Electron Microscopy of Giardia lamblia Cysts - Applied and ...
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1980, p. 821-832 Vol. 40, No. 4
0099-2240/80/10-0821/12$02.00/0
Electron Microscopy of Giardia lamblia Cysts
DANIEL L. LUCHTEL,* WILLIAM P. LAWRENCE, AND FOPPE B. DEWALLE
Department of Environmental Health, School of Public Health and Community Medicine, University of
Washington, Seattle, Washington 98195
The flagellated protozoan Giardia lamblia is a recognized public health prob-
lem. Intestinal infection can result in acute or chronic diarrhea with associated
symptoms in humans. As part of a study to evaluate removal of G. lamblia cysts
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from drinking water by the processes of coagulation and dual-media filtration, we
developed a methodology by using 5.0-,um-porosity membrane filters to evaluate
the filtration efficiency. We found that recovery rates of G. lamblia cysts by
membrane filtration varied depending upon the type and diameter of the mem-
brane filter. Examination of membrane-filtered samples by scanning electron
microscopy revealed flexible and flattened G. lamblia cysts on the filter surface.
This feature may be responsible for the low recovery rates with certain filters
and, moreover, may have implications in water treatment technology. Formation
of the cyst wall is discussed. Electron micrographs of cysts apparently undergoing
binary fission and cysts exhibiting a possible bacterial association are shown.
Exposure to the waterborne pathogen Giardia ably flexible and concluded that the interaction
lamblia is a current public health problem (10) of the flexible cyst wall in the filter pore may
as exemplified by recent outbreaks of giardiasis explain the different recovery rates on different
reported from Vail, Colo. (7), Berlin, N.H. (15), types and sizes of filters.
and Camas, Wash. (11). These outbreaks oc-
curred in municipalities that use surface water MATERIALS AND METHODS
for drinking purposes. Each of their seemingly Fecal material was collected from human giardiasis
adequate water treatment facilities failed to fol- patients in cooperation with the Washington State
low proper treatment procedures of the raw Parasitology Laboratory, Seattle. The material was
water. G. lamblia cysts were detected in the fixed in either 5% buffered Formalin or 2% glutaral-
finished water at both Berlin and Camas. The dehyde in 0.1 M cacodylate, which was done immedi-
percentages of stool specimens positive for G. ately after positive identification of G. lamblia cysts
lamblia cysts reported by U.S. state laboratories in the feces. A given quantity of the fecal material was
diluted 1:2 in distilled water, stirred into a liquid
in 1976 were 9.2 in California, 9.6 in Colorado, suspension, and filtered through three layers of gauze
10.6 in Minnesota, 9.5 in Maine, and 6.3 in Wash- that approximated a 50- to 80-,um-mesh sieve. The
ington (2). filtrate was centrifuged at 400 x g. After the super-
The work reported here is part of a study that natant was decanted, the sediment was emulsified with
determined the efficiency of a water treatment an equal amount of distilled water.
plant for removing G. lamblia cysts. Experi- We used the method of Sheffield and Bjorvatn (20)
ments showed that >99% of the cysts introduced to further separate the cysts from other fecal material.
into a water treatment pilot plant can be re- A 5-ml amount of the fecal suspension was added to a
moved by the processes of coagulation-floccula- discontinuous density sucrose gradient consisting of 5
ml each of 1.5, 1.0, 0.75, and 0.5 M sucrose solutions
tion, sedimentation, and dual-media filtration added successively to a 40-ml conical centrifuge tube.
