Contribution à l'étude de virus de mollusques marins apparentés ...

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0.2 g/I, KCI, No. P 1300'
2.77 g/I, Na,HPO" 12 H,O, No. S I 035'
0.2 g/I, KH,PO" No. PI560'
Distilled water.
Adjust the pH to 7.4.
Store aliquots at -20 oC.
Culture medium:
L- 15 (Leibovitz) medium, No. 074-01300'
Medium 199, No. 071-01200'
Basal three fold concentrated (3x) media:
5 liters unit of powdered medium L-15 or 199
0.35 g/I NaHCO" No. S8875', only for 199
medium
2.422 gll Tris, No. T1378'
Add 1.67 liter of distilled water.
Adjust the pH to 7.4 with HCI, No. A420'.
Sterilize by filtration through 0.22 J.1m.
Store at 4 oC.
Foetal calf serum (FCS):
FCS, No. 011-06290'
Decomplement at 56 oC for 30 min.
Store 50 ml aliquots at -20 oc.
Antibiotics stock solutions:
Penicillin-streptomycin solution:
10' Ulml, penicillin G, No. P3032'
0.1 g/m l, streptomycine sulfate, No.
S650l'
Distilled water.
Sterilize by filtration to 0.22 J.1m.
Store aliquots at -20 oC.
F1umequine solution:
30 mg/ml, fJumequine, No. 93 128'
Distilled water.
Add a few drops of 5N NaOH, No. S 1225',
to dissolve the powder.
Sterilize by filtration to 0.22 J.1m.
Aliquots are stored at - 20 oc.
B. Disposables and incidentals
Dissecting forceps
Cell culture 25 cm' fJasks, No_ 13139'0
0.22 J.1m sterile units of filtration, No.
SVGVBIOlO and No. SLGV025BS"
1 ml syringes , No. SPTI 12
18G needles, No. AJ4012 12
Scalpel blades, No. LBST24 12
Sterile Petri dishes, No. BPS9OGEp 12
24 x 36 mm glass coverslips, No. LC02436 12
Homogeneizer Dounce type and piston No. A
(76-152 J.1m), No. AI4.2oo.24"
Sterile centrifuge tubes, No. 84207"
1.5 ml microfuge tubes, No. 33605"
C. Equipment
Cell culture incubator"
Cell culture hood'6
Centrifuge type j-6M1E"
Microfuge type 12"
Inverted microscope
Transmission election microscope JEM 1200
EX"
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D. Animais and sea water
Pacific oysters, Crassos/rea gigas, were reared in
the Marennes-Oléron basin, France.
Sea water was pumped in the Marennes-Oléron
basin.
3_ Procedures
A. Preparation of oyster hemolymph and media
1. Crassas/rea gigas hemolymph (CGH)
a) Open carefully the oyster without damaging
the pericardic membrane.
b) Discard sea water and dry the animal with
a sheet of paper.
c) Immediately puncture slowly the hemo­
Iymph in the pericardic cavity. 0.5 to
2 ml hemolymph are expected for each
oyster.
d) Pellet the haemocytes (2500 rpm, 10 min.,
4 OC) and filter the cell free hemolymph
through a sterile 0.22 J.1m unit.
e) Store at 4 oC and use before two weeks.
Do not freeze.
2. Culture medium
Improvement of the medium:
a) L- 15 (3x) and 199 (3x) media and sea
water supplemented with 10% FCS.
b) L-15 (3x) mixed with sea water to
different ratio (4: 1,3:2,2:3 and 1:4) and
supplemented with 10% FCS.
c) L-15 (3x) and sea water to a ratio of 1:1,
supplemented with 0, l, 5 or 10% FCS ,
and 0, l, 5, or 10% CGH.
Optimized medium:
L- 15 (3x) and sea water to a ratio of 1: l ,
further supplemented with 10% FCS and
5% CGH.
Add 1: 1 000 penicillin-streptomycin and
fJumequine solutions to ail these media.
B. Preparation of culture surfaces
1. Cover the surface of a 25 cm' plastic cu lture
fJask with 5 ml of 0.1 mg/ml poly-D­
Iysin, then incubate at room temperature for
5 min. .
2. Rinse twice with 5 ml sterile distilled water
and allow to air dry.
3. Poly-D-Iysin coated fJasks could be stored at
4 oC, but were used before two weeks.
C. Decontamination of oysters
1. Wash and brush the shells under tap water,
then bath the oysters in 70% ethanol for
30 sec, and let them dry under a sterile
hood.
2. Carefully open the oysters without damaging
the pericardic membrane.
3. Wash the animaIs and the inner shells with
sea water-lween, rinse with autoclaved sea
water.