Exercise 1

TRANSLOCATION OF MATERIALS

1

Alcabasa, Janine Fortela, Estephen Garcia, Allaine Pozon, Alora Pamela Velina, Joubert Niel

ZOO 120 Group 3 A-2L

July 15, 2013

 ________________ 1 A scientific paper in partial fulfillment of the requirements in Zoology 120 Animal Physiology under nd Professor Maria Dalisay Giron-Maligalig Ph. D., 2 semester A. Y. 2011-2012.

INTRODUCTION

The transport of materials across the plasma membrane is essential to the life of a cell. Certain substances must move into the cell to support metabolic reactions while other substances produced by the cell for export or as cellular waste products must move out of the cell. The plasma membrane, a thin layer of lipids and protein, separate the contents of a cell from the surrounding extracellular fluid, whereas the membranes of organelles divide the intracellular fluid into several membrane-bound compartments (Widmaier, Raff, & Strang, 2003). Substances generally move across cellular membranes via two major transport processes, active or passive, depending on whether they require cellular energy. Active transport is the movement of substances across the plasma membrane via carrier proteins, which requires cellular energy (ATP  – adenosine triphosphate) to drive the substances against its concentration gradient. Another active process by which substances enter and leave the cell is through vesicle transport, w hich includes the endocytosis, exocytosis, and trancytosis of molecules. While passive transport moves substances across the plasma membrane using only the kinetic energy of the moving particles. There are three types of passive transport mechanism, namely, diffusion, osmosis, and filtration (Tortora & Derrickson, 2012; Scanlon & Sanders, 2007).

Diffusion is the movement of molecules from an area of higher concentration to an area of lower concentration  – with or along a concentration gradient . Diffusion occurs because molecules have free energy  – the kinetic energy; that is, they are always in motion. On the other hand, osmosis can be simply defined as the diffusion of water through a selectively permeable membrane, from an area with more water present to an area with less water. While filtration is a process which relies on the energy of mechanical pressure such that water and dissolved materials are forced through a membrane from an area of higher pressure to an area of lower pressure (Scanlon & Sanders, 2007; Sherwood, 2010).

The movements of molecules and ions between both the cell organelles and the cytosol  – the intracellular fluid (ICF), and the ICF and the extracellular fluid would depend on the properties of the membranes. Additionally, the rates at which different substances would move through the membranes vary in response to different conditions (Widmaier, Raff, & Strang, 2003; Tortora & Derrickson, 20 12). This study aims to demonstrate passive transport mechanism and show the effect of different factors on the movement of materials, specifically: (1) determine the factors that affect the diffusion of substances and plasma membrane permeability, (2) establish the haemolytic concentration of different solutions and account for the difference in the concentration of the substances required to produce hemolysis, and (3) differentiate isosmotic, hypoosmotic, and hy perosmotic solutions.

The study was conducted last June 21 and 26, 2013 at Room 405, College of Arts and Sciences Annex 1 Building, University of the Philippines Los Baños.

RESULTS AND DISCUSSION A.

DIFFUSION

Simple diffusion is a type of passive transport which allows molecules to pass through a solid (such as gellike agar), a liquid (such as water or blood), or a gas (such as the air) with kinetic energy as its driving force. The molecules would move about randomly, colliding and ricocheting off one another, while changing direction with each collision. This random collision of molecules is more commonly known as Brownian movement (Sherwood, 2010). Table 1. Average diffusion rate of dy es under different conditions.

Conditions Agar concentration Temperature Dyes

2% 5% o 28-30 C o 11-15 C Methylene Blue Congo Red Methyl Orange

Average diffusion rate (cm/hr) 0.113 0.103 0.113 0.068 0.089 0.067 0.116

The effect of different factors: (1) the agar concentration, (2) temperature, and (3) the type of dye used, on the diffusion of substances was observed. Likewise, the average diffusion rates of each set-up are shown in Table 1. In determining the effect of the agar concentration on the diffusion of substances, it was observed that the average diffusion rate of all dyes were faster in the 2% agar concentration than in the 5%, 0.113 cm/hr and 0.103 cm/hr, respectively. The pore size of a polymer solution (agar) decreases when its concentration is increased (Li, Zhang, and Gopalakrishnakone, 2008). Additionally, small pore size would decrease membrane permeability (Qtaishat, Khayet, & Matsuura, 2012). Therefore, the rate of diffusion is faster in the 2% agar, which is less concentrated than the 5% agar, due to higher membrane permeability. On the other hand, in determining the effect of the temperature on the diffusion of substances, it was observed that the average diffusion rate of al l dyes o

o

were faster in room temperature (28-30 ) than in cold temperature (11-15 C), 0.113 cm/hr and 0.068 cm/hr, respectively. This can be accounted for by the higher kinetic energy of molecules at high temperatures which results to greater collisions, thereby increasing the chance of a molecule to pass through the pores (Sherwood, 2010). Table 2. The different dyes used and their molecular weights in g/mol.