(W. P. Lawrence, Masters thesis, University of After centrifugation for 30 min at 1,000 x g, approxi-
Washington, Seattle, 1979). The efficiency of mately 4 ml was collected by capillary pipette from
cyst removal was evaluated by filtering the fin- both the water-0.5 M sucrose and 0.5 M-0.75 M su-
ished water from the pilot plant. In also evalu- crose interfaces. This suspension, consisting of cysts
ating the reproducibility of our filtration proce- and small noncyst particulate debris, was diluted 10-
dure with known concentrations of cysts, we fold with distilled water and centrifuged for 3 to 5 min
found that the recovery rates of cysts that were at 400 x g. The sediment, consisting of a high number
passed through two different types (Millipore of cysts relatively free of debris, was again diluted 10-
and Nuclepore) and diameters (47 and 293 mm) fold with distilled water and kept at 4°C until use. We
eliminated the final filtration, as recommended by
of membrane filters varied considerably. Sheffield and Bjorvatn (20), through a 20-/um filter to
Electron microscopy was used to determine remove any remaining debris.
the possible causes of these various rates. We Known quantities of cysts were added to an exper-
found that the cyst wall of G. lamblia is remark- imental water supply and tested in a pilot water treat-
821822 LUCHTEL, LAWRENCE, AND DEWALLE APPL. ENVIRON. MICROBIOL.
ment plant for the efficiency of cyst removal (W. P. filtered by gravity through 47-mm-diameter 5.0-gum-
Lawrence, Masters thesis, University of Washington, porosity Millipore or Nuclepore membrane filters. The
Seattle, 1979). It was necessary to develop a quanti- filters were air dried, and small pieces of the filters
tative method with a known recovery efficiency that were cut out and stuck onto stubs covered with double-
would retain any cysts still remaining in the finished stick tape. Other cyst suspensions were critical point
water after passing through the water treatment plant. dried to avoid membrane filtration and air drying. The
We developed a recovery method that used mem- aqueous suspensions were postfixed in 1% OS04 in 0.15
brane filters of 5-,um pore size to retain G. lamblia M cacodylate, dehydrated in ethanol, and critical-
cysts. We first tested two filters of a small diameter point dried with C02. After each step of the postfixa-
(47 mm). We soon found that it was necessary to test tion and dehydration procedure, the suspensions were
more expensive, larger-diameter filters (293 mm) to briefly centrifuged, and the fluid was decanted. For
maintain filtering efficiency for the relatively large the critical-point drying step, the suspensions were
encosed in BEEM capsules (Better Equipment for
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volumes of water from the treatment plant. The re-
covery efficiency of the filters was tested in the follow- Electron Microscopy, Inc., Bronx, N.Y.) capped on the
ing way. two ends with 5.0-gm-porosity Nuclepore filters (a
Aqueous suspensions of fixed G. lamblia cysts were modification of the procedure of Hayunga [8]). After
passed by vacuum through 5.0-,um-porosity Millipore critical-point drying, the BEEM capsules were opened,
(Millipore Corp., Bedford, Mass.) or 5.0-,um-porosity and the dried cysts were sprinkled onto stubs covered
Nuclepore (Nuclepore Corp., Pleasanton, Calif.) mem- with double-stick tape. The stubs were coated with
brane filters. Concentrations of cysts before and after gold-palladium in a Denton Vacuum Desk-1 sputter
filtration were determined by enumeration on a Clay- coater and viewed in a JEOL JSM-35 scanning elec-
Adams model 4011 Spencer Bright Line hemacytom- tron microscope (JEOL, Tokyo, Japan).
eter and collaborated with counts on a Coulter For the transmission electron microscopy studies,
Counter (Coulter Electronics, Hialeah, Fla.). Cysts aqueous suspensions of fixed G. lamblia cysts were
were removed from the 47-mm filters by immersing postfixed in osmium, dehydrated in ethanol, and
each ifiter in 10 ml of distilled water in a small flask embedded in Epon. Thin sections were stained with
and agitating gently by hand. The filter was then uranyl acetate and lead citrate and viewed with a
discarded, and the liquid was examined for presence JEOL JEM 100S electron microscope.
