NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
30
TABLE XI.
Serial
No.
Date.
Conditions of experiment.
Presence
of
amœbæ.
REMARKS.
1
12th August
1910.
Uninoculated Petri plate left out on
laboratory table without preliminary
exposure to air.
+18th August
1910.
Cysts of types (a) and ( b).
2
12th August
1910.
Same as above
. .
.
.
. —Up to 5th
September.
Medium overgrown by
moulds.
3
12th August
1910.
Uninoculated Petri plate left out on
laboratory table without preliminary
exposure to air.
—Up to 5th
September.
Ditto
ditto.
4
18th August
1910.
Plate first opened exposed to the air for
5 minutes then left out on laboratory
table as before.
—Up to 11th
September.
Ditto
ditto.
5
18th August
1910.
Similar to above but preliminary ex-
posure to air 10 minutes.
—Up to 5th
September.
Ditto
ditto.
6
18th August
1910.
Similar to above but preliminary ex-
posure to air 20 minutes.
—Up to 5th
September.
Ditto
ditto.
7
18th August
1910.
Similar to above but preliminary ex-
posure to air 30 minutes.
—Up to 5th
September.
Ditto
ditto.
8
18th August
1910.
Similar to above but preliminary ex-
posure to air 1 hour.
—Up to 5th
September.
Ditto
ditto.
9
18th August
1910.
Similar to above but preliminary ex-
posure to air 1½ hours.
+ 22nd August
Ditto
ditto.
10
18th August
1910.
Similar to above but preliminary ex-
posure to air 2 hours.
—Up to 5th
September.
Medium dried up.
11
18th August
1910.
Plate left out on window-sill of hospital
dysentery ward without preliminary
exposure to air.
—Up to 9th
September.
Medium dried overgrown
by moulds.
12
18th August
1910.
Similar to above
.
.
.
. —Up to 6th
September.
Ditto
ditto.
13
18th August
1910.
Plate left out on window-sill of hospital
general ward without preliminary
exposure to air.
+23rd August Cysts of type ( a) and (b).
14
18th August
1910.
Similar to above
. .
.
. +28th August
Ditto
ditto.
15
18th August
1910.
Plate left out on table of hospital
office without preliminary exposure to
air.
+22nd August
Ditto
ditto.
16
18th August
1910.
Plate left out on floor of "post dysen-
teric gang" ward without preliminary
exposure to air.
—Up to 11th
September.
Medium overgrown by
moulds.
17
18th August
1910.
Plate left out on floor of No. 1 jail
ward without preliminary exposure to
air.
+30th August Cysts of type (a) and (b).
18
18th August
1910.
Plate left out in cen tral tower of jail
without preliminary exposure to air.
—Up to 9th
September.
Medium dried up.
31
TABLE XI—contd.
Serial
No.
Date.
Conditions of experiment.
Presence
of
amœbæ.
REMARKS.
19 18th August
1910.
Plate left out on window ledge of tank
room without preliminary exposure to
air.
—Up to 6th
September.
Medium overgrown by
moulds.
20 18th August
1910.
Plate left out on table of jail office
without preliminary exposure to air.
—Up to 6th
September.
21 12th December
1910.
Uninoculated Petri plate left out on
laboratory table without preliminary ex-
posure to the air.
+3rd January
1911.
Cysts of types (a) and (b).
22 12th December
1910.
Petri plate first opened and exposed to
the air for 5 minutes, then left out on
laboratory table as before.
— Up to 3rd
February
1911.
Medium dried.
23 12th December
1910.
Similar to above but preliminary ex-
posure to air 10 minutes.
—Up to 3rd
February 1911.
Ditto.
24 12th December
1910.
Similar to above but preliminary ex-
posure to air, 20 minutes.
+3rd January
1911.
Cysts of type (a) and ( b).
25 12th December
1910.
Similar to above but preliminary ex-
posure to 30 minutes.
+3rd January
1911.
Ditto ditto.
26 12th December
1910.
Similar to above but preliminary ex-
posure to air 1 hour.
+3rd January
1911.
Ditto ditto.
27 12th December
1910.
Similar to above but preliminary ex-
posure to air 1½ hours.
+3rd January
1911.
Ditto ditto.
28 12th December
1910.
Similar to above but preliminary ex-
posure to air, 2 hours.
+13th January
1911.
Ditto. ditto.
29 12th December
1910.
Petri plate left out on window-sill in
hospital.
