Journal of Protistology
Online ISSN : 2433-412X
Regular Article
An alternative workflow for the extraction and study of testate amoebae (protist: Amoebozoa, Rhizaria, Stramenopiles) through vacuum filtering and grid-based low-vacuum scanning electron microscopy (SEM): Illustrated from scottish leaf litter
Jim Buckman Vladimir Krivtsov
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2022 年 54 巻 論文ID: e005

詳細
Abstract

The extraction and study of testate amoebae from peat, leaf litter, soils and water samples, typically involves wet-sieving, followed by hand picking of material for examination by optical and scanning electron microscopy (SEM). Herein, an alternative workflow is described for the extraction of testate amoebae, that has successfully been applied for a range of sample substrates (leaf litter, mosses, liverworts, lichens, sands and gravels), as well as water samples, which involves vacuum filtration of testate amoebae suspended in water and the methodical analysis of the dried filter paper in a grid-like search pattern using SEM. Leaf litter from the Riccarton Campus of Heriot-Watt University is used here to illustrate the workflow. The method benefits from its ease of use and speed, observation of all size ranges of testate amoebae present, including partial samples, and the placing of the recorded material within the context of other mineralised micro-organisms, micro invertebrates, as well as the size and composition of any detrital grains present. The method is unlikely to introduce user bias, is of relatively low cost, ensures a lack of contamination, and facilitates the study of testate amoebae within their larger environmental context. Comparison with wet-sieved material gives comparable results, with differences controlled by the size of the final sieve mesh size.

INTRODUCTION

The extraction of testate amoebae, a general term for a polyphyletic group of taxa belonging to Amoebozoa, Rhizaria and Stramenopiles (Kosakyan et al., 2016; Adl et al., 2019), is by no means a trivial matter. Many methods have been used for the collection and preparation of testate amoebae, predominantly associated with collecting from peat samples (Hendon & Charman, 1997; Charman et al., 2000; Booth et al., 2010; Zheng et al., 2019). Techniques typically involve soaking samples overnight in distilled water, and/or boiling for 10 min, often with the addition of an exotic marker such as Lycopodium spores where an estimate of density per cm3 is required (Booth et al., 2010). Material is then wet-sieved, with a large mesh sieve (300 µm) to capture oversize particles including organic detritus (leaves, macro-biota) and large particulate inorganics (sand), and a smaller mesh (10–15 µm) to selectively collect testate amoebae (Hendon & Charman, 1997; Charman et al., 2000; Beyens & Meisterfeld, 2001; Booth et al., 2010). Other authors (Payne, 2009; Wall et al., 2009) recommending the use of no micro-sieve in order to also collect the smaller (less than 10–15 µm) testate amoebae fraction. Various permutations of this process have been put forward, such as that of Zheng et al. (2019), which adds in an additional chemical processing stage with the aim of removing extraneous organic and minerogenic material. Samples are then typically selected, examined and characterized by optical microscopy, often with selected examples further studied by scanning electron microscopy (SEM) (Booth et al., 2010).

The workflows outlined above require the use of specialist fine mesh sieves (10 or 15 and 300 µm), that may not always be available. In addition, due to the inherent reusable nature of such sieves, even with careful cleaning between uses, a degree of cross-contamination and clogging may occur. Therefore, we devised a workflow that utilises readily available, single-use, filter papers, which is presented here as an alternative to wet-sieving. This method has been successfully applied by one of the authors in the examination of testate amoebae by SEM, from water samples (stream, pond and surface road gutter flow), plant material (leaf litter, mosses, liverworts, lichens) as well as beach sand and shingle samples. The new method is described herein and illustrated through an investigation of testate amoebae recovered from a sample of leaf litter. Detailed identification and discussion of the recorded taxa is beyond the scope of the current paper, although comparison of the recorded material with an identical aliquot processed by wet-sieving is discussed. In addition, the positive features, along with a few minor limitations, of the workflow are commented upon.