and quantity of G. lamblica cysts. The larger 293-mm
membrane filters were processed by using a two-step RESULTS
centrifugation process summarized in Fig. 1. Recovery
rates of cysts from the different types and diameters Since a subsequent part of the overall study is
of filters were then calculated. concerned with the efficiency of a water treat-
For the scanning electron microscopic studies, ment pilot plant for the removal of G. lamblia
aqueous suspensions of fixed G. lamblia cysts were cysts (Lawrence and DeWalle, manuscript in
preparation), we needed to develop and evaluate
20 liters of prefiltered (5.0 ,um) tap water a quantitative method with a known recovery
I efficiency that could be used to determine the
Pass through 293-mm (5.0-,um-pore size) number of cysts in a given volume of water.
Nuclepore filter at 10 lb/in2 with nitrogen Known quantities of cysts were filtered, and the
gas. 0.2-gum filter on nitrogen tank recovery efficiency was determined. Four differ-
I ent methods were checked against each other.
Filter removed and placed in shallow dish.
Cysts washed off by agitation of filter in 0.3 Recovery rates of G. lamblia cysts with the
liter of water (platform shaker, 47-mm-diameter 5.0-gum-porosity Millipore and
Toothmaster Co., Racine, Wis.) Nuclepore filters were comparable (Fig. 2). The
I same recovery rate, approximately 75%, was
Centrifuge retentate at 1,500 rpm for 10 found when the 293-mm-diameter Nuclepore fil-
min in eight 50-ml conical bottom tubes ter was used (Fig. 3). A significantly lower recov-
I ery rate, approximately 25%, was found after
Retain "sediment" (approximately filtering cysts with the 293-mm-diameter Milli-
10 ml x 8) pore filter. Coulter Counter and hemacytometer
4 counts of the filtrates showed that no cysts
Transfer to two 50-ml conical tubes and
recentrifuge passed through the filters. The reasons for the
4 less than 100% recovery from the filters and the
Retain "sediment" (approximately strikingly lower recovery on the large Millipore
5 ml x 2) filter were unclear. Therefore, it was decided to
4 study the filter surface with scanning electron
Enumerate on Coulter Counter and microscopy.
compare with initial concentration Cysts collected on either air-dried Millipore
FIG. 1. Summary of method used for the recovery or Nuclepore membrane filters exhibited dis-
of G. lamblia cysts from 293-mm-diameter 5.0-p,m- torted or flattened cyst walls (Fig. 4 to 11). The
porosity Nuclepore filters. pattern of such flattening of the cyst wall wasVOL. 40, 1980 ULTRASTRUCTURE OF GIARDIA CYSTS 823
100 __ __ _A
* -- lipore filters. Although there seemed to be fewer
-
cysts on the Millipore filters, it was more difficult
0
A to detect the cysts on the rough Millipore sur-
b-
U' face.
0
0 '-
We observed sectioned material by transmis-
10'0 - sion electron microscopy (Fig. 12) to confirm the
___, ____ presence of Giardia cysts. Cysts prepared via
40
a. critical-point drying were not flattened (Fig. 13;
A B see also Fig. 14 to 17). Rather, such specimens
appeared ovoid or spherical and agreed with the
01 transmission electron microscopic observations.
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103 04 s5 1O The possible forces that may act on cysts to
distort them during the processes of filtration
Cyst CoInc. (no,/m I and air drying are considered below.
Some additional observations were made on
FIG. 2. Recovery rates (A) of G. lamblia cysts from the material that had been prepared for electron
(A) 47-mm Nuclepore 5.0-ym -porosity filters and (B) microscopy. Some of the cysts appeared to show
47-mm Millipore 5.0-pum-poroosity filters. a process of division (Fig. 8 and 9). One
"stretched" cyst was found, apparently an arti-
4IiAn fact caused by the preparative procedures (Fig.
10).
With the scanning electron microscope, a va-
A riety of material was observed on the cyst wall.
