Grewal, P.S.

Horizon / Pleins textes

T H E  USE OF AGAR AS A  COVER-GLASS  SUPPORT  FOR MOUNTING  NEMATODES 
Parwinder S.  GREWAL 
Entomology and Insect  Pathology  Section, AFRC Institute of Horticultural 
Research,  Littleharnpton,  West  Sussex, BNI 7 6LP, U K .  
Morphometric  and  taxonomic  studies of nematodes 
involve the  handling  and measurement of large  numbers 
of specimens. During the preparation of permanent 
slides, glass-wool is widely used as a cover-glass support 
to avoid any  distortion  or  flattening of specimens (Hoo- 
per, 1986). Though glass-wool is very useful, difficulties 
can  arise during  the handling and selection of fibres of 
the correct diameter (i.e. those that exactly suit the 
nematode  body  diameter).  When  studying  morphologi- 
cal variations among different  nematode  populations  or 
the effects of methods of killing, f i i ng  or mounting 
nematodes  on  their  measurements  hundreds of nema- 
tode  specimens  may  be  used  and the glass-wool process 
can  become  tedious  and  time-consuming.  For  such 
studies and  for  other  routine observations of nematodes, 
a simple method for quick preparation of mounts of 
nematodes is described  here. 
Preparation of agar 
2 g of agar  (Technical  grade  No. 3, Oxoid Ltd.)  and 
50 mg of cupric  sulphate (CuSO4, 7Hz0) is dissolved 
in 100 ml of distilled  water by heating  gently  for 2 to 
3 min. It is then autoclaved at 121 “C for 15 min., 
allowed to cool and whilst still warm, poured  into 
Petri-plates. 
Preparation of nematode  mounts 
1. Place narcotised or killed specimens in a drop of 
mountant (narcotic, e.g. sodium azide, water, fixative, 
e.g. formalin  or TAF, lactophenol  or  anhydrous 
glycerol) on a glass slide. 
2. Take a  small  quantity of the  agar  (prepared as above) 
from  the Petri-plate  with clean forceps  and  place  it on 
the slide  adjacent to  the  drop of mountant  containing 
nematodes. 
3. Divide the agar into three to five similarly-sized 
portions with forceps and arrange them at equal 
intervals around the outer margin of the drop of 
mountant. 
4. Carefully  place the >cover glass on &e drop of 
mountant so that it rests evenly on al1 the pieces of 
agar. 
5. While  observing the slide under  a low power stereo- 
scopic microscope press the cover-glass gently with 
forceps, to the extent that the cover-glass almost 
touches  the nematodes. 
Revue  Nématol. 13 (1) : 121-122 (1990) 
6. A permanent preparation may now, if desired, be 
made by sealing the cover-glass with Thorne’s ce- 
ment  Zut ” or “ Glyceel ”), nail  varnish, or “ Aral- 
dite ” resin  (Hooper, 1986). 
Using  the above technique, 20 females  each of Cae- 
norhabditis  elegans and Tylenchus davainei were 
mounted  in glycerol (processed  according to Seinhorst, 
1959). Measurements of body  diameter  at  the  anus  and 
at the vulva showed that the two techniques did not 
differ  significantly  (Tab. 1). 
Table 1 
Comparative  mean  body  diameters  (Pm) at anus  and  vulva  of 
twenty  adult  female Caenorhabditis elegans and Tylenchus 
davainei mounted in glycerol using glass-wool or agar as 
cover-glass supports 
Nematode  Body  iam. Cover-glass  support 
Glass-wool  Agar 
C. elegans at  nus 20.6 * 3.2y’  0.5 * 2.9 
at vulva 59.8 k 7.4  60.1 k 6.9 
(19.0 - 28.4)** (18.6 - 29.0) 
(48 - 72) (50 - 73) 
T. davainei at  nus 16.1 & 3.4  15.9 k 3.5 
(12.4 - 20.2)  (12.2 - 20.6) 
at vulva 24.3 -t 5.5 24.4 & 5.6 
(20 - 33.4) (19.8 - 34.5) 
* Mean width k standard deviation, ** Range. 
Differences in mean width were not  significant  at  p < 0.01 
with glass-wool  and  agar  as  cover-glass  supports. 
Commonly  used  media  including Malt Extract Agar:, 
Nutrient Agar, Yeast Extract Agar, Potato Dextrose 
