327
Observations on the mastigonemes of two Chrysophyceae
using negative staining
By D. E. BRADLEY
(From the Department of Zoology, University of Edinburgh)
With 2 plates
Summary
This paper describes a negative staining procedure for small appendages on relatively
large organisms, and its application to the study of the ultrastructure of the mastigonemes of two chrysomonads. They have a remarkably complex structure, appearing
as tubes with a layer of ragged material on their surfaces. In spite of the large amount
of detail visible, it is not possible to propose a definite model of their structure.
Introduction
RELATIVELY little is known about the ultrastructure of the flagella of
members of the Chrysophyceae. Most organisms in the family possess
'Flimmergeissel' or flimmer-flagella (see Brown, 1945, for description and
bibliography). These consist of flagellar shafts with numerous thin filaments
or hairs radiating from them. These mastigonemes may only be 20 A or so
thick, but the most significant biologically are much larger (1 /x x 200 A). The
relatively little work that has been carried out on them is summarized by
Sleigh (1962) and Pitelka (1963). The flagellum of Ochromonas was illustrated
by Pitelka and Schooley (1955) using shadowing, and they showed that the
mastigonemes carried a pair of fine filaments at their tips. However, they
were unable to resolve any structure in them, and suggested that the mastigonemes were attached to the flagellum at the sheath. Manton (1956) took
a different view, suggesting that they were attached to the peripheral fibrils of
the shaft. There is thus some doubt about the position of attachment, but
there is none about the arrangement of flagellar fibrils within the shaft. The
' 9 + 2 ' system appears to be followed by flimmer and 'whip-lash' flagella alike.
This is shown well in the case of the organism Synura caroliniana, Whitford
( = S. petersenii, Korshikov, 1929) studied from a pure culture by Manton
(1955). The present paper describes observations on the fine structure of the
mastigonemes using negative staining.
The organisms used here, Synura spinosa (colonial) and Mallomonas
akrokomos (solitary), presented a particular problem in negative staining
because of the relatively large size of the cells. A procedure for this type of
specimen is described; it should be applicable to a wide range of organisms,
such as those examined by Pitelka and Schooley (1955).
[Quart. J. micr. Sci., Vol. 106, pt. 4, pp. 327-31, 1965.]
328
Bradley—Negative staining of Chrysophycean mastigonemes
Material and methods
Source of organisms
The choice of organisms depended entirely upon their availability in local
ponds and ditches since they are difficult to culture. In suitable conditions,
usually under ice, both genera often 'bloom' to become the dominant species
in a body of water. They can be quickly identified in the electron microscope
by the structure of their scales (see Petersen and Hansen, 1956, for Synura
and Harris and Bradley, i960, for Mallomonas taxonomy). The local occurrence of 'blooms' of Mallomonas akrokomos (Pascher, 1913) and Synuraspinosa
(Korshikov, 1929) provided an opportunity for the study of their flagella.
Methods of preparation
In order to ensure that theflagellaremain attached to the organisms, a simple
fixation procedure is carried out as follows. Approximately 500 ml of pond
water is placed in a large bottle and a few drops of buffered 1% osmium
tetroxide are added and mixed by gentle shaking. The bottle is then allowed
to stand overnight at 4 0 C. All the organisms will then have sedimented to the
bottom, whence they can be removed for negative staining. For this, the open
end of a o-i ml pipette is blocked with a finger and the pipette is inserted into
the bottle until the nozzle touches the bottom; 0-05 ml of liquid is allowed to
enter by releasing the finger, and a large number of organisms is carried in with
it. This suspension is blown out on to a glass slide and an equal volume of
neutral 2% potassium phosphotungstate (PTA) added. After mixing, 0*05 ml
is transferred to another slide. A specimen support grid previously coated
with a strong support film (e.g. collodion and carbon) is next placed film-down
on the liquid, and the slide is carefully inverted. It is held in this position
long enough to permit the organisms to settle on to the support film. For the
large colonies of Synura this occurs in a few moments, but several minutes are
required for the smaller Mallomonas akrokomos. The grid is next gripped with
forceps and pulled gently downwards from the surface of the drop. This
leaves a large droplet of liquid on the upper surface of the grid; most of this
must be removed for satisfactory negative staining. Removal is best carried
out from above with a piece of filter paper until only a thin film remains
covering the grid; care must be exercised to avoid disturbing the organisms
from the support film. If it is found that insufficient organisms are present,
a more concentrated initial suspension may be obtained by gentle centrifugation (1 to 2000 g) for a few minutes.
It has been found that this handling procedure is the simplest way to obtain
the correct concentration of PTA for the negative staining of appendages
FIG. I. The tip of the flagellum of Mallomonas akrokomos; phosphotungstate. (x6o,ooo)
F1G.2. ThetipofamastigonemeofaSj>««ras/)i«oja flagellum; phosphotungstate. (X 200,000)
FIG. 3. Mastigonemes of Synura spinosa; phosphotungstate. ( X 150,000)
FIG. 4. Fig. 3 enlarged, (x 333,000)
4
FIGS. 1-4
D. E. BRADLEY
FIGS. 5-9
D. E. BRADLEY
Bradley—Negative
staining of Chrysophycean mastigonemes
329
attached to objects which are much larger than those normally studied by the
method. The procedure works equally well with 2% uranyl acetate.
Results
As will be seen from the electron micrographs, the structure of the mastigonemes of Synura spinosa is similar to those of Mallomonas akrokomos;
observations on both organisms are therefore described together.
