Fixation: Primary fixation was done with 3% glutaraldehyde, in dark for two hours at room temperature. After primary fixation the cells were washed repeatedly with phosphate buffer and were subjected to secondary fixation with 1% osmium tetroxide in dark at room temperature for one hour followed by repeated washing with phosphate buffer.
Dehydration: Before proceeding for dehydration the cells were suspended in molten agar (2%). Small blocks of solidified agar (1sq.mm) were cut and passed through series of 30 %, 50%, and 90% ethanol v/v for 15 min each. The cells were further dehydrated with 100 % ethanol (30min x 3). The lower grades of ethanol were prepared in phosphate buffer.
Embedding: The dehydrated agar blocks were suspended in propylene oxide for 20 minutes at 4 deg C (2 changes), followed by treatment with 1:1 mixture of propylene oxide and Araldite 'A' for one hour at 60 deg C.
Araldite 'A': Araldite (resin) Cy-212 : 10ml
Dodecenyl succinic anhydride (hardener) : 12ml
Dibutyl-phthalate (plastisizer) : 1ml
The agar blocks containing cells were then incubated in Araldite A for one hour at 60 deg C followed by overnight incubation at room temperature. The next day Araldite 'B' was prepared freshly as follows:
Araldite A : 23ml
Trimethyl aminomethyl phenol : 0.4ml ( DMP-30 accelerator)
Araldite A was poured out and freshly prepared araldite B was added to the agar blocks. This was followed by incubation for two hours at 60 deg C for infiltration. The blocks were then finally removed and placed in refined beam capsule containing Araldite B and incubated for polymerization for 48-72 hrs at 60 deg C.
Block trimming and sectioning: The resin blocks were carefully trimmed to expose the underlying agar blocks. Sections of various thickness (200nm, 300nm, and 500nm) were cut using Leica Ultracut UCT microtome and transferred to 300 mesh copper grids.
Uranyl acetate staining: 10% alcoholic solution of uranyl acetate was prepared and centrifuged to remove any precipitate therein. The sections on copper grids were stained for 1 hour with uranyl acetate in dark at room temperature and then washed with distilled water thoroughly.
Transmission Electron Microscopy: Sections on grids were observed in Jeol (JEM 2000 FX ) electron microscope at 160 kV.