+3rd January
1911.
Ditto ditto.
30 12th December
1910.
Petri plate left out on window-sill in
hospital general ward, without pre-
liminary exposure to air.
—Up to 3rd
February
1911.
Medium dried.
31 12th December
1910.
Petri plate left out on table in hospital
office without preliminary exposure to
air.
+6th January
1911.
Cysts of type (a) and ( b).
32 12th December
1910.
Petri plate left out on floor of "post-
dysenteric gang" ward without preli-
minary exposure to air.
—Up to 3rd
February
1911.
Medium dried.
33 12th December
1910.
Petri plate left out on floor of No. 1
jail ward without preliminary ex-
posure to air.
—Up to 3rd
January 1911.
Ditto.
34 12th December
1910.
Petri plate left out on ledge near top of
central tower of jail, without preli-
minary exposure to air.
—Up to 3rd
January 1911.
Ditto.
35 12th December
1910.
Petri plate left out on window ledge of
tank room without preliminary ex-
posure to air.
—Up to 3rd
January 1911.
Ditto.
36 12th December
1910.
Plate left out on table of Superin-
tendent's office without previous ex-
posure to air.
—Up to 3rd
January 1911.
Ditto.
32
Subcultures of these amœbæ were readily obtained by taking a loopful of
the mixed growth of amœbæ, either vegetative forms or cysts, and bacteria,.
and stroking it on the surface of a fresh plate of Musgrave's medium.
The subcultures were generally kept in a moist atmosphere at a tempera-
ture of 22°-28° C. for the first day or two : the optimum temperature for their
multiplication probably lies within these limits.
Generally, after from 12 hours to two days, there is an abundant crop of
motile amœbæ along the needle-stroke of inoculation ; as a rule, after 5-6 days
many of the amœbæ are found to have become encysted.
In order to obtain a culture which would certainly represent only one
species of amœbæ, the following method was adopted.
A loopful of growth is inoculated at one point of the plate ; thence a
radiating stroke is produced on the surface of the medium with a fine platinum
needle. On examining this stroke under the low power several amœbæ are.
generally found. at the end of it. From this point a second stroke is made, and
so on, until, finally, one is selected which contains only a single amœba towards
its extremity : this single amœba is then cut off from the rest of the growth by
a stroke of carbolised vaseline painted on the surface of the medium after
the method of Walker 2 5
.
Pure cultures thus obtained were most easily preserved from contamination
by subculturing on test-tube " slopes " of Musgrave's agar.
With a Zeiss A A lens and a No. 12 eye-piece the main processes of the life-
cycle may be roughly followed on the inverted Petri plates, but, for examina-
tion under the higher powers of the microscope, the following method of sub-
culture was employed.
A slab of Musgrave's agar, half an inch square, is cut out of a fresh plate.
of the medium with a sterile knife and placed on a sterile slide.
A loopful is then taken from an old culture containing cysts and stroked
on the surface of the agar square ; a sterile cover glass is placed on top and its
edges are Sealed with melted paraffin wax. An air space should be left between
the edges of the agar slab and the paraffin wall.
Thus a microscopic moist chamber is provided, sealed against external
contamination. Such a preparation may be kept in a warm microscope cham-
ber at a temperature of 25° C. Examining such a slide-culture under the low
power, one picks out a spot towards the tail end of the inoculation stroke where
the cysts are thinly scattered ; a single one is then isolated under the oil
-immersion lens for continuous observation.
For permanent preparations, fixation after the method of V. Wasielewski
and. Hirschfeld 26 gave the best results.
The principal fixatives used were Osmic acid (2 per cent.) and corrosive
33
acetic-alcohol, followed by Giemsa's stain and Iron-hœmatoxylin-Eosin, respec-
tively.
The first method of fixation gives the best picture of the plasma, the second
brings out the nuclear material particularly well.
MORPHOLOGY AND LIFE CYCLE OF THE CULTIVATED AMŒBÆ.
A reference to table XI shows that, in 12 out of the 14 growths of amœbæ
obtained from the air in the manner described two types of cysts were distin
-guished.
The following description is concerned only with the growths of amœbæ
occurring on the single plate. (No. 24 in the table.)
Cysts of type ( a) (Fig. 1) have a diameter of 8-14, generally 10-12, micra ;
their shape is round, oval, more frequently trihedral, polygonal or stellate ;
the wall is about 0 . 25 micra in thickness and has a strongly marked double
contour ; the outer layer is generally wrinkled, reminding one of the outline of
an Ascaris ovum.