MATERIALS AND METHODS

The workflow involves a two-part process: i) vacuum filtration of water medium containing testate amoebae on to filter paper, ii) methodical examination by SEM in low-vacuum mode (Fig. 1). To illustrate the technique, a sample of leaf litter was obtained from the Riccarton Campus of Heriot-Watt University (September 2021). Less than 1 g of leaf litter was placed within a suitable container to which water was added. The sample was then vigorously agitated to separate and suspend any testate amoebae present, and the water immediately decanted into a vacuum filter device, and passed through a 0.4 µm (47 mm diameter) filter paper, which retained all testate amoebae that were present. The filter paper was then vacuum dried. Once dried, the sample was examined without sample coating using a Quanta 650 FEG SEM (FEI, Hillsboro), operated in low-vacuum mode (0.82 Torr), at an operating voltage of between 5 and 20 kV, utilising a backscattered electron (BSE) detector. The surface of the sample was examined in a grid-pattern (Fig. 2A), with a horizontal field of view of 500 µm (Fig. 2B). The grid-pattern search was carried out manually, utilising the full frame shift feature of the SEM. Where testate amoebae were observed in the field of view, detailed images were taken at a higher magnification (Fig. 2C–E) to determine taxonomic assignment and total numbers present.

Figure 1. Workflow for new sampling and analysis technique.

Red arrows indicate workflow followed in present study, black arrows are alternative procedures.

Figure 2. Schematic of scanning workflow and examples of results recorded.

(A) Theoretical distribution of testate amoebae, and areas scanned using manual grid method. Green dots represent testate amoebae that are observed, red dots are ones that would be missed. (B) Example of scanned tile, with examples of testate amoebae circled, white arrows indicate the positions of other testate amoebae. (C)–(E) Examples circled in (B), Trinema lineare, Euglypha tuberculata and Centopyxis indet.

In addition, to allow comparison between the current technique and commonly used wet-sieving techniques, a second identical aliquot was prepared using a combination of wet-sieving through 1 mm, 250 µm and 26 µm sieves. Material retained by the 26 µm sieve was transferred into water, and then passed through a 0.4 µm filter paper, which was then vacuum filtered and examined using the same SEM protocol as utilised in the filtration only method. To allow direct comparison between the two methodologies, a minimum of 100 detailed micrographs of observed taxa were recorded for both techniques. This was done to give an idea of what taxa would have been available for selection using a wet-sieving protocol.

RESULTS

New technique

The new method identified 111 individual testate amoebae referred to a minimum of 11 taxa (Table 1). Approximately 59% are siliceous scale forming taxa, from the superfamily Euglyphacea (Trinema lineare [34%], Trinema complanatum [1%], Tracheleuglypha acolla [8%] and 4 species of Euglypha [16%]), while 37% are agglutinated (Difflugia sp. [1%], Centropyxis indet [33%]) or organic (Arcella sp. [3%]) belonging to the superfamily Arcellacea, and 5% from the superfamily Cryptodifflugiaea (Cryptodifflugia sp.). Trinema lineare and Centropyxis are the dominant forms, with T. lineare showing an approximately 1 : 1 ratio with Centropyxis. It should also be noted that many of the Euglyphids were observed as components of long fecal pellets, 60–100×600 µm (Fig. 3A).

Table 1. Taxa recorded using both techniques
TaxaFiltered (n=111)Wet-sieved (n=133)
Trinema lineare3423
Trinema complanatum15
Puytoracia bergeri2
Tracheleuglypha acolla82
Assulina muscorum2
Corythion dubium1
Euglypha dickensii3
Euglypha hyalina1
Euglypha rotunda85
Euglypha tuberculata44
Euglypha compressa3
Difflugia sp.12
Difflugia pyriformis3
Centropyxis indet3344
Arcella sp.35
Cryptodifflugia sp.5

Values expressed as percentage rounded to nearest whole number.

Figure 3. SEM photomicrographs of testate amoebae in fecal pellets.

(A) Fecal pellet with Trinema, (B) with Trinema (white arrows) and Euglypha (red arrow).

