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Silver nitrate impregnation of ciliated protozoa
Arch. Protistenkd. 135 (1988) : 299-3 18 VEB Gustav Fischer Verlag l ena
Department of Zoology, British Museum (Natural History), London, England
Silver Nitrate Impregnation of Ciliated Protozoa By D. McL. ROBERTS and H. CAUSTON With 12 Figures Key words : Infraciliature ; CHATTON-LwOFF; Silver stain
Summary A modified silver nitrate impregnation procedure is described, based on the Chatton-Lwoff method. The major differences are ( I) the post-fixation in da Fane 's fixative is reduced to a single wash and (2) that the variability between preparations has been reduced by mixing da Fano' s fixative with the gelatine mountant prior to mixing with the cells. The total time required for the new protocol is 2 h from live culture to mounted slides.
Introduction Silver staining has been in use since 1843 (JONES 1973) and in that time has well earned its reputation for being capricious. PETERS (1958) divided the methods into two categories ; (1) those using simple silver salts and (2) those using protargol (a soluble protein/silver complex) (DAVENPORT et al. 1952). Both methods work in the same way but differ markedly in their sensitivities and the range of material stained: Fundamentally, the initial exposure of a cell to the silver solution results in the formation of either (1) minute metallic silver nuclei of a few silver atoms or (2) bound ionic silver which is reducible to metallic silver during development, at sites where cellular components have a high redox potential with silver ions (SAMUEL 1953b). Subsequent reduction in an alkaline developer, or by exposure to light, causes the deposition of much additional silver on these nuclei so that, as in photography, a latent image becomes a visual image. Carbonyl and thiol groups are thought to be important in the formation of silver nuclei (JONES 1973). Silver impregnation of protozoa was introduced in order to expose the location of pellicular components, particularly kinetosomal bases, for the purpose of taxonomic classification. Ciliated protozoa were stained by KLEIN (1926) using the "dry" method, so called because the cells were fixed by air drying onto a slide. Cells were then impregnated with silver nitrate and reducedby light. The main disadvantages of the methods were ( I) morphological distortion, introduced by air drying (for a modification, see FOISSN ER 1967), (2) the fact that marine cells could not be air dried (for a modification, see FOISSNER 1976a) and (3) that the surface of the cell adhering to the glass was not stained. CHATTON and LWOFF (1930, 1935, 1936) introduced chemical fixation followed by mounting in a gelatine film to overcome these difficulties and although the same structures were impregnated, the protocol became more protracted (for a modification, see FRANKEL and HECKMANN 1968; NELSEN and DEBAULT 1978). The silver carbonate method, introduced for ciliated protozoa by FERNANDEz-GALIANO (1966), used a formalin fixation followed by impregnation and development in the liquid phase. Impregnation revealed kinetodesmata with particular clarity, kinetosomes, cilia, nucleus and occasionally other pellicular organelles (e.g. trichocysts). The infraciliature revealed with the silver nitrate methods was not stained (FERNANDEz-GALIANO 1966, 1976; FERNANDEZ-GALIANO and RUIZ 1972; MADRAZO-GARIBAYand LOPEZ-OCHOTERENA 1973; AUGUSTIN et al. 1984). During
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the FERNANDEZ-GALIANO method the cellshaveto be removed and washed froma final volume of 45 ml, which is particularly difficult for small cells or sparse cultures although AUGUSTIN et al. (1984) have modified the method to use a drop of culture on a slide. The preparations are not permanent but can be stabilised at the cost of some clarity. The method works best with young cultures or freshly isolated cells. Protargol (silverproteinate) impregnation was one of the earliesthistological silver stains and the detailsfor its use wereestablished by BODIAN (1936, 1937) for tissuestaining;COLE and DAY (1940) recommended its usein protozoology, andit wasapplied to flagellated protozoans by KIRBY (1945), HONIGBERG (1947) and MOSKOWITZ (1950). KOZLOFF (1960), DRAGESCO (1962) and TuFFRAU (1967) usedthe method to stainhypotrichous ciliates,whichhad provedrefractory by the Chatton-Lwoff method. Cells stainedwith silver nitrategenerally appear as an assembly of black dots in a pale brown or clear matrix, the infraciliature showing as black lines. Cells stained by protargol usually reveal the nucleus and some cytoplasmic features in a brown tone and the kinetosomes in black. The cilia and sub-pellicular fibres are commonly stained, though the infraciliature is not(FLEURYet al. 1985;FOISSNER andSCHUBERT 1977;GRIM 1974). The ability to stainthenucleus indicates thattheredoxpotential of thesilvercomplex in protargol is different from that with pure silver ions, and that far more sites are able either to reduce the silver complex to metallic silveror to bind it for laterreduction by the developer. The advantage is that detailsof the nuclear morphology and the kinetosomal distribution can be obtained from the same preparation, but the stainednucleus can obscure somemorphological detail. In Uronema, for example, the oral apparatus is obscured by the nucleus, usually located just behindthe mouth. The objective of the present study was to develop a more consistent methodology for silver nitrateimpregnation.
Materials andMethods Cells werecultivatedfrom a wide varietyof freshwaterand marineenvironments,in both in mixedpopulationsand clonal cultures. For many of the experiments, an axenic species of Tetrahymena or a clonal culture of the hypotrich Englemanellia halsyi (kindly suppliedby Dr. JOHN DARBYSHIRE, MacaulayLand Use Research Institute) were used. Both marine and freshwater cultures were treated identically. . The basic impregnationregimen, referred to as the standard method and used as a control for each experimental procedure, was that of CORLISS (1953) with a few minor alterationsand modifiedfor use on 22 mm square coverslips manipulated in Columbia staining jars (ARNOLD R. HORWELL, 2, Grangeway, Kilburn High Road, London NW6 2BP, England). In the followingdistilled water is representedby "XH 20" and "formalin" means 40 % formaldehyde, unneutralised.
The Standard Method 1. Fix cells in Champy's Fluid for 3-5 min only. Champy's fluid consistsof7 parts cr03 (l % aq), 7 parts K2Cr207 (3 % aq) and4 partsOS04(2 % aq). The firsttwo componentsmay be stored as a mixture, but the osmium should be added immediately prior to use. The fixative should be used at room temperature.
2. Wash and post fix for at least 2 h or overnight. Washwith da Fano's fixativeuntil all trace of yellow colour is removed. Cells were normallyhandled in centrifuge tubes and washed 3 times. Da Fane's consists ofCoN0 3 I g; NaCll g; formalin 10 ml; XH20 90 mi. Formalin was allowed to stand for at least 3 days in its diluted form. 3. Mix with saline gelatine on a 22 mm coverslip. Mix one drop of cell suspensionin da Fano's fixative with one drop of saline gelatine (gelatine 10 g; NaCI0.05 g; XH20 100 ml) and spreadinto a thin layer using a mounted needle. A total volume of 20- 30 Itl will spread to the correct thickness. 4. Set at 5°C in a moist atmospherefor 3-5 min.
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5. CoverwithAgN0 3 solution at 5-10 °C for 30 min. TheAgN03 was3 % aqueous andwasre-used aslongasit remained clear.If step3 wascorrecta milky-white AgCl precipitate should be just visiblein the gelatine film whenviewed obliquely. 6. Expose the coverslips. The coverslips weretakenfromthe AgN03 without washing and covered withcold water.They wereexposed to UV light(or directsunlight) untilthe basalbodieswereclearly visible undermicroscopic inspection. 7. Wash3 timesin xHzO. 8. Dehydrate, clearand mount.
Modifications to the Standard Method Eachofthe 8 stageswereinvestigated; fixation was studied usingSusa's, Zenker's, Helly's, ZFA, Schaudinn's, Parducz'sand Bouin's fluid. Susa's Fluid: HgClz 4.5 g; NaCI 0.5 g; xHzO 80 mI; trichloroacetic acid2 g; glacialaceticacid4 ml; formalin 20 ml. The first 3 itemsmaybe held as a stocksolution. Zenker'sFluid:HgClz 5 g;KzCrZ07 2.5 g ; NazS041 g; XHzO 100 ml; glacial aceticacid5 ml. Thefirst4 items maybe held as a stocksolution, the acidaddedjust beforeuse. Helly's Fluid: The samestocksolution as Zenker's Fluid, but in placeof the aceticacid use 5 mI formalin. ZFA Fluid: Use the samestockas Zenker's Fluid, but add both 5 ml glacial aceticacid and 5 ml formalin. Schaudinn's Fluid: Sat. aq. HgClz 66 ml; 96% EtOH22 mI. Bouin's Fluid: Sat. aq. picricacid 75 ml; formalin 25 ml; glacial aceticacid 5 ml. Parducz'sFluid: 6 parts osmium tetroxide (2% aq); I part sat. aq. HgClz. Thegelatine wassupplied byRousselot Ltd., andwas250bloom,highclarity. Othersources ofgelatine wereused for comparison; (I) Grayslake gelatine (kindly supplied by Dr. J. Frankel, Dept. Zoology, University of Iowa, U.S.A.) and(2)leafgelatine supplied byHarrods Ltd., London. Solutions ofgelatine wereusedat 10% finalstrength, bothwith and without salt, and withand without Da Fano's fixative (see below). The coverslips wereset on a plastictraywith a moistpieceof absorbent paperfrozenon it, takendirectly froma freezerand the surface of the papermoistened with cold water. A smaller plastictray was usedfor a lid. UV lightwas from a 25 em tube (Sylvania F8T5/BLB) mounted 15 cm abovethe coverslips. Dehydration wascarried outfrom50% EtOH,theneitherthrough 70%, 95%, 100%, 100% withatleast5 minin eachsolution or automatically (ROBERTS andWARREN 1987). Clearing wascarried outusing2 bathsof xylene,withat least 10 min in each. Coverslips weremounted usingfiltered neutralCanada balsam. Allfigures wereproduced as detailed in the text. Alllowmagnification photographs takenin brightfield, all high powerphotographs takenwith Normarski interference contrast.
Results Variation of Fixative All preparations in this section were fixed for 3 min and washed with XR20. Control preparations were made using the standardmethod (Figs. 1,2). Helly's Fluid: Impregnation was finer than the control but the cytoplasm had a dark granular quality, although the lower surface was not obscured (Fig. 3). The infraciliature was visible on some cells. The gelatine showed a slight degree of rippling and was faint brown in colour. Parducz's Fluid: Impregnation was fine and consistentin both marine and freshwater cultures thoughonlythe uppersurfaceof marinecells stained. An amountof randomdeposition waspresent in all preparations. The cytoplasm was granularin appearance but the kinetosomes and mouthparts wereclear(Fig. 4). BothAZMand cirri werevisiblein marinehypotrichs. The gelatinewas lightly veined and pale orange. ZFA Fluid: In marine cultures, cells stainedyellow-brown with fine impregnation and delicate infraciliature, although the latter was obscuredby the colourof the cytoplasm. Freshwater cultures showeda heavy granular deposition, primarilyjust below the pellicle, such that the kinetosomes were not visible. The gelatine was rippled and light yellow/orange in colour.
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Fig. 1. Control slide of Tetrahymena produced with the standard method showing the nature of patterning of the gelantine. Seale bar = 200 urn.
