Fig 1.
Individually PI (A and D), Hoechst (B and E) and FDA (C and F) stained cercariae.
The PI and Hoechst-stained dead cercarial tails (A and B) show the staining of the nucleic acid. Both tails fluoresce strongest at the tail-head junction, as this is where most ruptured cell membranes are found resulting from the tail separation. The FDA-stained whole live cercaria (F) fluoresces strongest in the acetabular glands. Image D, E show a live cercaria stained with PI and Hoechst, respectively. Image C shows an FDA-stained dead cercaria.
Fig 2.
Higher magnification images of PI (A), Hoechst (B) and FDA (C) stained cercariae. Dead cercariae are stained with PI and Hoechst, and a live cercaria stained with FDA.
Fig 3.
Cercariae double stained with FDA-PI (B) and FDA-H (D).
Live, whole cercariae fluoresce green, whereas dead cercariae (often with their tail separated) fluoresce blue (Hoechst) or red (PI). Respective bright-field images of cercariae (A and C) shown for reference.
Fig 4.
Fluorescence of PI (A), Hoechst (B) and FDA (C) stained cercariae over time.
Each sample contains 100 ± 4 cercariae. Dead cercariae were heat-killed at 60°C for 5 minutes, and mixed samples contain 50% alive and 50% dead cercariae. Data points and error bars show the mean and SEM of nine wells (experiments run in triplicate and repeated three times).
Fig 5.
Fluorescence compared against cercaria number for the dyes considered in this study.
Live (FDA) and dead (PI and Hoechst) cercariae were serially diluted from 128 to 1 cercaria/well, with an additional well containing 160 cercariae. Fluorescence measurements were taken at 20 minutes. Data points and error bars show the mean and SEM of nine wells (experiments run in triplicate and repeated three times).
Fig 6.
Measured versus actual viability of cercariae as determined by the dyes used in this study.
Samples contained 100 ± 4 cercariae (ranging from 0–100% viable) and were stained with FDA-PI and FDA-H. Data points and error bars show the mean and SEM of nine wells (experiments run in triplicate and repeated three times). “Actual” viabilities are the known proportions of live and dead cercariae in each well, and “measured” viabilities are those quantified with the fluorescence data.
Fig 7.
Comparing the viability quantified with fluorescence assays and microscopy.
Samples containing 100 ± 4 cercariae with a viability of 30% ± 2% were stained with FDA-PI and FDA-H. Measurements were taken at 20 minutes. Three 50 μl aliquots of each sample were then examined under the microscope, and cercarial viability was recorded by counting moving (viable) and non-moving (non-viable) cercariae. Average quantified viabilities and SEMs are shown on the bar graph. The “actual” viability is the known proportion of live and dead cercariae in each well, chosen as 30%.