Quantitative Flotation(McMaster)

4 gram

56 ml

Stir Wait 30 min.

10 ml

Centrifuge 1200 g5-7 min

Gauze 30x30 cm

Remove supernatant with pipette

Add 3ml flotation liquid and mix with pipette

Fill a reading chamber

Wait 5 min.

Read 3 vertical linesat x200 magnificationand note species and Count numbersMultiplication factor: 47

3 mlFaeces (g) Tap water (ml)

1.0 141.5 212.0 282.5 353.0 423.5 494.0 56

x20

Added flotation liquid1,5 / 3 / 6 ml

EPG (Eggs Per Gram) = Count X Multiplication factorexample. EPG = 2 x 83= 166

Visual area

Objective Volume pellet is diluted in

(ml)

1 visual area

3 visual areas

1 column

3 columns

Total chamber

x4 1,5 377 126 56 19 10

3,0 306 102 50 17 10

6,0 253 84 46 15 10

x10 1,5 2358 786 139 46 10

3,0 1910 637 125 42 10

6,0 1578 526 114 38 10

x20 1,5 9431 3144 278 93 10

3,0 7639 2547 250 83 10

6,0 6316 2105 227 76 10

x40 1,5 37726 12575 556 185 10

3,0 30558 10186 500 167 10

6,0 25254 8418 455 152 10

x100 1,5 235785 78595 1389 463 10

3,0 190986 63662 1250 417 10

6,0 157840 52613 1136 379 10

3 ml

40

Eyepiece units(U) x5 (U x 20) x10 (U x 10) x20 (U x 5) x40 (U x 2,5) x100 (U x 1)1 20 10.0 5.0 2.5 12 40 20.0 10.0 5.0 23 60 30.0 15.0 7.5 34 80 40.0 20.0 10.0 45 100 50.0 25.0 12.5 56 120 60.0 30.0 15.0 67 140 70.0 35.0 17.5 78 160 80.0 40.0 20.0 89 180 90.0 45.0 22.5 910 200 100.0 50.0 25.0 1011 220 110.0 55.0 27.5 1112 240 120.0 60.0 30.0 1213 260 130.0 65.0 32.5 1314 280 140.0 70.0 35.0 1415 300 150.0 75.0 37.5 1516 320 160.0 80.0 40.0 1617 340 170.0 85.0 42.5 1718 360 180.0 90.0 45.0 1819 380 190.0 95.0 47.5 1920 400 200.0 100.0 50.0 2021 420 210.0 105.0 52.5 2122 440 220.0 110.0 55.0 2223 460 230.0 115.0 57.5 2324 480 240.0 120.0 60.0 2425 500 250.0 125.0 62.5 2526 520 260.0 130.0 65.0 2627 540 270.0 135.0 67.5 2728 560 280.0 140.0 70.0 2829 580 290.0 145.0 72.5 2930 600 300.0 150.0 75.0 3035 700 350.0 175.0 87.5 3540 800 400.0 200.0 100.0 4045 900 450.0 225.0 112.5 4550 1000 500.0 250.0 125.0 5055 1100 550.0 275.0 137.5 5560 1200 600.0 300.0 150.0 6065 1300 650.0 325.0 162.5 6570 1400 700.0 350.0 175.0 7075 1500 750.0 375.0 187.5 7580 1600 800.0 400.0 200.0 8085 1700 850.0 425.0 212.5 8590 1800 900.0 450.0 225.0 9095 1900 950.0 475.0 237.5 95100 2000 1000.0 500.0 250.0 100

Dimension in micrometers (mm)

U = 8

Sedimentation(heavy parasite eggs)

Fasciola hepatica

Dicrocoelium dendriticum

Fasciola buski

Opisthrchis noverca

3 g faeces + 50 ml water

gauzeFill with water

Wait 10 min Remove supernatant

Wait 10 min Resuspend in waterRemove supernatant

Add 1 drop of dye + mix

Add drop to slide + cover glass + read

Nematodirus spp.

Sedimentation („Trematode“ eggs)Method for heavier eggs that do not flotate in common flotation fluids

Baermann(lungworms & L3)

5-10 g faeces

Fill with water

Centrifuge1000 rpm, 2 min

Remove excess water

Baermann (lungworms & L3 larvae)Harvesting of larvae from faecal sample

water

Wait 24 hours

(no direct sun)

Fold gauze and put in

syringe

Remove faeces and

excess waterTransfer to 14 ml

centrifuge tube

Transfer to petri dish and

examine under

microscope

Baermann (lungworms)Harvesting of larvae from faecal sample

Dictyocaulus viviparus (390-450 µm)

Dictyocaulus filaria (550-585 µm)Larvae often curvedSluggish movement

Muellerius capillaris (300-320 µm)Posterior end of body often curled

Protostrongylus (320-400 µm)Posterior half of body may be curled

Cystocaulus (340-480 µm)Additional dorsal and ventral spines on middle part of tail

SheepCattle

Baermann (L3 larvae)Harvesting of larvae from faecal sample

Trichostrongylus (560-796 µm) 16 gut cells, Head tapered, Sheath forms short cone

http://www.rvc.ac.uk/review/parasitology/RuminantL3/Introduction.htm

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 BovineOstertagia

Strongyloides (650-850 µm)Oesophagus extends nearly half length of body, Sheath absent, Larval tail notched

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

Ziehl-Neelsen [acid fast staining]

(Cryptosporidium spp.)

Zeihl-Neelsen (Cryptosporidium spp.)Detection of C. spp. oocysts in faeces

Add small amount of feaces to slide

Add name with pencil

Smear feaces using second slide

Dry

Dry

Fixatemethanol (+2% HCl)

5 min

Color carbolic fuxine

20 min

Remove color10% H2SO4

10 sec

WashWater10 sec

Repeat 2 times

ColorBrilliant Green 2.5%

5 min

WashWater10 sec

Read with x400 magnification

0 = no oocysts+ = 1-5 oocysts

++ = 6-25 oocysts+++ = >25 oocysts

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

Species Size (µm) Hosts

Cryptosporidium parvum 5.0 x 4.5 Mammals, primarily cattle and humans

Cryptosporidium hominis 5.0 x 4.5 Humans, monkeys, also other animals

Cryptosporidium andersoni 7.4 x 5.5 Cattle and other ruminants

Cryptosporidium canis 5.0 x 4.7 Dogs, also other animals

Cryptosporidium felis 4.6 x 4.0 Cats, also other animals

Cryptosporidium suis 4.6 x 4.2 Pigs, also found in humans

Cryptosporidium baileyi 6.3 x 5.0 Birds

Cryptosporidium meleagridis 4.5 x 4.0 Birds

Cryptosporidium serpentis 6.2 x 5.3 Reptiles

http://courses.cit.cornell.edu/biomi290/microscopycases/crypto/index.htm