0
e- This was particularly evident on critical-point-
dried specimens (Fig. 13 to 17). Air-dried cysts
50i were usually free of such material (Fig. 6). Oc-
casionally, bacterium-like structures were asso-
40~
ciated with the cysts (at the upper right and
lower left of the double cyst shown in Fig. 10
and at the right of the cyst shown in Fig. 15).
One cyst in the sections prepared for transmis-
o sion electron microscopy showed a bacterium-
104 105 like structure associated with the cyst wall (Fig.
18).
Cyst Co nc. Ino./ml I Our transmission electron microscopic prepa-
rations usually showed a rather wide space be-
FIG. 3. Recovery rates (0) of G. lamblia cysts from tween the organism and the cyst wall (Fig. 12
(A) 293-mm Nuclepore 5.0-,1n L-porosity filters and (B) and 19). A peripheral array of vesicles was char-
293-mm Millipore 5.0-p,m-por-osity filters. acteristic for most organisms. A dense-staining
material coated the inside surface of these pe-
different for cysts collect ed on Millipore filters ripheral vesicles. A few larger peripheral lacunae
compared with cysts on INuclepore filters. The were seen (asterisk in Fig. 19). The inner surfaces
surface of the Millipore filter consists of inter- of the lacunae were lined with a dense-staining
meshed strands (Fig. 4 an d 5), and the diameter material. A dense material also coated the inner
of the individual strands is much smaller than surface of the cyst wall and the surface of the
the -5.0-Um
r-
-. r-. --. The distortion -
nore size. "'-scvst
of the .7 - encysted organism.
on the Millipore surface seemed to be deter-
mined to some extent by how it rested on the DISCUSSION
small individual strands (Fig. 5). For the cysts Information about the biology of Giardia or-
retained on the surface of a Nuclepore filter, the ganisms, the incidence of giardiasis, and the
pattern of distortion was distinctly different (Fig. ultrastructure of these parasitic protozoans is
6 and 7), apparently because of the smoothness reviewed in three recent publications (1, 10, 13).
of the Nuclepore surface. A fairly uniform, rim- Several scanning electron microscopy studies on
like structure was apparent around those cysts the trophozoite (4, 17, 23) complement transmis-
that rested on the flat surface of the filter (Fig. sion electron microscopy studies (3, 6, 18, 19;
7 and 8). Cysts that overlapped the filter pore additional references in 13). Previous ultrastruc-
were sharply bent into the pores (Fig. 6, 10, and tural studies of the cyst are those of Sheffield
11). Overall, more cysts per unit of area were and Bjorvatn (20), Sheffield (19), and Tombes
readily seen on the Nuclepore than on the Mil- et al. (21).824
N*
LUCHTEL, LAWRENCE, AND DEWALLE
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.,ep,
FIG. 4. A low-magnification view that shows three G. lamblia cysts (arrows) on a 5.0-,um-porosity Millipore
filter. Bar, 20 ,pm.
FIG. 5. A higher-magnification view of the middle cyst shown in Fig. 4. The cyst is flattened and distorted.
The distortions seem to depend on how the cyst rests on the contours of the filter surface. Bar, 5 ,um.
FIG. 6. A low-magnification view of a 5.0-tim-porosity Nuclepore filter that shows several cysts (arrows)
and some unidentified debris, presumably consisting of fecal material and ruptured cysts (arrowhead). Bar,
20 ,um.
FIG. 7. A higher-magnification view of a cyst, comparable to those shown in Fig. 6. The cyst is flattened on
the filter surface and typically shows a thin outer rim or flange. The central convex portion of the cyst is
caused by the encysted organism. Bar, 2 uin.
-&,%
.:Rt a)
APPL. ENVIRON. MICROBIOL.