Bouin's Fluid: Impregnation was achieved with both marine and freshwater cells but was difficult to observe due to heavy granularity in the cytoplasm (Fig. 5). The gelatine was finely crazed and light yellow in colour. Schaudinn's Fluid: Shape preservation was good in marine cells but staining was inconsistent. Some kinetosomes seemed to have fragmented and most cells had bright inclusions. For freshwater material, Tetrahymena did not stain and although Englemanellia stained weakly there was a random deposition of silver granules over the cell surface. The gelatine was rippled and light orange in colour. This fixative was also tested using Mayer's albumen as a mountant. Neither freshwater nor marine cells stained but acquired a granular quality. Susa's Fluid: Both marine and freshwater cells stained lightly so that the kinetosomes were just visible against the granular cytoplasm. The gelatine was very pale and lightly veined. Zenker's Fluid: Marine cells did not impregnate and were hyaline. Freshwater cells stained having dark kinetosomes but the cytoplasm was granular and stained somewhat. Many cells had fragmented. Da Fane's Fluid: Fixation was extended to 10 min and 2 h without pre-treatment in Champy's fluid. Cells were severely distorted, those fixed for 2 h were worst, and the impregnation was coarse. The infraciliature could not be observed. The gelatine had a faintly mottled appearance.
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Fig. 2. Control slide of Tetrahymena produced with the standard method showing the staining of an individual in an early division stage. Scale bar = 50 urn.
Variation of Post Fixation Treatment
Cultures of Tetrahymena were fixed in Champy's fluid for approximately 3 min and post fixed with Da Fane's fixative for 0 (single wash), 30, 60, 90 and 120 min. The quality of the impregnation deteriorated through the series, and although the intensity increased so did the granularity of the cytoplasm. The gelatine also changed from smooth through to the wavy appearance common with the standard method. All preparations were made with Da Fano's in the gelatine (see below). The use of marine Da Fane's (i.e. 3.5 g . 1-1 NaCl) in the standard protocol resulted in the gelatine acquiring a scalloped appearance, with granular dark redlbrown stipples. With marine cultures there was more random deposition of silver. In both cases cells were heavily overstained. TORCH and HUFNAGEL (1961) suggested that impregnation of marine cells was improved if the cells were washed in diluted seawater (1: 2) before embedding, and indeed with the standard method the impregnation was finer although the cytoplasm was more cloudy. When the gelatine contained Da Fano's (see below) it acquired a black and red streaked appearance. All cells were heavily overstained. Mixed cultures were washed in water without Da Fano's post treatment; the stain was less intense and the cells were distorted by pellicular folding.
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Fig. 3. An individual (Uronema sp.) from a mixed marineculturetreatedwith Helly's fixative. Scalebar
= 5011m.
Variations in Gelatine Mounting
1. The ratio of Da Fano's to gelatine Experience with the Chatton-Lwoff method indicated that the ratio of cell suspension in Da Fano's fixative to gelatine was important in achieving a good stain. Mixtures ofDa Fano's fixative and gelatine were made using 2, 4 and 6 ml DaFano's fixative in 10 ml of mixture. These solutions were made immediately prior to use and had setting times of approximately 25 min at 45°C. Cell suspensions were centrifuged, aspirated almost to dryness and resuspended in a gelatinelDa Fano's mixture. Cells mounted in the 2 ml mixture were finely impregnated although a little faint. The 4 rnl mixture gave the best result, with well stained cells, clearly visible mouthparts and a pale golden/ buff coloured background with slight rippling. The 6 rnl mixture gave a granular, brown/orange appearance to the gelatine, with a purple speckle and pale bifurcating lines running through it. The cells stained so heavily that the kinetosomes appeared blurred and enlarged, the infraciliature was not visible and the cytoplasm was granular. 1 rnl Da Fano's fixative was added to 2 ml non-saline gelatine. This ratio proved intermediate between the 2 ml and the 4 ml determined above and was used as the normal method in subsequent tests. 2. Salinity of the gelatine Since Da Fano's fixative contains salt, additional salt was omitted from the gelatine in the above
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Fig. 4. An individual Tetrahymena treatedwith Parducz's fixative . Scale bar = 50!!m.
test. Addition of salt (0.05 %) resulted in gelatine which had a darker colour and stronger rippling with wavy lines. 3. Drop size and Spreading
In previous experiments the quality of staining was found to depend on the thickness of the gelatine; cells lying deeper in the gelatine failed to stain as well as those nearer the surface or in thinner gelatine. Cells present in areas of very thin gelatine showed considerable distortion . A drop size of approximately 10 ul proved optimal for a coverslip 22 mm square. This can be spread using a mounted needle or another coverslip as for blood smears. 4. Source of gelatine Rousselotgelatine was used as the control and was compared with gelatine from two other sources (Grayslake and leaf) and also with an equal mixture of Rousselot gelatine and glycerol (cf. Mayer' s albumen ). All were prepared without salt and pre-mixed with Da Fane' s fixative . The test cells were a mixed marine culture washed once with Da Fano ' s fixative and once in distilled water prior to mounting. Grayslake gelatine produced a heavil y veined surface . The cells were well stained although both sides were not visible. Leaf gelatine gave the best results in terms of cell clarity, both surfaces were equally visible and well stained . The background had dark, scalloped lines, like fish scales , running through it which obscured the cell features in places . 20 Arch. Protistenkd. Bd. 135
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Fig. 5. An individual from a mixed marine culture treated with Bouin's fixative. Scale bar == 50flm.
The use of glycerinated gelatine gave much lighter cells, finely impregnated on a more rippled background. The amount of random deposition was greater than the control and the cells had additional lines due to pellicular folding, making interpretation difficult. 5. Albumen as an alternative mountant Mayer's albumen (egg white and glycerol), used in the protargol stain, was used as a mountant. Coverslips were prepared with a thin layer of albumen and air dried on a hot plate at approximately 40°C. Cells were fixed, washed in X H 20 and dehydrated through 70 % , 95 % and 100 % EtOH. A drop of cell suspension was dropped onto an inclined coverslip, so that the drop ran, spreading the cells. The mounted cells were left to air dry for 15 min on the hot plate then transferred to 3 % AgN0 3 in EtOH. Exposure to UV was also under alcohol, cooled to reduce evaporation by placing it on an ice tray. Both freshwater and marine cells were fixed in Champy's fluid,Schaudinn's fixative or Parducz's fixative. Neither of the cultures fixed with Champy's fluid exhibited appreciable staining and the cells showed heavy granularity. The result for Schaudinn's fixative was similar but the cells were a deep purple. Cells fixed with Parducz's fixative were a fairly uniform orange colour and had stained weakly but the colour of the cytoplasm obscured much of the detail. The background albumen was stained orange in the freshwater sample but not in the marine one.
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Fig. 6. The effect of heat drying on the gelantine film. Scale bar = 200urn.
Variation in the Setting Conditions Six regimens were investigated: 1. Cold, moist (control)
A shallow plastic tray lined with moist absorbant paper was refrigerated (at approximately - 20°C). Just before use the paper surface was moistened and a smaller plastic tray used as a lid.
2. Cold, dry As (1) above, but without a lid. 3. Room temperature, moist As (1) above, but not refrigerated. 4. Room temperature, dry As (3) above, but without a lid.
5. Hot, dry The coverslips were put onto a hot plate (approximately 40°C) for 5 min without a lid. 6. Very cold, dry Two methods were used to increase the cooling rate of the coverslips ; (1) they were set on a tray 20'
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Fig. 7. The effect of rapid cooling on the gelatine film. Scale bar = 20U urn.
over a mixture of dry ice and ethanol or (2) they were set on a smooth metal block pre-chilled to -20 °C. The results indicated that the most important factor in producing a light, smooth gelatine layer was to keep the coverslips cold, but not too cold. Coverslips from regimens 1 and 2 had an even gelatine coating with fine veined lines, those from 3 and 4 had fragmented into a network of taut, orange lines in a ladder pattern. The surface between these lines was speckled with maroon spots. The preparations set under moist conditions (i.e. using a lid) were marginally better than those without. The heat dried coverslips had a slightly greyer colour than the control, with a grainy texture and areas of deep red which stuck to the coverslips in taut, ragged sheets (Fig. 6). The dry ice/ethanol mixture produced covers lips to which the gelatine did not adhere well , most had become detached and the rest was in stippled patches . Setting on the metal block crazed the gelatine , more so where the gelatine was thicker (Fig . 7). Silver deposited in these cracks. The cells were well stained (Fig . 8). Variation in AgN0 3 Impregnat ion Using the original Chatton-Lwoff method, the time of impregnation was varied at 30, 60 and 120 min at 10 "C , At 120 min the impregnation was very dense and had a greater amount of random deposition . Shorter impregnation times were more satisfactory , but there was little difference between them. Using the modifications suggested by the preceding result s (see the methodology given below) ,
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Fig. 8. The effect ofrapid cooling on an individual Tetrahymena. Scale bar = 50[1m.
the impregnation time was varied again, using 1, 5, 10, 15,20,25 and 30 min. All slides stained well, although the shorter times produced more finely impregnated cells. The 1 min impregnation showed well stained infraciliature and mouthparts, the cells were sufficiently clear to allow the kinetosomes on the under surface to be seen. The 5 min impregnation revealed the infraciliature more strongly but there was some deterioration in the quality as the impregnation times increased. Silver nitrate was added to the gelatine directly to avoid impregnation all together. Neither Da Fano's fixative nor salt was added to the gelatine mixture. Tetrahymena were used as test cells and concentrations of 1, 3, 5, and 10 % AgN0 3 were used in addition to a control sample without silver impregnated in the normal manner. Cells were exposed, under cold water, to UV after mounting. The staining was faintest at the low concentrations, increasing to an acceptable level at 10 %, though random deposition was also worst on this slide, offsetting the advantage of the shortened protocol. The background gelatine was crystal clear at 1 % silver, but the stain was too faint to be of value (Fig. 9). Silver nitrate (3 % in EtOH) was used as fixative for Tetrahymena; some cells were mounted in Mayer's albumen and others exposed to UV in a suspension before being mounted in gelatine. Both preparations gave similar results, the cells had not stained and both the cells and the background were a granular, opaque white. Cells were treated with mercury orange (PEARSE 1972) to locate concentrations of thiol groups. Cells fixed with osmium or formalin showed no discemable differentiation.
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Fig. 9. Tetrahymena mounted gelatine containing 1% AgN03 • Scalebar = 200!!m.
Variations in Exposure/development Tetrahymena were stained using the modified protocol (see below) and exposed to UV for a range of times from 10 to 70 min increasing in 10 min intervals. Results showed that there was no change in the appearance of the preparations after 40 min; the gelatine was a darker, orange-tan colour and the kinetosomes appeared enlarged in comparison to the shorter exposure times (Figs. 10, 11). The infraciliature was visible only as a faint line but this was not consistent. Shorter exposure times gave better results; 10 min gave the clearest result, the cells were lightly coloured with visible mouthparts and kinetosomes and although not initially very clear had improved within a week so that both cell surfaces were equally clear and the infraciliature was easy to see. Attempts were made to develop the image chemically. Using a developer common in the protargol method (2.5 % hydroquinone, 12.5 % sodium sulphite, 20% sodium carbonate in x HzO) preparations made by the modified protocol were incubated for 5, 10, 15, 60, 120, 180 min and overnight at room temperature. None of the preparations developed as well as under UV; the cells were cream coloured opaque shapes with no differential staining (Fig. 12). Some deposition became apparent at the longest developmental period but kinetosomes could not be distinguished.
