Estonian University of Life Sciences
Institute of Veterinary Medicine
and Animal Sciences
Infectious Diseases
Responsible: Brian Lassen
Standard Operation Manual
Document nr:
Author: Brian Lassen
Version nr.: 1.1
Kinnitas:
Date: 10. veebr. 2012
Page. nr. 1 (7)
Quantitative flotation method using conentration flotation and chambers constructed of
microscopic slides or McMaster
1. GENERAL
The method is applied for quantitative egg counts retrieved from faecal samples using a chamber
constructed by microscopic slides. The advantages are: A) inexpensive (0.13 EUR/chamber
compared to 47 EUR for a McMaster chamber); B) higher clarity (0.1 mm cover slide compared to
thicker cover in McMaster chamber), C) allow higher magnification (McMaster chambers can not
always use x40). D) Allow more flexibility in volume exmined and more accurate calculations of
OPG/EPG. Disadvantages: breaks after around 10-20 uses and requires calculations
Protocol adapted from:
Modified from FAO Animal Health Manual 3, “Epidemiology, diagnosis and control of helminth
parasites of swine” p. 51-56
1.1. PERSONS RESPONSIBLE
Prepare work instructions Brian Lassen. Confirms the work instruction ................. Work
instructions and methodologies to be responsible for updating the file .......................
Place the code ..................... Location Guide confirms .....................
Place to monitor the timeliness of the Code ..................
1.2. STORAGE AND DISTRIBUTION
After washing, stored to dry. Must not be kept in liquid for long as it dissolves glue holding it
together.
2. Structure and content of work instructions
a) Collection of feacal sample
b) Preparation of faecal sample
c) Reading of sample
2.1. USAGE / PURPOSE OF THE INVESTIGATION
Quantitatively give information on the parasite burden of an animal based on eggs/oocysts shed in
faeces.
2.2. PRINCIPLE OF THE METHOD
A weighed sub-sample of faeces is dissolved in water, filtered, spun down, pellet resuspended in
flotation liquid, a subsample of solvent added to a reading chamber, and eggs counted with a
microscope when they have floated to the cover glass.
2.3. EQUIPMENT AND SUPPLIES
◦ Plastic bags/gloves for collection of individual faecal samples
◦ Water proof marker
 Plastic cup (supermarket) or clay crucible
 Plastic spoon (supermarket) or toughe spartel (Vet. Aptek or Kruuse-Dimela)












Dispenser with tap water or 100 ml measuring beaker with 1 ml intervals
Gauze (Vet. Aptek or Dimela)
14 ml centrifuge tubes
Centrifuge (MPW-350e)
Plastic pasteur pipettes (500 pc, Roth-Akrom-Ex, EA65.1)
Counting chamber (in house)
Microsopic splides, 76x26x1 mm (50 pc, Quantum)
Cover slides 50x24x0.1 mm (100 pc, Quantum)
Super glue
Flotation liquid (in house)
Click counter (Hounisen, 20863039)
Microscope (Zeizz, Axiostar plus)
2.3.1. Equipment calibration procedures
Construction of chamber (Henriksen and Korshom, 1984).
A) 1 x microscopic slide (76x26x1 mm)
B) 2 x pieces of microscopic slipe (26x26 mm cut from normal slide with a glass cutter)
Glue to attach B on A.
Cover slide (disposed after use)
Calibration of flotation liquid density
Weigh 1000 µl tap water in a glass 10 times [m:water]. Record weight.
Weigh 1000 µl flotation liquid in a glass 10 times [m:flotation]. Record weight.
Density (ρ) = [m, flotation] / [m, water] (ρ = g/cm3)
2.4. CULTURE MEDIA, CHEMICALS, REAGENTS
Sugar-salt flotation liquid (in house)
Description: flotation liquid: density 1.24
Warnings: corrosive to microscope – clean spills!