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is,
'VOL. 40, 1980
t| ULTRASTRUCTURE OF GIARDIA CYSTS 825
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ffFA c4
4 - - i3, ,-S - *|jO'e> f;i
i pj -
( r0
+ :'
' ~~ ~ _JJMT:
~~~~~~
4W ~ ~ ~ ~ ~ ~~~~M
FIG. 8-11. Air-dried cysts collected on Nuclepore filters. Apparently, encysted organisms are able to divide,
and the cyst, wall is then restructured to enclose separately each of the two newly formed organisms.
FIG. 8. A single cyst in which the organism inside appears to be in the process of dividing. Bar, 5 rim.
FIG. 9. A double cyst, apparently formed after an organism within a single cyst had divided. The arrows
indicate a line of demarcation that separates the two cysts. Presumably, this double cyst breaks apart to form
two separate cysts. Bar, 5 n.tm
FIG. 10. A double cyst that has been stretched artifactually during the preparation and filtration proce-
dures. Bar, 5 p5m.
FIG. 11. A cyst that has become distorted, apparently because of settling into a pore of the filter. Bar, ,im.
2
flattened shapes for Giardia cysts (Fig. 5 and 7) not filtering; air drying versus critical-point
are not consistent with the ovoid outlines of drying), most of the flattening is probably due
cysts shown by various light microscopic studies to the surface tension of water as the specimen
(13) and the transmission electron microscopy is being air dried. Some of the critical-point-
observations of Sheffield and Bjorvatn (20). We dried cysts were somewhat distorted (insert, Fig.
then confirmed that our material was Giardia 13), possibly due to some transient air drying
cysts by transmission electron microscopy (Fig. during the several fluid exchanges before the
12) and subsequently showed that ovoid cysts critical-point-drying step. But overall, although
could be prepared for scanning electron micro- the critical-point-dried cysts underwent several
scopic observation if the cysts are critical-point filtering and centrifugation steps, they retained
dried (Fig. 13). Although we did not check each their ovoid shape. On the other hand, filtration
set of variables independently (filtering versus had some effect on the cyst morphology as the~ ~
v
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826VOL. 40, 1980 ULTRASTRUCTURE OF GIARDIA CYSTS 827
cysts appeared distinctly different on the Milli- noted that there was no morphological effect on
pore surface (Fig. 5) compared with those on the the cysts with the sucrose flotation technique if
Nuclepore surface (Fig. 7). the cysts were removed immediately from the
Tombes et al. (21) studied the cysts of Giardia interface and placed in physiological saline. With
collected from a variety of mammals, including the methodology of Sheffield and Bjorvatn (20),
humans. The morphology of the cysts they col- the suspensions are diluted 10-fold with water
lected from humans is different from that ob- after collecting them from the interfaces.
served by us. The cysts they studied by phase Another possible effect of the sucrose flotation
microscopy had the typical elliptical shape; by method is that it may change the width of the
scanning electron microscopy, the cysts seemed space between the cyst wall and the organism.
to be distorted, having a cuboidal shape. Possible Sheffield (19) believes that these spaces are not
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reasons for our different results are difficult to caused by the different isotonic pressures of the
decide upon since Tombes et al. used a variety flotation solutions. We found a much wider
of fixation and preparative techniques, and for space between the cyst wall and the organism
any particular micrograph, the data are not than that shown by Sheffield and co-workers
given as to how the cysts were fixed, whether (19, 20) or the cyst shown by transmission elec-
the material was fixed immediately or after some tron microscopy in the study of Nemanic et al.
initial filtrations (sucrose flotation techniques (18). The material studied by Nemanic et al.
were not used), how long the material was stored (18) was not exposed to a sucrose flotation tech-
in aldehyde before drying, and whether the cysts nique as the organisms were prepared for elec-
were air dried or critical-point dried. Overall, tron microscopy by washing pieces of gut and
Tombes et al. noted no consistent differences in centrifuging the wash. Perhaps species differ-
cysts after air or critical-point drying. We found ences may be a factor in comparing our results
substantial differences in cyst morphology when with those of Nemanic et al. (18) but the reasons
cysts were air dried or critical-point dried. We for our results being different from those of
suggest that a possible procedural error that Sheffield and Bjorvatn (20) are not apparent
Tombes et al. mention in their discussion may unless they fixed the cysts after sucrose flotation.
