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I S O P Y C N O G R A P H Y
(Equilibrium Density Gradient Centrifugation)
(hint: the symbol of density or specific gravity is usually the Greek letter rho (ρ))

Many of the early advances that helped found molecular biology were based of isopynographic analyses of dsDNA, and DNA/RNA hybrids. Because this required ultracentrifuges, 72-hr runs, and expensive CsCl, it is beyond the scope of teaching labs. However, if larger particles are placed in preformed gradients of a much less expensive salt, slow-speed centrifuges and only a few minutes are required to demonstrate the power of isopycnography. Further, this power will allow its user to move into new realms of discovery. Here you will find that bacterial specific gravity can be an index of the cells' metabolic prowess at any given moment in time.

ITEMS NEEDED FOR PRACTICE

• A centrifuge: 3 krpm will take 30 minutes; 10 krpm will take 8 minutes
• Use one of the three following techniques
  • Alternate #1: A refractometer that directly measures the specific gravities of drop quantities from 1.0 to 1.5. However, refractometers for such high-density solutions are very expensive. These are NOT the devices normally employed to monitor salinities of seawater.
  • Alternate #2: various solutions of toluene and chloroform of known specific gravities. These should be made to range between 1.25 and 1.45) You must mix your own soltuions.
  • Alternate #3: Write to this author who will send you some variously colored threads that have different specified densities. You will cut off pieces about 2 mm long and use as internal density markers.) (Current samples: ρ_{black wool} = 1.358 g/cc; ρ_{red silk} = 1.414 g/cc; both with std dev = ± 0.003 g/cc. [When have you ever done work to almost 4 significant figures?-!] (Click for the tricks of cutting silk thread.)
• Round bottom GLASS centrifuge tubes of any size (15 ml size is used in protocol below)
• Rubber stoppers that make a tight fit inside the tube when inserted the "wrong way."
• 150 ml of a saturated aqueous solution of NaBr (specific gravity „ 1.5)
• Wooden board 3 ft x 6+ inches
• 10 ml pipets and a pipet pump
• 3 long-stem glass pasteur pipets and bulbs, two one-piece plastic droppers
• 2 broth cultures of E. coli (early log phase and stationary phase)

DIRECTIONS

-1. Inoculate some nutrient broth or equivalent and incubate at 37°C overnight.
0. To obtain the log-phase culture: 40 minutes before needed dilute some of the overnight culture 1:25 with new medium, and incubate that in a shaker bath at 37°C.
1. Place 0.5 ml of the saturated NaBr in each of two centrifuge tubes. (Obtain another rendition of this procedure WITH DIAGRAMS by clicking button. Some values will be different, but that indicates the latitude you have with this procedure.)
2. Add 0.2 ml of the overnight, and 6.5 ml of the log-phase culture to the tubes. Mix.
3. Add 6.3 ml of water to the overnight tube. Mix.
4. Make two long-stemmed funnels: Cut the rounded end off each of the plastic droppers.
5. Set the funnels into the tubes.
6. Add 7 ml of saturated NaBr to each funnel: the dense NaBr will form an underlayer. Note the interface between the bacterial layer and the dense underlayer.
7. Lift the funnel up such that its tip is at the interface.
8. Put your index finger over the open end of the funnel. Gently squeeze the funnel to force only the liquid out its tip. Do NOT let air bubbles come out. Lift funnel out of tube.
9. If the rubber stoppers do not have a small hole in them, cut a small groove up their sides with a razor blade.
10. Insert the stoppers with their wider ends in the tubes. Push them down to displace all the air in the tubes. (Air escapes via hole or groove.)
11. Place board on desk; put inch-thick props under the LONG edge that is away from you.
12. Place one of the tubes on the board by placing the rounded end lower than the stoppered end.
13. Adjust the height of the props such that the interface in the tube form a COMPLETE diagonal within the tube.
14. Right handers: slide the tube to near the left end of the board. Place right hand flat and firmly on the tube, and roll it to the other end of the board (0.5 seconds). Catch it as it comes off the end of the board, and IMMEDIATELY hold it vertically. You have a gradient!
15. Place the tubes in the centrifuge across from each other. Rotor may be swinging bucket or slanted. DO YOU NEED RUBBER SLEEVE-ADAPTORS? (Do not use hard plastic adaptors!)
16. If you do not have a refractometer and will be using the provided thread internal density markers, add two of each color bits of thread at this time to the tops of each of the gradients.
17. Centrifuge: 3 krpm for 30 minutes; or 10 krpm for 8 minutes Let the rotor come to a GENTLE stop by itself - DO NOT hand-deccelerate!
18. Gently lift the tubes from the rotor; you should see a fuzzy band of bacteria floating at their isopycnotic points in the gradients.

