Method for quantification of biological material in a sample

Abstract

Methods for the detection and/or quantification of a biological material in a sample. The method includes the steps of liquefying the sample (if necessary) and pouring the liquefied sample into the incubation vessel. The incubation vessel has a generally flat horizontal surface and the surface is divided into at least one incubation site. Each incubation site is adapted to hold an aliquot of liquid and is sized and shaped, and formed of a suitable material, to hold the aliquot within the well by surface tension. Any excess liquid from the liquefied sample is poured from the surface of the incubation vessel. The method then involves incubating that incubation vessel until the presence or absence of the biological material is determined. The presence of air bubbles can be dramatically reduced by the presence of a surface acting agent in the liquid sample deposited on the device surface. Such an agent can be added to the sterile diluent used to prepare the test reagent, can be separately added to the test reagent after it is prepared, or can be added to the test sample directly while it is being prepared for testing.

1 . A thin film culture incubation vessel kit for detecting a biological material in a sample, comprising: 
 a. a self supporting, waterproof substrate to which is adhered a rehydratable selective medium powder containing nutrients to promote growth of biological material;    b. a transparent cover sheet having a layer of adhesive consisting essentially of a substrate system having an effective amount of one or more nutrient indicators provided in an amount sufficient to produce a detectable characteristic signal in the medium during growth of the biological material, and said nutrient indicator being operable to alter a detectable characteristic of the sample if metabolized in the presence of said biological material;    c. a surface acting agent for use in reducing the introduction of air bubbles into the incubation vessel;    d. at least one gelling agent; and    e. instructions for using said kit.    
   
   
       2 . The kit of  claim 1 , wherein the surface acting agent is selected from the group consisting of anti-foaming agents, defoaming agents, detergents, surfactants, and bile acids.  
   
   
       3 . The kit of  claim 1 , wherein the surface acting agent is selected from the group consisting of sodium dodecyl sulfate, antifoam  204 , and antifoam  289 .  
   
   
       4 . The kit of  claim 1 , wherein the gelling agent is selected from the group consisting of xanthan gum, locust bean gum, rhamsan gum, guar gum, and gellan.  
   
   
       5 . The kit of  claim 1 , wherein said nutrient indicator alters the color of said selective media.  
   
   
       6 . The kit of  claim 5 , wherein said nutrient indicator alters the color of said selective media in the visible wavelength range.  
   
   
       7 . The kit of  claim 1 , wherein said nutrient indicator is a β-D-glucuronidase substrate.  
   
   
       8 . The kit of  claim 7 , wherein said β-D-glucuronidase substrate is selected from the group consisting of orthonitrophenyl-β-D-glucuronide, β-naphthalamide-D-glucuronide, α-naphthol-β-D-glucuronide, and 4-methylumbelliferyl-β-D-glucuronide.  
   
   
       9 . The kit of  claim 1 , wherein the nutrient indicator is a β-galactosidase substrate.  
   
   
       10 . The kit of  claim 1 , wherein said β-galactosidase substrate is selected from the group consisting of orthonitrophenyl-β-D-galactopyranoside and 4-methylumbelliferyl-β-D-galactopyranoside.  
   
   
       11 . The kit of  claim 1 , wherein said nutrient indicator is a β-glucosidase substrate.  
   
   
       12 . The kit of  claim 1 , wherein said nutrient indicator is a L-pyronidonyl aminopeptidase substrate.  
   
   
       13 . The kit of  claim 1 , wherein said nutrient indicator is a L-alanine aminopeptadase.  
   
   
       14 . The kit of  claim 1 , wherein said selective medium further comprises an antibiotic which prevents non-target microbes from metabolizing said nutrient indicator.