Fermentation flask for cultivating microorganisms, a growth media and method of use

Abstract

A container is provided for cell growth by artificial cultivation of selected biological material in a growth media for use in an oscillating incubator. The container has a circular bottom with rounded corners joining a sidewall that is inclined at an angle toward a longitudinal axis of the container to define a container volume. The bottom has six equally spaced baffles extending inward from the corners and upward toward a large diameter opening of the container centered on the longitudinal axis. The baffles have a triangular cross-sectional shape with an included angle of about 28-40°, and a height measured parallel to the longitudinal axis of about 15-25% of the usable container volume. The baffles end before the longitudinal axis. The container and baffles are blow molded from a polymer suitable for blow molding. An air permeable filter is placed over the container opening, with the filter having an adhesive on one side to stick to the container. A finger tab on the periphery of the filter helps to position and remove the filter. A growth media is placed in the container along with a biological material to be cultured and rotated from about 250 RPM to 450 RPM depending on the volume of the material and the container. The growth media comprises MgBr 2 , CuBr 2 , FeBr 2 , Tris, Tris-HBr, Tris-acetate, or Tris-HCl, KBr, adenine, palmitic acid, glycerol or D-(+)-glucose, or both, ATP, Protein Hydrolysate Amicase or other casamino acids, yeast or yeast extract, tryptone peptone, anti-foam and de-ionized water.

1 . A method for cell growth by artificial cultivation of selected biological material in a growth media for use in an oscillating incubator, the incubator including a container having a bottom with corners joining a sidewall inclined at an angle toward a longitudinal axis of the container to define a usable container volume, the container bottom having a plurality of baffles, comprising: 
 placing a selected biological material into the container;    placing a growth media in the container, the growth media comprising: 
 MgBr 2 ;  
 CuBr 2 ;  
 FeBr 2 ;  
 Tris, Tris-acetate, Tris-HCl, or Tris-HBr, buffered to pH 6-8  
 KBr;  
 adenine;  
 palmitic acid;  
 glycerol, or D-(+)-glucose, or both;  
 ATP;  
 Protein Hydrolysate Amicase or other casamino acids;  
 yeast or yeast extract;  
 tryptone peptone;  
 anti-foam, and  
 de-ionized water;  
   covering an opening of the container with an air-permeable filter; and    rotating the container at over 250 RPM for at least several hours.    
   
   
       2 . The method of  claim 1 , wherein the growth media and biological material comprise about 20% of the usable container volume and the rotational speed is between about 350 RPM to 450 RPM.  
   
   
       3 . The method of  claim 1 , wherein the growth media and biological material comprise about 40% of the usable container volume and the rotational speed is between about 250 RPM to 300 RPM.  
   
   
       4 . The method of  claim 1 , wherein the container has a circular bottom with six equally spaced baffles extending inward from the corners toward the longitudinal axis and upward toward a large diameter opening of the container centered on the longitudinal axis, the baffles having a triangular cross-sectional shape with an apex having an included angle of about 28-40°, and a height measured parallel to the longitudinal axis of about 15-25% of the usable container volume, the baffles ending before the longitudinal axis, the container and baffles being blow molded from a polymer suitable for blow molding.  
   
   
       5 . The method of  claim 1 , wherein the baffles extend radially inward.  
   
   
       6 . The method of  claim 1 , wherein the air permeable filter allows air to pass through substantially all of the opening to the container and the filter is removably fastened to the container by an adhesive.  
   
   
       7 . The method of  claim 1 , wherein, the growth media further comprises: 
 about 0.3 g to 3 g MgBr 2 ,    about 0.1 g to 0.5 g CuBr 2 ,    about 0.1 g to 1 g FeBr 2 ,    about 100 mL Tris, Tris-acetate, Tris-HCl, or Tris-HBr buffered to pH 6-8,    about 0.1 g to 1 g KBr,    about 10 g adenine,    about 0.1 g to 3 g palmitic acid,    about 1% by volume of D-(+)-glucose or glycerol, or both,    about 0.5 g ATP,    about 10-15 g Protein Hydrolysate Amicase or other casamino acids,    about 24 g yeast extract,    about 12 g tryptone peptone,    about 1:10,000 by volume of anti-foam, and    about 900 mL de-ionized water to form 1 L solution;    the growth media and a volume approximately 10-20 mL of the biological material are then added to the container.    
   