If you have a refractometer do 19 through 22 (otherwise jump to step 23):

19. Flame-heat and pull a capillary from one of the pasteur pipets. Break off the excess. Using a permanent marker, blacken the capillary tip of the pipet. Put bulb onto pipet.
20. Here comes the most CRITICAL PART of the operation! Without squeezing the bulb, insert the capillary tip into the tube about halfway between meniscus and bacterial layer. Use index finger of hand holding the tube to steady the pipet. Gently squeeze bulb to let out 3 bubbles of air. Lower tip into band, and release the squeeze on the bulb. Band goes into the pipet. Don't move if you see liquid still actively rising in the pipet.
21. Then rapidly lift the pipet out of the tube. Wipe its tip with something absorbant to prevent any of the upper, less-dense liquid from contaminating your reading.
22. Place drops into the refractometer, and get a reading. Get density from your comparison graph that you have made using known dilutions of the saturated NaBr. Now jump to step 27.

23. If you are not using internal thread density markers, you will put a small amount of your densest toluene/chloroform solution into a pasteur pipet.
24. Lower the pipet tip to just below the meniscus in the tube. Expel just one drop of the solvent mix. It will settle slowly to its isopycnotic level BELOW the bacterial band.
25. Do likewise with the other solvent mixes - going always from densest to less dense. You should find your bacterial band bracketted between two of the droplets.
26. Using a ruler, interpolate your bacterial band between the two bracketting droplets or bits of thread. Click button for hints on capturing and recycling your valuable thread markers.

27. Recycling NaBr. While NaBr is rather inexpensive, it is often out of stock, and at the very least it does take time to order, and receive it. Thus, if you have been saving the contents of used isopycnography tubes, you can recycle the somewhat impure NaBr for subsequent use:
    1. Half fill a teflon coated frying pan with your used NaBr solution.
    2. Boil away approximately 75% of the water. As the boiling commences, some of the bacterial proteins will congeal around the edges of the boiling area and can be spooned out and disgarded.
    3. After cooling, cakes of dirty, crystallized NaBr can be lifted out and broken to smaller pieces. Allow them to drain away excess fluid.
    4. Place the chunks in a glass casserole dish and place this in an oven that has a "self-cleaning" cycle. Such ovens lock and go to about 1000F (= 550c) to carbonize all organic materials (in your case, bacterial media and residual bacteria).
    5. Dissolve the gray/black chunks of burned NaBr in a minimum of water (you want a saturated solution.
    6. Filter this through filter paper or a tightly fibered paper napkin (not a paper towel). The carbon will be filtered out, and the filtrate will be only very slightly yellowish. Nonetheless, it will easily work for subsequent isopycnography.

28. QUESTIONS:
    1. What are other methods for making density gradients?
    2. Are log-phase and stationary-phase bacteria of different specific gravities?
    3. What would you get if you mixed both phases and centrifuged them in one tube?
    4. Why isn't the size of the bacteria a matter of concern?
    5. Why isn't the water content of the cells a matter of concern?
    6. Do the bacteria undergo a smooth transition of specific gravities as they go back and forth between log and stationary phases, or are there only quantum levels?
    7. Suppose you had a liter culture that didn't have a high enough concentration to be able to see a band of bacteria, how could you concentrate the bacterium without changing their phase of growth?
    8. Suppose you had a swirling log-phase culture, and you suddenly started bubbling nitrogen gas into the culture, what would happen to the cells' specific gravity?
    9. Suppose you had a swirling log-phase culture, and you suddenly added some T4 phage...
    10. Suppose... and you suddenly added some antibiotic...

29. Can you find any journal articles in which this technique has been used?
    What was done?
    What new questions can you think of that might be solved using this technique?

Cutting silk thread for this experiment and the care of scissors. Silk thread is composed of dozens of very fine microthreads of silk from the silkworm. When you cut 2 mm lengths for this experiment you do not want the bits to be frayed as that will likely result in the various marker threads' becoming ensnared with each other. The cuts must be clean. This is only possible by using a fresh razor blade, a very sharp knife on a hard cutting board, or a tight and new scissors of high quality. About scissors care: while it is convenient to have one scissors for doing all cutting, it should be noticed that those used to cut paper soon become too dull to cut fabric or hair. You should own at least two scissors - one exclusively for hair and cloth, and the other for paper. Paper dulls scissors (and knives) because various clays are used to make it smooth. Clays are rocklike and you wouldn't think of using your scissors to cut your sidewalk and yet retain its sharpness! Thus for cutting your bits of silk thread, DO NOT pick up the nearest scissors and expect to get good results.

The next thing you want to do is rid the bits of any skin oils. Place the bits in a test tube and pour in about an inch of alcohol and slosh them around for awhile to dissolve away the oils. Then dump them out onto a paper towel, which will absorb the alcohol and using a tweezers lift two or three of each color and place them into your centrifuge tube. (Click to return to where you were reading.)

Recapturing and recycling your thread markers. Place a paper towel (not filter paper!) in a funnel and dump the used isopycnography tubes into funnel. The bacteria and NaBr will flow rapidly through the paper towel leaving the threads behind. Then pour plain water through the funnel to rinse the threads free of NaBr. Flatten out the paper towel to dry. When dry, collect the threads and store for later use. (Click to return to where you were reading.)

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