Detection of respiratory enzyme activity in Giardia cysts and Cryptosporidium oocysts using redox dyes and immunofluorescence techniques.
The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epifluorescence microscopy. The reduction of CTC and p-iodonitrotetrazolium violet (INT), a non-fluorescent redox dye, was compared with propidium iodide (PI) and fluorescein diacetate (FDA) for the measurements of Giardia cyst viability over time. According to the PI and FDA staining techniques, nearly 60% of the cysts tested viable at the beginning of the observations; after 21 days their viability decreased to 5%. The redox dyes indicated that approximately 4-10% of the cysts were metabolically active 48 h after they were shed, followed by a decline in enzyme activity to near undetectable levels after 4 days. Spectral analysis on individual cysts indicated that the fluorescence emission of the reduced CTC and the fluorescein-labeled antibodies is distinctive for each compound and suitable for their simultaneous determination by microphotometry, flow cytometry and epifluorescence microscopy. The fluorescence signal remained without alteration when the cysts were transferred onto microscope slides coated with an optical embedding medium and stored at -20 degrees C. The fluorescence intensity of the reduced CTC, when properly standardized, can provide quantitative measurements of ETS activity of the cysts. This is the first report of a method to determine enzyme redox activity on intact cysts applicable to water, laboratory and animal samples.
Iturriaga
R
R
Department of Biological Sciences, University of Southern California, 90089-0371, Los Angeles, CA, USA. iturriag@mizar.usc.edu
Zhang
S
S
Sonek
G J
GJ
Stibbs
H
H
D000818:Animals; D019069:Cell Respiration; D002470:Cell Survival; D003458:Cryptosporidium / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D003592:Cytophotometry; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005872:Giardia / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010063:Ovum / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D011419:Propidium; D013050:Spectrometry, Fluorescence; D013777:Tetrazoles; D013778:Tetrazolium Salts
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013777:Tetrazoles; D013778:Tetrazolium Salts; C075453:5-cyano-2,3-ditolyltetrazolium; D011419:Propidium; C018506:diacetylfluorescein