Parazitologie LP

Methods in Parasitology
1. Preparing thin and thick

blood films with capillary or
venous blood
Swiss Tropical Institute, Basel
April 2005
1.0 Preparing thin and thick blood films
Contents
1.1 Preparatory steps
1.4 Using finger-prick blood
1.8 Preparing thin films
1.13 Preparing thick films
1.23 Using venous blood
Production Team:
Yvette Endriss, Elisabeth Escher & Birgit
Rohr Prof. Hanspeter Rohr (NeoCortex
Foundation) Prof. Niklaus Weiss (STI)

Materials for
finger pricks:
Disinfectant Swabs
Microscope slides
(cleaned with alcohol)
Sterile lancets
Special slide
as spreader
Gloves
(Pay attention to general precautions
when handling blood!)
Prepare
the microscope
slides
Use microscope
slides with or without
frosted end
Finger-prick
(capillary blood)
With the patient's left
hand, palm upwards, select
the third finger. (The big
toe can be used with
children. The thumb should
never be used for adults or
children). Use cotton wool
lightly soaked in alcohol to
clean the finger, using firm
strokes to remove grease
from the ball of the finger. Let
finger air-dry.
Finger-prick
(capillary blood)
With a sterile
lancet puncture the ball of
the finger using a quick
rolling
action.
Clip
Finger-prick
(capillary blood)
By applying
gentle pressure to the
finger express the first
drop of blood and wipe it
away with dry cotton
wool. Make sure no
strands of cotton remain
on the finger.
Finger-prick
(capillary blood)
Working quickly
with capillary blood
and handling clean slides
only by the edges, collect
the blood as follows: Apply
gentle pressure to the finger
and collect a single small
drop of blood about the size
on the end of the slide.
This is for thin film.
Thin
films (capillary
blood)
Using another clean slide as a
"spreader", and with the slide
with the blood drops resting
on a flat, firm surface, touch
the small drop with the
spreader (1) and allow the
blood to run along its edge.
Firmly push the spreader
along the slide (2), away
from the drops, keeping the
spreader at an angle of 45.
Make sure the spreader is in
even contact with the surface
of the slide. (Look at clip on
next slide)
Left
hand
Parasites
Thin films
(capillary blood)
Clip
This is correct!
Observe right angle!
Angle too flat
> film too long
Angle too
steep
> film too short
Mistakes:
Pressure on
spreader too strong
> waves"
Air bubble >
Uneven film
Thick films
(Capillary blood)
Apply gentle pressure to
the finger and collect two
larger drops, about a size ,
on the slide as shown in
the upper picture.
Handle the spreader" by
the edge, using the corner
to spread the blood in
a circular form with 3-
6 movements.
Thick films
Thick films
Check for the
right thickness:
You should be able
to read the
newspaper!
Thick films
Additional mistakes
Thick film too
thick
Thick film should
be round!
Labelling
With frosted
end use a pencil
Without frosted
end use diamond
pencil
Labelling
Faulty handling:
Do not use a ball
pen
Do not use a paper
label
Drying
Allow the thin
film and the thick
film to dry in a flat
level position
protected from flies,
dust and extreme
heat.
Drying
Optional:
Use special device
to dry thick or thin films
For example:
Small incubator 37C
Heating plate (not over
37c)
Drying
Do not use a hair
dryer or a fan!
(Collects the dust)
These slides look o.k.!
These slides look more
like art work
than useful blood
slides!
Venous blood can
be used instead of
capillary blood
Use vacutainers
with anticoagulant
(EDTA)
For preparing thin and
thick films use a glass
capillary to drop the
ETDA-blood.
Do not use a
plastic pipette!
Clip
Spare glass slides
Combination of a thin
and a thick film on the
same slide.
Spare glass slides
Combination of
a thin and a thick
film on the same
slide.
2. Staining blood films with Giemsa
stain
April 2005
2.0 Staining blood films with Giemsa
Contents
2.1 Materials
2.4 Fixation of thin films
2.5 Preparing Giemsa stain
2.10 Staining blood films
Production Team:
For perfect malaria staining, thin and thick
films should be made on separate slides. The pH of
the buffer solution should be 7.2 .
Thin films will be fixed with Methanol.
Thick films should not be fixed (to
allow haemolysis)!
Therefore avoid exposure of the thick films
to methanol vapour when thin and thick films are
on the same slide.
Materials:

Methanol (or
Ethanol) Staining
dishes Buffer
solution Giemsa
solution
(Giemsa powder can
also be used instead of
Giemsa solution)
Fixation for thin films only!
Fixation time for thin
films with methanol:
15 - 30 seconds
Important note: Fixation
time for ethanol is 20
minutes!
For perfect malaria staining
the pH should be
correct! (pH 7.2)
Commercially
available buffer tablets can
also be used.
Prepare your buffer by
using tablets of Soerensen (see
next slide)
Combine two stock solutions in
a certain proportion
KH
2
PO
4
and
Na
2
HPO
4
-2 H
2
O
Control the pH before use!
Stock solutions:
A: KH2PO4 (Merck Art. 4873) 9.08 g/l = 1/15 molar
B: Na2HPO4 2 H2O (Merck Art. 6580) 11,87 g/l = 1/15 molar (or
Na2HPO4 anhydricum (Merck Art. 6586) 9.47 g/l = 1/15 molar)
Buffer by Soerensen
Parasite
pH
20C
ml solution A ml solution B
6,4 73.3 26.7
6,5 68.2 31.8
6,6 62.5 37.5
6,7 56.5 43.5
Borrelia 6,8 50.4 49.6
6,9 44.6 55.4
Normal blood/Leishmania 7,0 38.9 61.1
7,1 33.4 66.6
Malaria / Babesia 7,2 28.0 72.0
7,3 23.2 76.8
7,4 19.2 80.8
Trypanosoma 7,45 17.5 82.5
Trichomonas 7,5 15.9 84.1
Prepare staining solution:
Take 94 ml buffer
solution pH 7.2
Then add
6 ml Giemsa
stock solution
Mix 94 ml buffer solution ph
7,2 with
6 ml Giemsa stock
solution >>
6% Giemsa solution
2.10 Staining blood films
Fill staining dish
with staining solution
Place thin film and
thick films into the
staining dish.
Stain blood slides for 45
minutes
(staining a large number of
thin films and thick films use
two separate staining dishes)
Thin films:
Wash the slides with
tap water
Caution:
Thick films need
careful rinsing!
(since they are not
fixed before staining)
Mistake:
Thick films are not rinsed
properly! Blood is lost!
Drying
For example:
Let air dry
Use small incubator
37C
Use heating plate (not
above
37c)
(do not use
excessive heat!)
3. Staining blood films with Field's stain
a) Thin films
b) Thick films (according to WHO)
April 2005
3.0 Field's stain for thin films
Contents
3.1 Fixing thin films
3.4 Staining thin films
3.9 Staining thick films
Production Team:
Rohr Prof. Hanspeter Rohr (Neocortex
A: Thin films
Materials:

Methanol
(absolute) Field's
stain A und B
Tube with
water Staining
dishes Filter paper
Fixation:
Fix thin film
with Methanol for 1
min.
Dry microscopic
slide on filter paper
Immerse slide in
Field's stain B (Eosin) for
5 seconds
Immediately wash
with tap water!
Immerse slide
in Field's stain
A (Methylene blue)
for 10 seconds
Immediately wash
with tap water
Dry thin films
3.9 Field's stain for thick films
B: Thick films
Materials:
Methanol No!
Field's stain A
und B
Tube with water
Filter paper
Immerse thick film in
Field's stain A (Methylene
blue) for 3 seconds
Do not forget: Thick films
need to be haemolysed
and are therefore not
fixed with methanol!
Rinse immediately
in tap water
Immerse thick film
in Field's stain B
(Eosin) for 3 seconds
Then rinse
immediately with tap
water
Let the
slide carefull
y dry
4. Concentration Method
for Microfilariae with
Formaldehyde
Solution
(+ Delafield's haematoxylin stain)
Swiss Tropical Institute, Basel April 2005
4.0 Concentration Method for Microfilariae
Contents
4.1 Materials
4.2 Concentrating blood
microfilariae
4.11 Staining microfilariae
Production Team:
Yvette Endriss, Elisabeth Escher & Birgit Rohr Prof.
Hanspeter Rohr (Neocortex Foundation) Prof. Niklaus Weiss
(STI)
Materials:
Centrifugation
tubes
2% Formalin
solution
Centrifuge
Microscopic
slides
Cover slips
Methylene blue
Pipettes
Gloves
Fill 2 centrifugation
tubes with 1 ml of anti-
coagulated blood
Add 9 ml of a
2% Formalin solution
to each tube
(final dilution: 0.2%)
Wait 5 minutes for
the red cells to
haemolyse
Mistake:
Blood
coagulated (left
tube)
Correct:
Complete
haemolysis (right
tube)
Centrifugation at 2000
rpm for 3 minutes
After centrifugation:
Sediment is visible
Decant the supernatant
Add a drop
of Methylene-blue to
one sediment and
mix carefully.
(for better visibility of
the microfilariae)
Prepare stained
sediment for
microscopy. Search and
count all microfilariae in
the whole sediment under a
cover slip (Objective 10x)
4.11 Haematoxylin stain for
If microfilariae are found:
Prepare thick film from the
second
sediment (containing no
Methylene- blue) for
identification by Delafield's
haematoxylin staining
4.12 Haematoxylin stain for Microfilariae
Carefully dry the
thick film
Fix the thick smear
with Methanol
Dry the thick smear
Stain the thick film with
filtered
Delafield's haematoxylin
for 30 minutes
Intensify the blue colour
by immersing the slide
in tap water for 10
minutes and let the
thick smear
dry afterwards.
5. Fresh Stool
Examination (Wet mount)
April 2005
5.0 Fresh Stool Examination
Contents
5.1 Materials
5.2 Preparing wet mount
5.7 Examining wet mount
Production Team:
Materials:
Microscope
slides
Cover slips
Sodium
chloride
solution in
small bottle with
pipette
Wooden stick
Fresh stool
Gloves
Use cleaned
microscope slides
Place a drop of saline
Take a small amount of
stool with a wooden
stick
Mistake:
Too much stool!
Place
coverslip Avoid air
bubbles!
Examination of helminth
ova :
Use 10x objective
Photo:
Schistosoma mansoni
Examination of amoebae:
Press cover slip
slightly, remove excess
liquid with paper towel
Clip
For amoebae:
Use 50x objective
with oil immersion
Example:
Entamoeba coli, cyst
6. Sedimentation und SAF*-
Ether Concentration
(*SAF: Sodium acetate-acetic acid-formalin
solution)
April 2005
6.0 Sedimentation & SAF-Ether Concentration
Contents
6.1 Materials
6.2 Sedimentation of stool
sample
6.8 SAF-Ether concentration
Production Team:
6.1 Sedimentation
Materials:

