Materials:
Poly-l-lysine coated cover slips. Use HMW(350,000-400,000 MW) poly-l-lysine at a concentration of 0.1%. Cover slips should be coated with fresh solutions only and should be used within 24 hours.
0.1? Nucleopore filters and 13mm filter holders.
Frozen(-200C) sample of Saccharopolyspora spinosa(S. spinosa) at a concentration of @2 x 109.
0.85% NaCl (physiologically compatible buffer)
2% Glutaraldehyde in 0.85% NaCl (GTA/NaCL)
2% Glutaraldehyde in 0.1M Na Cacodylate pH 7.2 (GTA/Cac)
Primary Fixation Methods:
Thaw S. spinosa sample and dilute in 0.85% NaCl to a concentration of 1 x 108:
A.
1. Pipette sample into a vial containing GTA/NaCL fixative with a poly-l-lysine coated cover slip on the bottom. Allow the fixed sample to settle onto cover slip for 30 minutes; final concentration of sample should be @1 x 107.
2. Transfer cover slips to fresh vials of GTA/Cac fixative and fix for a further 1.5 hours. at 40C.
B.
1. Pipette sample into a vial containing GTA/NaCL fixative; final concentration of sample should be @1 x 107.
2. Pass this solution slowly (5-10 mins.) through a 13mm 0.1? Nucleopore filter which was pre-wetted with 0.85% NaCl. Transfer wet filter to a petri dish containing GTA/Cac fixative and fix for a further 1.5 hours. at 40C.
C.
Pellet sample by centrifuging ( microfuge; 10,000g for 1sec.). Resuspend in GTA/Cac fixative and:
C1. Pipette sample into a vial containing GTA/Cac fixative with a poly-l- lysine coated cover slip on the bottom. Allow the fixed sample to settle onto cover slip for 30 minutes; final concentration of sample should be @1 x 107. Transfer cover slips to fresh vials of GTA/Cac fixative and fix for a further 1.5 hours at 40C.
C2. Pipette sample into a vial containing GTA/Cac fixative; final concentration of sample should be @1 x 107. Pass this solution slowly (5-10 mins.) through a 13mm 0.1? Nucleopore filter which was pre- wetted with GTA/Cac. Transfer wet filter to a petri dish containing GTA/Cac fixative and fix for a further 1.5 hours at 40C.
Secondary Fixation, Dehydration, CPD, Mount and Sputter Coating:
1. Rinse in 0.1M Na Cacodylate pH 7.2.
2. Fix: 2% OsO4 in 0.1M Na Cacodylate pH 7.2 for 1 hr. at room temperature.
3. Rinse in 0.1M Na Cacodylate pH 7.2.
4. Dehydrate in ethanol series (30%,50%,75%85%,95% and 3 x in 100%, 5 mins each).
5. Critical point dry.
6. Mount on Al stubs. Be careful not to "stretch" filters when mounting.
7. Sputter Coat with 100 ? AuPd.
Results:
B. Best method from protocols tested was B. S. spinosa were smooth-surfaced with no obvious artifacts from osmotic shock.
A and C1. The samples, unlike other similar preparations. did not stick to the poly-l-lysine coated cover slips. It's possible that the poly-l-lysine solution used to coat the cover slip was too old or not stored at a cold enough temperature. This protocol, using GTA/NaCl as the fix, should be repeated with coated cover slips, made from a poly-l-lysine solution made the same day or a one time thaw of an aliquot stored at -200C.
C2. Sample was clean. Centrifuging removes the amorphous material from the preparation. However, the surface of the bacteria was a little rough(i.e. a few blebs). This is caused either by fixing initially in GTA/Cac rather than the (supposedly) osmotically compatible GTA/NaCL or by the centrifuging process. So only use if you want to remove all the amorphous material, surrounding the bacteria and use GTA/NaCL as fixative of choice.
Conclusions:
Use 2% Glutaraldehyde in 0.85% NaCl (GTA/NaCL) as initial fix. Do not centrifuge sample unless a "clean" preparation is requested by research lab. Use 0.1? Nucleopore filters rather than Millipore, to avoid degradation of filter during sample preparation.
In the future: repeat protocols using poly-l-lysine coated cover slips (made from a fresh solution, since the cover slip method, if the samples will stick, would retain all possible material present in the sample.