be a significant factor in our different results. Perhaps selection of micrographs may be a con-
Sucrose flotation technique. We used the tributing factor as Sheffield, in a discussion after
sucrose flotation method of Sheffield and Bjor- his paper (19), states that a variety of cyst types
vatn (20) to prepare suspensions of cysts. They were seen; that is, cysts in which the cytoplasm
apparently fixed the cysts after the sucrose pro- was closely applied to the cyst wall, whereas
cedure. If so, they obtained remarkably good others showed large, open areas between cyto-
fixation after a lengthy concentration process. plasm and wall. We also saw sections of cysts in
We fixed the fecal material before the sucrose which the cytoplasm was closely applied to the
flotation. For laboratory diagnosis of giardiasis cyst wall, but since most of the sectioned cysts
in unfixed stools, the basic method is a zinc showed an open space (Fig. 12), our interpreta-
sulfate flotation method (13). With this tech- tion is that the organism does not occupy the
nique, the cytoplasm of the cells is plasmolyzed entire space of the cyst. The rimlike structure
by the hypertonic zinc sulfate solution, and the on air-dried cysts (Fig. 7) would also indicate
cytoplasm is characteristically concentrated at that the cyst wall collapsed into a space not
one side of the cyst (see Fig. 23 in reference 13). occupied by the encysted organism. We ob-
Although the cyst wall is apparently stable served that the cyst walls are usually 0.15 to 0.25
throughout the zinc sulfate flotation process, it pm thick, which is less than the 0.3-,um thickness
seems much more delicate when sucrose flota- observed by Sheffield and Bjorvatn (20).
tion is used. Levine (14) observed that Giardia Composition of cyst wall. The composition
cysts concentrated by sugar flotation shrivel and of the cyst wall is unknown. Filice (5) was not
become unrecognizable in a matter of minutes. able to obtain any positive histochemical infor-
Stevens, in a discussion after Levine's paper (14), mation, although he did show that it was Feul-
FIG. 12. A transmission electron micrograph of encysted Giardia organisms. The cyst walls usually form
smooth ovoid outlines, although a couple of examples of acutely folded cyst walls (arrows) can be seen (also
see insert of Fig. 18). Bar, 10 ,um.
FIG. 13. Smooth, ovoid cysts after critical-point drying. These cysts are embedded in a mat or clump of
debris, bacteria, and fecal material. A low-magnification micrograph of the entire clump is shown in the
lower right insert. The upper left insert shows examples of single, isolated cysts after critical-point drying.
Such cysts may show some moderate degree of distortion. Bar, 10 pim. Lower right insert bar, 100 ,um. Upper
left insert bar, 5 ,um.r
. I ra_
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w -^ .
# tsS -vS
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14
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I
A
9
.4
;a1 * Fj 4 Pb
;R floF
f.
4
a
,
*4t 4 s. -
FIG. 14-17. A variety of cyst morphologies as seen after critical point drying. Almost all cysts had some
sort of material or debris stuck on the cyst walL In some cases, structures that could be identified as bacteria
were attached to the cyst wall (Fig. 15). In other cases, unidentified fibrous forms were seen on the cyst walls
(Fig. 16 and 17). Bars, 2 pm.