Variation in Washing In all studies preparations were washed thoroughly prior to dehydration.
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Fig. 10. An individual Tetrahymena exposed to UV light for to min. Scale bar = 501!m.
Variation in Dehydration, Clearing and Mounting Preparations were routinely cleared by incubation in two xylene baths , with at least 10 min in each. Clove oil was used on a Tetrahymena preparation to see if the clarity of the cells could be improved. The results showed that there was a marginal improvement but not enough to warrant the increased cost of routinely using clove oil.
Discussion Fixation Correct fixation is the key to good staining. Of the fixatives used in this study, Champy's fluid produced the most consistent good results. The fixative properties of osmium tetroxide are thought to be due to its oxidation of unsaturated lipids and it has been suggested that it forms crosslinkages between lipid chains. This would account for the better shape preservation observed with osmium based fixatives. Chromium salts, which are oxidative, are thought to fix proteins and to react primarily with carboxyl groups forming coordinates with OH and HN z groups. Mercury ions behave in a similar fashion to chromium and have an affinity for the acid groups on proteins, especially carboxyl and hydroxyl. They differ, however, in that mercury ions have a high affinity for thiol groups (SH) (PEARSE 1980). Formalin is the classical fixative for proteins and a principal ingredient ofDa Fano's fixative.
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Fig. 11. An individual Tetrahymena exposed to UY light for 40 minutes. Scale bar = 50!lm.
Formaldehyde reacts with water to form its monohydrate, methylene glycol, and low molecular weight polymeric hydrates. These reactions are not rapid, which is why, for the sake of reproducibility, formalin solutions should be allowed to equilibrate for several days before use. The active ingredient is thought to be formaldehyde and not its hydrates or polymers. It is able to form crosslinkages in proteins, particularly involving amino, imino and amido, peptide, guanidy1, hydroxyl, thio1 and aromatic rings. It is thought to reduce S-S links to two SH groups which may then be linked by a methylene bridge (S-CH 2-S). This latter is readily ruptured by hydrolysis, possibly simply by washing (PEARSE 1980). The best results were obtained with Champy's, Helly's and Parducz's fluids, which are all heavy metal fixatives with chromium and/or mercury. XINBAI (1976, 1980a, b) found a mixture of saturated mercuric chloride and osmium tetroxide, as in PARDUCZ'S fixative but in different proportions, was efficient as a fixative prior to silver nitrate impregnation. The exact proportion of HgC12 : OS04 depended on the ciliate and its physiological condition. Other fixatives did not give such good results, particularly ZFA and Zenker's fluids, which are similar to Helly's fluid, except that Zenker's fluid contains acetic acid instead of formalin and ZFA contains acetic acid as well as formalin. Acetic acid therefore seems to interfere with the impregnation by silver nitrate. Susa's and Bouin's fluids, which gave poor results, also contain acetic acid, which is generally added to fixatives to improve their penetrating properties although it also causes swelling in most tissues (LEE 1937). Schaudinn's fluid, alcoholic mercuric chloride, allowed little impregnation. Ethanol causes protein precipitation and its fixative properties rely on the conformational changes induced (PEARSE 1980). It is important in silver amplification that the
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Fig. 12. An individual Tetrahymena impregna ted and treated with chem ical developer. Sca le bar = 50fl m.
nucleus of deposited silver atoms should not be too small or too dispersed and it is possible that the confonnationa1 changes separa te or occlude sites of potenti al deposition so that nuclei of silver atoms cannot form , Da Fano' s fixative , as a primary fixative, produc ed severe distortion in the cells, and staining was crude. The lack of shape retention is a recognised failing of the aldehyde fixatives which prim arily affect protein, not lipid . However , as a post fixative , the reducing properties of the formalin modified the oxid ative properties of the initial fixatives. The time required in Da Fano 's fixat ive was found to be sho rt, and in fact a single wash in a centrifuge tube proved to be effec tive . A second wash in distilled water removed any residual chromium salts. XINBAI (l980b) uses a post-fixation wash with NaCI + CoCJ2 to promote the staining of deeply sinking features . G e l a t ine Mo u n ti ng The principl e of silver staining is one of amplification; minute deposits of silver are made visible by deposition of more silver on that already present , as in photography. For additional silver to be deposited , silver ions must be present , and in photograph y this comes from unredu ced silver halide in the emulsion ; in histology care must be taken that sufficie nt silver is carried into the developer. Th is role was fulfilled by silver preci pitated by the Da Fano' s fixative mixed with the gelatine. The original Chatton-Lwoff method requ ired some experience to make it perfonn well, and in particular the amount of cell suspe nsio n in Da Fane ' s fixative that was mixed with the gelatine. To overcome
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this variability, the Da Fano's fixative and gelatine were mixed in predetermined proportion and added to the cells. When spread thinly, this method gave the correct quantity of deposited silver. The silver solution must diffuse through the gelatine, so that deeper lying cells often fail to stain while those above are perfectly impregnated. For this reason, thick layers should be avoided. The setting conditions were most important for a good, even gelatine film. When the cooling was too rapid, the gelatine did not set evenly and broke apart. When cooling was too slow, then dessication was a problem. Da Fano's fixative set the gelatine in about 25 min, but because the gelatine was warm it lost some water even in a moist chamber. Dessication can lead to premature deposition of silver ions and conformational changes in the cell proteins. XINBAI (1980b) used agar-agar instead of gelatine for the impregnation of deeply sinking features, though the outer cortical features did not impregnate well. The role of the gelatine and the agar-agar in this case is unclear. Impregnation SAMUEL (1953 a) studied the effect of impregnation time and pH on the impregnation of ganglion sections. He concluded that sections could be over impregnated and that high pH values (8.7) gave undifferentiated impregnation. Low pH values (6.8) gave rise to random deposition especially for shorter impregnation times. No attempt was made to control the pH of the impregnation solutions in this study. Prior to impregnation, the pH of the silver nitrate solutions was in the range 7.0 to 7.8. The biochemical composition of the protozoan cell has not yet been established with any degree of certainty. Silver was consistently deposited, with both the inorganic and protargol methods, at the basal bodies of cilia. This may have been due to thiol groups located at the ends of the microtubular bundles forming the cilium or from the binding sites for Cat ' or Mgt" which are known to affect ciliary beat (SLEIGH 1973; STEBBINGS and HYAMS 1979). The infraciliature was stained by silver nitrate, its location corresponded to that of fibrils in or below the pellicle in most ciliates. There appear to be several kinds of micro-fibrils which are generally classified by size, only the microtubules being reasonably well defined. In Euplotes physical features which correspond to the argentophilic dorsal network have been called "rugae", which RUFFOLO (1976) suggested were tubular extrusions of the pellicular membrane. This is difficult to accept since the rugae occur in pairs, whereas the argentophilic structure is a single line. FOISSNER and SIMONSBERGER (1975 a) demonstrated that in the dry silver technique, silver did not accumulate in folds of the pellicle. It is more plausible that the sites of deposition are the cylindrical fibrils lying underneath the inner alveolar membrane (GRIM et al. 1980; FOISSNER 1978; FOISSNER 1976b; FOISSNER and SIMONSBERGER 1975a, b). In Tetrahymena the longitudinal line between rows of cilia align with a microtubular bundle (GRIM et al. 1980). In Paramecium, the infraciliature is revealed by deposition of silver at the underside of the junction of adjacent alveolar membranes, where no cellular component was differentiated, and in the uppermost third of pellicular ridges, where a fibro-granular material was located (FOISSNER 1977). No differentiated cell component was found at the site of the infraciliature in Colpidium colpoda (FOISSNER and SIMONSBERGER 1975b; FOISSNER 1981). Silver nitrate can be used to demonstrate the presence of disulphide bonds in keratins (see PEARSE 1972 for review), provided the pH of the impregnation medium is sufficiently well controlled. It was claimed that at high pH values, thiol groups were not detected. Thiol groups, common in fibrous structural proteins, are a prime candidate for the effective site of silver deposition. However it is likely that more than one site was responsible for silver reduction; silver will be reduced at any site where the redox potential is correct. XINBAI (1976, 1980a, b) has developed a series of methods in which the cortical depth of impregnation is controlled. The fixation, post-fixation and mountant are tailored to the particular cells being impregnated. These methods are particularly useful in revealing deeply sinking
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structures, such as the oral apparatus in Paramecium (XINBAI 1980 b). In general agar-agar inhibits the development of the stain in the outer cortical layers. Increasing the proportion of OS04 in the fixative increases the penetration of the stain. Furthermore, actively feeding cells require more OS04 in the fixative than do hungry cells. The methodologies for deeper features also require postfixation with NaCl + CoCh. Further studies are required to resolve the histochemical identity of the groups active in silver deposition. Reduced bound silver can be differentiated from deposited metallic silver nuclei by sulphite washing (SAMUEL 1953 b); calcium binding sites can be identified by treatment with potassium-ferricyanide and sodium thiosulphate (PLENK 1975). Illumination Whilst being reduced by light, the gelatine must be protected from detaching from the glass of the coverslip and from dessication. Infra-red light causes heating of the preparations which will soften the gelatine layer. UV light sources, low in infra-red output, were therefore used to expose the slides which were under chilled water to absorb any residual infra-red. FRANKEL and HECKMANN (1968) found that the quality of the stain was critically dependent upon the initial exposure to light and found it convenient to continue to expose the cells through the dehydration phase. The results presented here were conducted under standard conditions of normal room light followed by UV, although some trials were made using tungsten illumination in place of UV, which developed the stain just as well. The heating effect with tungsten meant that the covering water had to be replaced during the exposure, so the UV light was used for its greater convenience. The process of silver deposition is reversible and will amplify the image so long as there is a sufficient pool of available silver ions. Should this pool become limited, photo erosion of the image will occur; fine features will be lost and the silver deposited on the strong features. It is therefore important not to overexpose the slides, although if sufficient silver was deposited in the gelatine matrix this will not be a practical limitation. SAMUEL (1953 a) developed silver nitrate impregnations of ganglion sections using an hydroquinone/sulphite developer. It is unclear why chemical reduction does not develop the latent image in the case of ciliated protozoa. Dehydration, Clearing and Mounting Soluble reaction products must be removed from the gelatine matrix because the reactions are reversible and fading would result. Thorough washing must preceed dehydration. Experience has shown that synthetic mounting media are not as durable as Canada balsam, which may be softened and re-mounted many tens of years after a preparation was originally made. Slides in the BM(NH) collections dating back well into the last century are still in their original balsam mounts, whereas synthetic media have often required re-mounting due to deterioration as little as 5 years after preparation. General comments Slides often seem to improve with storage and although it is not possible to perform direct comparisons those which have not stained particularly well are worth re-examining a few weeks after staining. The mechanism for this is unclear, but may be associated with the slow penetration of the balsam clearing the cells more effectively, and making previously indistinct features visible. During an active experimental procedure stocks will be frequently subcultured to ensure sufficient material for staining. In consequence, most tests are carried out on cultures with high vitality which generally impregnate most easily. Old cultures often fail to stain as well, if at all (observations from this laboratory; FOISSNER, pers. comm.; FRANKEL and HECKMANN 1968).
