FACTORS AFFECTING THE DETERMINATION OF RESPIRATORY ACTIVITY ON THE BASIS OF CYANODITOLYL TETRAZOLIUM CHLORIDE REDUCTION WITH MEMBRANE FILTRATION

Deficiencies in traditional bacterial enumeration techniques which rel y on colony formation have led to the use of total direct counting met hods, such as the acridine orange direct count technique for the enume ration of planktonic bacteria, As total direct counts provide no infor mation on the viability or activity of the organisms, demonstration of respiratory activity with the fluorochrome cyanoditolyl tetrazolium c hloride (CTC) has been employed. We ha,e modified this technique by pe rforming filtration prior to CTC incubation Cells captured on a polyca rbonate membrane were incubated on absorbent pads saturated with mediu m containing CTC. Following counterstaining with DAPI (4',6-diamidino- 2-phenylindole) total and respiring cells were enumerated by epifluore scence microscopy. Factors affecting CTC reduction by Klebsiella pneum oniae, Salmonella typhimurium, and Escherichia coli K-12 were investig ated. With K. pneumoniae, nutrient additions to the CTC medium did not increase the number of respiring cells detected, CTC reduction by all three organisms decreased in response lo an increase of the pH of the CTC medium above pH 6.5. Increasing phosphate concentrations contribu ted to this inhibitory effect. CTC-membrane filter counts of ii. pneum oniae, S. typhimurium, and E. coli K-12 and of bacteria in well water corresponded closely with plate counts (r = 0.987), The results show t hat careful attention should be given to the composition of CTC-contai ning media which are used to enumerate respiring bacteria. With an app ropriate medium, reliable enumeration of respiring bacteria can be ach ieved within a few hours.