Doug Eernisse, California State University Fullerton

Last updated: September 2003

Goal: Fix selected dissected specimens for anatomical study

a) small animals intact or removed from "shell"
b) small animals dissected to obtain samples of particular tissue

• testes "plucks" for sperm ultrastructural comparison
• ovaries large pieces - further sliced after 1 hr fix
• brooded embryos if any
• other selected tissues as requested by specific collaborators, e.g., radula, jaws, digestive tract, gills, etc.
• specific tissue subsamples in alcohol for molecular studies

General procedures

a) sodium bicarbonate buffered 2.5% glutaraldehyde in filtered seawater

b) 10% formalin in same buffer and sea water

• useful for any technique where autofluorescence of glutaraldehyde is a problem
• preferrable to Bouin's fix only for safety reasons (picric acid)

Considerations

a) sodium bicarbonate (Na2CO3) buffer selected as superior to alternatives.

• does require adjustment to final pH 7.3 but standard solution recipe possible
• sodium cacodylate buffer might have better properties but poses health risks
• phosphate buffers tend to precipitate
  

b) gluteraldehyde best if made fresh (10 ml ampules of 25% glut./case of 10)

• advantage -- recipe simple and fresh: 10:40:50 = 100 ml fix
• 10 ml 25% glut.; 40 ml 2.5% sodium bicarbonate; 50 ml filtered seawater
• 25mm 0.45µm mesh Millipore syringe filters convenient for ambient seawater (capacity to 100 ml)
  

c) preparation of fix should be in fume hood or possibly in open air on deck

d) fix should be stored at 5°C until use and for minimum of 1 hour (or overnight) after fix

e) time considerations depend on use

• small "intact" animals or animals only removed from shell(s): 5 min/specimen
• testes grab: 5-10 min/specimen
• ovary section, later slice: 10-15 min/specimen
• other tissues could be collected at this stage, e.g., for molecular studies
• full dissection: 1 hr/specimen
  --would require sylgard resin in petri plate, 0, 00, 000 insect pins
  --fine dissection tools and dissecting scope in hood ideal
• if ample material, animals could be observed in filtered seawater for spawning
  --separate males, collect "dry" sperm, store at 5°C
  --spawned eggs washed, then sperm at various dilutions introduced
  --gluteraldehyde fixes performed at regular intervals on subsamples
  --light microscope observations/photos would be ideal
  --some embryos allowed to develope could be fixed at regular intervals
  --these studies might require a trained embryologist

f) routine procedure might keep specimens in fix overnight, then dehydration series

• regular histology: dH20 rinse, 30, 50, 70% ethanol - store here
• Formalex to neutralize aldehyde fixes (1:9 dilution)
• best if delivered to researchers within a week if possible
  --80, 90, 95, 100, 100, 100, xylene + plastic (performed by researcher)
• Osmium post-fix for ultrastructural work or SEM (performed by researcher)

Approximate Costs per sample

a) chemicals relatively inexpensive

• glutaraldehyde (EM grade): approx. $10/100 ml fix in bulk
• Millipore disposible filter: approx. $1/100 ml fix in bulk
• other chemical costs minor
• 100 ml fix should be sufficient for multiple specimens

b) containers relatively inexpensive

• many of sampled tissues could be fixed in 1.5 ml microcentrifuge tubes
• empty plastic "formalin" containers in various sizes for larger animals/tissues
• bottles for fix with 100+ ml capacity
• dissection trays: petri plates + sylgard resin and insect pins
  

c) dissection equipment could be moderately costly

• fine scalpels needed but could be reused ($1 to $15 each)
• scissors ($70), watchmakers forceps ($20), etc.

d) fume hood with stereo scope, possibly on boom stand, fiber optic lights

e) headband magnifiers for sorting material ($25)