Wet sieving technique

Wet-sieving identified 133 individual testate amoebae referred to a minimum of 13 taxa (Table 1). Approximately 47% are siliceous scale forming taxa, from the superfamily Euglyphacea (T. lineare [23%], Trinema complanatum [5%], Puytoracia bergeri [2%], Tracheleuglypha acolla [2%], Assulina muscorum [2%], Corythion dubium [1%] and three species of Euglypha [12%]), while 54% are agglutinated (Difflugia sp. [2%], Difflugia pyriformis [3%], Centropyxis indet [44%]) or organic (Arcella sp. [5%]) belonging to the superfamily Arcellacea. T. lineare and Centropyxis are the dominant forms, with nearly twice the number of Centropyxis than T. lineare). Six taxa were only observed from the wet-sieving technique: P. bergeri, A. muscorum, C. dubium, Euglypha compressa and Difflugia pyriformis, which account for 13% of the total recorded testate amoebae density. Clear examples of Euglyphid bearing fecal material were observed (Fig. 3B).

DISCUSSION

For both sampling methods, all smaller Trinema (those below 40-45 µm) were assigned to T. lineare, rather than the similar but typically larger species of Trinema enchelys, which is in general agreement with other authors (see Ogden & Headley, 1980; Todorov & Bankov, 2019); although no exact demarcation in terms of size is agreed upon for differentiation of the species. It is possible that some examples of T. lineare from the present study may represent T. enchelys, and given the variability of the form noted for T. lineare (here and in the literature) it is possible that the species could be further divided. Nevertheless, such points are not critical to the present paper, and although a major review of the genus would be worthwhile, this is not covered herein. With the exception of Centropyxis (see below), no other major taxonomic problems were encountered during the present investigation.

Comparison of taxa using different methodologies

Although there are differences in the details of the taxa recorded, both methodologies recorded similar occurrences and densities. Both sampling methods produced results where the taxa were dominated by Trinema and Centropyxis. Main differences noted are in the ratio of Trinema versus Centropyxis, with T. lineare relatively more abundant in comparison using the new filtered workflow, and Centropyxis with the wet-sieved preparation technique. Lower levels of T. lineare in the wet-sieved material, could be attributed to the use of a 26 µm mesh for the collection of material, as T. lineare from the present study has a size of up to 45 µm by 15 µm which could pass through the collection sieve. In addition, it should also be noted that most of T. lineare recorded from the wet-sieved material were noted as components within fecal pellets (Fig. 3B), which were too large to pass through the 26 µm sieve. The inclusion of T. lineare within fecal pellets is of particular interest, as these are unlikely to be hand-picked and would therefore potentially be missed using more traditional preparation and selection techniques. The wet-sieved material lacks Cryptodifflugia sp., which again could be attributed to the use of a 26 µm mesh sieve, as Cryptodifflugia (recorded herein) has a typical diameter of 10–15 µm which would easily pass through the collection sieve. Both of these examples highlight the advantage of the new workflow (using filter paper rather than sieving), and agrees with the limitations imparted by wet-sieving, and selection of the final sieve mesh size, as noted by previous authors (Payne, 2009; Wall et al., 2009).

Minor differences, such as the additional presence of P. bergeri, A. muscorum, C. dubium, E. compressa, Difflugia pyriformis within the wet-sieved material, but absent from the direct filtration method, are likely explained by the larger sample size of the former (n=133 as opposed to n=111), or may reflect minor variations in the composition of the leaf litter. The latter is also suggested by the occurrence, in small numbers, of Euglypha hyalina and Euglypha dickensii only within the sample prepared using the new workflow.

These results give confidence that the new workflow can be utilised in the study of testate amoebae, and that comparison to other areas that used techniques such as wet-sieving will be valid. The technique has also been successfully applied by the authors to testate amoebae from a range of other environments: water samples (ponds, streams, overland flow), mosses, liverworts, lichens and beach sands, and was developed in our previous studies of sustainable urban drainage systems (SUDS) and blue-green infrastructure (BGI) (Krivtsov et al., 2020a; 2020b).

Advantages of new technique

One of the chief advantages of the new workflow is that, compared to other methods that use wet-sieving, there should be significantly less sample bias in selection of material; as it is possible to image all material within the sample, including smaller and broken material which may otherwise be lost during sieving, as well as larger oversized taxa that may otherwise be retained by a 300 µm filtering sieve, or taxa contained within larger fecal pellets (Fig. 3) which may be overlooked during sample picking. The workflow also allows the observation of other associated microorganisms (Fig. 4), or indeed could be applied to the study of such material in their own right. Where relevant the testate amoebae can be placed within their sedimentological context (grain size, shape and composition), or direct association with organic plant material. In addition, the workflow utilizes field emission SEM analysis, rather than optical microscopy, which has higher resolution and therefore aids in taxonomic analysis; with the easier identification of the character of test plates, occurrence and nature of pores, presence of spines, as well as overall test shape and size. Finally, sample preparation should be far faster and less time consuming.

