1. 8- Osmotic Changes in Red Blood Cells.
Lab Teaching staff (Boys) Dr. Syed Najam Dr. Muhammad Irfan Mr. Muhammad Amir Mustufa
good●morning,●my●name●is●doctor●muhammad●irfan●and●today●we●gonna●discuss●experiment●number●8●Osmotic●Changes●in●Red●Blood●Cells.●all●experiments●are●present●in●the●web●site●box.net/firstyear

2. Learning Objectives
By the end of the practical students should be able to:
Demonstrate process of osmosis experimentally using RBCs from a human subject.
Define terms like, active and passive transport, osmosis, hypo, hyper and isotonic solutions.
Explain the swelling and shrinkage of RBCs in hypo and hypertonic medium.
good●morning,●my●name●is●doctor●muhammad● irfan●and●today●we●gonna●discuss●experiment●n umber●8●Osmotic●Changes●in●Red●Blood●Cells.● all●experiments●are●present●in●the●web●site●bo x.net/firstyear
By●the●end●of●the●practical●students●should●be●able●to:●1.●Demonstrate●process●of●osmosis●experimentally●using●RBCs●from●a●human●subject.●2.●Define●terms●like,●active●and●passive●transport,●osmosis,●hypo,●hyper●and●isotonic●solutions,●and●3.●●Explain●the●swelling●and●shrinkage●of●RBCs●in●hypo●and●hypertonic●medium.●

3. Requirements Slides with cover slips Test tubes Droppers Lancet
Alcohol swab
Needle or tooth pick
Human subject
Isotonic, hypotonic and hypertonic solutions.
Microscope
Sedarwood oil
the●requirements●for●today's●experiment●are:●
Slides●with●cover●slips,●Test●tubes,●Droppers,●Lancet,●Alcohol●swab,●Needle●or●tooth●pick,●Human●subject,●Isotonic,●hypotonic●and●hypertonic●solutions,●Microscope●and●●Sedarwood●oil●

4. Transport across the cell membrane
Two types of processes are involved in transport across the cell membrane.
Passive Transport
Movement of molecule from higher concentration to lower concentration without utilizing the energy “ATP”.
Active Transport
Movement of molecule from lower concentration to higher concentration by carrier protein, utilizing the energy (ATP).
before●beginning●the●discussion●let's●discuss●different●modes●of●transport●across●the●cell●membrane.●actually●,●Two●types●of●processes●are●involved●in●transport●across●the●cell●membrane.●1.●Passive●Transport,●which●is●the●Movement●of●molecule●from●higher●concentration●to●lower●concentration●without●utilizing●the●energy●“ATP”,●and●2.●Active●Transport,●that●is●the●Movement●of●molecule●from●lower●concentration●to●higher●concentration●by●carrier●protein,●utilizing●the●energy●(ATP).●

5. Simple Passive Diffusion Facilitated (with carrier) Osmosis Exosmosis
(without carrier)
Facilitated
(with carrier)
Osmosis
Exosmosis
(out side)
Endosmosis
(inside)
This slide shows different types of passive transport. there are mainly two types of passive transport, diffusion and osmosis. diffusion is again divided in to simple diffusion which is carried out with using any carrier protein , and facilitated transport, which is carried out with the help of  carrier protein. osmosis is again divided in to ex-osmosis which is the movement outside the cell a nd end-osmosis which is the movement of molecules inside the cells.  

6. Type of solutions Solution = Solute + Solvent e.g., (Salt + Water)
Isotonic Having concentration of solute similar to the cytoplasm.
Hypotonic Having concentration of solute lower than the cytoplasm.
Hypertonic Having concentration of solute higher than the cytoplasm.
solution●is●actually●a●combination●of●a●solute●that●we●mix,●like●salt●in●salt●solution●and●solvent●in●which●we●mix●the●solute●,●means●water●in●a●salt●solution.●this●slide●shows●different●Type●of●solutions.
1.●is●Isotonic●Having●concentration●of●solute●similar●to●the●cytoplasm.
2.●Hypotonic●Having●concentration●of●solute●lower●than●the●cytoplasm,●
3.●Hypertonic●Having●concentration●of●solute●higher●than●the●cytoplasm,●and,●4.hypotonic●solution●contains●less●solute●(thus,●more●water)●than●the●cytoplasm●of●the●cells.●Thus,●placing●normal●RBCs●into●a●hypotonic●solution●causes●water●movement●into●the●cells,●causing●swelling●and●lysis.●As●the●RBCs●swell,●the●increased●turgor●(pressure)●results●in●rupture●(lysis)●of●the●plasma●membrane.

