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ROSENOW'S "RECIPES" FOR:

DIVERSE DISEASES: ISOLATION AND PRODUCTION OF STREPTOCOCCI [58R1]
DIVERSE DISEASES: SIMILAR ORGANISMS, DIFFERENT DISEASES [58R1]
DIVERSE DISEASES: PREPARATION OF "THERMAL" ANTIBODY [58R1]

58R1: Rosenow, E.C., Studies on specific prevention and treatment of diverse diseases shown due to specific types of nonhemolytic streptococci, Am. Practitioner and Digest of Treatment (Philadelphia), 9(5), May 1958, p. 755-761:
ISOLATION AND PRODUCTION OF STREPTOCOCCI (DIVERSE DISEASES) [58R1]
"Serial dilution cultures at steps of 10-1, 10-6, and 10-10 were made of nasopharyngeal swabbings, with cotton-wrapped aluminum wire swabs, of the nasopharynx of persons having diverse chronic disease. The material thus obtained was inoculated into tall (8-10 cm.) columns of 0.2 percent dextrose broth adjusted to pH 7.0, to which pieces of fresh calf or beef brain were added, comprising approximately one part of brain substance to six or seven parts of broth, and autoclaved at 17 pounds' pressure for 20 minutes. Cultures were made in this freshly prepared medium directly or soon after sterilization, but after prolonged storage the test tubes containing the tall columns of medium were heated in a boiling water bath for 15 minutes to remove absorbed oxygen."
"The nasopharyngeal swabbings were obtained without touching the tongue. The material on the swabs was washed off in 2 ml. of NaCl solution, and then a tube of dextrose-brain broth was inoculated with the swab, representing an estimated dilution of the material swabbed of 10-2. Two serial transfers were then made, with a nicrome wire, the length of the column of dextroxe brain- broth sterilized in a Bunsen flame at each step, representing dilutions of inocula of 10-6 and 10-10 respectively. Growths after incubation at 35 degrees C. for 18 to 24 hours, at a dilution of 10-2, consisted of predominating numbers of gram- staining, short-chained streptococci and, usually, of moderate numbers of gram-staining micococci and sometimes also gram-negative, gas-producing bacilli. Growth at the dilution of 10-6 usually consisted of a pure culture of short-chained, alpha- type streptococci. Growths usually did not occur at dilutions of 10-10, but if positive such growth consisted of a pure culture of streptococci.
One milliliter of pure cultures from such end points of growth in dextrose-brain broth were inoculated into [previously warmed, per 54R1] 200 ml. or gallon lots of 0.2 percent dextrose broth, and the streptococci, thus grown at 35 degrees C. for 18 hours in the smaller lots, were harvested in a cup-type centrifuge. Pure cultures of streptococci in the gallon lots of 0.2 percent dextrose broth were harvested in the revolving bowl of the Sharples super-centrifuge. The sedimented streptococci from the cup-type centrifuge were in turn suspended in dense suspension of an estimated 200 billion streptocci per milliliter, and those from the larger lots from the bowl of the super centrifuge at 1000 billion per milliliter of two parts of chemically pure glycerol and one part of saturated NaCl solution and stored in the dark in the refrigerator at 10 degrees C. Some of the streptococci in this menstruum remained viable for months. All remained gram-positive and antigenically specific for many months on storage at 10 degrees C. Such storage made it readily possible to maintain serologic and other specific properties of streptococci as isolated in diverse diseases.[58R1]

SIMILAR ORGANISMS, DIFFERENT DISEASES [58R1]
--"The streptococci isolated in dextrose-brain broth from nasopharyngeal swabbings of persons having diverse diseases were much alike in size, chain formation and staining reactions. All were gram-positive and none produced zones of clear hemolysis on blood-agar, but instead, small colonies surrounded by a narrow green or indifferent zone were formed. The freshly isolated strains from nasopharyngeal swabbings of persons who had diverse acute or chronic disease on intravenous injection into mice and rabbits, localized and produced lesions "electively" in the organ or organs corresponding to those involved in patients from whom the streptococci were isolated."

PREPARATION OF "THERMAL" ANTIBODY (DIVERSE DISEASES) [58R1]
"... Thermal antibody solutions ... were prepared by autoclaving the respective specific and control NaCl solution suspensions containing 20 billion streptococci per milliter for 96 hours, and diluting the supernatant of such autoclaved suspensions with an equal volume of NaCl solution and adding 0.2 percent phenol as a preservative."

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