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Amoebae
are removed from the agar plate by glass rod and concentrated by
bench-top MSE centrifuge at 3 Krpm for 10 minutes.
The cell pellet is then washed with Neff’s, 75% seawater, or
other solution depending on the amoeba.
Amoeba are then allowed to settle on coverslips in a moist
chamber (usually, a petri dish with damp tissue paper), until they adopt
a normal locomotionary morphology.
Some amoeba require the coverslips to be NaOH treated before.
This treatment is a 1% NaOH bath heated to 80oC for
10minutes followed by several changes of distilled water.
Nissenbaum’s Fixative.
(Nissenbaum, 1953). Must
be prepared fresh.
Working
Concentration
1.9mls
HgCl2
saturated aqueous solution
1.0mls
Glacial
acetic acid
200µls
Formalin
(38-40% formaldehyde)
200µls
Tertiary
butyl alcohol (tert-butanol)
500µls
1).
Allow amoeba to settle on the coverslip (as above) and pipette on
Nissenbaum’s.
2).
Leave 5minutes or so. Wash with acidified HgCl2 (10mls
saturated HgCl2 ,0.5mls glacial acetic acid) for 5-10
minutes.
3).
Depending on the staining step, either wash in 50%, 35%, 15% ethanol,
and distilled H20, about 5 mins each, or leave in 50%
ethanol.
Lugol’s Iodine.
Iodine/Potassium iodide solution (Sigma L-6146).
For fixing and counting flagellates.
(Potassium iodide (KI) 6g, iodine (I2) 4g in 100mls
distilled H20). Add a drop to the culture and view.
Formalin Seawater.
Promising results were obtained with SLB with the following
simple fixative.
Working
Concentration
1.2mls
75% seawater
1mls
Formalin
(38-40% formaldehyde)
200µls
1).
Allow amoeba to settle on the coverslip and pipette on fixative
2).
Leave for 10minutes (possibly longer).
3).
Washed in 75% seawater.
Carnoy’s Fixative.
Used (Enzien et
al 1989)
to fix Paratetramitus jugosus
prior to staining nuclei with mithramycin
Working
Concentration
0.80mls
70%
ethanol 600µls
Glacial
acetic acid
200µls
1).
Allow amoeba to settle on the coverslip and pipette on fixative
2).
Leave for 10minutes, wash in 70% ethanol.
Staining
of Amoebae
Dye
Stains
Heidenhain’s iron
Haematoxylin
A method for staining mitotic figures in amoebae, after fixation
in Nissenbaum’s or Seawater formalin (Page, 1988). (method successfully checked with H. clara ).
1).Incubate
1-2 hours in 2% ammonium ferric sulphate (NH4Fe(SO4)2.12H2O).
2).Rinse
first in distilled H20 and then tap water.
3).Incubate
1-2 hours in 0.5% haematoxylin (Sigma H-9627) in 2% ammonium ferric
sulphate.
4).Wash
in tap water and mount
Kernechtrot (Nuclear Red)
A method
for staining mitotic figures in amoebae, after fixation in
Nissenbaum’s. (Page,
1988).
(method successfully checked with SLB, H.
clara and Acanthamoeba sp.).
This stain seems to fade quite quickly.
1).
0.5g Aluminium sulphate in 10mls H2O, warm in heating bath
set at 80oC.
2).
Add 10mgs Kernechtrot (Nuclear Fast Red, 60700
Fluka) and dissolve in the warm Aluminium sulphate solution.
3).
Filter the solution and cool to room temperature.
This should last about a month.
4).
For staining, fix the cells on a coverslip with Nissenbaum’s as above.
Remove the DDH2O that the cells should now be in and
add the Kernechtrot solution.
5).
Incubate for about 15 minutes (this may have to be varied), wash 2x in
water and mount.
Modified Field’s Stain.
Originally
used to stain malarial parasites in thick blood films, this method has
been modified by (Pirehma
et al, 1999)
to stain Acanthamoeba.
Klein’s Silver relief
stain. This
method shows surface features in relief and seems to be useful for
amoebal cysts.
1).
Wash the cysts free of bacteria 2x as the bacteria stain heavily and
obscure the field.
2).
Fix cysts in Nissenbaum’s or in ethanol: glacial acetic acid (9:1).
3).
Wash in water.
4).
Add 2% silver nitrate soln. Leave 20minutes.
5).
Wash well with water
6).
30minutes to several hours on UV light box
7).
Air dry, mount and view.
Fluorescent
Stains
Mithramycin.
High background staining has been reported with DAPI stained
protists (Coleman,
1978) which turns
out to be due to polyphosphates. Better results were obtained with mithramycin for Paratetramitus
jugosus (Enzien et al, 1989). N.B.
Mithramycin is very toxic. (and expensive).
1).The
amoeba are first fixed in Nissenbaum’s, Formalin Seawater or
Carnoy’s, as above and rehydrated.
2).
The amoeba are RNAse treatment for 30 minutes to decrease background. To
the approximately 200µl of water on the coverslips, add 10µl of
10mg/ml RNAse (in 0.1M NaAc pH 5.0. boil 15-20').
3).
Mithramycin (Sigma M-6891) at 50µg/ml in 20mM phosphate buffer pH7.0,
10mM MgSO4 is added to the coverslips and incubated for 1-2
hours.
4).
Coverslips were then rinsed in water and mounted on slides.
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