Fegan, N.

Hayward, A.C.

Stirling, G.R.

Webb, R.I.

Horizon / Pleins textes

Fundarn. appl. NernalOl., 1993,16 (4), 309-313
Cryofixation and cryosubstitution as a method for preparing
Meloidogyne javanica eggs for electron microscopy
Narelle FECAl" *, Richard 1. WEBB **, A. Chris HAYWARD * and Graham R. STIRJJNC **
*' Department ofMicrobiolog)', * Centre fryr Microscopy and Microanalysis,
The University of Queensland, Brisbane 4072, Austmlia,
and ** Qyeensland Department ofPrimar)' Industries,
Meiers Road, IndoOToopilly, 4068, Austmlia.
Accepted for publication 12 October 1992.
Surnmary - Transmission electron microscopy (TEM) of nemarodes eggs is difficult because me lipid layer of me egg shell
prevents penetration of me chemicals used for fixation and embedding. Conventional chemical fixation of intact eggs does not allow
observation of me internaI egg components as mey are damaged during processing. Cryofixation and cryosubstirution was used as
an alternative memod for me preparation ofinract nemarode eggs for sectioning and resulted in sections of eggs mat were well fixed
and free from me artefacts caused by chemical fixation. The integriry of embryos and larvae within eggs was mainrained. The
nematode eggs can now be srudied as an intact system by using mis memod.
Résumé - Cryofixation et cryosubstitution : une méthode de préparation des œufs de Meloidogyne javanica pour
l'examen en microscopie électronique - L'érude en microscopie électronique à transmission des œufs de nématodes est rendue
difficile par la présence de la couche lipidique de la coque qui empêche la pénétration des produits chimiques utilisés pour la fixation
et l'inclusion. La fixation d'œufs intacts avec les produits chimiques usuels ne permet pas en effet l'observation des composants
internes de l'œuf, ceux-ci étant détériorés durant le processus. La cryofixation/cryosubstirution est utilisée comme une mémode de
remplacement pour la préparation d'œufs de némarodes intacts en vue de coupes fmes. Cene mémode permet d'obtenir des coupes
correctement fixées et dépourvues des artefacts causés par les fixateurs chimiques. Les embryons et les larves contenus dans l'œuf
sont conservés intacts. En utilisant cene mémode, les œufs de némarodes peuvent maintenant être érudiés sans que leur intégrité ne
soit aneinre.
Keywords: Cryofixation, cryosubstirution, electron microscopy, Meloidogyne javanica, nemarode eggs.
Nematode eggs are difficult to fIx for electron mi-
croscopy because the inner lipid layer of the shell pro-
vides an impenneable barrier to chemical fIxatives
(Bird, 1971; Wharton & Jenkins, 1978). This problem
has been overcome partially by using long incubations
in fIxatives (Bird, 1968), by cracking the eggs open to
allow penetration of the fIxatives (Bird & McClure,
1976; Perry el al., 1982; Perry & Trett, 1986; Wharton
& Bone, 1989), or by freezing, sectioning on a cryotome
and then fIxing (Wharton, 1979). Recently a microwave
fIxation technique has been used to aid the penetration
of chemical fIxatives into nematode eggs with suitable
results for exarnining the ultrastructure of unhatched
nematodes Gones & Gwynn, 1991). CryofIxation and
cryosubstitution procedures have been successfuUy used
as a way of observing living material without the use of
chemical fIxatives (Menco, 1986). Freezing samples
rapidly in a cryogen cooled by liquid nitrogen, instanta-
neously arrests aU activity and captures cel! components
without the artefacts associated with chemical process-
ing, such as shrinkage and dissolution of metabolites.
CryofIxation has been used to study fungal-infected
eggs under scanning electron microscopy (SEM) with
good results (Lopez-Llorca & Duncan, 1988, 1991) but
these eggs were damaged through fungal penetration of
the egg sheU and this may have aided processing. The
aim of this work was to compare chemical fIxation with
cryofIxation and cryosubstitution as methods for study-
ing whole, intact nematode eggs.
Materials and methods
NEMATODEEGGS
Eggs of Meloidogyne javanica were inoculated onto
tomato plants grown in pots and 6 to 8 weeks later, eggs
from the next generation of nematodes were harvested
by gently agitating roots in 0.5 % NaGCl (modifIed
from McClure el al., 1973).
CONVENTIONAL CHEMICAL FIXATION FOR ELECTRON
MICROSCOPY
Nematode eggs were fixed in 3 % glutaraldehyde in