Agar  (at  recommended  concentrations)  where  tried and 
were found equally  effective for  routine  preparation of 
temporary  nematode  mounts. But there is the danger of 
microbial  contamination if the slides  are  to  be  kept for 
long. Distilled water agar did not give the required 
degree of gel as the pieces of agar slipped out of the 
cover-glass rather than  spreading below. So a number of 
salts including copper sulphate, sodium ethylmercuri- 
thiosalicylate Thimerosal ”) and sodium hypochlorite 
were tried to break the agar  gel to  the extent  required 
and at the same time be toxic to microbes. In these 
respects  copper  sulphate  proved  ideal. 
121 
P. S. Grewal 
Ësser (1988) embedded  nematode cysts in agar blocks 
to examine  the vulva area and  the present  paper provides 
an additional use of agar that overcomes some com- 
monly-encountered  problems during preparation of 
slides of nematodes. The wax-ring method of sealing 
mounts  (De  Maeseneer & D’Herde, 1963) is very useful 
for lactophenol or glycerol based permanent mounts, 
and  can probably  be  adapted foi temporary  mounts  (in 
water  or fixatives) but  the heat  required to melt  the wax 
could  be  detrimental to specimens especially narcotised 
ones; it might also affect comparative studies of me- 
thods of killing and  fiing. 
Other advantages of the described agar method are 
that  it removes the need  for  additional cover-glass 
supports when the area  occupied by the mountant 
exceeds a  quarter of the cover-glass area  (Hooper, 1986) 
as it often does in the case of aqueous or fixative 
mountants.  Furthermore, oil-immersion objectives may 
be  more easily used as thick  mounts, which can  result 
from  the wax-ring method  (Hooper, 1986), can  be 
avoided using  agar pieces. 
Permanent nematode mounts prepared by the agar 
method were very successful.  Esser (1973) recom- 
mended  Thorne’s  cement Zut ”) as a cover-glass 
support  for  preparation of permanent  ematode 
mounts. Agar would be more useful because of the 
problems  associated with the viscosity of cement (which 
is a  critical  factor). Also cement  firmly seals the cover- 
Accepté pour publication le 29 avril 1989. 
glass to  the slide making  it  difficult to recover/remount 
specimens.  Agar  did not provide  any such problems  to 
remounting. Moreover, this method is simple,  quick and 
inexpensive. 
ACKNOWLEDGEMENTS 
The author is  indebted to Paul N. Richardson  for  encoura- 
gement  and  reading  the  manuscript. 
REFERENCES 
DE MAESENEER, J. & D’HERDE, J. (1963).  Méthodes  utilisées 
pour l’étude des anguillules libres du sol. Revue Agric., 
ESSER,  R. P. (1973). Zut as a cover-glass support for nema- 
todes. Nematologica, 19 : 566-567. 
ESSER, R. P. (1988).  A  simple  method for examination of the 
vulva  area  of  mature  cysts  of Heterodera spp. J.  Nematol., 
HOOPER,  D. J. (1486).  Handling, fiing, staining  and  mounting 
nematodes. In : Southey, J. F. (Ed.) Laboratoy Methods for 
Work with Plant and Soi1 Nematodes. London, Her Ma- 
jesty’s  Stationery  Office : 59-80. 
SEINHORST, J. W. (1959). A rapid  method for the  transfer  of 
nematodes  from  fixative  to  anhydrous  glycerin. Nematolo- 
gica, 4 : 67-69. 
BZUX., 16 : 441-447. 
20 : 497-498. 
Dates  de  publication  du  volume 12 
Fascicule 1 (p. 1 à 104) ................. 13 janvier 1989 
Fascicule  4  (p. 329 à 440) ............ 30 octobre 1989 
Fascicule 2 (p. 105 à 216) ............ 13 avril 1989 
Fascicule 3 (p. 217 à 328) ............ 13 juillet 1989 
122 Revue Nématol. 13 (1) : 121-122 (1990) 


The use of agar as a cover-glass support for mounting nematodes

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