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Fixation Methods for Pseudomonas

1. Parducz fixative (Science 1969 pp.1064-65)-- is used with protozoan ciliates, as an instantaneous fix of ciliary motion, in order to give life-like appearance. It is assumed, that in the case of dinoflagellates, the fixative will also keep flagella from falling off.

METHOD 1A:

Allow pseudomonas to settle and attach to poly-l-lysine(Sigma 300,000MW )coated cover slips (@ 30 mins.).

a. Carefully add a saturated solution of HgCl2(in ddH2O) to a stock solution of 2% OsO4 (in ddH2O) in the ratio of 6 parts 2% OsO4 to one part HgCL2. Fix pseudomonas in this soln. for 30 mins. at 4oC.

b. Rinse 3 times with ddH2O.

c. Dehydrate with Ethanol: 5 mins. each in 30%, 50%, 70% and 95% ETOH

d. Three 10 mins. washes with 100% ETOH.

e. Critical Point Dry with liquid CO2.

f. Mount cover slips onto Al stubs with silver paint. Paint to narrow conductive strips from cover slip to stub.

g. Sputter Coat -- 100 ? of AuPd.

METHOD 1B:

a. Same as Method 1A, except that the pseudomonas are fixed before attachment to coated cover slips: Pipette drop of bacteria into fixative vial, containing fixation soln, which has a poly-l-lysine (Sigma 300,000MW) coated cover slip on the bottom; the pseudomonas will settle and attach to coated cover slips during fixation time.

2. The conventional method of fixation is to fix first in a glutaraldehyde(GTA) solution, that is osmotically compatible to the media in which the biological specimen normally exists. A second fix of OsO4 is then used to stabilize the specimen for SEM viewing.

METHOD 4A:

Allow pseudomonas to settle and attach to poly-l-lysine(Sigma 300,000MW ) coated cover slips (@ 30 mins.)

a. Fix for 1 hour at 4oC in 2% GTA in 85% ZMB seawater media. Always use a fresh GTA solution.

b. Rinse with ZMB seawater medea

c. Rinse with 0.1M PO4 buffer, pH 6.0

d. Fix for 15 mins., on ice, in 1% OsO4 in 0.1M PO4, pH 6.0

e. Rinse with ddH2O.

f. Dehydrate with Ethanol: 5 mins. each in 30%, 50%, 70% and 95% ETOH

g. Three 10 mins. washes with 100% ETOH.

h. Critical Point Dry using liquid CO2 as the transition fluid.

i. Mount cover slips onto Al stubs with silver paint. Paint to narrow conductive strips from cover slip to stub.

j. Sputter Coat -- 100 ? of AuPd.

METHOD 4B:

a. Same as Method 1A, except that the pseudomonas are fixed before attachment to coated cover slips: pipette drop of bacteria into fixative vial, containing fixation soln., which has a poly-l-lysine (Sigma 300,000MW) coated cover slip on the bottom; the pseudomonas will settle and attach to coated cover slips during fixation time.

OBSERVATIONS

1A: Flagella retained in rigid, straight position. Both smooth and rough surfaced bacteria present. Many more rough-surfaced (blebbed) bacteria present than smooth surfaced. Osmotic damage to bacteria.

1B: Flagella retained rigid, straight position. Both smooth and rough surfaced bacteria present. Many more smooth surfaced bacteria present than rough-surfaced (blebbed). Osmotic damage to bacteria.

4A: Flagella retained in both straight and flexible position. Both smooth and rough surfaced bacteria present. Many more rough-surfaced (blebbed) bacteria present than smooth surfaced. No osmotic damage to bacteria.

4B: Flagella retained in both straight and flexible position. Some flagella loss. Many more smooth surfaced bacteria present than rough-surfaced (blebbed) . No Osmotic damage to bacteria.

CONCLUSIONS

Since I am not sure of the differences between the smooth and rough surfaces of bacteria, both methods of attachment (before and after fixation) should be used when and if you need to fix more of these samples.

The Parducz fixative works well in retaining the flagella, but there is osmotic damage. I would suggest modifying this method with the buffers used in methods 4A &4B. Therefore, for future fixations, I would suggest the following fixation method:

METHOD 1:

1. Allow pseudomonas to settle and attach to poly-l-lysine(Sigma 300,000MW ) coated cover slips (@ 30 mins.).

a. Carefully add a saturated solution of HgCl2(in ddH2O) to a stock solution of 2% OsO4 (in ZMB seawater media.) in the ratio of 6 parts 2% OsO4 in ZMB seawater media to one part HgCL2. Always use a fresh soln. of OsO4. Fix pseudomonas in this soln. for 30 mins. at 4oC.

b. Rinse 3 times with 85% ZMB seawater media.

c. Dehydrate with Ethanol: 5 mins. each in 30%, 50%, 70% and 95% ETOH

d. Three 10 mins. washes with 100% ETOH.

e. Critical Point Dry with liquid CO2.

f. Mount cover slips onto Al stubs with silver paint. Paint to narrow conductive strips from cover slip to stub.

g. Sputter Coat -- 100 ? of AuPd.

METHOD 2:

a. Same as Method 1, except that the pseudomonas are fixed before attachment to coated cover slips: Pipette drop of bacteria into fixative vial, containing fixative solution., which has a poly-l-lysine (Sigma 300,000MW) coated cover slip on the bottom; the pseudomonas will settle and attach to coated cover slips during fixation time. Fixative solution: Carefully add a saturated solution of HgCl2(in ddH2O) to a stock solution of 2% OsO4 (in ZMB seawater media.) in the ratio of 6 parts 2%OsO4 in ZMB seawater media to one part HgCL2

POLY-L-LYSINE (PL) COVER SLIPS

Solution: 1mg/ml poly-l-lysine(>300,000 MW) in ddH2O. Use a fresh soln. each time. Store the PL in the freezer.

1. Clean cover slips with 95% ETOH and dry with chhesecloth.

2. Spread cover slips out in petri dish.

3. pipette drop of PL soln. onto each cover slip so as to cover entire surface; withdraw excess.

4. Cover petri-dish and let stand for 30 mins.

5. Rinse cover slips with ddH2O and stand on edge of filter paper to dry: Coated-side up.

NOTE: ONLY USE FRESHLY PREPARED COVER SLIPS

Last Updated: 10/2/08