0.1 M sodium cacodylate buffer (pH 7.2) for 24 h at
room temperature and the peUeted eggs were then
washed twice in the same buffer. The eggs were enrobed
in 2 % agarose Type VII (low geUing temperature, Sig-
ma), washed again with 0.1 M sodium cacodylate buffer
and then post-fIxed in 1 % osmium tetroxide in 0.1 M
sodium cacodylate buffer for 2 h at room temperature.
ISSN 1164-5571/93/04/309 05 S 2.50/ © Gauthier-Villars - ORSTOM 309
N. Fegan et al.
After mree 10 min washes in 0.1 M sodium cacodylate
buffer me eggs were dehydrated mrough a graded ema-
nol series (50 %, 70 %, 85 %, 95 % and 100 %) for
20 min each.
INFiLTRATION, E1V1BEDDING AND SECTIONING
Eggs were infùtrated wim Spurr's resin (SPUIT, 1969)
over a 7 day period on a rotator at room temperature,
wim 24 h in each of me foUowing emanol:Spurr's resin
mixtures; 3:1, 2:1, 1:1, 1:2 and 1:3. The last two in-
cubations were in pure Spurr's resin for 24 h each, wim
fresh resin being added every 12 to 16 h. Samples were
placed under vacuum for 10 min and transferred to a
rotator at 37 oc. After 2 h, fresh resin was added and me
specimens were placed at 37 oC for anomer 2 h before
blocking. Polymerisation took 16 h at 50 oc. Blocks
were trimmed and sectioned wim a diamond knife on a
Reichert-Jung Ultracut E LÙtramicrotome and placed
onto formvar coated copper slot grids. Sections were
stained using lead citrate (Reynolds, 1963) and uranyl
acetate (Daddow, 1986).
CRYOFIXATION AND CRYOSUBSTITUTION
A drop of nematode eggs suspended in distiUed water
was placed onto a min layer of 2 % agarose and aUowed
ta dry until me eggs adhered to me agarose. Small blocks
of agarose, about 3 mm in diameter were cut and placed
onto a metal pin for plunge freezing. Liquid nitrogen
was used to cool propane to - 187 oC in a Reichert-Jung
KF80 immersion cryofixation system. When me tem-
perature of me propane was between - 187 oC and
- 180 ·'C me eggs were plunged into me cryogen, men
stored under liquid nitrogen. A dry-ice/acetone bam was
used to cool 2 % osmium tetroxide in acetone (dried
over molecular sieve) of which 1.5 ml was added to each
cryotube containing only one or two blocks of agarose
wim eggs. The eggs were kept in me osmium-acetone
solution for 3 days at - 79 oC using a dry-ice/acetone
bam ta maintain me temperature. Samples were aUowed
to warm up to - 20 oC overnight, men brought ta 4 oC
for 4 h before being placed at room temperature for
mree 20 min washes in dry acetone. The specimens
were men infiltrated, embedded and sectioned as previ-
ously described using acetone instead of emanol for in-
filtration. Grids were stained and examined under
TEM.
SCANNING ELECTRON MICROSCOPY
Samples for SEM were prepared following me cryo-
fixation and cryosubstitution procedure until me last
acetone wash at room temperarure. Specimens were
subjected to critical point drying using a Polaron critical
point dryer, mounted and coated wim platinum before
observation.
310
LrGHT MfCROSCOPY
Thick sections were cut from polymerised blocks and
heat fixed onto a glass microscope slide. Sections were
stained wim toluidine blue on a hot plate for 2 min,
rinsed and viewed under bright field microscopy.
EXAMINATION 01' SPECIMENS
Samples prepared for transmission electron micros-
copy were viewed using a Hitachi H-800 transmission
electron microscope. Scanning electron microscopy was
performed using a }EOL G400F scanning electron mi-
croscope and an Olympus phase contrast microscope
wim camera was used for light microscopy.
Results
CHEMICALLy FlXED EGGS
Eggs mat were processed using a standard chemical
fixation protocol were unsuccessfuUy fixed, surviving
for 24 h in glutaraldehyde and 1 h in osmium tetroxide.
Sections of eggs stained wim toluidine blue showed loss
of internaI contents mat also resulted in distortion and
shrinkage of me egg shape (Fig. 1 A). Blocks were diffi-
cult to cut as me eggs feU out of embedding material.
Orily me egg sheU remained partially intact as was evi-
dent when viewed under TEM (Fig. 1 B).
CRYOFlXED AND CRYOSUBSTITUTED EGGS
Cryofixed and cryosubstituted egg sections viewed
under me light microscope showed mat eggs had re-
mained intact and internai contents had been successful-
ly fixed (Fig. 1 C). Eggs observed under me scanning
electron microscope displayed a smoom surface of me
egg shell (Fig. 1 D). Bom embryonic and larval stages of
