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Fixing  and Staining Amoebae

Page updates 2/3/03

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.

 

References:-

Coleman A.W., Maguire, M.J., Coleman, J.R. (1981). Mithramycin

Nissenbaum, G. (1953). A combined method for the rapid fixation and adhesion of ciliates and flagellates. Science 118; 31-32.

Nissenbaum, G. (2001) Behind the discovery of "Nissenbaum's" fixatives. J. Euk.Microbiol. 48, 3-9.

Page, F.C. (1988) A new key to freshwater and soil gymnamoebae. FBA NERC. P26.

Page, F.C. (1983). Marine gymnamoebae. NERC. P9.

Pirehma, H., Suresh, K., Sivanandam, S., Khairul Anuar, A., Ramakrishman, K. and Suresh Kumar, G. (1999). Field’s stain – a rapid staining method for Acanthmaoeba spp. Parasitol.Res. 85; 791-793.

 
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