Development of a cell wash buffer that minimizes nucleic acid loss from Clostridium perfringens 10543 A.
Autolytic activity and nucleic loss from Clostridium perfringens 10543 A was demonstrated during successive cell washes in hypotonic TES buffer. Autolysis increased nearly sixfold and nucleic acid loss nearly twofold when 10 mM EDTA was added to 0.3 M Tris-sucrose buffer. Attempts to minimize both autolysis and nucleic acid loss from C. perfringens during routine washing steps were unsuccessful when the effects of sucrose concentration, pH, CaCl2 addition, or wash temperature were examined independently. However, autolytic activity was eliminated and nucleic acid loss reduced to less than 5% when C. perfringens cells were washed at 4 or 25 degrees C in 1.0 M sucrose, 50 mM Tris--HCl, and 25 mM CaCl2 at pH 5.7.
H P
HP
Blaschek
M A
MA
Klacik
D001431:Bacteriological Techniques; D001433:Bacteriolysis; D002122:Calcium Chloride / Q000494:pharmacology; D003016:Clostridium perfringens / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007038:Hypotonic Solutions; D009696:Nucleic Acids / Q000378:metabolism; D009997:Osmotic Pressure; D012329:RNA, Bacterial / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology; D014325:Tromethamine / Q000494:pharmacology
D004269:DNA, Bacterial; D007038:Hypotonic Solutions; D009696:Nucleic Acids; D012329:RNA, Bacterial; D014325:Tromethamine; D013395:Sucrose; D004492:Edetic Acid; D002122:Calcium Chloride