Agar Plate 24 Well Plate Haemocytometer

Fig. 3.5. Subsampling flow chart for enumeration of active protists. The moisture content from oven-dry weight for each sample is required. Weighed subsamples are then placed in various containers for enumeration of taxa at the microscope (see text for explanation).

Table 3.4. Typical abundances of active protists and total species in soil samples. Taxa listed by motility group based on extraction method.

Extraction method

Taxa

Active species

Total species

Numbers (g dry soil) Cells/g Species/g

Naked amoebae Testate amoebae Small flagellates Flagellates Ciliates Hyphae Reticulate Yeasts

Non-nutrient agar plate Soil dilution, litter sections Soil dilution Soil dilution Soil dilution Ergosterol:chitin ratio Agar plate & membrane Radioactivity

Nutrient agar over time 103-106 1 0-100

Litter bait agar plate 10-104 10-20

Dilution, nutrient amendment 104-106 10-50

Non-flooded Petri plate over time 1-102 <10

Non-flooded Petri plate over time 0-102 10-30

Nutrient agar or bait m/g <10

Agar plate bait ? <10

Nutrient agar or bait 0-104 <5

The amoeboid species in the Amoebozoa (protozoa) (except the Testacealobosea) and the Labyrinthulea (Chromista) can be obtained from non-nutrient agar plates. This preparation also reveals Actinobacteria, Myxobacteria and sometimes other bacterial taxa, which can be identified from reproductive structures or colony morphology. Soil droplets, with or without dilution, are placed on a thin layer of 1.5% non-nutrient agar. The plates are incubated overnight and observed with an inverted microscope under phase contrast, at appropriate magnifications (this is not possible if the agar is too thick). The active amoebae can be visualized at the periphery of the soil droplets, as they crawl looking for food. As some cells will remain in the soil, this only provides an estimate of the number of individuals which are active. The number of cells remaining within the soil can be reduced by decreasing the quantity of soil used in each droplet. These eventually will encyst. The advantage of this technique is to provide sufficient moisture for amoeboid species, but not for swimming species to disperse. It also provides a clean preparation of living amoebae required for species identification. Amoeboid organisms are recognized by their overall shape, type of pseudopodial locomotion and details of pseudopodia.

The Testacealobosea and most Filosea (protozoa) need to be separated from the litter or particulate organic matter. These organisms are best observed in soil smears, collected by filtration on membranes, or by dissecting through litter and detritus. The filamentous or hyphal species (such as Thraustochytrid, Oomycetes and Hyphochytrea (chromista), the Chytridiomycetes and other fungi) cannot be obtained intact or active from collected soil. These species are not motile, except for the zoospore dispersal stages, or not at all. Similarly, the reticulate interstitial species of amoeboid organisms cannot be obtained intact. These are normally damaged during sampling, caused by movement and friction between soil particles. However, some can be isolated from relatively undisturbed soil by placing a wet membrane or tissue of large mesh on a soil sample. The organism will crawl through the pores or mesh, and can be separated from the soil beneath. Although some of these species are known, their abundance cannot be estimated, as they are not usually assayed.

The Ciliophora species in the soil consist of species that swim in the gravimetric water, as well as species that crawl along surfaces and on to microdetritus. They can be identified by their characteristic swimming and locomotion. Less abundant, larger species of other taxa that swim in the gravimetric water can be observed along with the ciliates in this preparation (such as Sarcomonadea and Euglenoida). This preparation requires the soil subsample to be water saturated, then decanted into a Petri plate several times. The wash water will contain mostly the larger free-swimming species (>20 ^m) that tend not to hold on to particles. Alternatively, decigram quantities of soil can be disaggregated in a Petri plate with a large quantity of deionized water and observed. For identification, the preparation is scanned with a compound microscope at appropriate magnifications. Most species will require staining of the cil-iature or, for smaller species, SEM observations are necessary.

All other taxa of swimming species (with one or more cilium), in the chromista and protozoa, are best observed in centigrams of soil subsam-ples in water, with an inverted microscope or compound microscope, and phase contrast (Fig. 3.6). These species tend to be about the size of small soil particles (4-12 ^m long and smaller diameter), and tend to hug particles with a cilium. It is best to scan between soil particles, at the bottom of wells, and to look for movement. Transects of known dimensions can be scanned across the wells, or an ocular grid can be used to scan known areas of the well bottom. If the abundance of the nanofla-gellates is >5 X 104 individuals/ml in the suspension, the Neubauer grid of a haemocytometer can be used to count cells in smaller grid areas. (The haemocytometer grid is also useful to enumerate bacterial cells in soil suspensions, as their abundance is sufficiently high.) These small protist species are difficult or impossible to identify without a compound microscope. In general, SEM of fixed preparations is recommended to avoid misidentification and guessing. Many species of the soil nanofla-gellates' are still unknown.

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