Quantitative Flotation (McMaster)

1. Quantitative Flotation (McMaster)

2. 56 ml Gauze 30x30 cm Stir Wait 30 min. 4 gram Remove supernatant with pipette Fill a reading chamber Wait 5 min. Read 3 vertical lines at x200 magnification and note species and Count numbers Multiplication factor: 47 Add 3ml flotation liquid and mix with pipette  10 ml 3 ml Centrifuge 1200 g 5-7 min

3. x20 Added flotation liquid 1,5 / 3 / 6 ml 3 ml Visual area EPG (Eggs Per Gram) = Count X Multiplication factor example. EPG = 2 x 83= 166

4. 40 U = 8

5. Sedimentation (heavy parasite eggs)

6. Sedimentation („Trematode“ eggs) Method for heavier eggs that do not flotate in common flotation fluids Nematodirus spp. Fasciola hepatica Dicrocoelium dendriticum Fasciola buski Opisthrchis noverca 3 g faeces + 50 ml water Fill with water Wait 10 min Remove supernatant gauze Add drop to slide + cover glass + read Add 1 drop of dye + mix Remove supernatant Wait 10 min Resuspend in water 

7. Baermann (lungworms & L3)

8. Baermann (lungworms & L3 larvae) Harvesting of larvae from faecal sample Fold gauze and put in syringe Fill with water Wait 24 hours (no direct sun) 5-10 g faeces water Remove faeces and excess water Transfer to 14 ml centrifuge tube Centrifuge 1000 rpm, 2 min Transfer to petri dish and examine under microscope Remove excess water

9. Baermann (lungworms) Harvesting of larvae from faecal sample Muellerius capillaris (300-320 µm) Posterior end of body often curled Dictyocaulus viviparus (390-450 µm) Cattle Dictyocaulus filaria (550-585 µm) Larvae often curved Sluggish movement Sheep Cystocaulus (340-480 µm) Additional dorsal and ventral spines on middle part of tail Protostrongylus (320-400 µm) Posterior half of body may be curled

10. Baermann (L3 larvae) Harvesting of larvae from faecal sample Trichostrongylus (560-796 µm) 16 gut cells, Head tapered, Sheath forms short cone Cooperia (666-956 µm) 16 gut cells, Head square with refractile bodies, Sheath tail medium tapering (Cooperia oncophora), or finely pointed and refractile (Cooperia curticei type) Ovine Bovine Ostertagia Oesophagostomum (726-923 µm) / Chabertia (650-789 µm) Approx. 32 gut cells, Head broad and rounded, Sheath tail filamentous, Larval tail not notched or lobed Strongyloides (650-850 µm) Oesophagus extends nearly half length of body, Sheath absent, Larval tail notched http://www.rvc.ac.uk/review/parasitology/RuminantL3/Introduction.htm

11. Ziehl-Neelsen [acid fast staining] (Cryptosporidium spp.)

12. Zeihl-Neelsen (Cryptosporidium spp.) Detection of C. spp. oocysts in faeces Add small amount of feaces to slide Smear feaces using second slide Dry Fixate methanol (+2% HCl) 5 min Color carbolic fuxine 20 min Add name with pencil Repeat 2 times Wash Water 10 sec Color Brilliant Green 2.5% 5 min Wash Water 10 sec Remove color 10% H2SO4 10 sec Dry Read with x400 magnification 0 = no oocysts + = 1-5 oocysts ++ = 6-25 oocysts +++ = >25 oocysts

13. Ziehl-Neelsen (Cryptosporidium spp.) Detection of C. spp. oocysts in faeces Read with x400 magnification 0 = no oocysts + = 1-5 oocysts ++ = 6-25 oocysts +++ = >25 oocysts http://courses.cit.cornell.edu/biomi290/microscopycases/crypto/index.htm