FIG. 18. A transmission electron micrograph showing a structure, presumably bacterial in nature, attached
to the cyst walL The fibrous coat of the attached structure seems to interact with the fibrous cyst wall. The
insert shows a low magnification view of the entire cyst and attached structure. Bar, I pm. Insert bar, 2 pm.VOL. 40, 1980 ULTRASTRUCTURE OF GIARDIA CYSTS 829
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*,~
* 1ST
a e_a
jw-
FIG. 19. An encysted organism with its typical array of peripheral vesicles (also see Fig. 12). N, nuclei; A,
axonemes of the flagellae; S, microtubule-ribbon complexes of the fragmented sucking disk. The arrow points
to a portion of the cyst wall that has apparently retained a staining density similar to the staining density of
the inner surface of the cyst wall. The asterisk is in a peripheral lacuna. Bar, 1 pm.
gen stain negative; it did not stain with a lipid stand the surface tension of water during air
stain, Sudan IV, and it did not seem to be drying, and its flexible nature, even after fixa-
affected by various enzyme digestions (pepsin, tion, may lower the filtering efficiency of various
trypsin, and papain). In any case, the cyst wall water filtration plants. The nature of the cyst
is not fixed adequately with aldehydes to with- wall needs to be taken into consideration when830 LUCHTEL, LAWRENCE, AND DEWALLE APPL. ENVIRON. MICROBIOL.
various cyst model systems are being tested. For filters (sand and anthracite) before any turbidity
example, Logsdon et al. (16) used 9-,m-diameter breakthrough, indicating that the filter column
radioactive microspheres as a model for Giardia was still intact. Although an equivalent pore size
cysts because the cysts are difficult to obtain, cannot be determined in dual-media filters, that
detect, and count, whereas the radioactive mi- G. lamblia cysts can somehow penetrate the
crospheres are similar in size to Giardia cysts dual-media filter again indicates their flexible
and are easy to trace. Our observations suggest nature.
that such microspheres would be filtered more Some cysts are probably lost because they are
efficiently than Giardia cysts in pilot water fil- destroyed during the preparative steps. What is
tration plants. probably a remnant of a cyst is shown in Fig. 6.
Loss of cysts during membrane filtration. Overall, destruction of cysts probably accounts
The maximum rate of recovery obtained from for most of the 25% loss of cysts during the
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the Millipore and Nuclepore membrane filters recovery procedure on membrane filters. A fur-
was 75%. A number of factors may account for ther loss occurs with the Millipore filter, proba-
the 25% loss. Cysts may remain attached or bly because of the embedding of cysts in the
embedded in the filter after the recovery proce- filter.
dure, adhere to nonfilter surfaces of the filtration Formation of cyst wall. The staining den-
assembly, pass through the filter, or be de- sity of the material lining the inner surfaces of
stroyed during the filtration or centrifugation the peripheral vesicles and lacunae is similar to
process or both. the staining density of the material on the sur-
The filters were agitated by hand as vigorously face of the encysted organism and the inner
as possible without destroying the filters. It was surface of the cyst wall (Fig. 18 and 19). The
later suggested that perhaps a better method vesicles thus seem to contain a secretory mate-
would be to vigorously and systematically wash rial that eventually is used to make the cyst
the fiter surfaces with strong streams of distilled wall. The cyst wall has a fibrous substructure,
water with 0.01% Tween 20 from a capillary as if it were formed by successive layers of
pipette. We did not test such a washing proce- material. We suggest that the successive layers
dure. Compared with the unidimensional surface arise from successive waves of vesicles that co-
of the Nuclepore filter, the convoluted fibrous alesce to form enlarging peripheral lacunae.
structure of the Millipore filter may permit cysts Eventually, one giant lacuna in effect completely
and other material to become embedded within surrounds the organism, and, in the process,
the depth of the filter, and, by our recovery another layer of the cyst wall has been laid
procedure, the cysts would not be readily washed down. By some type of maturational process,
out. Such differences in the filter characteristics the newly formed layer of the cyst wall then
may explain the difference in recovery rates loses much of its staining density. Occasionally,
between the 293-mm-diameter Millipore filter however, the staining density is retained, as in-
and the 293-mm-diameter Nuclepore filter. dicated by the arrow in Fig. 19.