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Conclusions The methodology which takes the above points into account is more rapid than the ChattonLwoff method as specified by CORLISS (1953). Routinely in this laboratory, the procedure takes about 2 h from live cells to finished preparation. It may be summarised as a working schedule:
1. Fix cells in Champy's Fluid for 3 min. Champy's fluid consists of7 parts Cr03 (1 % aq), 7 parts K2Cr207 (3 % aq) and 4 parts OS04 (2 % aq) all at room temperature. The first two components may be stored as a mixture, but the osmium should be added immediately prior to use. Centrifuge the cells, aspirate as much culture fluid as practical, disperse, add approximately 1-2 ml of fixative.
2. Wash once with Da Fano's fixative. DaFano's consists ofCoN0 3 1 g; NaCll g; formalin 10 ml; XH20 90 ml. Formalin should be unneutralised, and left for at least 3 d in its diluted form. Fill the centrifuge tube with Da Fano's fixative. 3. Wash once with XH 20.
Centrifuge the cells, aspirate, disperse and re-fill the tube with XH 20. Leave for approximately 1 min.
4. Mount cells in GelatinelDa Fano's mixture. Immediately before use, mix 2 ml gelatine (15 %) with 1 ml Da Fano's fixative. Add 3- 5 drops of the mixture to the aspirated cells in a centrifuge tube. Take a small drop of the cell suspension (10 ul) and spread with a mounted needle on a 22 mm square coverslip.
5. Set the gelatine on an ice tray for 3-5 min. Moisten a piece of absorbant paper (e.g. filter paper) on a tray and freeze in the ice-box of a domestic refrigerator or domestic freezer. Just before use, moisten the surface with a little water spread with the fingers. Place the coverslips on the tray beneath a lid. 6. Incubate in AgN0 3 at 10 °C for 5 min. The AgN0 3 is 3 % aqueous and may be re-used so long as it remains clear.
7. Develop the coverslip. Expose to UV for 10 min under chilled XH20. 8. Wash thoroughly in XH 20. Wash at least 3 times in cold XH20. 9. Dehydrate, clear, mount. Dehydrate through the alcohols, 50 %, 70 %,95 %, 100 %, 100 %,5 min in each. Clear in two changes of xylene, 10 min in each. Mount in Canada balsam.
Acknowledgements We wish to express our gratitude to Dr. W. FOISSNER, University of Salzburg, and Dr. S. XINBAI, Harbin Pedagogical College for their informed discussion and to the Trustees of the British Museum (Natural History) for providing a vacation studentship for Ms. CAUSTON to carry out this work at the Museum.
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Literature AUGUSTIN , H., FOISSNER, W., and ADAM , H. (1984) : An improvedpyridinated silver carbonate methodwhich needs few specimens and yields permanent slides of impregnated ciliates (Protozoa, Ciliophora). Mikroskopie 41: 134-137. BODlAN , D. (1936): A newmethodforstaining nerve fibersandnerveendings inmountedparaffinsections. Anal. Rec. 65: 89-97 . - (1936): The staining of paraffinsections of nervous tissueswithactivatedprotargol. The role of fixatives . Ibid. 69: 153-162. CHATTON, E., et LWOFF, A. (1930): Impregnation, pardiffusionargentique, de I'infraciliature de ciliesmarins et d'eau douce, apres fixation cytologiqueet sans dessication. C. r. Seanc, Soc. BioI. 104: 834-836. - (1935): Lesciliesapostomes. Morphologie, cytologic, ethology, evolution, systematique. I. Apercuhistorique et general. Etude rnonographique des genres et des especes. Archs. Zool. expo gen. 77: 1-453. - (1936): Techniques pour l'etude des Protozoaires, specialement de leurs structures superficielles (cinetome et argyrome) . Bull. Soc. fr. Microsc. 5 : 25-39 . COLE , R. M., and DAY, M. F. (1940): The use of silveralbumose(protargol) in protozoological technique. J. Parasit. Suppl. 26: 29-30. CORLISS, J . O. (1953): Silver impregnation of ciliated protozoaby the CHATTON-LwOFF technic. StainTechno!' 28: 97-100. DAVENPORT, H. A., PORTER, R. W. and MYHRE, B. A. (1952): Preparation and testing of silver-protein compounds . Ibid. 27: 243-248. DRAGESCO, J. (1962): L'orientationactuelle de la systematique des cilieset la techniqued' impregnation au proteinate d'argenl. Bull. Microsc. appl. 12: 49-58 . FERNANDEz-GALIANO, D. (1966): Une nouvelle methode pour la mise en evidence de I'infraciliature des cilies. Protistologica 2: 35-38. ( 1976): Silver impregnation of ciliated protozoa: Procedure yielding goad results with the pyridinated silver carbonate method. Trans. Am. microsc. Soc. 95 : 557- 560. et RUIZ, S. (1972): Descriptiond' une nouvelle espece de cilie, Colpidiumuncinatum. Protistologica8 : 295- 298. FLEURY, A., IITODE, F., DEROUX, G., ct FRYD-VERSAVEL , G. (1985) : Unite et diversite chez les hypotriches (Protozoaires, cilies) II. - Elements d'ultrastructure comparee chez divers represenrants du sous-ordre des Euhypotrichina. Ibid. 21: 505-524. FOISSNER, W. (1967): Wimpertiere im Silberpraparat. Ein .rrockenes" Verfahren zur Darstellung des Silberliniensystems. Mikrokosmos 56: 122-1 26. (1967a): Erfahrungen miteiner trockenen Silberimpragnationsrnethode zurDarstellungargyrophilerStrukturenbei Protisten. Verh. zoa!.-bot. Ges. Wien ll5 : 68-79. (1976b) : FunfzigJahre Forschung am Silberliniensystem der Ciliaten. Naturk. Jb. Stadt Linz 22: 103-11 2. (1977): Elektronenmikroskopische Untersuchung tiber die Lage und Natur des Silberliniensystems von Paramecium. Mikroskopie 33: 260-276 . (1978): Euplotes moebiusi f quadricirratus (Ciliophora, Hypotrichida) I. Die Feinstrukturdes Cortex und der argyrophilen Strukturen. Arch. Protistenkd. 120: 86-117. (1981): Das Silberliniensystem der Ciliaten: Tatsachen, Hypothesen, Probleme. Mikroskopie 38: 16-26. undSCHUBERT, G. (1977): Morphologie der Zooideund Schwarmervon Heteropolaria colisarumgen. nov., spec. nov. (Ciliata,Peritrichida),einer symphoriontenEpistlidaevon Colisafas ciata (Anabantoidei, Be1ontiidae). Acta Protozool. 16: 231-247. und SIMONSBERGER, P. (l975a) : Vergleichende licht- und rasterelektronenmikroskopische Untersuchungen an trockenpriiparierten Silberliniensyslemen von Ciliaten (Protozoa). Mikroskopie 31: 193-205. - (1975b): ElektronenmikroskopischcrNachweisder subpelliculiiren Lage desSilberliniensystemsbei Colpidium colpoda (Ciliata, Tetrahymenidae). Protoplasma 86: 65-82. FRANKEL, J. , and HECKMANN , K. (1968): A simplified Chalton-Lwoff silver impregnation procedure for use in experimental studies with ciliates. Trans. Am. microsc. Soc. 87: 317-321. GRIM , J. N. (1974): A protargol study of the fiber systemof the ciliate Haltaria. Ibid. 93: 421-425 . - HALCROW, K. R., and HARSHBARGER, R. D. (1980) : Microtubules beneath the pellicles of two ciliate protozoaas seen with the SEM. J. Protozoal. 27: 308-310. HONIGBERG, B. (1947):Thecharacteristicsof theflagellateMonocercomonas verrens, sp. n. from Tapirusmalayanus. Univ. Calif. PubIs Zoo!. 53: 227- 236. JONES, D. B. (1973) :Silverstains. In: P. GRAY (ed.), The EncyclopediaofMicroscopyandMicrotechnique. NewYork.
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KIRKBY, H. (1945): The structureof the common intestinaltrichomonad of man. J. Parasit. 31: 163-174. KLEIN, B. M. (1926): Uber eine neue Eigentiimlichkeit der Pelliculavon Chilodon uncinatus EHRBG. Zoo!' Anz. 67: 160-162. KOZLOFF, E. N. (1960): Morphological studies on holotrichous ciliates of the family Hysterocinetidae I. Hysterocineta eiseniae BEERS and Ptychostomum campelomae sp. nov. J. Protozoo!. 7: 41-50. LEE, A. B. (1937): The Microtomist's Vade-mecum (eds. GATENBY, J. B., & PAINTER, T. S.). 10thed. London. MADRAZO-GARIBAY, M., et LOPEZ-OCHOTERENA, E. (1973): Nouvellesprecisionobtenuesa I' aidede la techniquede FERNANDEZ-GALIANO et concernantentre autre I'infrastructureciliairedans Ie genre Paramecium. Protistologica 9: 481-485. MOSKOWITZ, N. (1950): The use of protein silverfor stainingprotozoa. Stain Techno!. 25: 17-20. NIELSEN, E. M., and DEBAULT,L. E. (1978): Transformation in Tetrahymena pyriformis: descriptionof an inducible phenotype. J. Protozoo!' 25: 113-1l9. PEARSE, A. G. E. (1972): Histochemistry Theoretical & Applied. Volume2. 3rd ed. Edinburgh. - (1980): Histochemistry Theoretical & Applied. Volume I. Preparative and Optical Technology. 4th ed. Edinburgh. PETERS, A. (1958): Staining of nervous tissue by protein-silvermixtures. Stain Techno!' 33: 47-53. PLENK, V. H. (1975): Differentiation of silver-calciumsalt staining methods using a photographic developer. Mikroskopie 31: 73-76. ROBERTS, D. McL., and WARREN, A. (1987): An inexpensive apparatusfor automaticcontinuousdehydration. Stain Techno!. 62: 211-215. RUFFOLO, J. J. (1976): Fine structureof the dorsalbristlecomplexandpellicleof Euplotes. J. Morph. 148: 469-488. SAMUEL, E. P. (1953a): Impregnation and developmentin silver staining. J. Anat. 87: 268-277. - (l953b): The mechanismof silver staining. Ibid 87: 278-287. SLEIGH, M. A. (1973): The Biologyof Protozoa. London. STEBBINGS, H., and HYAMS, J. S. (1979): Cell Motility. London. TORCH, R., and HUFNAGEL, L. (1961): Differentiation of speciesof Diophrys by meansof silverstaining.Bio!. Bull. mar. bio!' Lab., Woods Hole 121: 411 (Abstr.). TUFFRAU, M. (1967): Perfectionnement et pratiquede la techniqued' impregnation au protargoldes infusoirescilies. Protistologica 3: 91-98. XINBAI, S. (1976): Studies on conjugation in Stylonychyia mytilus II. Morphogenesis of argentophilic system. Acta zoo!. sin. 22: 71-83 + 8 plates (In Chinese). (1980a): Morphology and morphogenesis in Pseudomicrothorax dubius. Ibid. 26: 71-79 + 2 plates (In Chinese). (1980b): The morphology and morphogenesis of the buccal apparatus in Paramecium and their phylogenetic implications. I. Morphology of buccal apparatus. Ibid. 26: 205-212 + I plate (In Chinese). Authors' addresses: D. McL. ROBERTS, Dept. Zoology, British Museum (Natural History), Cromwell Road, London SW7 5BD, England; Ms. H. CAUSTON, School of Biology, Kings Building, University of Edinburgh, EdinburghEH9 3JT, Scotland.
