Biological Membranes

Dialysis, Osmosis and Diffusion

1. Diffusion
1.1 Diffusion of solutes in water
1.1.1 Prepare a series of four test tubes containing ~10 mL the
following:
1.1.1.1 Distilled water
1.1.1.2 Tap water
1.1.1.3 Hot distilled water (70 OC or higher)
1.1.1.4 Cold distilled water (on ice)
1.1.2 Label the tubes accordingly.
1.1.3 To each test tube place a dot amount of KMnO 4 crystals.
1.1.4 Take note of the time at which the KMnO 4 crystals were
completely dissolved in the water.
1.1.5 Repeat the determination.
1.2 Diffusion in a colloid
1.2.1 Melt gelatine/agar according to manufacturers procedures a.
1.2.2 Pour ~10 mL of the melted gelatine in test tubes and allow it to
solidify.
Prepare 8-10 gelatine-test tubes.
1.2.3 Overlay ~0.5 mL of the KMnO4 in one of gelatine-tube.
1.2.4 Add the same amount of Congo Red and Methelyne Blue in
separate gelatine-test tubes.
1.2.5 Perform two sets of gelatine-tubes per group.
1.2.6 Measure the migration of the dye starting at the top level of the
gelatine.
1.2.7 Obtain the measurements for 120 minutes at 10-minute intervals
or until the dye reaches the bottom of the tube.
2. Osmosis
2.1 Osmosis (Longanisa Casing Model)
2.1.1 Place ~10 mL of saturated sugar solution in a test tube.
2.1.2 Cover the mouth of the test tube with 1-ply of Longanisa casing.
Secure the cover with a rubber band.
2.1.3 Obtain the weight of the covered test tube (using a triple beam
balance or analytical balance).
2.1.4 Suspend the test tube upside down in a 250-mLbeaker containing
distilled water. Use an Iron clamp to fasten the test tube.
2.1.5 Obtain the weight of the test tube at 10-minute intervals for 120
minutes. Tabulate the results.
2.1.6 Prepare two set-ups per group.
2.2 Osmosis in RBC
2.2.1 Prepare 3 clean glass slides and cover slips.
2.2.2 Place a drop or two of blood on each glass slide.
a
b

Manufacturers gelatin recipe could be diluted further (by half) as needed.

Ask the laboratory techs to prepare the solutions.

* Use a STERILE lancet to make a puncture at the donors finger.

Sanitize the finger with 70% alcohol before and after the
puncturing.
2.2.3 Apply a drop of Isotonic-Normal Saline Solution (0.85 M NaCl) in
one glass slide.
2.2.4. Apply a drop of Hypertonic Salt Solution (3.00 M NaCl) and to
Hypotonic Salt Solution (0.30 M NaCl) to the remaining glass slides.
2.2.5 Observe the RBCs under the microscope at 40X-objective. Take
pictures and compare the sizes of the RBCs of the different slides.
3. Dialysis
3.1 Fill a test tube with 3 mL Methylene Blue Solution and 5 mL Saturated
CaCl2 solution.
3.2 Cover the mouth of the test tube with 1-ply of Longanissa casing. Secure
the cover with a rubber band.
3.3 Suspend the test tube upside down in a beaker (250 mL) containing
distilled water. Use an Iron clamp to fasten the test tube.
3.4 Test the presence of Methylene Blue, Chloride and Calcium in the beakerwater after every minute for 30 minutes.
3.4.1 The presence of Methylene Blue in the beaker-water can be
identified by visual inspection. A faint blue color in the beaker-water
indicates the presence of Methylene Blue.
3.4.2 The presence of Chloride is identified by adding a drop of Silver
nitrate/Silver sulphate Test Solution to 1-mL aliquot of the beaker-water.
3.4.3 The presence of Calcium is identified by adding a drop of Oxalic
acid Test Solution to 1-mL aliquot of the beaker-water.
3.5 Tabulate the results. Which Ion and/or molecule passed through the
longanissa skin first?
3.6 Perform the experiment in duplicates.

Materials (per group):

(5) 250 mL Beakers
(10) test tubes
(1) test tube rack
(1) thermometer
(4) iron clamp
(1) iron stand
(1) ruler
(1) stopwatch
(5) Glass slides with cover slip
(5) sterile Lancets
(1) 70% alcohol with cotton balls
Droppers (for each liquid reagent)
(2) Waste Bottles (small)
(1) microscope
(1) analytical balance (we have to go to the weighing room to do the weighing)
(1) electric stove

a
b

Manufacturers gelatin recipe could be diluted further (by half) as needed.

Ask the laboratory techs to prepare the solutions.

Reagents (for the class)

100 mL Normal Saline Solution (0.85% NaCl)
100 mL Isotonic Solution (0.30 % NaCl) b
100 mL Hypertonic Solution (1.80 % NaCl) b
Saturated Calcium chloride solution. b
Ice

Congo Red solution b

Methylene Blue Solution b
KMnO4 solution b
KMnO4 crystals
Gelatine (to be provided by the class)
Distilled Water (to be provided by the class)
White sugar (to be provided by the class)
Longanisa skin
Suggested schedule of activities:
Minute
Activity
Preparation of Longanisa
Set-Ups
Longanisa Set Up
Determinations
Diffusion with Gelatine
Diffusion with Water
RBC Osmosis
Consolidation of Results
Presentation of Results

3
0

6
0

x
x

9
0

12
0

15
0

18
0

21
0

24
0

27
0

30
0

33
0

36
0

Time
Allotme
nt
(min)
60

x
x

120
X
X

60
x
x

60
x

180
x

180
x

60

NOTES:
Everyone is expected to wear complete PPEs (lab gowns, goggles and hair nets for
the ladies). Non-compliance will be given deductions.

CCD 2013
Draft 1

a
b

Manufacturers gelatin recipe could be diluted further (by half) as needed.

Ask the laboratory techs to prepare the solutions.