In general, methods to retrieve ciliates from soil samples can be divided into two direct and indirect methods (Foissner 1987). Direct methods are only appropriate for estimation of ciliates abundance if fresh, moist soil is used. Active ciliates in soil are low, as they depend on soil moisture (Adl and Coleman 2005). Alternatively, the indirect methods allow ciliate cysts formed owing to dry conditions in the environment to be made active, thus the potential diversity of ciliates in different types of soil can be retrieved. Most probable number (MPN) are indirect methods in which water, organic substances, mineral elements, and/or bacterial food sources are added to enrichment media to encourage the growth of soil protozoa (Foissner 1987). However, MPN methods have negative aspects, for example such methods are well known to select of some particular protozoan species if only one type of bacterial food is added. Moreover, the results of MPN methods vary depending on the enrichment protocol (Foissner 1987). Finlay et al. (2000) developed a method to estimate the potential abundance of free-living protozoa in soil, which overcomes some limitations and problems of the direct and most probable number methods. The key advantage of this method is that nothing more than adding filtered rain water to the soil is required (Finlay et al. 2000). It is suitable for the comparison of protozoa between different soils. In the present study, the method of Finlay et al. (2000) was followed to investigate abundance and richness of soil ciliated protozoa. The details of the method are described below:
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Sample treatment
After immediate return to the River Laboratory, soil samples were removed from the original plastic bag and the soil was then homogenised by thorough mixing in a clean 30 cm-diameter glass bowl. A sub-sample of soil (~50 g) was then taken and spread out as a layer in 15 cm diameter glass Petri dish and dried at room temperature (18-22 oC) for six days. After the soil for determination of ciliates was dried, it was passed through a 4 mm sieve to remove large objects such stones. This material was now called “air-dried soil”. This soil was used for ciliate-related work. The remaining soil was used to analyse soil characteristics
Incubation
To facilitate the development of ciliates, 5 g of air-dried soil was placed into a 5 cm diameter sterile plastic Petri dish. This was replicated three times for each sample. A measured volume of filtered rain water was then added to samples - sufficient to produce a slurry. Whatman® syringe filters with a 0.2 µm diameter pore were used to filter rain water to exclude ciliates and other microbes. Slurry samples were then incubated in the dark at 15 oC and ciliates were investigated after 4 days and 10 days of incubation.
Enumeration of ciliates
After 4 days and 10 days of incubation, the wet soil prepared as above was pressed to release water. A 50 µl sample from the water runoff was taken and placed on to a glass Sedgewick-Rafter Chamber. Ciliate numbers in the 50 µl subsample were determined under a compound microscope at a magnification of 40-125 X. This was repeated five times for each Petri dish of soil.
Calculations
The number of ciliate cells g-1 oven-dried weight of soil = a x (V1+ V2)/(50 x W) Where:
a: The number of ciliate cells counted in 50 µl sample from the water runoff V1: Volume of rain water added into 5 g of air-dried soil
V2: Volume of water determined in 5 g of air-dried soil W: oven- dried weight of 5 g of air-dried soil
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During the counting process, the species richness of ciliates was determined by identifying observed ciliates using taxonomic works such as Kahl (1935); Foissner (1987, 1993); Foissner et al. (1991, 1992, 1994, 1995); Foissner et al. (2002); Foissner (2016), and other specialised taxonomic literature. After counting, samples were used for enrichment cultures in order to obtain later populations of ciliates, which enabled further identification; enrichment cultures also facilitate growth of species that were not observed during the counting process. This enrichment is also useful to provide sufficient cells for detailed observations and for silver impregnations. Enrichment cultures are prepared by putting a soil inoculum (approximate 5 g of rewetted soil) into 50 mL sterile culture flasks that contained medium as described in Table 2.2 below.
Table 2.2 Medium used to prepare enrichment cultures of soil ciliates.
Component per/flask
SES* 5 mL
Mineral water 20 mL
Boiled Wheat Grain 1/2 grain
Soil from the Petri dish ~ 5 g of rewetted soil
*SES (Soil Extract added Salts) components (http://www.ccap.ac.ukmedia/SES.pdf):
Component per litre
Stock solutions (1-3)* 20.0 mL each
Soil extract 100.0 mL
Distilled 840.0 mL
*Stock solutions per litre
(1) K2HPO4 1.0 g
(2) MgSO4.7H2O 1.0 g
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All ciliates that grew in the flask were identified under a compound microscope using a combination of glass micro-pipetting and Silver-staining techniques (see below).
