Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-06-28
2002-08-27
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S968000, C435S004000
Reexamination Certificate
active
06440689
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of measurement of microbiological activity in highly scattering systems. Specifically, this patent application is in the field of fluorescent measurement of microbiological activity in opaque mediums such as slurries and colloids and certain Metal Working Fluids.
BACKGROUND OF THE INVENTION
Microbial contamination in opaque mediums such as slurries and colloids and certain Metal Working Fluids is a significant problem in many industries. In papermaking, additives such as kaolin slurry, precipitated calcium carbonate suspensions or starch solutions can harbor large microbial populations, which serve as inocullum for the papermnachine. Mining companies are required to supply industries such as paper and ceramics with treated and preserved additives and also need to monitor microbial contamination. Certain Metal Working Fluids are also susceptible to microbiological contamination.
The conventional method of controlling microbial growth is through the use of biocides. Biocides are chemicals that inhibit microbial growth by destroying the cell wall or cellular constituents of microorganisms. Physical conditions such as temperature, radiation, or interactions with treatment chemicals contained within a system can have a negative impact on the effectiveness of the biocide. To compensate for the reduced effect, biocides can either be added continuously or intermittently on an “as-required” basis. The minimal possible use of biocides is encouraged since biocides are both expensive and toxic. Thus, to prevent waste, constant monitoring and testing of the slurry or colloid or Metal Working Fluid is required to determine the proper quantity of biocide for controlling microbial growth.
Most slurries and colloids and certain Metal Working Fluids are opaque, which means they are not transparent to the passage of light. Another way to describe opaque media is that they are highly light scattering media. For the purposes of this patent application the term, “opaque” is used to refer to any medium which when placed in a 1 cm cuvette in the path of a non-absorbing visible light beam, acts to reduce the intensity of the light by 20% or more due to scattering.
When media are opaque, it is not possible to know what is inside an opaque media simply by looking at it. This means that it is impossible to tell if there is microbiological contamination of an opaque slurry or an opaque colloid or an opaque Metal Working Fluid by looking at it. Therefore, conventional, known optical methods of detection of microbiological contamination (such as optical density measurements and ATP measurements) cannot give results for opaque media. This is because light cannot pass through the sample, as light loss becomes inversely proportional to the extent of light scattering. Therefore, other methods of detecting microbial contamination in an opaque media must be used.
At present samples of opaque slurries or opaque colloids or opaque Metal Working Fluids are typically monitored for microbiological contamination using standard “plate-count” methods. Standard “plate-count” methods are typically referred to as “plating”. Plating of samples requires trained personnel, equipment and a 48 hours incubation period during which microbes in the slurry can reproduce rampantly. The actual method of plate counting involves withdrawing a sample, diluting the sample, and applying the sample to the surface of a Nutrient agar medium. After incubation for 24 to 48 hours, the sample is checked for the presence of microorganisms and, where appropriate, the organisms are counted by manual or video means.
Some industrial situations require the use of High Pressure Liquid Chromatographs (HPLC) to determine if there is residual biocide left in the sample. HPLC can only measure biocide concentration and not microbial activity. HPLC also requires expensive equipment and trained personnel for routine measurements. Since HPLC only measures residual biocides, it cannot measure biocide resistant strains of microbiological organisms that are developing in the opaque media.
In addition to grab sampling, other on-site sampling techniques are available, such as Dip slide and Adenosine Triphosphate (ATP) tests. Unfortunately, such tests are not practical to use when measuring microbiological contamination in opaque medium because ATP tests require a transparent sample and therefore do not work in opaque medium and Dip slides require 24 to 48 hours for test results to develop. Thus, neither test is suitable for field evaluation of microbiological contamination.
Additional methods for monitoring the microbial populations in various mediums are described and claimed in the following references.
U.S. Pat. No. 5,206,151 describes and claims a method to measure the minimum inhibitory concentration of biocides by adding various amounts, types and combinations of biocides to aliquots of sample containing bacteria, adding an oxidation-reduction dye such as Resazurin or tetrazolium violet and Nutrients and monitoring the change in color.
U.S. Pat. No. 5,413,916 describes and claims a method for determination of toxicity of an environmental sample to bacteria by the addition of Resazurin and glutaraldehyde and bacteria to the sample and measuring absorbance (at 603 nm) as compared to a blank.
U.S. Pat. No. 5,336,600 describes and claims a method for detection of micro-organisms consisting of mixing a Resorufin (or Resorufin derivative not including Resazurin) and Nutrient medium and measuring a decrease in the fluorescence.
U.S. Pat. No. 5,523,214 describes and claims a method of identification of microbes using methylene blue and Resazurin stabilized with potassium ferricyanate or iron salts mixed with potassium ferrocyanate or sodium tungsate or tartrazine yellow or reactive red 4 or similar compounds. The patent claims substantial increase in sensitivity using this mixture as compared to using either dye alone.
Aliquots of sand filters were assessed using Resazurin reduction method, in an experiment described in an article entitled: “Resazurin reduction tests as an estimate of coliform and heterotrophic bacterial numbers in environmental samples” Can. Bull. Environ. Contam. Toxicol. 49, 354, 1992.
An article entitled, “Resazurin reduction as a function of respiratory burst in bovine neutrophils is an article in Am. J. Vet. Res. 58, 601, 1997, describes a technique of fluorometrically monitoring the end-point of Resazurin (Resorufin) as a measure of respiratory burst.
The article, “Automation of the Resazurin Reduction Test using Fluorometry of Microtitration Trays”, by Ali-Vehmas, Louhi and Sandholm,
J. Vet. Med.,
B 38, 358-372 (1991) describes the automation of the fluorescent Resazurin-to-Resorufin reduction test for monitoring bacterial numbers in broth cultures and milk. The reduction of Resazurin (blue color) to Resorufin (a pink color) and finally and reversibly to dihydroresorufin (colorless) is well known in the art of determining mircrobiological contamination in milk. This method involves taking contaminated samples of milk in microtitration plates, adding a Nutrient medium and measuring the fluorescence corresponding to the Resorufin peak at 5 min intervals. This continuous measurement of the same sample makes this an automated measurement. The Resorufin intensity peaks when the increase in intensity due to conversion from Resazurin is offset by the decrease due to formation of the non-fluorescent dihydroresorufin. In this work, the sample population is increased significantly by addition of Nutrient medium (which is a necessary part of the method).
U.S. Pat. No. 6,060,318, entitled, “Tracing of Process Additives in Industrial Ceramics Applications”, claims a fluorometric method for monitoring concentration of chemicals in ceramic slurries and powders having an external surface. In this patent, a solid-state fluorometer, (in the surface fluorescence configuration) is used to monitor the concentration of fluorescence molecules in ceramic slurries. Applications within ceramic slurries include mo
Banks Rodney H.
Casselman Nancy L.
Chattoraj Mita
Davis Ronald V.
Fehr Michael J.
Breininger Thomas M.
Brumm Margaret M.
Leary Louise N.
Ondeo Nalco Company
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