The mastigonemes are shown attached to the flagellar shaft in fig. r. This
micrograph shows the tip of the flagellum of Mallomonas akrokomos with a few
widely spaced mastigonemes penetrating the flagellar membrane. Something
of the flagellar fibrils can be seen within the membrane (arrowed) and the
mastigonemes appear to be attached to them in some way. They have a sharp
kink a short way from their point of attachment, a feature also present in
Synura spinosa.
The tip of a mastigoneme of Synura spinosa is shown in fig. 2; there are
two or three fine filaments radiating from it. Here the negative staining is not
good enough to show anything more than the apparently tubular form of the
hairs. Fig. 3, though at a lower magnification, illustrates a number of points.
Firstly, the diameter of the mastigonemes can be measured to be about 230 A,
and also the nature of the filaments is shown up more clearly, with a definite
periodic structure at the arrowed point. This area is shown enlarged in fig. 4.
Each filament consists of a central portion appearing hollow since it is penetrated by the PTA. This is surrounded by a well-defined wall which sometimes appears as a regularly broken line producing the periodic structure.
An ill-defined ragged mass of material surrounds the tube. Part of a mastigoneme of Mallomonas akrokomos is shown in fig. 5 and, though rather better
preserved, is clearly similar to those already described. A suggestion of
periodic structure in the wall is visible at the top.
The use of uranyl acetate as a negative stain provides a rather different
picture. In fig. 6, the intact left-hand mastigoneme has a clearer, narrower
central region and the material surrounding it is less ragged. The right-hand
filament has broken down into 3 well-defined and narrower fibrils, about
45 A in width. While this appearance was not frequent, it was found several
times.
A particularly interesting feature of the Mallomonas akrokomos preparation
was the way in which many of the mastigonemes broke down into a mass of
very fine fibrillar elements (fig. 7). While it is not obvious from this micrograph, two or three distinct bundles of these elements are associated with each
intact mastigoneme. Some of these are shown in figs. 8 and 9. The fibrillar
elements are 15 to 20 A in width.
FIG. 5. Part of a mastigoneme of Mallomonas akrokomos; phosphotungstate. (X 333,000)
FIG. 6. Synura spinosa mastigonemes; uranyl acetate. ( X 333,000)
FIG. 7. Mastigonemes of Mallomonas akrokomos breaking down into fibrils; phosphotungstate. (X 333,000)
FIGS. 8 and 9. Fibrils of Mallomonas akrokomos mastigonemes; phosphotungstate. (x 333,000)
33°
Bradley—Negative staining of Chrysophycean mastigonemes
Discussion
In fig. i, the mastigonemes appear to arise from the flagellar fibrils, though
one cannot see them actually branching. This tends to support the theory of
Manton (1956). There is then an apparently rigid portion before a sharp kink
in the flimmer filament; this projects straight out from the shaft and perhaps
serves to prevent the mastigonemes from becoming entangled with one
another as would be the case if they were fully flexible throughout their whole
length. There is some doubt as to whether the mastigonemes are themselves
capable of movement; there is no reason why this should not be the case since
bacterial flagella, of about the same diameter, move a great deal to propel the
cell. Alternatively, movement could be transmitted from the flagellar fibrils,
provided the mastigonemes were in fact joined to them. As will be evident
from the ensuing discussion, the fine structure of the mastigonemes does not
obviously throw any light on this matter.
The twin filaments radiating from the tips of the mastigonemes in fig. 8
are clearly similar to those figured by Pitelka and Schooley (1955) for Ochromonas, though they are longer in both Synura and Mallomonas.
The mastigoneme structure is complex, its appearance differing according
to the negative stain used. It must be remembered that different negative
stains will undoubtedly cause the mastigonemes to break down in different
ways because of varying chemical effects and pH changes during drying
(Bradley, 1962). There are 3 basic structures visible: the tube with the
broken wall, the 3 emerging filaments (fig. 6), and the very fine filaments
(figs. 7, 8 and 9). It is difficult to reconcile the triple filaments with the
tubular appearance. All that can be said is that they are probably bound
together by the material on the mastigoneme surface, which is perhaps
arranged in a helical manner as suggested by the periodic nature of the tube
wall (figs. 3, 4 and 5). It would appear that the 3 filaments themselves are
made up of finer strands.
The structure of the mastigonemes is far more complex than expected and
these observations are of a preliminary nature. Clarification might best be
attained by studying other flagellates by the technique described to see
whether the basic appearance is similar. It might be found possible to clarify
the relationship between the mastigonemes and the flagellar fibrils by means
of sectioning, but this would be extremely difficult because of the very small
size of the point of attachment. The negative staining of more stable flagella
might be the most profitable approach to the problem. The present study
indicates that chrysophycean mastigonemes are not stable structures.
The technique used here, which might be described as a sedimentation /
negative-staining procedure, is of wider application and could be used on any
large objects with appendages which are sufficiently transparent to electrons
for negative staining.
The author is grateful to Dr. D. C. Barker and Miss Ruth E. Schmitter for valuable
discussion, and to Miss C. A. Dewar for technical assistance. Low magnification
Bradley—Negative staining of Chrysophycean mastigonemes
331
electron micrographs were taken with a Philips EM 75 electron microscope provided
by D.S.I.R. grant No. 3/9/1725.
References
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PASCHER, A., 1913. Die Susswasser-Flora Deutschlands, Osterreichs und der Schweiz, z, 7.
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