NaCI 0.9%
SAF-solution
Ether
Centrifugation tubes Sieve
Funnel/gauze Rubber stopper
Pasteur pipette with rubber
bulbs Wooden spatula Beaker
(50ml)
Conical glass / plastic
bottles Wire mesh
Microscope slides, cover
slips Gloves
6.2 Sedimentation
Carefully mix fresh
stool with saline in a
glass beaker
6.3 Sedimentation
Filter stool
suspension through a
sieve with two layers of
gauze into a conical glass
6.4 Sedimentation
Leave 1 hour
for sedimentation
6.5 Sedimentation
Carefully pour off
the supernatant fluid
until the sediment is
visible
6.6 Sedimentation
Take up part of
the sediment using
a Pasteur
pipette. Leave to stand
for 2 - 3 minutes (Mini-
sedimentation in the
pipette)
6.7 Sedimentation
Prepare 3 slides
for microscopy
Search for
helminth eggs and
larvae
6.8 SAF-Ether Concentration
Pour the rest of
the sediment into a
conical centrifugation
tube
Centrifuge for S
min at 2000 rpm
Decant supernatant
Fill the tube with
7 ml of SAF solution and 3
ml ether
(Caution: inflammable!)
Mix with wooden stick
Centrifuge for
S minutes at 2000 rpm.
After
centrifugation: 4
layers can
be observed
ETHER
DETRITUS
SALINE
SEDIMENT
Remove all
layers except the
sediment using a
pipette on a pump
(Alternatively:
loosen the detritus with
a wooden stick
and decant)
Mix sediment and place a
drop on a
microscope slide. Add
cover slip
Examine the
whole sediment
7. SAF* method for stool specimen
(*SAF: Sodium acetate-acetic acid-formalin solution)
April 2005
7.0 SAF method for stool specimen
Contents
7.1 Materials
7.2 SAF
method
Production Team:
Materials:
SAF-, NaCl-solution
Ether
Centrifuge tubes
Centrifuge stand
Funnel
Gauze
Spatula
Rubber stoppers
Pipettes
Microscope slides,
cover slips
Place stool
sample (approx. 1g, size
of a hazelnut) into a
tube containing 10 ml of
SAF
Mix stool thoroughly
Agitate tube vigorously
Filter stool solution using a
funnel with gauze
Centrifuge for 1
minute at 2000 rpm
Remove supernatant with a
pipette
or
Decant the supernatant!
Add 7ml
saline
Mix with a wooden stick
Close tubes with
rubber stoppers, shake
well
keeping the thumb on the
stopper
Remove rubber
stopper carefully
(pressure!) and centrifuge
tubes for 5 minute at
2000 rpm.
After centrifugation: 4
layers are detectable
ETHER
DETRITUS
SALINE
SEDIMENT
Remove 3
layers (Ether/Detritus/NaCl)
with a pipette
or
loosen the detritus with a
wooden stick and decant
Sediment should be less than
1 ml (left side)
(if more (right side):
repeat the concentration
with saline/ether)
Mix the sediment
and place a drop on
a microscope slide
Add cover slip
Slide ready for
microscopy: Search first for
helminth eggs (10x
objective).
Then, for protozoa,
press cover slip slightly,
remove excess liquid with
paper towel and use 50x
objective with oil immersion
8. KATO-Katz technique for helminth eggs
April 2005
8.0 KATO-Katz technique for helminth eggs
Contents
8.1
Materials
8.2
Procedure
Production Team:
Materials:

Kato-set (Template with
hole, screen, nylon or plastic,
plastic spatula)
Newspaper or glazed
tile Microscope
slides Cellophane as cover
slip,
soaked in Glycerol-
malachite green solution
Fresh stool Gloves
Prepare the layer
Glazed tile or newspaper
Place the template
with hole in the centre of
a microscope slide
Use gloves !
Place a small amount
of faecal material on
the newspaper or the
glazed tile.
Press the screen on top
so that some of the
faeces filters through and
scrape with the flat spatula
across the upper surface to
collect the filtered faeces.
Add the collected faeces
in the hole of the template
so that it is completely
filled.
Remove the
template carefully so that the
cylinder of faeces is left on
the slide.
Cover the faecal material
with the pre-soaked
cellophane strip.
Invert the microscope slide and
firmly press the faecal sample
against the cellophane strip on a
smooth hard surface such as a
tile. The material will be
spread evenly.
Carefully remove the slide
by gently sliding it sideways
to avoid separating the
cellophane strip. Place the slide
with the cellophane upwards.
The smear should be examined in a
systematic manner and the eggs of each species
reported. Later multiply by the appropriate number
(see inlet-information of the Kato-set) to give
the number of the eggs per gram faeces.
9. Adhesive tape method for the detection
of pinworm eggs
April 2005
9.0 Adhesive tape method
Contents
9.1
Materials
9.4
Procedure
Production Team:
Materials:
Clear adhesive
tape
Microscope slides
Gloves
(Xylene or Butyl
acetate)
Mistake:
Adhesive tape is
not clear!
Use gloves
! (pinworm eggs
are infectious!)
Prepare the tape
Take sample on peri-
anal skin in the morning
Stick tape on
microscope slide
4-6 negative tapes
are needed to rule out
a pinworm infection!
Clip
Picture under microscope: ^
r
Tape without Butyl
acetate /Xylene produces
many air bubbles!
Place a drop of
Xylene on the edge of
the tape to remove
air bubbles
Picture under mikroscope:
0
Without air bubbles

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Methods in Parasitology

1. Preparing thin and thick

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