DROSOPHILA SALIVARY GLAND CHROMOSOMES ©David B. Fankhauser, Ph.D. Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
Dissected out  salivary glands	This page has been accessed  times since 12 February 2002.  16 January 1992, latest revision 16 Jan 1996, 6 Jan '97, 21 Jan '97, 5 Jan '98, 4 Jan 02	banded chromosomes  1000x

NOTE: Five points are awarded when you show the instructor your banded chromosomes!

Drosophila melanogaster (the common fruit fly) not only was a superb organism for Thomas Hunt Morgan to have picked to perform genetic studies, but this organism also offers an outstanding opportunity to study chromosome structure due to the polytene nature of the chromosomes found in its salivary glands.  In these glands, many multiple DNA replications have occurred without mitosis.  The multiple DNA strands remain closely associated yielding large chromosome structutres with specific banding patterns along their length.  Thus chromosomal alterations such as deletions, transpositions, duplications, etc can be visually verified using the unique banding as identifying landmark features.

We will isolate salivary glands from third instar larvae (the stage just before pupation).  These will be fixed, stained and squashed and examined at 1000x (oil immersion).
Review the proper use of a Dissecting Scope before performing this experiment.

PROCEDURES  

Click on picture  to enlarge	Procedural step
	1. Pick several third instar larvae from a culture flask by picking up with a disecting needle (or paint brush with rolling motion), place a few in drop of Ringer's solution on a microscope slide.  Do not put too much liquid or the specimen will jump around when needles touch the liquid.  Of course, don't use too little or the specimen will dry out. Pictured below to the left are two larvae in reflected light.  The head is tapered, the caudal end is more blunt. Here is a labeled image of intact larvae. The image (of the head of a larva) to the right is taken with transmitted light, showing the inner organs better.  Here is a view which shows the entire length of the larva . Here is a diagram of the location of organs within the larva (salivary glands and other organs). Here is an photograph of the internal components labeled
	2. Isolate a pair of salivary glands by dissecting out with two needles as demonstrated. (They are attached close to mouth, transparent, and paired). Push all debris to edge of drop and remove with Pasteur pipette. Clean glands of extra material (fat bodies, etc) using needles like scissors. Here is a labeled image of the anterior components of a larva after initial dissection. Here is a blackboard diagram of  isolated salivary glands attached to the head. Note that the ganglia (looking very much like a penis and testes) is attached very close to the origin of the two salivary glands.
	3. Fix in 45% HOAc: Remove most of Ringer's solution to the waste tube with a Pasteur pipette, replace with 45% HOAc to fix the tissue. Make sure glands are surrounded with/and immersed in HOAc. Trim off any superfluous material which may still be present. Let sit in HOAc for 5 minutes. Meanwhile, illustrate dissected-out glands.
	4. Remove HOAc with Pasteur pipette, discard in "waste" test tube. Wash again with 45% HOAc if floating particles are still visible. Always keep in view. Never let the glands dry out.
	5. Stain with aceto-orcein stain, covering the fixed glands Make sure glands are surrounded by and immersed in stain. Warm slightly over burner. (Do not dry!) Watch under dissecting scope. It may take 20 minutes for the nuclei to become dark.
	6. Remove stain, wash with 45% HOAc: When glands look quite dark, carefully remove excess stain with a Pasteur pipette. (Keep a close eye on where the glands are--you don't want to lose them...) Position glands in center of slide. Wash 2x with HOAc, remove and discard each successive wash. There should be no visible stain or other material (except in the glands) following the second wash. Leave a small amount of colorless HOAc in which the glands are immersed.
	7. Cover with coverslip: With glands in center of slide, lie slide on white paper, lower coverslip carefully over the glands as if hinged. Note the red smudge formed by the flattened gland.
inadequate squash:           Could be squashed more:	8. Squash the chromosomes: Holding the coverslip in place from the edges to prevent movement, pound firmly several times straight down with a pencil eraser over the flattened gland to squash it out. If it spreads well, examine with high dry to see chromosomes. If they look like balls of yarn, they need more spreading. Repeat tapping. Do not smear by moving the cover slip, it will shear the chromosomes.
	9. Examine under the microscope: After adequate spreading is obtained, scan field at 100x looking for well-spread and banded chromosomes. Switch to 400x, pick best banding. At 1000x (oil immersion), illustrate in your book. Take a photograph if possible.  Here is a page with several more picture of nicely banded polytene chromosomes from this protocol, 2002. And more from 2005.
	10. To preserve your slide, seal edges of coverslip with a bead of fingernail polish, let completely dry, label slide: Drosophila salivary chromosomes, your name, and date. Clean up your microscope and carefully store it according to your training.

Solutions used:
 

Ringers: 860 mg NaCl 30 mg KCl 35 mg CaCl2 Dissolve in 100 mL d H2O. Store at 4C.

Aceto-orcein stain (freshly filtered): 1 g orcein stain 50 mL 45% HOAc Heat to dissolve, let cool, freshly filter through Whatman #1 filter paper.

 
 
 

Problems 1997:
Students did not watch carefully the dissection of the glands, had trouble locating glands.
Critical point is just after pulling the head off--if you dig around blindly and lose the glands then you may not find them again.
Use the fat bodies as indicators of the glands--be sure that you have glands before cleaning them up
Aceto-orcein was FULL of crap--filtered, and it seemed to work.
Don't forget to warm during the staining.

Problems 1996:
Offered 10 points for well banded chromosomes. Need to offer at beginning of class. (should be 5 points?)
Students could not find glands (many)
Some needles were dull.
students lost glands during staining (some)
Some students quit before 4:00, did not have chromosomes
squashed glands looked flattened, distorted.