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Table 17.4 Mean percentage recovery of C. parvum oocysts seeded directly into extractants and recovered by IMS (Trial A)
Extractant
Mean % oocyst recovery (± standard deviation) (n [ 3)
0.1 M HEPES, pH 5.5
89.4 ( 5.1)
1 M Sodium bicarbonate, pH 6.0
88.2 ( 9.8)
1 M Glycine, pH 5.5
105.3 ( 18.4)
1 M Bicine, pH 5.6
81.1 ( 12.5)
1% Sodium dodecyl sulfate (þ Antifoam A)
90.2 ( 5.5)
PBS, 150 mM, pH 7.2
11.4 ( 5.7)
0.1 M Tricine, pH 5.4
9.8 ( 2.5)
Extraction buffer (EB a)
5.9 ( 4.5)
a EB is the buffer recommended in the US EPA’s method 1623 (Anon., 1998) for the extraction of Cryptosporidium oocysts and Giardia cysts from capsule filters. Its composition is: 0.01 M Tris pH 7.4, 0.1% sodium dodecyl sulfate, 0.005 M EDTA, 150 ppm antifoam A.
prior to IMS to determine the degree of compatibility between the extractant, foodstuff, and IMS. The results are presented in Table 17.5. Sodium bicarbonate eluates when dried onto microscope slides left extensive debris, producing slides that were difficult to read because of oocyst occlusion. Extraction in HEPES and bicine generated lower recoveries. The trial was repeated, and tripotassium phosphate, glycine, glycine þ sodium dodecyl sulfate þ antifoaming agent were also tested. Extraction in HEPES, bicine, and tricine generated larger standard deviations and lower recoveries, and were deemed too expensive based on their performance. 1M glycine (pH 5.5) was chosen as the most effective extractant. In Trial C, we investigated seeding lettuce leaves with known numbers of oocysts, extracting the oocysts with selected extractants, and determined the efficiency of recovery (Table 17.6). This lower recovery, compared to previous results, indicated that not all oocysts were extracted from the lettuce and/or recovered by IMS. The data indicated that non-covalent interactions between oocysts and lettuce surfaces, while complex, could be minimized by judicious choice of extractant. Minimizing the oocyst surface charge reduces the attractive forces between oocysts and matrix enabling them to be released into the extractant.