7. Solutions that contain the same concentration of solutes as the cytoplasm are called isotonic solutions e.g., normal saline. No net movement of water molecules ,so RBC maintain their shape and size.
this●slide●shows●a●picture●of●slide●with●isotonic●Solutions●that●contain●the●same●concentration●of●solutes●as●the●cytoplasm●for●example,●●●normal●salyne.●you●can●appreciate●that●if●u●place●red●cell●in●●this●solution,●they●maintain●their●shape●and●remain●normal.●this●is●physiological●and●normal●solution

8. A hypotonic solution contains less solute (thus, more water) than the cytoplasm of the cells. Thus, placing normal RBCs into a hypotonic solution causes water movement into the cells, causing swelling and lysis. As the RBCs swell, the increased turgor (pressure) results in rupture (lysis) of the plasma membrane.
this●slide●shows●a●picture●of●slide●with●A●hypotonic●solution●that●contains●less●solute●(thus,●more●water)●than●the●cytoplasm●of●the●cells.●Thus,●placing●normal●RBCs●into●a●hypotonic●solution●causes●water●movement●into●the●cells,●causing●swelling●and●lysis.●As●the●RBCs●swell,●the●increased●turgor●(pressure)●results●in●rupture●(lysis)●of●the●plasma●membrane.●

9. A hypertonic solution contains more solute than water in comparison with the cytoplasm of the cells. Thus, placing normal RBCs into a hypertonic solution causes water movement out of the cells resulting in their shrinkage
this●slide●shows●a●picture●of●slide●with●A●hypertonic●solution●that●contains●more●solute●than●water●in●comparison●with●the●cytoplasm●of●the●cells.●Thus,●placing●normal●RBCs●into●a●hypertonic●solution●causes●water●movement●out●of●the●cells●resulting●in●their●shrinkage●●and●lysis

11. Effect of Concentration of Solute on movement of Water.
Solute concentration
(outside the cell)
(inside the cell)
Isotonic
Solute %
isotonic
Hypotonic solution
LOW SOLUTE %
(high solvent)
Hypertonic cell
Hypertonic solution
HIGH SOLUTE %
(low solvent)
Hypotonic cell
0.9 %
0.9 %
in●this●slide,●you●can●see●the●Effect●of●Concentration●of●Solute●on●movement●of●Water.●●on●side●of●the●table●shows●the●concentration●of●solute●outside●the●cell,●while●the●other●side●shows●solutes●inside●the●cells.●in●isotonic●solution,●like●0.9%●saliine●solution●in●which●we●add●0.9●grams●of●sodium●chloride●in●100●milliliter●of●water.●in●this●case●the●concentration●of●solute●is●same●on●both●side●of●the●cell●membrane,●so●there●is●no●net●movement●of●molecules.●second●is●the●example●of●hypotonic●solution,●that●is●0.45%●salt●solution.●in●this,●as●the●concentration●is●more●inside●of●the●cell●than●outside,●there●is●a●movement●of●water●form●outside●to●inside●the●cell●,●resulting●in●cell●swelling●death●.●tha●last●one●is●hypertonic,●that●is●2%●salt●solution,●here●the●movement●of●water●is●towards●outside●
0.9 %

12. this●slide●is●showing●the●effect●of●different●solution●upon●the●shape●of●red●cell.●if●u●place●red●cells●in●isotonic●solution,●the●middle●one,●the●shape●is●maintained,●while●if●u●place●them●in●either●hyper●or●hypotonic●solution,●they●die●as●a●result●of●swelling●or●shrinkage●

13. this●slide●again●is●showing●the●same●phenomenon,●see●the●shape●in●different●solutions.