Department of Zoology, British Museum (Natural History), London, England
Silver Nitrate Impregnation of Ciliated Protozoa By D. McL. ROBERTS and H. CAUSTON With 12 Figures Key words : Infraciliature ; CHATTON-LwOFF; Silver stain
Summary A modified silver nitrate impregnation procedure is described, based on the Chatton-Lwoff method. The major differences are ( I) the post-fixation in da Fane 's fixative is reduced to a single wash and (2) that the variability between preparations has been reduced by mixing da Fano' s fixative with the gelatine mountant prior to mixing with the cells. The total time required for the new protocol is 2 h from live culture to mounted slides.
Introduction Silver staining has been in use since 1843 (JONES 1973) and in that time has well earned its reputation for being capricious. PETERS (1958) divided the methods into two categories ; (1) those using simple silver salts and (2) those using protargol (a soluble protein/silver complex) (DAVENPORT et al. 1952). Both methods work in the same way but differ markedly in their sensitivities and the range of material stained: Fundamentally, the initial exposure of a cell to the silver solution results in the formation of either (1) minute metallic silver nuclei of a few silver atoms or (2) bound ionic silver which is reducible to metallic silver during development, at sites where cellular components have a high redox potential with silver ions (SAMUEL 1953b). Subsequent reduction in an alkaline developer, or by exposure to light, causes the deposition of much additional silver on these nuclei so that, as in photography, a latent image becomes a visual image. Carbonyl and thiol groups are thought to be important in the formation of silver nuclei (JONES 1973). Silver impregnation of protozoa was introduced in order to expose the location of pellicular components, particularly kinetosomal bases, for the purpose of taxonomic classification. Ciliated protozoa were stained by KLEIN (1926) using the "dry" method, so called because the cells were fixed by air drying onto a slide. Cells were then impregnated with silver nitrate and reducedby light. The main disadvantages of the methods were ( I) morphological distortion, introduced by air drying (for a modification, see FOISSN ER 1967), (2) the fact that marine cells could not be air dried (for a modification, see FOISSNER 1976a) and (3) that the surface of the cell adhering to the glass was not stained. CHATTON and LWOFF (1930, 1935, 1936) introduced chemical fixation followed by mounting in a gelatine film to overcome these difficulties and although the same structures were impregnated, the protocol became more protracted (for a modification, see FRANKEL and HECKMANN 1968; NELSEN and DEBAULT 1978). The silver carbonate method, introduced for ciliated protozoa by FERNANDEz-GALIANO (1966), used a formalin fixation followed by impregnation and development in the liquid phase. Impregnation revealed kinetodesmata with particular clarity, kinetosomes, cilia, nucleus and occasionally other pellicular organelles (e.g. trichocysts). The infraciliature revealed with the silver nitrate methods was not stained (FERNANDEz-GALIANO 1966, 1976; FERNANDEZ-GALIANO and RUIZ 1972; MADRAZO-GARIBAYand LOPEZ-OCHOTERENA 1973; AUGUSTIN et al. 1984). During
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the FERNANDEZ-GALIANO method the cellshaveto be removed and washed froma final volume of 45 ml, which is particularly difficult for small cells or sparse cultures although AUGUSTIN et al. (1984) have modified the method to use a drop of culture on a slide. The preparations are not permanent but can be stabilised at the cost of some clarity. The method works best with young cultures or freshly isolated cells. Protargol (silverproteinate) impregnation was one of the earliesthistological silver stains and the detailsfor its use wereestablished by BODIAN (1936, 1937) for tissuestaining;COLE and DAY (1940) recommended its usein protozoology, andit wasapplied to flagellated protozoans by KIRBY (1945), HONIGBERG (1947) and MOSKOWITZ (1950). KOZLOFF (1960), DRAGESCO (1962) and TuFFRAU (1967) usedthe method to stainhypotrichous ciliates,whichhad provedrefractory by the Chatton-Lwoff method. Cells stainedwith silver nitrategenerally appear as an assembly of black dots in a pale brown or clear matrix, the infraciliature showing as black lines. Cells stained by protargol usually reveal the nucleus and some cytoplasmic features in a brown tone and the kinetosomes in black. The cilia and sub-pellicular fibres are commonly stained, though the infraciliature is not(FLEURYet al. 1985;FOISSNER andSCHUBERT 1977;GRIM 1974). The ability to stainthenucleus indicates thattheredoxpotential of thesilvercomplex in protargol is different from that with pure silver ions, and that far more sites are able either to reduce the silver complex to metallic silveror to bind it for laterreduction by the developer. The advantage is that detailsof the nuclear morphology and the kinetosomal distribution can be obtained from the same preparation, but the stainednucleus can obscure somemorphological detail. In Uronema, for example, the oral apparatus is obscured by the nucleus, usually located just behindthe mouth. The objective of the present study was to develop a more consistent methodology for silver nitrateimpregnation.
Materials andMethods Cells werecultivatedfrom a wide varietyof freshwaterand marineenvironments,in both in mixedpopulationsand clonal cultures. For many of the experiments, an axenic species of Tetrahymena or a clonal culture of the hypotrich Englemanellia halsyi (kindly suppliedby Dr. JOHN DARBYSHIRE, MacaulayLand Use Research Institute) were used. Both marine and freshwater cultures were treated identically. . The basic impregnationregimen, referred to as the standard method and used as a control for each experimental procedure, was that of CORLISS (1953) with a few minor alterationsand modifiedfor use on 22 mm square coverslips manipulated in Columbia staining jars (ARNOLD R. HORWELL, 2, Grangeway, Kilburn High Road, London NW6 2BP, England). In the followingdistilled water is representedby "XH 20" and "formalin" means 40 % formaldehyde, unneutralised.
The Standard Method 1. Fix cells in Champy's Fluid for 3-5 min only. Champy's fluid consistsof7 parts cr03 (l % aq), 7 parts K2Cr207 (3 % aq) and4 partsOS04(2 % aq). The firsttwo componentsmay be stored as a mixture, but the osmium should be added immediately prior to use. The fixative should be used at room temperature.
2. Wash and post fix for at least 2 h or overnight. Washwith da Fano's fixativeuntil all trace of yellow colour is removed. Cells were normallyhandled in centrifuge tubes and washed 3 times. Da Fane's consists ofCoN0 3 I g; NaCll g; formalin 10 ml; XH20 90 mi. Formalin was allowed to stand for at least 3 days in its diluted form. 3. Mix with saline gelatine on a 22 mm coverslip. Mix one drop of cell suspensionin da Fano's fixative with one drop of saline gelatine (gelatine 10 g; NaCI0.05 g; XH20 100 ml) and spreadinto a thin layer using a mounted needle. A total volume of 20- 30 Itl will spread to the correct thickness. 4. Set at 5°C in a moist atmospherefor 3-5 min.
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5. CoverwithAgN0 3 solution at 5-10 °C for 30 min. TheAgN03 was3 % aqueous andwasre-used aslongasit remained clear.If step3 wascorrecta milky-white AgCl precipitate should be just visiblein the gelatine film whenviewed obliquely. 6. Expose the coverslips. The coverslips weretakenfromthe AgN03 without washing and covered withcold water.They wereexposed to UV light(or directsunlight) untilthe basalbodieswereclearly visible undermicroscopic inspection. 7. Wash3 timesin xHzO. 8. Dehydrate, clearand mount.
Modifications to the Standard Method Eachofthe 8 stageswereinvestigated; fixation was studied usingSusa's, Zenker's, Helly's, ZFA, Schaudinn's, Parducz'sand Bouin's fluid. Susa's Fluid: HgClz 4.5 g; NaCI 0.5 g; xHzO 80 mI; trichloroacetic acid2 g; glacialaceticacid4 ml; formalin 20 ml. The first 3 itemsmaybe held as a stocksolution. Zenker'sFluid:HgClz 5 g;KzCrZ07 2.5 g ; NazS041 g; XHzO 100 ml; glacial aceticacid5 ml. Thefirst4 items maybe held as a stocksolution, the acidaddedjust beforeuse. Helly's Fluid: The samestocksolution as Zenker's Fluid, but in placeof the aceticacid use 5 mI formalin. ZFA Fluid: Use the samestockas Zenker's Fluid, but add both 5 ml glacial aceticacid and 5 ml formalin. Schaudinn's Fluid: Sat. aq. HgClz 66 ml; 96% EtOH22 mI. Bouin's Fluid: Sat. aq. picricacid 75 ml; formalin 25 ml; glacial aceticacid 5 ml. Parducz'sFluid: 6 parts osmium tetroxide (2% aq); I part sat. aq. HgClz. Thegelatine wassupplied byRousselot Ltd., andwas250bloom,highclarity. Othersources ofgelatine wereused for comparison; (I) Grayslake gelatine (kindly supplied by Dr. J. Frankel, Dept. Zoology, University of Iowa, U.S.A.) and(2)leafgelatine supplied byHarrods Ltd., London. Solutions ofgelatine wereusedat 10% finalstrength, bothwith and without salt, and withand without Da Fano's fixative (see below). The coverslips wereset on a plastictraywith a moistpieceof absorbent paperfrozenon it, takendirectly froma freezerand the surface of the papermoistened with cold water. A smaller plastictray was usedfor a lid. UV lightwas from a 25 em tube (Sylvania F8T5/BLB) mounted 15 cm abovethe coverslips. Dehydration wascarried outfrom50% EtOH,theneitherthrough 70%, 95%, 100%, 100% withatleast5 minin eachsolution or automatically (ROBERTS andWARREN 1987). Clearing wascarried outusing2 bathsof xylene,withat least 10 min in each. Coverslips weremounted usingfiltered neutralCanada balsam. Allfigures wereproduced as detailed in the text. Alllowmagnification photographs takenin brightfield, all high powerphotographs takenwith Normarski interference contrast.
Results Variation of Fixative All preparations in this section were fixed for 3 min and washed with XR20. Control preparations were made using the standardmethod (Figs. 1,2). Helly's Fluid: Impregnation was finer than the control but the cytoplasm had a dark granular quality, although the lower surface was not obscured (Fig. 3). The infraciliature was visible on some cells. The gelatine showed a slight degree of rippling and was faint brown in colour. Parducz's Fluid: Impregnation was fine and consistentin both marine and freshwater cultures thoughonlythe uppersurfaceof marinecells stained. An amountof randomdeposition waspresent in all preparations. The cytoplasm was granularin appearance but the kinetosomes and mouthparts wereclear(Fig. 4). BothAZMand cirri werevisiblein marinehypotrichs. The gelatinewas lightly veined and pale orange. ZFA Fluid: In marine cultures, cells stainedyellow-brown with fine impregnation and delicate infraciliature, although the latter was obscuredby the colourof the cytoplasm. Freshwater cultures showeda heavy granular deposition, primarilyjust below the pellicle, such that the kinetosomes were not visible. The gelatine was rippled and light yellow/orange in colour.
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Fig. 1. Control slide of Tetrahymena produced with the standard method showing the nature of patterning of the gelantine. Seale bar = 200 urn.