Figure 4. SEM photomicrographs of examples of other microorganisms that can also be recorded using the technique.

(A) Diatom, (B) chrysophacean cyst, (C) charophyte, (D) golden algae, (E) group of chrysophacean cysts, (F) heliozoan, (G) ciliate Coleps sp., and (H) dinoflagellate. Note material recorded from other samples, locations and environments.

Drawbacks and limitations of new technique

Due to the nature of the workflow, a number of factors need to also be addressed. Samples cannot be selectively positioned in terms of top, bottom, side view, therefore, what you see is what you get (Fig. 3). This proved to be a particular problematic area when trying to identify and quantify species within Centropyxis. Although material referred to Centropyxis indet., has not been further subdivided (using either method), it is likely that amongst others C. aerophilia, C. cassis, C. orbicularis and C. plagiostoma are represented. However, it was not possible to identify this material to species level in every case, so to simplify matters in the present case all Centropyxis were lumped together. This did not prove to be such a problem with other taxa observed. As samples were examined in low-vacuum SEM, and therefore not coated with a conductive coating (gold, palladium, carbon, etc.), the observed specimens could have been re-mobilised in water, washed and separated, concentrated and picked out using a dissecting microscope, allowing selective picking and sample orientation, which in the case of Centropyxis would have aided in clearer species differentiation. Due to the need to avoid undue levels of image overlap, and consequently double or triple counting of taxa, not all testate amoebae present will be observed (Fig. 2A). Testate amoebae on filters may be masked by clays, silt or fine sand sized detrital particles and organics. No transmitted light optical characterization can be performed, therefore details such as cell structure (i.e. nucleus shape, size, position), nature of pseudopodia and observations on colour cannot be made. Nevertheless, the reprocessing of material captured on the filter paper could be undertaken for optical analysis.

Finally, the technique clearly does not allow for the observation of live individuals. Where such observations are required it is recommended that an aliquot be separated for optical examination prior to sample preparation.

Further workflow modifications

The workflow can be modified to use a semi-automated method of image collection, forming montages from a collection of image tiles, through use of software such as “MAPS” (Fig. 5A). In which case, large areas of filter paper can be scanned using an overlapping grid pattern, with additional detailed micrographs taken at the end of the montage scan, or by pausing the collection of tile images during the collection of the montage. This would provide the additional benefit of generating a permanent record of the whole surface of the filter paper, which can later be reviewed to extract additional information. The slight overlap (10%) of each image ensures that the whole surface is examined, which should maximise the number of individual testate amoebae recorded (compare Fig. 5B with Fig. 2A), while minimising double counting of taxa through unnecessary image overlap. For further details on the grid imaging software used and its application see previous works (Buckman et al., 2014; 2020). It would theoretically also be possible to syringe water containing testate amoebae through smaller diameter cartridge filters, or use larger diameter filter papers folded to line a simple funnel apparatus.

Figure 5. Alternative automated grid search method.

(A) Screenshot of alternative automated grid search method, with backscattered electron images, using software such as “MAPS” (FEI, Hilsboro). Field of view of each scanned tile is approximately 1 mm. (B) Theoretical distribution of testate amoebae, same as in Fig. 2A, illustrating total coverage and no missing taxa (all green).

ACKNOWLEDGEMENTS

We acknowledge use of the scanning electron microscopy facilities at the Centre for Environmental Scanning Electron Microscopy (CESEM), Institute of GeoEnergy Engineering, Heriot-Watt University. We also thank staff at the Lyell Centre (Ryan Pereira and team) for loan of vacuum filtering equipment and provision of filter papers.

References
 
© 2022 Jim Buckman and Vladimir Krivtsov / © 2022 Japan Society of Protistology. This is an open access article under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
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