Exercise 5 - Cellular Transport Mechanisms

STUDENT OBJECTIVE

Students study how materials move in and out of cells. Brownian movement, diffusion, and osmosis are observed and measured. Students study the pumping rates in the water expulsion vesicle of Paramecium under experimental conditions. Turgid and plasmolyzed plant cells are observed.

EQUIPMENT AMOUNT (Class of 24 with 8 groups)
Compound microscopes 1/student
Balance (0.1 g sensitivity) 2/lab


MATERIALS

Osmometer (see lab manual fig. 5.3) Demonstration
  Thistle tube Beaker
  Animal membrane (CBS#68-4030)* Ring, clamp
  Meter stick Stand
Gas diffusion tube (see lab manual fig. 5.4) Demonstration
   Glass tube, 40 cm long x 30 mm OD
   Rubber stoppers, #6
   Glass jars/lids, 2 oz
   Applicator sticks with cotton on end
Lens paper 8 pkg/lab
Slides 48/lab
Coverslips, #1 small square glass 1/2 oz/lab
Elodea (CBS) 5 sprigs/lab
Paramecium caudatum culture (CBS) 1/lab
Glass rod, 12 cm x 7 mm (for crushing Elodea) 12/lab
Alcohol lamps 4/lab
Dialysis tubing (Spectra/Por 2.5 cm width gives 2 ml/cm, 12,000-14,000
    molecular wt. cut off: order Fisher #08-667B) 8/lab
Marking pencil 8/lab
Test tubes, 10 ml 64/lab
Rack, small test tube 8/lab
Albustix reagent strips (Fisher) pkg/lab
Beaker, 250 ml 8/lab
Glass jars, 2 oz with lid (one each for I2 crystal and KI crystal) 2/lab
Blotting tissue 1 box/lab
Petri plates containing 5 mm of 1% plain agar 8/lab
Cork borer, 5 mm 8/lab

SOLUTIONS

Distilled water
India ink, dilute (1:5)
Methyl cellulose or protoslo (from lab #2)
1% albumin (Sigma)* in 3% NaCl
3% sodium chloride (NaCl)
1% sodium sulfate (Na2SO4)
0.25% soluble starch in 1% sodium sulfate (Na2SO4)
1 M silver nitrate (AgNO3)
2% barium chloride (BaCl2)
Iodine/iodide solution (I2KI)
2 mM Sodium azide
2% FeSO4
2% KFeCN
0.1 N H2SO4

PREPARATION

Several Weeks before Lab

  1. Paramecium caudatum should be ordered for delivery several days before lab.

  2. Elodea should be ordered and delivered several days before lab. Upon arrival, place in a large aerated aquarium with strong light.

One Week Prior to Lab

  1. India ink preparation:

    1 ml ink/5 ml water

    Mix ink into water and store in small dropper bottles.                 Shelf life: one month

  2. Make glass rods by cutting 7 mm diameter rod into 12 cm lengths. Fire polish the ends.

  3. Agar preparation:

    1 g agar in 99 ml water
    Heat to dissolve; pour into plates before it gels; can be heated in boiling water to reliquify.

  4. Sodium chloride preparation:
    3% NaCl 15 g NaCl/485 ml distilled water
  5. Albumin-sodium chloride preparation:
    1% albumin in 3% NaCl 5 g albumin/500 ml 3% NaCl
    Sprinkle albumin on the salt solution; let set for 30-60 minutes. Stir gently without splashing. Store in the refrigerator.

  6. Sodium sulfate preparation:
    1% Na2SO4 15 g Na2SO4/1500 ml distilled water
    Stir solution until salt dissolves.

  7. Soluble starch in sodium sulfate preparation:
    0.25% soluble starch in 1% Na2SO4 3.25 g starch/1500 ml Na2SO4
    While stirring, add starch to sodium sulfate solution. Heat until starch is dissolved. Resulting solution will be clear with a slight opalescence. Store in the refrigerator.
    Shelf life: one month in refrigerator

  8. Silver nitrate preparation:
    1 M AgNO3 8.6 g AgNO3/distilled water to make 50 ml
    Stir to dissolve and pour into dropper bottles. CAUTION: Solution will stain skin and clothes.

  9. Barium chloride preparation:
    2% BaCl2 1.0 g BaCl2/distilled water to make 50 ml
    If cloudy, filter. Package in dropper bottles.

  10. Iodine/Iodide solution preparation:

    5 g I2
    10 g KI
    100 ml distilled water

    Dissolve the potassium iodide in the water. Add the iodine crystals and stir to dissolve. Pour into eight brown-colored dropper bottles and store in the dark. (Clear bottles can be wrapped in foil to prevent exposure to light.)
    Shelf life: one month

  11. Potassium ferrocyanide preparation:
    2% KFeCN 2 g KFeCN/98 ml distilled water
    Package in dropper bottles.

  12. Ferrous sulfate preparation:
    2% FeSO4 2 g FeSO4/98 ml distilled water
    Package in dropper bottles.

  13. Sodium azide
    2 mM NaN3 (azide) 13 mg NaN3/100 ml distilled water
    Packaged in dropper bottles.
    Warning: This is a deadly poison. Avoid inhaling dust and skin contact.
  14. Sucrose preparation:
    35% sucrose 105 g sucrose/195 ml water

    Dissolve sucrose in water, add a few crystals of Congo red, and stir. Package 100 ml by putting equal amounts in eight dropper bottles. Use the rest for the thistle tube of the osmometer.

