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

*Please refer to the Appendix for name and address of supplier.

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

DEMONSTRATION

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% 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

    
    Package 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:

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 400¥, 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.
7. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

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 freshwater 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