eggs could be clearly identified wimin intact sheUs as
observed under electron microscopy (Fig. 2 A, C). Sec-
tions of whole eggs containing embryonic ceUs showed
me developing ceUs and nuclei, containing nucleoli, very
clearly (Fig. 2 B). The DNA wimin me nuclei did not
form aggregates, which can occur when chemical fixa-
tives are used. The layers of me egg sheU could be clearly
identified. Sections of larvae wimin eggs showed me
cuticle striations and internai contents of me larvae.
Closer examination showed me layers of me cuticle (Fig.
2 D).
Discussion
Our results confirmed mose of omers who have
shown mat intact nematode eggs cannot be prepared for
electron microscopy by conventional chemical fixation.
It has always been necessary to damage eggs in sorne
way to allow penetration of me chemical fixatives (Bird
& McClure, 1976). However, in me present srudy intact
nematode eggs could be readiJy examined using cryofix-
ation and cryosubstitution. Samples were easiJy proc-
essed, and procedures for preparation were less invasive
man for conventional chemical fixation.
Fundam. appl. NemalOl.
C7yofixalion and oyosUbS[ÙUlion in eleclron mzCroscopy
A
Fig. 1. A : Lighr microscopy of a toluidine blue srained section of a chemical!y tlxed .ègg; B . TEM of a chemical!y proccssed cgg; C :
Lighr microscopy of a toluidine blue srained section of cryo-processed nematode ..:ggs; 0 : SEM of cryoprocessed eggs. (Bars
equ'ivalenr : A, C =40 iJ.m; B =5 iJ.m; D =15 iJ.m.)
Jones and G\vynn (1991) suggested that methods
with quick fixation times were best because artefacts
from physiological processes such as autolysis could oc-
cur in larvae that were still living in the fixative. Our own
observations have shown that larvae within eggs can
survive 24 h in glutaraldehyde and 1 h in osmium te-
troxide. The fixation time can be substantially reduced
by cryofixation, because only fractions of a second are
required for fixation. This aUows the technique to be
appLied to the capture of very rapidly occurring physio-
logical or metabolic processes.
The major disadvantage of cryofixation is the forma-
tion of ice crystals which can cause major structural
damage to cells. lee crystal damage may occur if the
sampIe is left to warm at any time before substitution is
complete. Care must therefore be taken ta maintain the
temperature of the samples below - 40 oC for more than
Vol. 16, n° 4 - 1993
10 min (Steinbrecht, 1985). lce crystals may form in the
specimens while f[cezing as only the leading edge of the
sampIe is instantly frozen, producing vitrified water
without crystal growth. However minimal amage was
found in the nematode egg sy~têm and cryofixation
was superior to chemical fixation because ultra-
structural detail was apparent and membranes and other
structures were berrer preservcd. A cryogcn such as pro-
pane was used because the transfer of heat between the
specimen and the cryogen was much faster, cooling
the specimen more rapidly than if a less dficient medi-
um were used.
Substitution is used ra dehydrare a specimen and
serves as a valuable link berwecn cryofixation and fur-
ther processing at room tempcrature ( teinbrecht
& Müller, 1987). Acetone is used to dissolve the iee and
serves to rem ove the water from the specimen. The
311
N. Fegan et al.
Fig. 2. Cryoprocessed nemarode eggs. A: An early developmental stage egg showing a cluster of embryonic cells; B : Higher
magnification of an embryonic cell showing weil preserved cellular components and nucleus (Nu), as well as the chitin (Ch) and
vitelline (arrow) layers of the egg shell; C : A larva within an egg; D : Higher magnification of C showing weil preserved contents and
developing cuticle (Cu). (Bars equivalent : A, C = 5/-Un; B, D = l,.un.)
chemical fixative, osmium tetroxide is included to aid in
stabilisation of ultrastfUcture, which can begin at the
lowest temperature in which the fixative is reactive
(Steinbrecht & Müller, 1987) usually around subzero
temperatures. The substitution was left for three days,
so there was time for the osmium tetroxide to penetrate
the egg sheU and prevent dererioration of tissue. The
penetration of this fixative may have been aided by the
solvent which may have penetrated the egg shell better
than water in wruch most chemical fixatives are dis-
solved. Less marerial is washed out of sections with
cryosubstitution (Menco, 1986) so the processing does