Bouin's Fluid: Impregnation was achieved with both marine and freshwater cells but was difficult to observe due to heavy granularity in the cytoplasm (Fig. 5). The gelatine was finely crazed and light yellow in colour. Schaudinn's Fluid: Shape preservation was good in marine cells but staining was inconsistent. Some kinetosomes seemed to have fragmented and most cells had bright inclusions. For freshwater material, Tetrahymena did not stain and although Englemanellia stained weakly there was a random deposition of silver granules over the cell surface. The gelatine was rippled and light orange in colour. This fixative was also tested using Mayer's albumen as a mountant. Neither freshwater nor marine cells stained but acquired a granular quality. Susa's Fluid: Both marine and freshwater cells stained lightly so that the kinetosomes were just visible against the granular cytoplasm. The gelatine was very pale and lightly veined. Zenker's Fluid: Marine cells did not impregnate and were hyaline. Freshwater cells stained having dark kinetosomes but the cytoplasm was granular and stained somewhat. Many cells had fragmented. Da Fane's Fluid: Fixation was extended to 10 min and 2 h without pre-treatment in Champy's fluid. Cells were severely distorted, those fixed for 2 h were worst, and the impregnation was coarse. The infraciliature could not be observed. The gelatine had a faintly mottled appearance.
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Fig. 2. Control slide of Tetrahymena produced with the standard method showing the staining of an individual in an early division stage. Scale bar = 50 urn.
Variation of Post Fixation Treatment
Cultures of Tetrahymena were fixed in Champy's fluid for approximately 3 min and post fixed with Da Fane's fixative for 0 (single wash), 30, 60, 90 and 120 min. The quality of the impregnation deteriorated through the series, and although the intensity increased so did the granularity of the cytoplasm. The gelatine also changed from smooth through to the wavy appearance common with the standard method. All preparations were made with Da Fano's in the gelatine (see below). The use of marine Da Fane's (i.e. 3.5 g . 1-1 NaCl) in the standard protocol resulted in the gelatine acquiring a scalloped appearance, with granular dark redlbrown stipples. With marine cultures there was more random deposition of silver. In both cases cells were heavily overstained. TORCH and HUFNAGEL (1961) suggested that impregnation of marine cells was improved if the cells were washed in diluted seawater (1: 2) before embedding, and indeed with the standard method the impregnation was finer although the cytoplasm was more cloudy. When the gelatine contained Da Fano's (see below) it acquired a black and red streaked appearance. All cells were heavily overstained. Mixed cultures were washed in water without Da Fano's post treatment; the stain was less intense and the cells were distorted by pellicular folding.
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Fig. 3. An individual (Uronema sp.) from a mixed marineculturetreatedwith Helly's fixative. Scalebar
= 5011m.
Variations in Gelatine Mounting
1. The ratio of Da Fano's to gelatine Experience with the Chatton-Lwoff method indicated that the ratio of cell suspension in Da Fano's fixative to gelatine was important in achieving a good stain. Mixtures ofDa Fano's fixative and gelatine were made using 2, 4 and 6 ml DaFano's fixative in 10 ml of mixture. These solutions were made immediately prior to use and had setting times of approximately 25 min at 45°C. Cell suspensions were centrifuged, aspirated almost to dryness and resuspended in a gelatinelDa Fano's mixture. Cells mounted in the 2 ml mixture were finely impregnated although a little faint. The 4 rnl mixture gave the best result, with well stained cells, clearly visible mouthparts and a pale golden/ buff coloured background with slight rippling. The 6 rnl mixture gave a granular, brown/orange appearance to the gelatine, with a purple speckle and pale bifurcating lines running through it. The cells stained so heavily that the kinetosomes appeared blurred and enlarged, the infraciliature was not visible and the cytoplasm was granular. 1 rnl Da Fano's fixative was added to 2 ml non-saline gelatine. This ratio proved intermediate between the 2 ml and the 4 ml determined above and was used as the normal method in subsequent tests. 2. Salinity of the gelatine Since Da Fano's fixative contains salt, additional salt was omitted from the gelatine in the above
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Fig. 4. An individual Tetrahymena treatedwith Parducz's fixative . Scale bar = 50!!m.
test. Addition of salt (0.05 %) resulted in gelatine which had a darker colour and stronger rippling with wavy lines. 3. Drop size and Spreading
In previous experiments the quality of staining was found to depend on the thickness of the gelatine; cells lying deeper in the gelatine failed to stain as well as those nearer the surface or in thinner gelatine. Cells present in areas of very thin gelatine showed considerable distortion . A drop size of approximately 10 ul proved optimal for a coverslip 22 mm square. This can be spread using a mounted needle or another coverslip as for blood smears. 4. Source of gelatine Rousselotgelatine was used as the control and was compared with gelatine from two other sources (Grayslake and leaf) and also with an equal mixture of Rousselot gelatine and glycerol (cf. Mayer' s albumen ). All were prepared without salt and pre-mixed with Da Fane' s fixative . The test cells were a mixed marine culture washed once with Da Fano ' s fixative and once in distilled water prior to mounting. Grayslake gelatine produced a heavil y veined surface . The cells were well stained although both sides were not visible. Leaf gelatine gave the best results in terms of cell clarity, both surfaces were equally visible and well stained . The background had dark, scalloped lines, like fish scales , running through it which obscured the cell features in places . 20 Arch. Protistenkd. Bd. 135
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Fig. 5. An individual from a mixed marine culture treated with Bouin's fixative. Scale bar == 50flm.
The use of glycerinated gelatine gave much lighter cells, finely impregnated on a more rippled background. The amount of random deposition was greater than the control and the cells had additional lines due to pellicular folding, making interpretation difficult. 5. Albumen as an alternative mountant Mayer's albumen (egg white and glycerol), used in the protargol stain, was used as a mountant. Coverslips were prepared with a thin layer of albumen and air dried on a hot plate at approximately 40°C. Cells were fixed, washed in X H 20 and dehydrated through 70 % , 95 % and 100 % EtOH. A drop of cell suspension was dropped onto an inclined coverslip, so that the drop ran, spreading the cells. The mounted cells were left to air dry for 15 min on the hot plate then transferred to 3 % AgN0 3 in EtOH. Exposure to UV was also under alcohol, cooled to reduce evaporation by placing it on an ice tray. Both freshwater and marine cells were fixed in Champy's fluid,Schaudinn's fixative or Parducz's fixative. Neither of the cultures fixed with Champy's fluid exhibited appreciable staining and the cells showed heavy granularity. The result for Schaudinn's fixative was similar but the cells were a deep purple. Cells fixed with Parducz's fixative were a fairly uniform orange colour and had stained weakly but the colour of the cytoplasm obscured much of the detail. The background albumen was stained orange in the freshwater sample but not in the marine one.
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Fig. 6. The effect of heat drying on the gelantine film. Scale bar = 200urn.
Variation in the Setting Conditions Six regimens were investigated: 1. Cold, moist (control)
A shallow plastic tray lined with moist absorbant paper was refrigerated (at approximately - 20°C). Just before use the paper surface was moistened and a smaller plastic tray used as a lid.
2. Cold, dry As (1) above, but without a lid. 3. Room temperature, moist As (1) above, but not refrigerated. 4. Room temperature, dry As (3) above, but without a lid.
5. Hot, dry The coverslips were put onto a hot plate (approximately 40°C) for 5 min without a lid. 6. Very cold, dry Two methods were used to increase the cooling rate of the coverslips ; (1) they were set on a tray 20'
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Fig. 7. The effect of rapid cooling on the gelatine film. Scale bar = 20U urn.
over a mixture of dry ice and ethanol or (2) they were set on a smooth metal block pre-chilled to -20 °C. The results indicated that the most important factor in producing a light, smooth gelatine layer was to keep the coverslips cold, but not too cold. Coverslips from regimens 1 and 2 had an even gelatine coating with fine veined lines, those from 3 and 4 had fragmented into a network of taut, orange lines in a ladder pattern. The surface between these lines was speckled with maroon spots. The preparations set under moist conditions (i.e. using a lid) were marginally better than those without. The heat dried coverslips had a slightly greyer colour than the control, with a grainy texture and areas of deep red which stuck to the coverslips in taut, ragged sheets (Fig. 6). The dry ice/ethanol mixture produced covers lips to which the gelatine did not adhere well , most had become detached and the rest was in stippled patches . Setting on the metal block crazed the gelatine , more so where the gelatine was thicker (Fig . 7). Silver deposited in these cracks. The cells were well stained (Fig . 8). Variation in AgN0 3 Impregnat ion Using the original Chatton-Lwoff method, the time of impregnation was varied at 30, 60 and 120 min at 10 "C , At 120 min the impregnation was very dense and had a greater amount of random deposition . Shorter impregnation times were more satisfactory , but there was little difference between them. Using the modifications suggested by the preceding result s (see the methodology given below) ,
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Fig. 8. The effect ofrapid cooling on an individual Tetrahymena. Scale bar = 50[1m.
the impregnation time was varied again, using 1, 5, 10, 15,20,25 and 30 min. All slides stained well, although the shorter times produced more finely impregnated cells. The 1 min impregnation showed well stained infraciliature and mouthparts, the cells were sufficiently clear to allow the kinetosomes on the under surface to be seen. The 5 min impregnation revealed the infraciliature more strongly but there was some deterioration in the quality as the impregnation times increased. Silver nitrate was added to the gelatine directly to avoid impregnation all together. Neither Da Fano's fixative nor salt was added to the gelatine mixture. Tetrahymena were used as test cells and concentrations of 1, 3, 5, and 10 % AgN0 3 were used in addition to a control sample without silver impregnated in the normal manner. Cells were exposed, under cold water, to UV after mounting. The staining was faintest at the low concentrations, increasing to an acceptable level at 10 %, though random deposition was also worst on this slide, offsetting the advantage of the shortened protocol. The background gelatine was crystal clear at 1 % silver, but the stain was too faint to be of value (Fig. 9). Silver nitrate (3 % in EtOH) was used as fixative for Tetrahymena; some cells were mounted in Mayer's albumen and others exposed to UV in a suspension before being mounted in gelatine. Both preparations gave similar results, the cells had not stained and both the cells and the background were a granular, opaque white. Cells were treated with mercury orange (PEARSE 1972) to locate concentrations of thiol groups. Cells fixed with osmium or formalin showed no discemable differentiation.
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Fig. 9. Tetrahymena mounted gelatine containing 1% AgN03 • Scalebar = 200!!m.
Variations in Exposure/development Tetrahymena were stained using the modified protocol (see below) and exposed to UV for a range of times from 10 to 70 min increasing in 10 min intervals. Results showed that there was no change in the appearance of the preparations after 40 min; the gelatine was a darker, orange-tan colour and the kinetosomes appeared enlarged in comparison to the shorter exposure times (Figs. 10, 11). The infraciliature was visible only as a faint line but this was not consistent. Shorter exposure times gave better results; 10 min gave the clearest result, the cells were lightly coloured with visible mouthparts and kinetosomes and although not initially very clear had improved within a week so that both cell surfaces were equally clear and the infraciliature was easy to see. Attempts were made to develop the image chemically. Using a developer common in the protargol method (2.5 % hydroquinone, 12.5 % sodium sulphite, 20% sodium carbonate in x HzO) preparations made by the modified protocol were incubated for 5, 10, 15, 60, 120, 180 min and overnight at room temperature. None of the preparations developed as well as under UV; the cells were cream coloured opaque shapes with no differential staining (Fig. 12). Some deposition became apparent at the longest developmental period but kinetosomes could not be distinguished.