The plasma is compact and coarsely granular and a circular or oval
eccentric nucleus is as a rule, more or less distinctly visible in unstained
specimens.
Cysts of type ( b) (Fig. 19), on the other hand, are much smaller, having
a diameter of 3-8 micra ; the shape is round or oval, with little or no tend-
ency towards the modified forms often assumed by cysts or type (a) ; the
surface is smooth and the wall delicate with a faint double contour The plasma
is hyaline with thinly-scattered highly-retractile granules ; a nucleus is hardly
to be distinguished.
Cysts of both types stain an intense purple black with Iron-hœmatoxylin.
In order to determine whether these two well-defined types or cysts repre-
sent two different species or simply polymorphic forms belonging to a single
species attempts were made to obtain cultures originating from a single cyst
of each type by Walker's method as above described.
In many cases, however, the cysts isolated failed to germinate ; it was there-
fore found more practicable to select a single motile individual as the starting-
point of a strain.
(1) Amœbæ of type ( a).
In this way cultures of amœbæ were obtained which were found to form
cysts of type (a) only.
If such a culture be allowed to encyst, the cysts be transferred to a slide
preparation, as above described, and one of them be isolated under the 1-12 lens,
its development may be followed in detail.
F
34
After 1-3 hours (at a temperature of 22°—28°) a faint streaming movement
of the contained granules becomes evident and a bright vacuole appears in the
plasma. After a very veriable period, from 10 min.—35 min., the vacuole
suddenly shuts and either disappears entirely or leaves only a minute dark
speck to mark its site ; then, very slowly and gradually, it re-appears. This
process is repeated at diminishing intervals until the vacuole may contract after
irregular periods of 40-180 seconds the streaming of the granules becoming
more and more active and the nucleus more clearly apparent in the meantime.
After 3-7 hours, as a rule, from the time of insemination, during active
movements of the contents, a small knob of protoplasm is seen to thrust
suddenly through the cyst wall at one point (Fig. 2.)
Streaming through the narrow outlet, the active amœba rapidly increases
in size, so that a considerable portion of it may escape without any coincident
shrinking of the contained part away from the inner wall of the cyst.
The amœba may become completely clear of the cyst within 5-20 minutes,
of the rupture, and may by that time have reached a diameter of 15-25 micra.
The nucleus stands out distinctly as a circular or oval body with a dark
centre and a clear peripheral halo ; the total diameter is about 2 . 5 micra
One contractile vacuole is invariably present which, at this stage, generally
contracts, somewhat irregularly, once in 20-80 seconds and there may, more
rarely, be two or more; clear circular areas about 1-2 micra in diameter, which
remain uncontracted, are often seen in the plasma.
Sometimes the distinction between ectoplasm and endoplasm is remarkably
clear, but often the protoplasm is uniformly granular. In this respect the same
amœba shows varying characters at different times.
The pseudopodia vary much in form, may be broad and blunt or spinous
simple or branched ; the commonest form seems to be a single, rather broad
blunt process, the margin of which is fringed with short spikelets. Several
pseudopodia may be protruded in different directions at the same time ; many
bacteria may be ingested. In specimens fixed with Osmic acid and stained by
Giemsa's solution the blunter pseudopodia are well demonstrated (Fig. 14).
After its escape from the cyst the amœba continues to move more or less
actively over the surface of the fresh medium, leaving a trail of bacteria in its
wake.
Actively motile amœbæ of this type have been watched on twelve occasions
for periods of from 1-6 hours without showing any radical change in their
behaviour.
On the other hand, on nine occasions an amœba under examination was
observed to shrink and assume a rounded or oval form (Fig. 6), then the body
undergoes an. hour-glass constriction and finally divides into two daughter
35
amœbæ, each approximately half the size of the parent (Figs. 7 and 8) ; the
whole process from the first evidence of constriction till complete division takes
about two minutes.
The process of nuclear division was not followed in fresh specimens ; indeed
I have found the behaviour of these amœbæ, while undergoing fission, to be in.
this respect similar to that observed by Liston in one strain of amœbæ isolated
by him from liver abscess pus. As in the amœbæ described by Liston, on the
condensation of the plasma previous to division, the nucleus becomes obscure,
nor in the new daughter amœbæ is a nucleus immediately to be seen ; it takes
shape gradually and not .till 2-5 minutes after division does it become clearly
visible.