Day before Lab
  1. The animal membrane should be soaked in warm water prior to stretching over the osmometer opening. Dialysis tubing can be used in place of animal membrane by cutting tube lengthwise to make a sheet.

  2. The dialysis tubing should be cut into individual 15 cm lengths and soaked in distilled water.

Day of Lab

Osmometer setup:

Slip a two-inch piece of rubber tubing over the small end of a thistle tube; close with a clamp. Use 35% sucrose with Congo red indicator to fill the thistle tube over the sink, adjusting the clamp so that the sugar solution fills only the reservoir. This operation is simplified if thistle tubes with side arms are used. Stretch the previously soaked animal membrane over the mouth of the reservoir and wrap heavy cotton thread several times around the lip. All wrinkles in the membrane should be removed and a final wrap of a tight rubber band made over the thread. With a wide rubber band, clamp the filled osmometer to a meter stick on a ring stand. Immerse the reservoir in a beaker of water. A long piece of glass tubing can be joined to the thistle tube using the rubber tubing as a coupling. The level of the fluid in the tube should rise several millimeters every ten minutes. The flow can be ``turned off'' by removing the tube from the water and immersing the end of the tube in the stock solution of 35% sucrose.

NOTES

Animal membranes from the osmometer can be reused if thoroughly rinsed and dried before storing. It will be stained with Congo red but this does not affect function.

CLASSROOM SUGGESTIONS

  1. Experiment on dialysis bags should be started first. This will allow enough time for measurable changes in water gain and salt diffusion.

  2. If there is inadequate crushing of the Elodea, students will be unable to see the organelles suspended in the water. Most students are only accustomed to zeroing in on a fairly large object at 400x, therefore, finding the small vibrating particles eludes them. It would be very helpful to prepare demonstration slides of the suspended particles.

  3. Diffusion in gases should be done as a demonstration to avoid irritating fumes in the lab room. Obtain two small jars that can be closed with #6 rubber stoppers. Work in a fume hood and wear goggles to add about 5 ml concentrated HCl to one jar and 5 ml of concentrated NH4OH to the other stopper. When performing the experiment, dip the applicator stick with cotton on the end in one or the other solution and place in the diffusion tube (fig. 5.4).

  4. Audiovisual aids on transport in cells can be used as a summary for the laboratory.

  5. A minimum homework assignment would be a short essay on the differences between diffusion, osmosis, and active transport.

  6. Because there are several activities, this is a busy lab and students need to be reminded of the inter-relatedness of the experiments. If all experiments are done count on three hours.

ANSWERS TO CRITICAL THINKING QUESTIONS

  1. Isotonic saline has the same tonicity as blood plasma, blood cells and body tissues. Administering anything other than an isotonic solution would cause either crenation (as a result of hypertonic saline) or hemolysis (a result of hypotonic saline) of the red blood cells. This rapid destruction of red blood cells is extremely serious, if not life threatening. Isotonic saline would have no osmotic effect on the red blood cells.

  2. As the temperature of any phase (gas, liquid or solid) is raised, the kinetic energy of the molecules increases. The rate of diffusion is directly proportional to temperature. The rate of osmosis would also increase with an increase in temperature, until the osmotic equilibrium was reached. Simultaneous osmosis and diffusion rates would also increase.

  3. Increased concentration of a albumin inside dialysis tubing bag would increase the final weight of bag due to increased osmotic uptake of water.

  4. The salt osmotically draws the water out of the cabbage. This covering of liquid creates a perfect anaerobic environment for the fermentation process that produces the sauerkraut.

  5. In fresh water fishes, the blood salt concentration is much less than the salt concentration in their environment. Therefore, water tends to enter their bodies osmotically, and salts are lost by diffusion outward. This osmosis and diffusion occurs across the membranes of the gills. Conversely, fish living in a marine environment, where the salt concentration in the water is much higher than that in fish, have quite the opposite problems. They tend to lose water and gain salt.

  6. Because of low serum protein levels, there is osmotic movement of water out of the circulation and into the tissue spaces. This accumulation of fluid in the abdominal cavity is termed `ascites' and is responsible for the distention.

SUPPLEMENTAL MATERIALS

Bio Sci II, videodisc contains diagrams and photos applicable to this exercise. See appendix.
Diffusion and Osmosis, video filmstrip. Rochester, NY: Ward's. #78W340
Diffusion and Osmosis, 14-minute film. Chicago, IL: Encyclopaedia Britannica Educational Corp.

BACK