Variation in Washing In all studies preparations were washed thoroughly prior to dehydration.
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Fig. 10. An individual Tetrahymena exposed to UV light for to min. Scale bar = 501!m.
Variation in Dehydration, Clearing and Mounting Preparations were routinely cleared by incubation in two xylene baths , with at least 10 min in each. Clove oil was used on a Tetrahymena preparation to see if the clarity of the cells could be improved. The results showed that there was a marginal improvement but not enough to warrant the increased cost of routinely using clove oil.
Discussion Fixation Correct fixation is the key to good staining. Of the fixatives used in this study, Champy's fluid produced the most consistent good results. The fixative properties of osmium tetroxide are thought to be due to its oxidation of unsaturated lipids and it has been suggested that it forms crosslinkages between lipid chains. This would account for the better shape preservation observed with osmium based fixatives. Chromium salts, which are oxidative, are thought to fix proteins and to react primarily with carboxyl groups forming coordinates with OH and HN z groups. Mercury ions behave in a similar fashion to chromium and have an affinity for the acid groups on proteins, especially carboxyl and hydroxyl. They differ, however, in that mercury ions have a high affinity for thiol groups (SH) (PEARSE 1980). Formalin is the classical fixative for proteins and a principal ingredient ofDa Fano's fixative.
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Fig. 11. An individual Tetrahymena exposed to UY light for 40 minutes. Scale bar = 50!lm.
Formaldehyde reacts with water to form its monohydrate, methylene glycol, and low molecular weight polymeric hydrates. These reactions are not rapid, which is why, for the sake of reproducibility, formalin solutions should be allowed to equilibrate for several days before use. The active ingredient is thought to be formaldehyde and not its hydrates or polymers. It is able to form crosslinkages in proteins, particularly involving amino, imino and amido, peptide, guanidy1, hydroxyl, thio1 and aromatic rings. It is thought to reduce S-S links to two SH groups which may then be linked by a methylene bridge (S-CH 2-S). This latter is readily ruptured by hydrolysis, possibly simply by washing (PEARSE 1980). The best results were obtained with Champy's, Helly's and Parducz's fluids, which are all heavy metal fixatives with chromium and/or mercury. XINBAI (1976, 1980a, b) found a mixture of saturated mercuric chloride and osmium tetroxide, as in PARDUCZ'S fixative but in different proportions, was efficient as a fixative prior to silver nitrate impregnation. The exact proportion of HgC12 : OS04 depended on the ciliate and its physiological condition. Other fixatives did not give such good results, particularly ZFA and Zenker's fluids, which are similar to Helly's fluid, except that Zenker's fluid contains acetic acid instead of formalin and ZFA contains acetic acid as well as formalin. Acetic acid therefore seems to interfere with the impregnation by silver nitrate. Susa's and Bouin's fluids, which gave poor results, also contain acetic acid, which is generally added to fixatives to improve their penetrating properties although it also causes swelling in most tissues (LEE 1937). Schaudinn's fluid, alcoholic mercuric chloride, allowed little impregnation. Ethanol causes protein precipitation and its fixative properties rely on the conformational changes induced (PEARSE 1980). It is important in silver amplification that the
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Fig. 12. An individual Tetrahymena impregna ted and treated with chem ical developer. Sca le bar = 50fl m.
nucleus of deposited silver atoms should not be too small or too dispersed and it is possible that the confonnationa1 changes separa te or occlude sites of potenti al deposition so that nuclei of silver atoms cannot form , Da Fano' s fixative , as a primary fixative, produc ed severe distortion in the cells, and staining was crude. The lack of shape retention is a recognised failing of the aldehyde fixatives which prim arily affect protein, not lipid . However , as a post fixative , the reducing properties of the formalin modified the oxid ative properties of the initial fixatives. The time required in Da Fano 's fixat ive was found to be sho rt, and in fact a single wash in a centrifuge tube proved to be effec tive . A second wash in distilled water removed any residual chromium salts. XINBAI (l980b) uses a post-fixation wash with NaCI + CoCJ2 to promote the staining of deeply sinking features . G e l a t ine Mo u n ti ng The principl e of silver staining is one of amplification; minute deposits of silver are made visible by deposition of more silver on that already present , as in photography. For additional silver to be deposited , silver ions must be present , and in photograph y this comes from unredu ced silver halide in the emulsion ; in histology care must be taken that sufficie nt silver is carried into the developer. Th is role was fulfilled by silver preci pitated by the Da Fano' s fixative mixed with the gelatine. The original Chatton-Lwoff method requ ired some experience to make it perfonn well, and in particular the amount of cell suspe nsio n in Da Fane ' s fixative that was mixed with the gelatine. To overcome
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this variability, the Da Fano's fixative and gelatine were mixed in predetermined proportion and added to the cells. When spread thinly, this method gave the correct quantity of deposited silver. The silver solution must diffuse through the gelatine, so that deeper lying cells often fail to stain while those above are perfectly impregnated. For this reason, thick layers should be avoided. The setting conditions were most important for a good, even gelatine film. When the cooling was too rapid, the gelatine did not set evenly and broke apart. When cooling was too slow, then dessication was a problem. Da Fano's fixative set the gelatine in about 25 min, but because the gelatine was warm it lost some water even in a moist chamber. Dessication can lead to premature deposition of silver ions and conformational changes in the cell proteins. XINBAI (1980b) used agar-agar instead of gelatine for the impregnation of deeply sinking features, though the outer cortical features did not impregnate well. The role of the gelatine and the agar-agar in this case is unclear. Impregnation SAMUEL (1953 a) studied the effect of impregnation time and pH on the impregnation of ganglion sections. He concluded that sections could be over impregnated and that high pH values (8.7) gave undifferentiated impregnation. Low pH values (6.8) gave rise to random deposition especially for shorter impregnation times. No attempt was made to control the pH of the impregnation solutions in this study. Prior to impregnation, the pH of the silver nitrate solutions was in the range 7.0 to 7.8. The biochemical composition of the protozoan cell has not yet been established with any degree of certainty. Silver was consistently deposited, with both the inorganic and protargol methods, at the basal bodies of cilia. This may have been due to thiol groups located at the ends of the microtubular bundles forming the cilium or from the binding sites for Cat ' or Mgt" which are known to affect ciliary beat (SLEIGH 1973; STEBBINGS and HYAMS 1979). The infraciliature was stained by silver nitrate, its location corresponded to that of fibrils in or below the pellicle in most ciliates. There appear to be several kinds of micro-fibrils which are generally classified by size, only the microtubules being reasonably well defined. In Euplotes physical features which correspond to the argentophilic dorsal network have been called "rugae", which RUFFOLO (1976) suggested were tubular extrusions of the pellicular membrane. This is difficult to accept since the rugae occur in pairs, whereas the argentophilic structure is a single line. FOISSNER and SIMONSBERGER (1975 a) demonstrated that in the dry silver technique, silver did not accumulate in folds of the pellicle. It is more plausible that the sites of deposition are the cylindrical fibrils lying underneath the inner alveolar membrane (GRIM et al. 1980; FOISSNER 1978; FOISSNER 1976b; FOISSNER and SIMONSBERGER 1975a, b). In Tetrahymena the longitudinal line between rows of cilia align with a microtubular bundle (GRIM et al. 1980). In Paramecium, the infraciliature is revealed by deposition of silver at the underside of the junction of adjacent alveolar membranes, where no cellular component was differentiated, and in the uppermost third of pellicular ridges, where a fibro-granular material was located (FOISSNER 1977). No differentiated cell component was found at the site of the infraciliature in Colpidium colpoda (FOISSNER and SIMONSBERGER 1975b; FOISSNER 1981). Silver nitrate can be used to demonstrate the presence of disulphide bonds in keratins (see PEARSE 1972 for review), provided the pH of the impregnation medium is sufficiently well controlled. It was claimed that at high pH values, thiol groups were not detected. Thiol groups, common in fibrous structural proteins, are a prime candidate for the effective site of silver deposition. However it is likely that more than one site was responsible for silver reduction; silver will be reduced at any site where the redox potential is correct. XINBAI (1976, 1980a, b) has developed a series of methods in which the cortical depth of impregnation is controlled. The fixation, post-fixation and mountant are tailored to the particular cells being impregnated. These methods are particularly useful in revealing deeply sinking
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structures, such as the oral apparatus in Paramecium (XINBAI 1980 b). In general agar-agar inhibits the development of the stain in the outer cortical layers. Increasing the proportion of OS04 in the fixative increases the penetration of the stain. Furthermore, actively feeding cells require more OS04 in the fixative than do hungry cells. The methodologies for deeper features also require postfixation with NaCl + CoCh. Further studies are required to resolve the histochemical identity of the groups active in silver deposition. Reduced bound silver can be differentiated from deposited metallic silver nuclei by sulphite washing (SAMUEL 1953 b); calcium binding sites can be identified by treatment with potassium-ferricyanide and sodium thiosulphate (PLENK 1975). Illumination Whilst being reduced by light, the gelatine must be protected from detaching from the glass of the coverslip and from dessication. Infra-red light causes heating of the preparations which will soften the gelatine layer. UV light sources, low in infra-red output, were therefore used to expose the slides which were under chilled water to absorb any residual infra-red. FRANKEL and HECKMANN (1968) found that the quality of the stain was critically dependent upon the initial exposure to light and found it convenient to continue to expose the cells through the dehydration phase. The results presented here were conducted under standard conditions of normal room light followed by UV, although some trials were made using tungsten illumination in place of UV, which developed the stain just as well. The heating effect with tungsten meant that the covering water had to be replaced during the exposure, so the UV light was used for its greater convenience. The process of silver deposition is reversible and will amplify the image so long as there is a sufficient pool of available silver ions. Should this pool become limited, photo erosion of the image will occur; fine features will be lost and the silver deposited on the strong features. It is therefore important not to overexpose the slides, although if sufficient silver was deposited in the gelatine matrix this will not be a practical limitation. SAMUEL (1953 a) developed silver nitrate impregnations of ganglion sections using an hydroquinone/sulphite developer. It is unclear why chemical reduction does not develop the latent image in the case of ciliated protozoa. Dehydration, Clearing and Mounting Soluble reaction products must be removed from the gelatine matrix because the reactions are reversible and fading would result. Thorough washing must preceed dehydration. Experience has shown that synthetic mounting media are not as durable as Canada balsam, which may be softened and re-mounted many tens of years after a preparation was originally made. Slides in the BM(NH) collections dating back well into the last century are still in their original balsam mounts, whereas synthetic media have often required re-mounting due to deterioration as little as 5 years after preparation. General comments Slides often seem to improve with storage and although it is not possible to perform direct comparisons those which have not stained particularly well are worth re-examining a few weeks after staining. The mechanism for this is unclear, but may be associated with the slow penetration of the balsam clearing the cells more effectively, and making previously indistinct features visible. During an active experimental procedure stocks will be frequently subcultured to ensure sufficient material for staining. In consequence, most tests are carried out on cultures with high vitality which generally impregnate most easily. Old cultures often fail to stain as well, if at all (observations from this laboratory; FOISSNER, pers. comm.; FRANKEL and HECKMANN 1968).