Storage: room temperature in plastic bottle
Making of batch: see 2.9
2.5. PROCEDURE
2.5.1. Pretreatment of samples
Samples should be taken from rectum of animal to avoid contamination of other animals faecal
samples or earth nematodes if taken on pasture. If not possible, then take from a freshly shed
sample, but only the top of the sample (<1 hour).
Put sample in a plastic bag and mark it with: farm name, age of animal, earmark/name, date, faecal
consistency.
Scoring of faecal consistency (should be scored at the farm since the consistency may change in the
bag over time):
Binary scale: diarrhoeic (definition: runs out of bag at room temperature)/non-diarrhoeic
Five scale: 0 = hard and dry, 1= normal, 2= soft, 3= thin, 4= watery, 5= watery and bloody
All equipment should be thoroughly washed before use, and poured with hot water after washing
(or autoclaving).
Before taking sub-sample to be examined, mix sample in the bag by gently squeezing the material
around to homegenize the material and thus eggs in the sample.
2.5.2. The reagents and testing of
It is possible to include a negative if suspecting cleaning procedure of the method is faulty (water in
plastic cup – no faeces).
2.5.3. PROCEDURE
[faecal samples may contain zoonotic parasites (eg. Giardia, Cryptosporidium, Toxocara canis etc.).
1. Thus, take care to avoid consumig faecal matters: wear gloves and wash hands frequently.]
2. Mark a plastic cup with the sample number
3. Weigh 4 grams of faeces with a plastic spoon into the plastic cup
4. Add 56 ml tap water (14 ml per gram feaces = 14 ml water + 1 ml volume per gram feaces;
so 15 ml = 1 gram faeces) using the dispenser (avoid air bubbles in the dispenser) and mix.
5. Leave sample for half an hour.
6. Mix sample and filter suspension through a single layer of into a 14 ml centrifuge tube up to
the 10 ml mark (= 10/15 = 2/3 g faeces).
7. Centrifuge sample 7 min. at 234 rcf (1200 rpm)
8. Remove the supernatant by tilting the centrifuge tube at a 45 degree angle and carefully
removed supernantant with a pasteur pipette sucking from the surface to the bottom.
9. [at this point samples can be stored up to a week in a refridigator, but should be read as early
as possible]
10. Resuspended the pellet with saturated sugar and salt solution (ρ = 1.26 g/cm3) up to the 4 ml
mark 5 min prior examination (= 3 ml flotation liquid + 1 ml pellet).
11. Fill a counting chamber with mixed sample (total reading volume [Vr]: 24 mm x 24 mm x 1
mm = 576 mm3 = 0.576 ml).
12. Read 3 vertical rows (alternatively entire chamber) of the chamber at x200 magnification
and count eggs/oocysts [COUNT] using a click counter. See APPENDIX for creating
multiplication tables. If method is performed as described above the multiplication factor is
calculated: [MF] = 6 / [Vr]
2.6. RESULTS
2.6.1. Calculation of results: calculation formulas and explanations of the symbols
Calculation of multiplication factor, altered quantitative flotation:
Multiplication factor [MF] is described in APPENDIX and tables.
2.6.2. Expression of results and interpretation
Results should be related to age of the animal to determine its relevance.
It should be taken into account that a low count may indicate either: maximum, an increasing, or
decreasing amount of eggs.
The classical methods assume 1 g faeces = 1 ml. The actual volume may vary depending on
contents of the faecal matter. This creates an uncertanty of the final count (not studied).
It has to be taken into account that the mass of faeces with different consistency (diarrhoeic or
normal) contain different amounts of water and create a considerable underrepensentation of
diarrhoic EPG/OPG counts (less faecal material per gram). This can be accounted for by drying a
subsample to determining dry matter content (2 days in 70 degrees Celsius in an incubator).
2.6.3. Reliability:
Specificity – depends on egg type
Sensitivity: 20 eggs per gram faeces (calculated for 2 chambers of 0.30 ml of a McMaster chamber,
otherwise depends on volume read in home made chamber).
