Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2001-11-19
2003-10-28
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S255700, C435S256800, C435S404000, C435S431000, C435S034000
Reexamination Certificate
active
06638755
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a simple culture medium useful for, for example, detection, identification, and transportation of various microorganisms, and more particularly to a simple culture medium in which a test fluid inoculated thereto diffuses very rapidly and inoculation of a sample is possible even in an inclined state, an inverted state, or a gravity-free state.
BACKGROUND ART
Among conventional methods for culturing various microorganisms for detection and identification thereof, spread plate method, pour plate method, liquid medium method, etc. are generally employed. However, these methods require sterilization of culture medium, apparatus, etc. before culture of microorganisms, and inoculation of a test sample into the medium requires skilled operation such as spreading or pouring. Furthermore, when a conventional culture medium is sterilized after preparation of the medium or after hermetic packaging of the medium, maintaining the quality and performance of the medium during transportation and storage thereof is difficult; i.e., sterilization of the medium encounters difficulty.
Therefore, extensive studies have been performed on a simple culture medium which enables culture to be carried out easily, and a variety of culture devices and culture media have been proposed, including (1) a culture device in which an adhesive layer, a layer of a cold-water-soluble powdery gelling agent containing nutritive ingredients, and a cover sheet are successively laminated on the upper surface of a waterproof substrate (Japanese Patent Application Laid-Open (kokai) No. 57-502200); (2) a culture device in which a layer of a mixture of a cold-water-soluble gelling agent and a microorganism culture medium, and a fibrous water-absorbent sheet are successively laminated on the surface of a waterproof flat plate (Japanese Patent Application Laid-Open (kokai) No. 6-181741); and (3) a simple culture medium in which a medium composition containing an adhesive, a gelling agent, and a bacterial nutritive ingredient is carried with a fibrous water-absorbent sheet having a mesh larger than the particle size of the gelling agent (Japanese Patent Application Laid-Open (kokai) No. 9-19282).
However, the culture device in (1) above requires operations including careful application of an upper sheet and spreading of a test sample over a certain area by means of a spreader, and these operations must be carried out carefully on a flat plane such as a table; i.e., the culture device involves the problem of cumbersome inoculation operation. The culture device in (2) above involves problems in terms of preparation; i.e., insertion of the fibrous water-absorbent sheet after coating of the medium ingredients is troublesome, and the coating area of the medium ingredients must be greater than the area required for detection of microorganisms. In addition, since the fibrous water-absorbent sheet is not fixated, during inoculation, careful operation is required so as not to cause the sheet to move. The simple culture medium (3) involves problems in that the medium must be allowed to stand until a test fluid permeates and diffuses throughout the sheet, since the test fluid diffuses in the sheet slowly, and in that, even when the water-absorbent sheet is laminated and affixed onto a waterproof flat plate, the sheet is exfoliated from the plate very easily.
Accordingly, an object of the present invention is to provide a simple culture medium in which a test fluid inoculated thereto diffuses very rapidly, the medium containing a waterproof flat plate and a water-absorbent sheet reliably adhering onto the plate; enabling inoculation in an inclined state, an inverted state, or a gravity-free state, as well as in an even state; enabling detection and identification of microorganisms through a simple operation; and facilitating transportation, sterilization, etc. of the medium.
DISCLOSURE OF THE INVENTION
The present inventors have performed extensive studies in order to improve the aforementioned simple culture medium (3) (Japanese Patent Application Laid-Open (kokai) No. 9-19282), from the viewpoints of enhancement of the diffusion rate of a test fluid and reliable fixation of a sheet. As a result, the present inventors have found that, as compared with the case where an adhesive is incorporated in a large amount and a sheet impregnated with an alcohol suspension is dried in a usual manner, when the amount of an adhesive in an alcohol suspension containing a medium composition is reduced, a water-absorbent sheet is sufficiently impregnated with the suspension, and the resultant sheet is gradually dried, unexpectedly, the sheet can be caused to adhere onto a waterproof flat plate very strongly; and that there can be obtained a simple culture medium in which a test fluid diffuses at a very high rate; i.e., the test fluid instantaneously diffuses when inoculated into the medium. The present invention has been accomplished on the basis of these findings.
Accordingly, the present invention provides a simple culture medium produced by impregnating a fibrous water-absorbent sheet with a suspension formed by suspending in an alcohol (a) an adhesive (0.01-0.4 wt. %) which is soluble both in water and alcohol, (b) a gelling agent which is soluble in water and insoluble in alcohol, and (c) a bacterial nutritive ingredient, the fibrous water-absorbent sheet having a mesh larger than the particle size of the gelling agent and being placed on a waterproof flat plate, and by drying the resultant sheet while controlling rapid evaporation of the alcohol, to thereby cause the water-absorbent sheet to adhere onto the waterproof flat plate; and a method for producing the medium.
BEST MODE FOR CARRYING OUT THE INVENTION
An adhesive which serves as ingredient (a) of a medium composition must be soluble in water and alcohol. Examples of the adhesive include hydroxypropyl cellulose, polyvinyl pyrrolidone, and polyethylene oxide. Of these, hydroxypropyl cellulose is particularly preferred.
A gelling agent which serves as ingredient (b) must be soluble in water and insoluble in alcohol. Examples of the gelling agent include naturally occurring gelling agents such as xanthan gum, locust bean gum, guar gum, and carrageenan; and synthetic gelling substances such as hydroxyethyl cellulose. Of these, xanthan gum is particularly preferred. Such a gelling agent is preferably used in the form of powder having a mean particle size of 500 &mgr;m or less, particularly 0.5-50 &mgr;m.
A bacterial nutritive ingredient suitable for growth of a microorganism to be detected is chosen as ingredient (c). For example, when a variety of microorganisms are proliferated, typical nutritive medium ingredients are used, and when a specific microorganism is selectively proliferated, selected medium ingredients are used.
Preferably, a composition containing ingredients (a), (b), and (c) (hereinafter the composition will be referred to as “medium composition”) further contains an appropriate color-developing agent for facilitating observation of colonies. Examples of the color-developing agent include dyes which color colonies, such as triphenyltetrazolium chloride, 3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; enzyme substrates such as 5-bromo-3-indolyl-&bgr;-D-galactoside; and pH indicators such as Bromothymol Blue and Neutral Red.
Examples of alcohols for suspending the medium composition include C1-C5 alcohols such as ethanol and 2-propanol.
The fibrous water-absorbent sheet in which the aforementioned medium composition is to be carried must allow an inoculated test fluid to diffuse readily by means of a capillary phenomenon for carrying thereon, and have a network structure capable of holding the gelling agent contained in the medium composition. To this end, the fibrous water-absorbent sheet must have a mesh larger than the particle size of the gelling agent, and a thickness larger than the particle size. For example, the fibrous water-absorbent s
Kodaka Hidemasa
Mizuochi Shingo
Shibata Hideaki
Afremova Vera
Nissui Pharmaceutical Co., Ltd.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Saucier Sandra E.
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