not affect the immunoreactivity of the specimens (Ichi-
kawa el al., 1989), allowing specifie components to be
traced.
The embedding protocol used was long, but success-
312
fuI. Shorter times were tried and tended to give similar
results, but best results were obtained over the longer
time periods. Cryofixation and cryosubstitution does
not involve expensive and complex equipment or harm-
fui chemical fixatives such as the aldehydes, and is a very
useful method for material that is difficult to fix. We
developed the method to study the interaction between
nematode eggs and bacterial antagonists that do not
damage the egg sufficiently for chemical processing.
AcknowledgeInents
We thank Mrs Helen Hosmer for her assistance with pro-
duction of the electron micrographs. The financial support of
the Rural Industries Research and Development Corporation
is acknowledged.
Fundarn. appl. NernalOl.
References
BIRD, A. F. (1968). Changes associated with parasitism in
nematodes. III. U1trastructure of the egg shell, larva1 cuti-
cie, and contents of the subventra1 esophageal glands in
Meloidogyne javanica, with sorne observations on hatching.
J. Parasit., 54: 475-489.
BIRD, A. F. (1971). The SlntClUre of nemalOdes. New York,
Academie Press, 318 p.
BIRD, A. F. & MCCLURE, M. A. (1976). The tylenchid (Ne-
matoda) egg shell : structure, composition and permeability.
ParasilOlogy, 78: 19-28.
DADDOW, L. Y. M. (1986). An abbreviated method of the
double lead stain technique. J. submicrosc. Cytol., 18 : 221-
224.
IcHIKAWA, M., SASAKI, K. & IcHIKAWA, A. (1989). Optimal
preparatory procedures of cryofixation for irnmunocyroche-
mistry. J. Elear. Microsc. Techn., 12 : 88-94.
JONES, ]. T. & GWYNN, 1. A. (1991). A method for rapid
fixation and dehydration of nematode tissue for transmis-
sion electron microscopy. J. Microsc., 164 : 43-51.
LOPEZ-LLORCA, L. V. & DUNCAJ'1, G. H. (1988). A study of
funga1 endoparasitism of the cereal cyst nematode (Helero-
dera avenae) by scanning electron microscopy. Canad. J.
Microbiol., 34: 613-619.
LOPEZ-LLORCA, L. V. & DUNCAN, G. H. (1991). Effects of
fungal parasites on cereal cyst nematode (Helerodera avenae
Woll.) from naturaUy infested soil - a scanning electron
microscopy study. Canad. J. Microbiol., 37 : 218-225.
MCCLURE, M. A., KRuK, T. H. & MJSAGHI, 1. (1973). A
method for obtaining quantities of c1ean Meloidogyne eggs.
J. NemalOl., 5 : 230.
Vol. 16, n° 4 - 1993
Cryofixalion and cryosllbsliLUlion in eleclron microscopy
MENCO, B. Ph. M. (1986). A survey of ultra-rapid cryofixa-
tion methods with particular emphasis on applications to
freeze-fracturing, freeze-etching, and freeze-substitution. J.
Eleclr. Microsc. Techn., 4: 177-240.
PERRY, R. N. & TRETT, M. W. (1986). Ultrastrucrure of the
eggsheU of Helerodera scmuhlii and H. glycines (Nematoda :
Tylenchida). Revue NémalOl., 9: 399-403.
PERRY, R. N., WHARTON, D. A. & CLARKE, A.]. (1982). The
strucrure of the egg-sheU of Globodera roslOchiensis (Nemato-
da: Tylenchida). lnt. J. Parasit., 12: 481-485.
REYNOLDS, E. S. (1963). The use oflead citrate at high pH as
an electron-opaque stain in electron microscopy. J. Ce!!
Biol., 17 : 208-212.
SPURR, A. R. (1969). A low-viscosity epoxy resin embedding
for electron microscopy. J. Ullraslr. Res., 26 : 31-43.
STEINBRECHT, R. A. (1985). Recrystallization and ice-crystal
growth in a biologicaJ specimen, as shown by a simple freeze
substirution method. J. Microsc., 140 : 41-46.
STEINBRECHT, R. A. & MüLLER, M. (1987). Freeze-sub-
stirution and freeze drying. ln: Steinbrecht, R. A. & Zierold,
K. (Eds). Cryolechniqlles in biological eleclron microscopy,
Berlin & Heidelberg, Springer-Verlag : 149-171.
WHARTON, D. A. (1979). The strucrure of the egg-shell of
Aspiculun's lelraplera Schu1z (Nematoda: Oxyuroidea).
Parasitology, 78: 145-154.
WHARTON, D. A. & BONE, L. W. (1989). Baci!!lIs lhuringiensis
israelensis toxin affects egg-sheU ultrastrucrure of Tricho-
slrongylus colubriformis (Nematoda). lnverlebrale Reprod. &
Develop., 15: 155-158.
WHARTON, D. A. &JENKlNS, T. (1978). Structure and chem-
istry of the egg-sheU of a nematode (Trichuris suis). Tissue &
Ce!!, 10: 427-440.
313


Cryofixation and cryosubstitution as a method for preparing Meloidogyne javanica eggs for electron microscopy

Abstract

Similar works

Full text

Available Versions

Horizon / Pleins textes