In stained specimens some amœbæ were seen in which the nucleus, while
taking the stain deeply, showed an appearance suggestive of simple fission
(Fig. 19) ; in none was evidence of karyokinesis seen. In one stained specimen
(Fig. 15), an amœba was seen showing an hour-glass constriction in the middle
with a clear vacuole at each pole without distinct nuclear staining.
In fresh specimens, on many occasions, a small portion of a motile amœbæ
was seen to become detached ; these fragments showed no active movement
and ultimately disappeared from view after half an hour or more. On four
occasions, however, true motile buds, 2-5 micra in diameter, were seen to emerge
from the parent body.
In specimens fixed by corrosive-alcohol-acetic acid and stained by Iron-
hœmatoxylin, an amœba is often seen to contain 1 to 12 or more clear rounded
spaces, 1 to 4 micra in diameter, with or without a central or eccentric dot
which takes the chromatin stain faintly (Figs. 16, 17 and 18) ; these apparently
correspond to the "internal buds," or " merozoits " of Noc.
Many small protoplasmic masses, one or more micra in diameter, some
containing a distinct nucleus, others without any chromatin staining, are also
seen in stained films from the cultures, sometimes collected into groups.
The small bodies without any visible chromatin possibly represent the
detached. fragments of protoplasm seen in fresh specimens.
It is, therefore, evident that multiplication by unequal budding also takes
place in these amœbæ
A third striking modification in the activity is very commonly seen in
these cultures.
An amœba may throw out highly refractile globular or blunt branched
motile processes, 2-4 micra in diameter, at one or more points on its surface.
The whole of the original body may in a few minutes be completely overlaid
by or absorbed in these amœboid excrescences which adhere to one another, the
original nucleus being entirely lost to view.
F 2
36
Thus a writhing lobulated mass is produced which may assume the most
bizarre forms, moruloid, moniliform or cochleate ; the lobules composing it,
some of which often show contractile vacuoles, may lie one on top of the other,
so that all cannot be focussed at once (Figs. 11-13).
This process has been noted to occur in more than twelve cases while
individual amœbæ were under observation.
Such bodies have been consecutively observed under the higher powers of
the microscope on twenty recorded occasions with the following results :—
(i) In four cases the lobulate body has moved about for periods of from
35 minutes to 5 hours and changed its shape continuously but
has not undergone any alteration in character.
(ii) In eleven cases one of the elements composing the mass has been
seen to absorb the others into itself, gradually acquiring a visible
nucleus, until an amœba, of the ordinary vegetative type resulted.
This amœba was observed ( a ) in three cases, to retain its characters
unaltered for periods up to 2 hours ; ( b) in 5 cases, to reassume the lobulate
form after periods of from 3 minutes to 1½ hours ; ( c ) i n o n e c a s e , t o g i v e o f f a
bud, 5 minutes after formation ; (d) also, in one case, to divide into two, 3
hours 15 minutes after formation.
(iii) In two cases the lobulate body has been observed to divide into
two similar masses.
(iv)In three cases one of the processes has been observed to become
detached, acquiring a visible nucleus and all the characters of the
usual vegetative type. No forms definitely corresponding to
these lobulate bodies have been found in stained specimens.
The significance of this form of activity is not clear ; it may perhaps
represent either a modification of the process of budding or a third distinct
method of reproduction by multiple division.*
* Major Liston informs me that he has observed similar appearances in his cultures ; he attributes them to
the fact that the amœbæ concerned "are simply penetrating the surface of the agar." He points out that
amœbæ grow below the surface of the agar, and that, in old cultures, cysts way be focussed in various
planes of the medium.
I find that this is apparently a constant phenomenon in cultures of type ( a).
It is very difficult to interpret the active movements of these burrowing amœbæ, as different points of
their surface are at different levels, so that the whole body cannot be focussed at one time. Moreover, another
amœbæ might suddenly come into view in the field of the microscope, having entered it from a higher or lower
plane, and thus give rise to an appearance of division on the part of the amœba originally under observation.
It seems at least probable that the activities of these lobulate bodies here described may be due to modified
processes of fission and budding going on below the surface of the agar.
The fact that these forms occur only below the surface accounts for their absence in fixed and stained films.
37
Three or four days after insemination some of the amœbæ generally
become encysted.
