannot be detected unless the ATP is first ectracted from within the cells. Typically, this is accomplished by mixing the sample with an extraction reagent (releasing reagent) which extracts the ATP from within the cells. The extracted ATP then can be mixed with the luciferase/luciferin reagent to produce the observable reaction. It is important that the extraction reagent chosen does not inactivate the luciferase/luciferin reagent. Nor should the extraction reagent be toxic if it is used on a food preparation surface, for example.
The luciferase/luciferin reagent cannot be stored with the extraction reagent as it will inactivate the luciferase and/or the luciferin over time. If either is inactivated, no light will be produced when combined with ATP. Therefore, the luciferase/luciferin reagent and extraction reagent must be stored separately until the time the test is conducted.
The bioluminescent reaction of ATP and luciferase/luciferin has traditionally been conducted using two basic types of systems: vial systems and all-in-one swab devices. A vial system uses a series of vials containing the reagents necessary to conduct the ATP tests. An all-in-one swab device provides all of the reagents and the swab in a self-contained apparatus.
In a vial system, for example, a first vial contains the extraction reagent, a second vial contains dried luciferase/luciferin reagent, and a third vial contains a buffered solution. At the time of the test, the luciferase/luciferin reagent is hydrated by adding the appropriately buffered solvent from the third vial to the vial containing the luciferase/luciferin reagent.
A sample is collected by wiping a pre-wetted swab across the testing surface. Typically, the swab is pre-wetted with saline. The swab containing the sample is placed in a test tube. Next, the proper amount of extraction reagent from the first vial is pipetted into the test tube containing the swab. After sufficient time has passed to ensure ATP extraction, the buffered solution containing hydrated luciferase/luciferin reagent is pipetted into the test tube and the luciferase is allowed to react with the ATP. The test tube is then placed into a luminometer where the amount of light produced by the reaction is measured. If more than one sample is taken, each sample is placed in its own test tube.
While vial systems produce correct results, there are deficiencies. One large problem is that the quantity of luciferase/luciferin solution prepared must be used within a short time perio
Andreotti Peter E.
Berthold Fritz
Banner & Witcoff , Ltd.
Beisner William H.
Berthold Fritz
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