15. Procedure
Take three slides and mark them A, B and C for isotonic, hypotonic and hypertonic, respectively.
II. Put one or two drops of isotonic, hypotonic and hypertonic solutions at the centre of these three slides, respectively.
III. After cleaning your finger with alcohol swab, prick it with the help of lancet to draw blood. Add one drop of blood in each of the solutions poured on three slides.
now●let's●discuss●the●procedure.
1.●Take●three●slides●and●mark●them●A,●B●and●C●for●isotonic,●hypotonic●and●hypertonic,●respectively.●
2.●Put●one●or●two●drops●of●isotonic,●hypotonic●and●hypertonic●solutions●at●the●centre●of●these●three●slides,●respectively.●
3.After●cleaning●your●finger●with●alcohol●swab,●prick●it●with●the●help●of●lancet●to●draw●blood.●Add●one●drop●of●blood●in●each●of●the●solutions●poured●on●three●slides. ●

16. Procedure
IV. Mix the blood with solution with the help of separate match stick or common pin on all the three slides.
V. Take care to mix the blood with respective solutions on three slides as quickly as possible so that there should not be any clotting of blood.
VI. Wait for five minutes.
VII. Put cover slip over each of the slide having blood solution mixture and observe the shape and size of RBCs, under 4x, 10x, 40x and 100x objectives.
4.●Mix●the●blood●with●solution●with●the●help●of●separate●match●stick●or●common●pin●on●all●the●three●slides.●
5.●Take●care●to●mix●the●blood●with●respective●solutions●on●three●slides●as●quickly●as●possible●so●that●there●should●not●be●any●clotting●of●blood.●
6.●Wait●for●five●minutes.●
7.●Put●cover●slip●over●each●of●the●slide●having●blood●solution●mixture●and●observe●the●shape●and●size●of●RBCs,●under●4x,●10x,●40x●and●100x●objectives.●

17. Precautions Make sure to sterilize your finger before prick.
II. Drop of blood should be of appropriate size.
III. Mix the blood and the solution quickly to avoid clotting of blood.
IV. Use different droppers to pour different solutions on slides.
V. Air bubble should not appear under the cover slip.
here●are●some●of●the●precautions●that●u●must●take●while●performing●the●experiment.●
1.●Make●sure●to●sterilize●your●finger●before●prick.●
2.●Drop●of●blood●should●be●of●appropriate●size.●
3.●Mix●the●blood●and●the●solution●quickly●to●avoid●clotting●of●blood.●
4.●Use●different●droppers●to●pour●different●solutions●on●slides.●and,●
5.●Air●bubble●should●not●appear●under●the●cover●slip.●

18. Placing RBC in hypotonic solution will cause:
a. Shrinkage of cell.
b. No change in shape and size.
c. Swelling of cell.
d. decrease in turgor pressure within the cell.
E. Change only in shape not in size.
now●there●are●a●few●question●for●your●revision●of●the●topic●.●question●number●1●is●Placing●RBC●in●hypotonic●solution●will●cause:●a. Shrinkage●of●cell.●b. No●change●in●shape●and●size.●c. Swelling●of●cell.●d. decrease●in●turgor●pressure●within●the●cell.●E. Change●only●in●shape●not●in●size.●

19. In Isotonic solution concentration of Sodium Chloride is:
b. 5%
c. 0.9%
d. 0.5%
e. 0.2%
here●'s●the●second●one●.● ●In●Isotonic●solution●,●●concentration●of●Sodium●Chloride●is,●a.● 2%●,●●b.● 5%●,●●c.● 0.9%●d.● 0.5%,●or●●e. 0.2%●

20. References Guyton Text book of medical Physiology 13th edition.
Biology by Solomon, Berg, Martin and Villee.
this●concludes●our●experiment●for●today.●please●go●through●the●slides●again●and●again●.●some●questions●can●come●form●this●experiment.●good●luck●for●your●future●and●stay●safe.●bye