Encystment seems often to be immediately preceded by remarkably
active multiplication : this results in the formation of clumps of amœbæ which
may become massed together, giving the appearance of plasmodia.
In the end each amœba becomes rounded, the body shrinks, becomes more
granular in appearance and acquires a thin envelope ; this is at first single but
later presents the characteristic appearance of double contour, the outer layer
being wrinked. The original circular outline may be modified to become
trihedral polygonal or stellate, the nucleus becomes less distinct, the contractile
vacuole ceases to open, until it becomes apparent that the youngest generation
has reached the cystic stage from which its ancestors set out.
In stained specimens many of the amœbæ undergoing encystment are
seen to contain numerous internal buds (Fig. 18)
Not all the amœbæ encyst about the same time ; indeed many are often
found still motile in cultures more than a month old.
(2) Amœbaof type (b) .
Attempts to obtain a culture the progeny of a single individual of this
type did not succeed, but ultimately a culture originating from a number of
small motile forms was obtained which, in subculture, was invariably found to
yield cysts of the small type only.
Germination of the cysts has not been directly observed in this strain ; on
six occasions a single cyst on fresh medium was kept under the oil-immersion
lens for from 10 hours to 3 days without showing any signs of germination.
Empty cyst shells have not been seen in unstained specimens, but in
stained films from the cultures they appear as rounded shrivelled bodies about
3-5 micra in diameter. With Giemsa's stain they take a brownish or greenish
-colour, with Iron-hœmatoxylin they are light yellow.
The vegetative amœbæ of this type seldom measure more than 12 micra.
Many small forms are also found, measuring as little as 2 micra.
The vegetative form (Fig. 21), is generally very filmy and delicate, showing
a faint hyaline plasma, containing scattered granules without any definite
distinction between ectoplasm and endoplasm. Pseudopodia are commonly
single and rather broad and blunt ; spinous pseudopodia were not seen in this
form.
The nucleus is generally hard to distinguish in unstained specimens but
may appear as a faint pinkish circular area 1-2 micra in diameter. One
contractile vacuole is generally present in the active amœba and contracts once
in 25-70 seconds.
38
Movement is, as a rule, more active than in type (a) , the whole body
glid–ing rapidly, often without any appreciable formation of pseudopodia.
Fission has been observed in fresh specimens 24 times (Figs. 22 and 23).
The amoeba does not lose its motility and become condensed before fission
as in type (a) but, while actively motile, it suddenly assumes a dumb-bell shape
and divides into two daughter-amœbæ within one minute.
The formation and detachment of a bud about 1-4 micra in diameter has
been observed in fresh specimens on four occasions.
Nothing has been observed in this strain approaching the formation of the
lobulate bodies which are so characteristic in cultures of type (a), but, on the
other hand, the following curious modifications of activity have been noticed.
An active amœba may suddenly become constricted in the middle, the
two halves separating and giving a transitory appearance of fission ; they, how-
ever, immediately come together again, the amœba resuming its former habit.
In the same way a small portion of the amœba may appear to separate off
from the main body to become immediately reabsorbed.
These appearances are suggestive of abortive fission and gemmation,
respectively.
On two occasions in cultures of this type, a motile amœba has been ob-
served to become spherical and acquire a double-contoured envelope. One of
them was a daughter amœba, the product of fission one hour old when encyst-
ment commenced ; in the other case the amœba had given off a bud 33 minutes
before.
In stained specimens (Fig. 25) the vegetative forms have a diameter of
2-14 micra ; the nucleus, which in a well-grown form measures about 2 micra,
stains less intensely than in the amœba of type (a) ; Osmic acid fixation
followed by Giemsa staining fails to show a clear distinction between ectoplasm
and endoplasm.
Fission forms (Fig. 27) and forms containing endogenous buds (Fig. 26)
are seen, as also vacuolated bodies without any well-defined nucleus but with
more or less scattered irregular chromatin masses or granules.
The details of the life-cycle of these two types of amœbæ have been
studied in cultures on solid agar only ; however, both_ types " (a) " and "
have been found to multiply to some extent in diluted broth (1 in 10, 1 in
100), though this medium does not seem to be at all so favourable to their
growth as is Musgrave's agar.
Flagellated forms, such as V. Wasielewski and Hirschfeld 6 f o u n d t o
develop in growths of " Straw-amœbæ " transferred from agar to weak
broth, were not observed in either type. In the fluid cultures no contractile
vacuole was observed in the vegetative phase.