By Jeff Blyth, Institute of Biotechnology , University of Cambridge Tennis court rd. Cambridge CB2 1QT

What follows is in the form of a worksheet based on the paper published in :

The Imaging Science Journal Vol 47 pp 87- 91 1999

1)Presubbed glass plates . (You can use old holographic plates with the gelatin removed with the aid of household bleach.)

2).Gelatin of "bloom strength" between 250 and 300 (eg. 300bloom from Aldrich cat no. 27,162-4) , (You can use culinary gelatin without any sugar or flavourings.)

( 3)ascorbic acid or Vitamin C,

4) Silver-nitrate , (A 1N volumetric standard solution is a useful form)

5) Lithium bromide "99%".

6) Chromium acetate , (you can use "chrome alum" instead)

7) dye(s): Pinacyanol Chloride (for HeNe 633nm exposure) or

1,1 ‘-diethyl -2,2’ cyanine iodide for exposure at 532nm .

8)Sodium hydroxide

9) (for new glass plates) 3-amino-propyltriethoxysilane

Silver nitrate : 6% w/v in (DI) water , (or the " 1N volumetric standard solution" diluted by 1volume to 2 volumes DI water.)

Stock dye solns : for 633nm 1g. /1000 ml Methanol, but for 532 nm dye, 1g. per 500 ml methanol

Lithium bromide 3% w/v in 50/50 methanol /water

Chromium acetate solution 1% or Chrome Alum 2%

Gelatin solution. 15%: (see next paragraph)

Ascorbic acid 1% solution in water , adjusted to around pH 5 with any alkali.

Preparation of plates . Glass plates usually need a pre-treatment step or the gelatin coating may peal off. You can use old holographic plates by simply giving them a 10 min. soak in neat domestic bleach solution and then rub off the old gelatin layer under tap water. After a final rinse in distilled water, no further subbing step is usually required.

However with freshly cut glass plates, I leave them soaking overnight in a strong (10%) caustic soda solution to both degrease them and activate the surface. (Don’t do this without eye protection and thick rubber gloves as splashing this dangerous liquid is almost unavoidable.) They are then washed in DI water and dried .

After the plates are dry I rub them over with a 1% solution of 3-amino-propyltriethoxysilane in acetone on a tissue until it has evaporated, and leave them in air to interact with the silane for at least two hours before coating. (The silane solution has to be freshly prepared for each batch of plates.)

Add 30g gelatin to 170ml cold water and mark the liquid level on the beaker. Place beaker in a water bath and heat while stirring constantly until gelatin solution is between 70 and 80^o C.

Stir until all granules have cleared. Top up level to the mark . To remove skin and surface foam, pour through a fine mesh (nylon stocking works fine) into a preheated beaker. Then immediately proceed to next step:

Hold the beaker in your right hand and with you left incline the presubbed glass plate (preheated to around 60^oC) at an angle of about 30^o to the vertical with its bottom edge in a clean tray. Pour the gelatin in a line about 1 cm from the top of the plate. The pouring rate must be continuous until the furthest edge of the plate is reached . (You may have to accept the tendency of the coating to not completely cover the lower part closest to the furthest edge. ) Lean plate against something for a few minutes while coating gels. Run a knife along thick layer at the bottom to free plate rather than risk tearing the delicate coating. (Since no hardener is involved yet the gel can be readily scooped up and recoated if you are not satisfied.). Put plate in cold solution of chromium acetate for 1 minute. Shake off drips and then (without washing away that salt ) blow plate with cold air until dry. Once the layer is dry leave the plate to complete the chrome hardening effect overnight in a warmer. (Preferably at around 60^oC for several hours). Rinse the hardened plate in DI water and dry in a warm air flow. If you want to cut plate up for the next step then after scoring the glass on the back and cracking it, it is best not to pull sections apart before running a scalpel blade along the gelatin side first so that it is cut and not torn apart..

1. For a 5 x 4" plate place approx 3 ml 6% silver nitrate soln. in the centre and at once squash it with a clean flat cover plate (preferably transparent plastic so that you can see the air bubbles are squeezed out). Leave for 3 minutes. Safelighting is not necessary here but the lighting should be subdued.

2. Remove cover plate and immediately remove the excess silver solution on its surface by gently brushing over the plate with the edge of a piece of filter paper .

3. Blow dry plate with cool air.

5. Under safelight conditions , add 2.5 ml of dye solution per 100ml of lithium bromide solution , add about 0.2 ml of 1% ascorbic acid solution (this is the same solution as is used in the final sensitizing bath ) agitate the bath and plunge plate in while maintaining the agitation for about 4 minutes.

6. Rinse well under running tap water (any AgBr only on the surface can be removed

by gently rubbing with ungloved finger.) Plates usually come out this bath beautifully clear under the green safe light, without any surface deposit.

The plate can be immersed for 1 minute in 1% ascorbic acid solution adjusted to pH 5 using a little sodium carbonate or hydroxide . Alternatively the well known triethanolamine pre-swelling technique can be used with the advantage of increased brightness at a shorter wavelength. (prolonged settling period may then be necessary however to avoid creep while the exposure is being made).

After exposure the plate is then treated conventionally.