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on the cultivation of taxa 
can also be found in the chapters in this book 
devoted to different classes. While an attempt 
is made to culture some individuals, other cells 
should be more carefully observed cytologically 
and by molecular techniques. 
 3.1.2 Morphology and Multivariate 
Morphometrics
 Cytological observations should begin with 
living cells, if possible. However, this may not 
be possible if only fixed environmental samples 
are available. For living cells, various aspects of 
their behaviour can be observed – the nature and 
speed of swimming. Other initial observations can 
include gross morphological features: body shape; 
body size (e.g., length, width); the kind and extent 
of ciliation; the general placement of the oral area 
(e.g., prostomial , ventrostomial ); and details of the 
oral ciliature (Fig. 3.2). Other features that might 
be observed in living cells include: kinds of prey 
items in food vacuoles; types of food reserves; 
pigmentation; kinds of endosymbionts; kind, 
number, and distribution of contractile vacuoles; 
and kind, number, and distribution of extrusomes 
(e.g., mucocysts , toxicysts ) (Fig. 3.2). While a 
great deal can be learned by careful observation of 
living cells, as demonstrated by the detailed obser-
vations of earlier microscopists (e.g., Bütschli, 
1887–1889; Kahl, 1930–1935; Kent, 1880–1882; 
Stein, 1854, 1859), and especially today with 
differential interference contrast microscopy, it is 
essential for modern descriptions to also use staining 
techniques.
 Descriptions of the four common methods of 
silver staining – the dry silver nitrate method , 
the wet silver nitrate method , protargol or silver-
proteinate staining , and silver carbonate staining 
– can be found in Protocols in Protozoology (Lee 
& Soldo, 1992) and in Foissner (1991). A modified 
method of protargol staining, which uses cellulose 
acetate filters, is particularly useful for describing 
and quantitatively enumerating samples from the 
plankton (Montagnes & Lynn, 1993) and from 
soils (Acosta-Mercado & Lynn, 2003). Electron 
microscopical examination of silver-stained ciliates 
has demonstrated silver deposits in morphologically
Fig. 3.1. Life history of the predatory apostome ciliate Phtorophrya as an example of the richness of characters that 
can be derived from a study of the life cycle. Phtorophrya is a “hyperparasite” feeding on the exuviotrophic apos-
tome ciliate Gymnodinioides, which itself feeds on the exuvial fluids of its crustacean host. After Gymnodinioides
encysts as a phoront on the crustacean host’s cuticle (stippled area), the tomite of Phtorophrya encysts as a phoront 
on Gymnodinioides! Phtorophrya then penetrates the Gymnodinioides phoront wall and transforms to a young tro-
phont that grows to a mature trophont by feeding upon the cytoplasm of Gymnodinioides. The mature trophont of 
Phtorophrya then becomes a tomont, dividing many times in palintomy to form multiple tomites, which excyst to 
find a new host. (Modified from Chatton & Lwoff, 1935a.)
3.1 At the Genus-Species Level 77
78 3. Characters and the Rationale Behind the New Classification
significant structures, such as the boundaries 
between cortical alveoli, microtubular organelles 
of the cortex and deeper cytoplasm, nuclear struc-
tures, and extrusomes (Foissner, 1975, 1977; Tellez, 
Small, Corliss, & Maugel, 1982; Zagon, 1970). 
 In general, the body shape and size of the fixed 
and stained ciliates should also be measured as 
fixation and staining can introduce artifacts (e.g. 
see Lynn & Berger, 1972, 1973). The gross mor-
phological features observed for the living cells 
– such as, the kind and extent of ciliation and the 
general placement of the oral area (e.g., prostomial , 
 ventrostomial ) and details of the oral ciliature – can 
be confirmed. In addition, these silver methods reveal
characters of the argyrome that can be taxonomically
useful (Fig. 3.3). Even more generally useful, are 
features of the kinetome – the organellar system 
composed of all kinetids covering the body of a 
given ciliate. Here, a large number of characters 
can be measured, depending upon the ciliate. 
These could include qualitative characters, such 
as, whether and where there are monokinetids , 
 dikinetids , or polykinetids and what is the nature 
of their ciliation (Fig. 3.2). As well, quantita-
tive characters can be measured, such as the total 
number of somatic kineties , the number of kinetids 
in a particular kinety, the number of postoral kine-
ties , and the number of somatic kineties on the left 
side and right side of the body. How the kineties 
converge to form suture lines or secant systems 
can also be important (Fig. 3.3). Ultimately, refer-
ence to the pertinent recent literature that properly 
describes new species will provide an exhaustive 
set of characters. It is important to obtain measure-
ments on at least a statistical minimum number of 
cells, ideally at least 30 (Berger, 1978). 
 It is generally essential to use stained prepara-
tions to discover the details of the oral ciliature. 
 Prostomial ciliates often have simple oral struc-
tures, like monokinetids and dikinetids , which are 
not easily visible in living specimens (Fig. 3.2). 
 Ventrostomial ciliates may have significantly larger 
oral structures and many more of them. Although 
their detailed structure may be concealed from 
view in the living specimen, well-stained speci-
mens can provide a wealth of information. Again, 
in addition to qualitative features of the shape of 
each unit and the pattern of their organization, 
measurements can be made, for example, on the 
number of oral structures, the numbers of rows and 
numbers of kinetosomes in each structure, and the 
length and width of each structure. If an actively 
growing population was discovered or cultivation 
has been possible, dividing individuals may be 
discovered and the detailed characterization of 
 division morphogenesis may reveal features that 
could distinguish the species, but these features are 
more typically used at the genus level and above 
(see 3.2.2 MORPHOGENETIC PATTERNS). 
 Finally, as Corliss (1979) emphasized, nuclear 
features can be extremely important. Nuclear cytol-
ogy can be revealed by protargol staining and also 
by the Feulgen nucleal stain (Lee & Soldo, 1992). 
The shape, size, number, and placement of both 
Fig. 3.2. A ventrostomatous and a prostomatous ciliate 
with morphological features labelled that are significant 
in the taxonomic description of morphological species. 
Reference should be made to the Glossary (Chapter 2) 
for definitions of these structures and for a more com-
plete list of significant features
 micronuclei and macronuclei should be recorded. 
As well, the distribution of chromatin can be impor-
tant and whether the macronucleus can be classified 
as homomerous or heteromerous (Raikov, 1982). 
While large chromatin aggregates in the macro-
nucleus may indeed be nucleoli , they cannot be 
concluded to be so without objective evidence that 
they are the actual site(s) for the production of 
ribosomes (e.g., Postberg, Alexandrova, & Lipps, 
2006). Without this evidence, they must only be 
referred to as chromatin bodies. 
 If there is a sufficient database on related species,
some assessment, often using statistical approaches, 
can determine whether the new isolate is significantly
different morphologically. For example, Berger 
(1965) and Lynn and Berger (1972, 1973) used a 
univariate statistical approach to demonstrate that 
demes of the echinoid endocommensal ciliates 
Plagiopyliella and Thyrophylax were significantly 
different on a number of morphological charac-
ters. Biometric characterization has now become 
standard practice, enabling future researchers to 
compare new isolates. Multivariate morphometric 
techniques have been applied with some success 
to distinguish the cryptic species of Paramecium
and Tetrahymena

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