Purpose: To stain the skeletal elements of early to late
developmental stages to compare the distribution of cartilage and
bone deposition during development.
     Incubate chick embryos and remove them at various stages for
treatment as below for cartilage or bone stain. Since we are only
here for one laboratory session, we will have embryos at various
stages of the preparation so that you can do the steps. The
procedure below is for the process using the same embryos. You
will do the following abbreviated stains:
  1. Open egg and separate embryo from yolk and membranes.
For cartilage:
  2. Take an embryo which has been already fixed and soaked in
alcohol; remove viscera, feathers and skin and fat. Place in
stain for cartilage.
   3. Take an embryo which has been stained and start destain.
Take a jar and some methyl salicylate to finish the job by
yourself.
For bone:
   4. Take a fixed embryo  that has been placed in KOH for a day
and eviscerate and deskin, defeather.
   5. Prepare a stain solution to take with you to finish the
process by yourself. You will be back in the lab for several days
doing other things, so we will leave the solutions out for you so
you can finish your stains, just making the transfers when
necessary. Bring in a baby food or jelly jar or two so that you
can take your final specimens with you.

These techniques are written up in Experimental Embryology by
Roberts Rugh, 1965, Burgess Publ. Co. Minneapolis, Minn.

LUNDVALL TECHNIQUE FOR STAINING OF CHICK EMBRYO CARTILAGE: REF:
Anat. Anzeiger 25 and 27, 1905 and 1906
     This technique may be used for embryos of any stage, but
works best before bone formation which starts at 12 days. If only
one stage is to be done, use 9-10 days.
1. Remove embryo from yolk and extraembryonic membranes, wash
away any yolk. Fix in 10% formaldehyde for 48 hrs. Caution
students not to breathe the vapor- just put the embryo in and
close the lid.
2. Transfer to 70% alcohol for 2 hrs.
3. Using forceps, remove skin, feathers, and all fatty tissue.
4. Stain for 2-3 days (the longer time for larger embryos) in
0.25% methylene blue or toluidine blue made up in 70% alcohol to
which 3% ( by volume) HCl has been added. This will overstain. 
5. Destain in several changes of 70% alcohol for about 48 hrs.
Don't proceed unless you see blue cartilage stained.
6. Destain for 4 hrs in 95% alcohol. The softer tissues will
become transparent and destained. A common cause of failure is to
have too much water in the specimen so that it won't take up the
solvent in the next step, so if you have a specimen older than 12
days you will have to let it sit in 95% for a longer time to
penetrate. 
7. Transfer the embryo to methyl salicylate (oil of wintergreen)
to which has been added 25% by volume benzyl benzoate. Use a
glass bottle (melts plastic). The embryo will clear completely
and may be stored for years. 

MODIFIED SPALTEHOLZ METHOD FOR STAINING SKELETAL ELEMENTS:
     This procedure is excellent for chick embryos past the 10th
day of incubation.
1. Remove embryo from yolk and extraembryonic membranes, wash
away any yolk. Eviscerate. Fix in 10% formaldehyde for 48 hrs.
Caution students not to breathe the vapor- just put the embryo in
and close the lid.
2. Transfer to 1% KOH for 24 hrs.
3. Transfer to tap water and with forceps pick off as much fleshy
material as possible (skin, muscle).
4. Transfer to 95% alcohol, change once in 24 hr period.
5. Transfer to ether under a chemical hood for 1-2 hr to dissolve
away any fat, or use acetone if there is little or no fat. (Do
this in a glass bottle, not plastic)
6. Transfer to 95% alcohol for 6 hrs, change once.
7. Transfer to 1% KOH for 6 days (or until very soft-don't
overdo- more for bigger).
8. Put in fresh alazarin red "S" for 12 hrs. (0.001 g alazarin
red in 100 ml 2% KOH: if your balance doesn't measure a
milligram, it is about as much powder on a paper to make a circle
the size of a pencil eraser.) 9. Transfer to 1% KOH for 24 hr.
10. Place in solution of 1/2 1% KOH and 1/2 glycerine for 24 hrs.
11. Store in 100% glycerine.