Silver-staining techniques
There are many different impregnation and staining techiques to reveal typical characteristics of ciliates, such as vital staining, protargol, silver-carbonate (Fernández-Galiano 1994), and the dry/wet silver nitrate method (Lee and Soldo 1992; Foissner 2014). The ammoniacal silver carbonate impregnation method (Fernández-Galiano 1994) can reveal the kinetosomes, some cortical and cytoplasmic features of ciliates, especially the kinetodesmal fibre and the nuclear apparatus. The method is also quick to do, which is useful as it makes the identification of ciliate species quicker. Consequently, the ammoniacal silver carbonate impregnation method (Fernández-Galiano 1994) was followed in the present study.
The procedure followed was adapted from Fernández-Galiano (1994). The following components are added to a glass beaker (Volume 50 mL)*:
5 mL of distilled water
2 mL of the sample to be studied 2-3 drops of formalin
18-20 drops of bacto-peptone 5 % 10 drops of pure pyridine
1 mL of ammoniacal silver carbonate solution 20 mL of distilled water
*Order of components added as listed from top to bottom
The beaker was then stirred to mix the components, and placed into a water bath at 55 °C. The beaker was left in the water bath until the liquid changes into a brandy- like colour. At this point, the liquid was poured into a glass evaporation dish that had about 50 mL of distilled water in it. The impregnated suspension was left to settle, and then swirled slightly around to concentrate the cells in the centre of the capsule. Finally, a glass micropipette was used to pick up and place the impregnated cells on a microscope slide, which was then covered with a coverslip and observed under a compound microscope.
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This method is not good for some ciliates, for example hypotrichs, as the formalin used for cell fixation makes them burst (Foissner 2014).Therefore, for hypotrichs and other fragile ciliates the protargol method described in Foissner (2014) was followed. The procedure followed was adapted from Foissner (2014):
Ciliate cells in the sample to be studied were fixed in commercial Bouin’s fluid in a glass beaker for 20 minutes. The ratio of the fixative and the sample fluid was 1:1. The solution contained the fixed ciliate cells were then poured into a 15 mL polypropylene centrifuge tube. The fixed ciliate cells in the centrifuge tube were concentrated and washed three times with tap water to remove Bouin’s fluid by centrifugation at 2000 rpm for two minutes. It should be noted in this washing step that centrifuged cells were added enough tap water to bring to 10 mL and shaken vigorously. After centrifuging, the supernatant was carefully decanted, but 1 mL in the tube was kept with taking great care (not to disturb the loosely pelleted ciliates). At the final washing time, about 0.3 mL of concentrated and fixed cells was remained in the tube. One small drop of albumin-glycerol was placed on the centre of a very clean glass slide, and then a small drop of concentrated and fixed cells in the tube was added to the slide. The drops were mixed well with a mounted needle and spread over the middle-third of slide. Usually, eight very clean glass slides were used for a sample. After that, the slides were dried overnight at room temperature. When the slide dried, they were placed in an eight-slot staining jar filled with ethanol 95 % for 20 minutes. Also, an another eight-slot staining jar contained protargol solution 0.5 % was placed in oven at 60 oC to warm the solution up before the slides contained fixed ciliate cell were place into. After slide dripping for 20 minutes in ethanol 95 %, the slides were rehydrated though ethanol 70 % and washed two tap water steps for 5 minutes each. The slides were then placed in 0.2 % potassium permanganate for 2 minutes before they were transferred to 2.5 % aqueous oxalic acid for 3 minutes. The slides were collected to a staining jar filled with tap water and washed two times with tap water and one time with distilled water for 3 minutes each. The slides were transferred to the warm protargol solution (60 oC) and impregnated for 40-60 minutes, depending on specific ciliate species. The staining jar contained protargol solution and the slides were removed from the oven. One slide was removed from the jar and dipped into tap water for 1-2 seconds. Then 1-2 drops of ordinary developer were added and ciliate cells were immediately observed for development under a compound microscope. When oral structures of ciliate cells become visible (but not very dark), remove the slide from the stage of microscope, pour off fluid and dip the slide in and out of a container with 500 mL of tap water. To
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interrupt the development of ciliate infraciliature, the slide was dripped in 5 % sodium thiosulphate for 5 minutes. The same steps were carried out for the remaining slides. After that, the slides were washed three times with tap water for 3 minutes each. The slides were alternately transferred though ethanol 70,100 and 100 % to dehydrate before they were cleared by two 10 minutes transfers through xylene. Finally, the slides were mounted by synthetic neutral medium and applied cover glass (24x40 mm). They need to leave overnight to harden before any observations.