   
       8 . The method of  claim 7 , wherein the growth media and biological material comprise about 20% of the usable container volume and the rotational speed is maintained at between about 350 RPM to 450 RPM for a total time in excess of 10 hours, with the biological material forming substantially no aggregations and remaining at least 85% soluble.  
   
   
       9 . The method of  claim 7 , wherein the growth media and biological material comprise about 20% of the usable container volume and the rotational speed is maintained at between about 350 RPM to 450 RPM until the optical density exceeds 24 for an 18 hour culture.  
   
   
       10 . The method of  claim 7 , wherein the growth media and biological material comprise about 40% of the usable container volume and the rotational speed is maintained at between about 250 RPM to 300 RPM for a total time in excess of about 10 hours with the biological material forming substantially no aggregations and remaining at least 85% soluble.  
   
   
       11 . The method of  claim 1 , wherein the growth media and biological material comprise about at least 20% of the usable container volume and the rotational speed is maintained at between about 350 RPM to 450 RPM until the optical density exceeds 20 for an 18-24 hour culture.  
   
   
       12 . The method of  claim 1 , wherein the growth media and biological material comprise about at least 20% of the usable container volume and the rotational speed is maintained at between about 350 RPM to 450 RPM for over 24 hours with the material forming a protein with substantially no aggregations and remaining at least 90% soluble.  
   
   
       13 . The method of  claim 1 , wherein the biological material includes a Histidine tag.  
   
   
       14 . A growth media for use in reproducing biological cell material, comprising: 
 MgBr 2 ;    CuBr 2 ;    FeBr 2 ;    Tris, Tris-acetate, Tris-HCl, or Tris-HBr, buffered to pH 6-8    KBr;    adenine;    palmitic acid;    glycerol, or D-(+)-glucose, or both;    ATP;    Protein Hydrolysate Amicase or other casamino acids;    yeast or yeast extract;    tryptone peptone;    anti-foam, and    de-ionized water.    
   
   
       15 . The growth media of  claim 14 , further comprising de-ionized water in an amount to dissolve the ingredients to form a growth media, and wherein about 100 mL of Tris, Tris-acetate, Tris-HCl, or Tris HBr, is added to buffer the growth media to a pH of about 6-8.  
   
   
       16 . The growth media of  claim 14 , wherein the ingredients comprise: 
 about 0.3 g to 3 g MgBr 2 ,    about 0.1 g to 0.5 g CuBr 2 ,    about 0.1 g to 1 g FeBr 2 ,    about 100 mL Tris, Tris-acetate, Tris-HCl, or Tris-HBr buffered to pH 6-8,    about 0.1 g to 1 g KBr,    about 10 g adenine,    about 0.1 g to 3 g palmitic acid,    about 1% by volume of glycerol or D-(+)-glucose, or both,    about 0.5 g ATP,    about 10-15 g Protein Hydrolysate Amicase or other casamino acids,    about 24 g yeast extract,    about 12 g tryptone peptone,    about 1:10,000 by volume of anti-foam, and    about 900 mL de-ionized water.    
   
   
       17 . The growth media of  claim 14 , wherein the ingredients per liter of de-ionized water, comprise: 
 about 0.3 g to 3 g MgBr 2 ,    about 0.1 g to 0.5 g CuBr 2 ,    about 0.1 g to 1 g FeBr 2 ,    about 100 mL Tris, Tris-acetate, Tris-HCl, or Tris-HBr buffered to pH 6-8,    about 0.1 g to 1 g KBr,    about 10 g adenine,    about 0.1 g to 3 g palmitic acid,    about 1% by volume of glycerol or D-(+)-glucose, or both,    about 0.5 g ATP,    about 10-15 g Protein Hydrolysate Amicase or other casamino acids,    about 24 g yeast extract,    about 12 g tryptone peptone, and    about 1:10,000 by volume of anti-foam.    
   
   
       18 . The growth media of  claim 14 , further comprising a biological material mixed with the growth media.  
   
   
       19 . The growth media of  claim 14 , further comprising a biological material mixed with the growth media in a ratio of about 1:1.