What is still puzzling are the comparable recov- Hemmes and Hohl (9) ventured a similar hy-
ery rates of the 47-mm-diameter Millipore and pothesis for encystment of Phytophthora par-
the 47-mm-diameter Nuclepore filters. However, asitica zoospores. Encystment involved the fu-
a 293-mm-diameter filter has approximately 39 sion of peripheral vesicles with the plasma-
times more surface area than a 47-mm-diameter lemma, followed by the release of glycoprotein
filter. Thus, although there may be some differ- and possibly other cell wall precursor materials.
ence in recovery rates for the 47-mm-diameter Friend (6) suggested that the location of the
Millipore and Nuclepore filters, perhaps we were peripheral vacuoles in the trophozoites of Giar-
not able to detect this difference until the larger dia muris were consistent with a secretory func-
surface area of the large-diameter filter (with its tion, perhaps the secretion of the cyst wall.
larger number of cysts) made it apparent. Mucocytes in other protozoa are rows of globular
Part of the overall 25% loss could be attrib- elements beneath the pellicle that discharge ge-
utable to cysts passing through the filters. The latinous or mucoid secretions (6). Finally, Filice
flexibility of the cysts is suggestive evidence for (5) observed that, in living organisms, the cyst
how cysts could pass through individual pores of forms first on the dorsal surface of the tropho-
smaller diameter than the size of the cysts (Fig. zoite from refractile granules in the peripheral
11). With the optical and Coulter Counter meth- cytoplasm. On the other hand, Sheffield (19)
ods used, however, no cysts were detected in the discounted the role of the peripheral vesicles as
filtrates. In our removal study (W. P. Lawrence, secretory vesicles involved in cyst wall formation
Masters thesis, University of Washington, Se- since he noted their abundance after wall for-
attle, 1979), some G. lamblia cysts were found mation. Although our observations also showed
to pass through a 4-ft column of dual-media this abundance of vesicles (Fig. 12 and 19), theirVOL. 40, 1980 ULTRASTRUCTURE OF GIARDIA CYSTS 831
density was usually decreased where the organ- air-dried specimens. Whether material is depos-
ism was apposed against the cyst wall. We sug- ited on the cyst wall during the procedure of
gest that such an area of apposition represents critical-point drying, washed off the cyst wall
a zone where a number of vesicles had been during filtering, or removed by aqueous surface
secreted just before fixation, and that the re- tension during air drying is unknown. We sug-
maining vesicles would have subsequently ex- gest that the association is real since the purpose
panded this zone of secretion as the process of of critical-point drying is to preserve the delicate
laying down another layer of the cyst wall would details of biological structure.
have proceeded. If our interpretation is correct, Conclusions. The physiological nature of the
the fascinating question still remains of how G. lamblia cyst wall remains unclear. Flexibility
these vesicles are generated since there does not of the cyst wall has resulted in experimental
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appear to be a vesicle-generating system present, difficulties with membrane filtration of the cysts
such as a Golgi apparatus. in aqueous suspension. These findings point to
Double cysts. We observed a variety of dou- potential difficulties in removing cysts from wa-
ble cysts (Fig. 8 to 10). Kofoid and Swezy (12) ter with present water treatment technology.
concluded that such cysts resulted from binary
fission of a zooid. In their discussion, it is not ACKNOWLEDGMENTS
explicitly clear whether the "sister zooids" This study was funded in part by U.S. Environmental
formed by the process of binary fission are en- Protection Agency grant R 806127.
We thank John Boykin, Department of Environmental
cysted after division but before separation (and Health, University of Washington, for his help with photog-
then separate after the joined zooids each form raphy and Yvonne Fichtenau, formerly of the State of Wash-
a cyst) or an encysted zooid undergoes binary ington Parasitology Lab, for her cooperation in providing
fission, and then each sister zooid becomes en- specimens.
closed in a separate cyst. Apparently, Giardia LITERATURE CITED
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