2.6.4. Workability characteristics
2.7. Differences to this methodology
Some methods of use 1-5 grams of sample.
2.8. ToR documents or document and Literature
Link: Original McMaster method; guided presentation:
http://www.rvc.ac.uk/review/parasitology/EggCount/Purpose.htm
1. Henriksen S., Korsholm H., 1984. Parasitologisk undersøgelse af fæces-prøver.
Konstruktion og anvendelse af et enkelt opbygget tællekammer. Dansk Vet. Tidskr. 67,
1193-1196.
2. Modified from FAO Animal Health Manual 3, “Epidemiology, diagnosis and control of
helminth parasites of swine” p. 51-56
2.9. SOLUTIONS FOR PREPARATION INSTRUCTIONS
Sugar-salt flotation liquid
1. Dissolve 500 g sugar per liter of tap water under constant heating and stirring (DO NOT
BURN SUGAR – will give brown liquid)
2. Add salt until saturated (continue heating)
3. Cool down and bottle.
4. Write down: name of maker of solvent, batch date, density, what kind of flotation liquid on
bottle.
APPENDIX: Volume calculations
APPENDIX: Multiplication factor calculations
APPENDIX: MULTIPLICATION FACTORS AND CALCULATIONS
Objective area in mm =
Visual index in mm
Magnification of objective x Tubus factor
Visual index: 18 mm (Zeiss) and 22 (Nikon H550L), Tubus factor: 1
Objective area:
Magnification of objective
Microscope
Ceti Topic T w. phase
contrast (mm)
Objective area (mm3)
V = r2 x ¶ x 1 mm
Volume of objective
area (ml)
x4
x10
x20
x40
x100
4.5
5.0
5.5
15.90
19.64
23.76
1.59 x 10-2
1.97 x 10-2
2.38 x 10-2
1.8
2.0
2.2
2.55
3.14
3.80
2.55 x 10-3
3.14 x 10-3
3.80 x 10-3
0.9
1.0
1.1
0.64
0.79
0.95
6.36 x 10-4
7.85 x 10-4
9.50 x 10-4
0.45
0.50
0.55
0.16
0.20
0.24
1.59 x 10-4
1.96 x 10-4
2.38 x 10-4
0.18
0.20
0.22
0.03
0.03
0.04
2.55 x 10-5
3.14 x 10-5
3.80 x 10-5
Visual
index
18 (Zeiss)
20
22 (Nikon)
18 (Zeiss)
20
22 (Nikon)
18 (Zeiss)
20
22 (Nikon)
Multiplication Factors for Henriksen and Korsholm chamber
Concentration Technique (Visual index: 18, 20, and 22)
Magnification
Visual
Index
Volume
pellet is
diluted
in (ml)
1 visual
area
3 visual
areas
1 column
3 columns
Total chamber
x4
18
20
22
18
20
22
18
20
22
18
20
22
18
20
22
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
377
306
253
2358
1910
1578
9431
7639
6316
37726
30558
25254
235785
190986
157840
126
102
84
786
637
526
3144
2547
2105
12575
10186
8418
78595
63662
52613
56
50
46
139
125
114
278
250
227
556
500
455
1389
1250
1136
19
17
15
46
42
38
93
83
76
185
167
152
463
417
379
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
x10
x20
x40
x100
Eye ocular converter to micrometer
Zeiss Axiostar Plus
Magnification
Micrometer bars
(1/100mm)
1000 (x100)
18
400 (x40)
45
200 (x20)
90
100 (x10)
180
50 (x5)
356
40 (x4)
Objective area
(mm)
0.1800
0.4500
0.9000
1.8000
3.6000
10 ocular bars
(1/100 mm)
1
2.5
5
10
20
1 ocular bar
(mm)
0.0010
0.0025
0.0050
0.0100
0.0200
1 ocular bar
(micrometer)
1.0
2.5
5.0
10.0
20.0