-Adults T. infestans                         -Dentist putty                         -Stove at 60* C

-Dissecting materials                        -Solid Vaseline                      -Locking device

-1cc Syringe                                   -Ringer solution                      -Series of alcohol

-Small Petri dishes                          -2.5% solution of Cl2Co         -Methyl salycilate

-Trap                                             -(NH4)2S Solution                  -Canadian balsam

-Play dough                                    -Revealing solution                 -Cover

Procedure

The filling of axons with the Cobalt Chloride has the following steps:

1.Application                                                                   4.Fixation

2.Diffusion                                                                       5.Intensification

3.Precipitation                                                                 6.Clarifying and montage

Application (1/2 hours)

Put the insect to sleep by placing in a freezer for a few minutes.  Immobilize it with a bit of play though, by holding tight the neck area.  Remove the cornea from the ocellus with the tip of the scalpel.  Place a drop a distilled water, leave for approximately one minute.  Remove the retinal cells with the tip of the scalpel of a needle.  Dry the drop of water with absorbing paper.

Diffusion (2hours)

Make cup of solid Vaseline around the ocellus.  Be very careful and avoid any Vaseline entering the ocellus.  Place a drop of Cobalt Chloride solution in the cup covering the ocellus.  Cover the drop completely with solid Vaseline.  Leave to diffuse for about 2 hours at room temperature.  Afterwards, clean the zone with absorbing paper.

Precipitation (20-30 minutes)

Glue the insect's back to a small plate being careful with its neck.  Place the small plate in a Petri dish and fix it in place with some play dough.  Dissect with the insect facing up until the nervous system is exposed (brain and ganglia, see figure).  Wash with Ringer solution.  Apply Ammonium Sulfide solution drop by drop over the whole nervous tissue and make sure that it remains submerged in the liquid for about 3 to 5 minutes.  Afterwards, wash with Ringer solution.

Fixation (2 hours)

Place the insect, stuck on the small plate inside a bottle with the locking device.  Leave for 2 hours at room temperature.

Hydration (50 minutes)

Remove the inset from the locking device and take it to the 70% alcohol solution.  Finish the dissection in the 70% alcohol solution, removing the whole nervous system.  Continue the hydration in decreasing order of alcohols: 50% - 30% - Distilled water, for 10 minutes each.  From then on, handle the preparation with a plastic pipette with the tip cut at an angle.

Intensification (2 hours)

Place the preparation in a small Petri dish with revealing solution base preheated at 60* C and leave for an hour in a dark place at 60* C.  Then place the preparation in revealing solution with silver preheated at 60* C and take to stove.  Check every 10 minutes the color of the preparation until it takes on a light tobacco brown color.  After 30 minutes, renew the solution.  Once it has taken on the desired color, remove and place the preparation in distilled water previously preheated at 60* C an leave for 10 minutes.

Dehydration (1h 10 minutes)

Put the preparation through a series of increasing alcohols: 30% - 50% - 70% - 80% - 90% - 100% x 2, each for about 10 minutes.

Montage (30 minutes plus observation time)

Clear the solution in methyl salycilate for 20 minutes.  At this point, you may place it in a holder for observation.  For the montage, place the tissue in a holder with methyl salycilate - Canadian balsam 1:1 and cover it up.  Leave to dry on stove.

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