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Conclusions The methodology which takes the above points into account is more rapid than the ChattonLwoff method as specified by CORLISS (1953). Routinely in this laboratory, the procedure takes about 2 h from live cells to finished preparation. It may be summarised as a working schedule:
1. Fix cells in Champy's Fluid for 3 min. Champy's fluid consists of7 parts Cr03 (1 % aq), 7 parts K2Cr207 (3 % aq) and 4 parts OS04 (2 % aq) all at room temperature. The first two components may be stored as a mixture, but the osmium should be added immediately prior to use. Centrifuge the cells, aspirate as much culture fluid as practical, disperse, add approximately 1-2 ml of fixative.
2. Wash once with Da Fano's fixative. DaFano's consists ofCoN0 3 1 g; NaCll g; formalin 10 ml; XH20 90 ml. Formalin should be unneutralised, and left for at least 3 d in its diluted form. Fill the centrifuge tube with Da Fano's fixative. 3. Wash once with XH 20.
Centrifuge the cells, aspirate, disperse and re-fill the tube with XH 20. Leave for approximately 1 min.
4. Mount cells in GelatinelDa Fano's mixture. Immediately before use, mix 2 ml gelatine (15 %) with 1 ml Da Fano's fixative. Add 3- 5 drops of the mixture to the aspirated cells in a centrifuge tube. Take a small drop of the cell suspension (10 ul) and spread with a mounted needle on a 22 mm square coverslip.
5. Set the gelatine on an ice tray for 3-5 min. Moisten a piece of absorbant paper (e.g. filter paper) on a tray and freeze in the ice-box of a domestic refrigerator or domestic freezer. Just before use, moisten the surface with a little water spread with the fingers. Place the coverslips on the tray beneath a lid. 6. Incubate in AgN0 3 at 10 °C for 5 min. The AgN0 3 is 3 % aqueous and may be re-used so long as it remains clear.
7. Develop the coverslip. Expose to UV for 10 min under chilled XH20. 8. Wash thoroughly in XH 20. Wash at least 3 times in cold XH20. 9. Dehydrate, clear, mount. Dehydrate through the alcohols, 50 %, 70 %,95 %, 100 %, 100 %,5 min in each. Clear in two changes of xylene, 10 min in each. Mount in Canada balsam.
Acknowledgements We wish to express our gratitude to Dr. W. FOISSNER, University of Salzburg, and Dr. S. XINBAI, Harbin Pedagogical College for their informed discussion and to the Trustees of the British Museum (Natural History) for providing a vacation studentship for Ms. CAUSTON to carry out this work at the Museum.
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Literature AUGUSTIN , H., FOISSNER, W., and ADAM , H. (1984) : An improvedpyridinated silver carbonate methodwhich needs few specimens and yields permanent slides of impregnated ciliates (Protozoa, Ciliophora). Mikroskopie 41: 134-137. BODlAN , D. (1936): A newmethodforstaining nerve fibersandnerveendings inmountedparaffinsections. Anal. Rec. 65: 89-97 . - (1936): The staining of paraffinsections of nervous tissueswithactivatedprotargol. The role of fixatives . Ibid. 69: 153-162. CHATTON, E., et LWOFF, A. (1930): Impregnation, pardiffusionargentique, de I'infraciliature de ciliesmarins et d'eau douce, apres fixation cytologiqueet sans dessication. C. r. Seanc, Soc. BioI. 104: 834-836. - (1935): Lesciliesapostomes. Morphologie, cytologic, ethology, evolution, systematique. I. Apercuhistorique et general. Etude rnonographique des genres et des especes. Archs. Zool. expo gen. 77: 1-453. - (1936): Techniques pour l'etude des Protozoaires, specialement de leurs structures superficielles (cinetome et argyrome) . Bull. Soc. fr. Microsc. 5 : 25-39 . COLE , R. M., and DAY, M. F. (1940): The use of silveralbumose(protargol) in protozoological technique. J. Parasit. Suppl. 26: 29-30. CORLISS, J . O. (1953): Silver impregnation of ciliated protozoaby the CHATTON-LwOFF technic. StainTechno!' 28: 97-100. DAVENPORT, H. A., PORTER, R. W. and MYHRE, B. A. (1952): Preparation and testing of silver-protein compounds . Ibid. 27: 243-248. DRAGESCO, J. (1962): L'orientationactuelle de la systematique des cilieset la techniqued' impregnation au proteinate d'argenl. Bull. Microsc. appl. 12: 49-58 . FERNANDEz-GALIANO, D. (1966): Une nouvelle methode pour la mise en evidence de I'infraciliature des cilies. Protistologica 2: 35-38. ( 1976): Silver impregnation of ciliated protozoa: Procedure yielding goad results with the pyridinated silver carbonate method. Trans. Am. microsc. Soc. 95 : 557- 560. et RUIZ, S. (1972): Descriptiond' une nouvelle espece de cilie, Colpidiumuncinatum. Protistologica8 : 295- 298. FLEURY, A., IITODE, F., DEROUX, G., ct FRYD-VERSAVEL , G. (1985) : Unite et diversite chez les hypotriches (Protozoaires, cilies) II. - Elements d'ultrastructure comparee chez divers represenrants du sous-ordre des Euhypotrichina. Ibid. 21: 505-524. FOISSNER, W. (1967): Wimpertiere im Silberpraparat. Ein .rrockenes" Verfahren zur Darstellung des Silberliniensystems. Mikrokosmos 56: 122-1 26. (1967a): Erfahrungen miteiner trockenen Silberimpragnationsrnethode zurDarstellungargyrophilerStrukturenbei Protisten. Verh. zoa!.-bot. Ges. Wien ll5 : 68-79. (1976b) : FunfzigJahre Forschung am Silberliniensystem der Ciliaten. Naturk. Jb. Stadt Linz 22: 103-11 2. (1977): Elektronenmikroskopische Untersuchung tiber die Lage und Natur des Silberliniensystems von Paramecium. Mikroskopie 33: 260-276 . (1978): Euplotes moebiusi f quadricirratus (Ciliophora, Hypotrichida) I. Die Feinstrukturdes Cortex und der argyrophilen Strukturen. Arch. Protistenkd. 120: 86-117. (1981): Das Silberliniensystem der Ciliaten: Tatsachen, Hypothesen, Probleme. Mikroskopie 38: 16-26. undSCHUBERT, G. (1977): Morphologie der Zooideund Schwarmervon Heteropolaria colisarumgen. nov., spec. nov. (Ciliata,Peritrichida),einer symphoriontenEpistlidaevon Colisafas ciata (Anabantoidei, Be1ontiidae). Acta Protozool. 16: 231-247. und SIMONSBERGER, P. (l975a) : Vergleichende licht- und rasterelektronenmikroskopische Untersuchungen an trockenpriiparierten Silberliniensyslemen von Ciliaten (Protozoa). Mikroskopie 31: 193-205. - (1975b): ElektronenmikroskopischcrNachweisder subpelliculiiren Lage desSilberliniensystemsbei Colpidium colpoda (Ciliata, Tetrahymenidae). Protoplasma 86: 65-82. FRANKEL, J. , and HECKMANN , K. (1968): A simplified Chalton-Lwoff silver impregnation procedure for use in experimental studies with ciliates. Trans. Am. microsc. Soc. 87: 317-321. GRIM , J. N. (1974): A protargol study of the fiber systemof the ciliate Haltaria. Ibid. 93: 421-425 . - HALCROW, K. R., and HARSHBARGER, R. D. (1980) : Microtubules beneath the pellicles of two ciliate protozoaas seen with the SEM. J. Protozoal. 27: 308-310. HONIGBERG, B. (1947):Thecharacteristicsof theflagellateMonocercomonas verrens, sp. n. from Tapirusmalayanus. Univ. Calif. PubIs Zoo!. 53: 227- 236. JONES, D. B. (1973) :Silverstains. In: P. GRAY (ed.), The EncyclopediaofMicroscopyandMicrotechnique. NewYork.
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KIRKBY, H. (1945): The structureof the common intestinaltrichomonad of man. J. Parasit. 31: 163-174. KLEIN, B. M. (1926): Uber eine neue Eigentiimlichkeit der Pelliculavon Chilodon uncinatus EHRBG. Zoo!' Anz. 67: 160-162. KOZLOFF, E. N. (1960): Morphological studies on holotrichous ciliates of the family Hysterocinetidae I. Hysterocineta eiseniae BEERS and Ptychostomum campelomae sp. nov. J. Protozoo!. 7: 41-50. LEE, A. B. (1937): The Microtomist's Vade-mecum (eds. GATENBY, J. B., & PAINTER, T. S.). 10thed. London. MADRAZO-GARIBAY, M., et LOPEZ-OCHOTERENA, E. (1973): Nouvellesprecisionobtenuesa I' aidede la techniquede FERNANDEZ-GALIANO et concernantentre autre I'infrastructureciliairedans Ie genre Paramecium. Protistologica 9: 481-485. MOSKOWITZ, N. (1950): The use of protein silverfor stainingprotozoa. Stain Techno!. 25: 17-20. NIELSEN, E. M., and DEBAULT,L. E. (1978): Transformation in Tetrahymena pyriformis: descriptionof an inducible phenotype. J. Protozoo!' 25: 113-1l9. PEARSE, A. G. E. (1972): Histochemistry Theoretical & Applied. Volume2. 3rd ed. Edinburgh. - (1980): Histochemistry Theoretical & Applied. Volume I. Preparative and Optical Technology. 4th ed. Edinburgh. PETERS, A. (1958): Staining of nervous tissue by protein-silvermixtures. Stain Techno!' 33: 47-53. PLENK, V. H. (1975): Differentiation of silver-calciumsalt staining methods using a photographic developer. Mikroskopie 31: 73-76. ROBERTS, D. McL., and WARREN, A. (1987): An inexpensive apparatusfor automaticcontinuousdehydration. Stain Techno!. 62: 211-215. RUFFOLO, J. J. (1976): Fine structureof the dorsalbristlecomplexandpellicleof Euplotes. J. Morph. 148: 469-488. SAMUEL, E. P. (1953a): Impregnation and developmentin silver staining. J. Anat. 87: 268-277. - (l953b): The mechanismof silver staining. Ibid 87: 278-287. SLEIGH, M. A. (1973): The Biologyof Protozoa. London. STEBBINGS, H., and HYAMS, J. S. (1979): Cell Motility. London. TORCH, R., and HUFNAGEL, L. (1961): Differentiation of speciesof Diophrys by meansof silverstaining.Bio!. Bull. mar. bio!' Lab., Woods Hole 121: 411 (Abstr.). TUFFRAU, M. (1967): Perfectionnement et pratiquede la techniqued' impregnation au protargoldes infusoirescilies. Protistologica 3: 91-98. XINBAI, S. (1976): Studies on conjugation in Stylonychyia mytilus II. Morphogenesis of argentophilic system. Acta zoo!. sin. 22: 71-83 + 8 plates (In Chinese). (1980a): Morphology and morphogenesis in Pseudomicrothorax dubius. Ibid. 26: 71-79 + 2 plates (In Chinese). (1980b): The morphology and morphogenesis of the buccal apparatus in Paramecium and their phylogenetic implications. I. Morphology of buccal apparatus. Ibid. 26: 205-212 + I plate (In Chinese). Authors' addresses: D. McL. ROBERTS, Dept. Zoology, British Museum (Natural History), Cromwell Road, London SW7 5BD, England; Ms. H. CAUSTON, School of Biology, Kings Building, University of Edinburgh, EdinburghEH9 3JT, Scotland.