Dye Methylene blue Methyl orange Congo red

Size (MW in g/mol) 319.85 327.33 696.66

Colloid or Non-colloid Non-colloid Colloid Colloid

Lastly, in determining the effect of the type of dye used on the diffusion of substances, it was observed that among the three different dyes, methyl orange have the fastest rate of diffusion followed by methylene blue and congo red at 0.116 cm/hr , 0.089 cm/hr , and 0.067 cm/hr, respectively (Table 1). Among the three, (as shown in Table 2) methylene blue has the lowest molecular weight followed by methyl orange, and congo red at 319.85 g/mol , 327.33 g/mol, and 696.66 g/mol, respectively

(Khurana, 2005; Sabnis, 2010). Theoretically, the smaller the

molecular weight, the faster the rate of diffusion. Since kinetic energy is directly related to both mass and velocity 2

(KE=1/2mv ), smaller molecules tend to move faster (Sherwood, 2010). Additionally, the colloidal property of each dye must be considered (also in Table 2) since colloids appear homogenous like solutions, but are consist of  comparatively large particles dispersed throughout another substance (Ebbing & Gammon, 2011). Consequently, the smaller the size of a particle, the faster is the rate of diffusion. Therefore, non-colloids would diffuse faster than colloids (Ahluwalia & Raghav, 1997). And t hus, methylene blue should have the fastest rate of diffusion followed by methyl orange, and Congo red (Sherwood, 2010). Inconsistency of the data can be accounted for by contamination of the dye used.

B.

Movement Across Membrane Table 3. Relative number of stained yeast cells exposed to different treatments.

Test Tube A B C

Treatment Formalin Heat Control

Cells stained + +++ ++

Figure 1. Yeast cells treated with A) formalin B) heat and C) control with all set-ups stained using Congo red dye.

In the experiment, more yeast cells were stained when heated until boiling point compared to when treated with formalin (Table 3 and Figure 1). Formalin or formaldehyde is the simplest of crosslinking reagents

(fixatives). In addition to amide groups, it also reacts with imines, guanidyls, hydroxyls, sulfhyrdyls, and carboxyls. Fixation immobilizes cell components and decreases membrane permeability. Therefore, too little or none at all of  the dye diffused into the cell. On the other hand, heating the solution increased the rate of diffusion. That is, as the temperature is raised, the kinetic energy of molecules increases thereby increasing the number of collisions with the cell membrane (Ahluwalia & Raghav, 1997; Exbrayat, 2013; Goswami & Pal, 2006). Additionally, heating alters the physiology of the cell, such that excessive heat stress may lead to cell death. These alterations constitute the heat shock response of the cell, which is an array of metabolic changes characterized by the impairment of major cellular functions and by the adaptive reprogramming of the cell metabolism. Increased membrane fluidity or membrane stretch often results as the primary response to heat shock due to several fatty acid changes (adaptation of membrane lipids) and covalent modifications of proteins through methylation, glycosylation, ubiquitination, and acetylation. Changes in the metabolic activity of the cell would alter membrane permeability as well (Bensaude, Bellier, Dubois, Giannoni, & Nguyen, 1996). Moreover, membrane injury from sudden heat stress events may result either from denaturation of membrane proteins or from melting of m embrane lipids. Membrane lipids are highly susceptible to changes in temperature and consequently to changes in membrane fluidity, permeability, and cellular metabolic functions. Lipid saturation level typically increases, whereas unsaturated lipids decrease with increasing temperature. However, high temperature fluidizes by melting the lipid bilayer, thereby increasing membrane permeability, and increasing the leakage of ions and other cellular compounds from the cell (Krishnan, Ramakrishnan, Raja Reddy &Reddy, 2011). Therefore, more yeast cells were stained when heated due to the increase in membrane fluidity and permeability as well as the high kinetic energy of the molecules. C.

Osmosis in Biological Membranes

Osmosis is the diffusion of water across a semipermeable membrane. Water moves from an area of  higher concentration (low solute concentration) to an area of lower concentration (high solute concentration), thereby creating a net movement of the solvent through the semipermeable membrane. Osmosis occurs only when a membrane is permeable to water but is not permeable to certain solutes. There are two ways by which water molecules pass through the plasma membrane: (1) by moving between neighbouring phospholipid molecules in the lipid bilayer  – simple diffusion; and (2) by moving through aquaporins  – integral membrane proteins that function as water channels (Scanlon & Sanders, 2007; Tortora & Derrickson, 2012). As water moves through osmosis across a semipermeable membrane, the shape and volume of cells change  – increases or decreases. The ability of a solution to change the volume of cells through altering their water content is known as tonicity. There are three types of solutions based on their tonicity: isotonic, hypotonic, and hypertonic. Given a cell, an isotonic solution contains solute concentrations that cannot cross the plasma membrane and are equal to that inside the cell (isosmotic cell). The water molecules enter and exit at the same rate, therefore the cell keeps its normal shape and volume. On the other hand, a hypotonic solution has lower solute concentrations compared to that inside the cell (hyperosmotic cell), resulting to a greater influx of water into the cell. The cell would then swell and eventually burst. Oppositely, a hypertonic solution has higher solute concentrations compared to that inside the cell (hypo-o smotic cell), thus the water molecules move out of the cells faster than they enter such that the cells would shrink (Tortora & Derrickson, 2012).

Table 4. Weight of eggs placed in different solutions for 48 hours

Solution Water Vinegar Syrup

Weight (g) Initial 116.45 109.53 109.17

Day 1 120.80 117.00 102.25

Day 2 122.45 118.15 103.35

Figure 2. Eggs placed in solutions of different solute concentrations (S-syrup, V-vinegar, W-tap water).

Initially, the shell-less eggs have semi-transparent  – judging from the silhouette of the egg yolk  – whiteyellowish outer membranes. The eggs sunk to the bottom of the plastic cups containing vinegar and water, whereas the egg in the syrup solution floated (Figure 2. Initial). Whether a solid object sinks or floats when dropped into a solution depends on its density. Density is defined as the ratio of the mass of a substance to the volume occupied by that mass. When an object is less dense than the solution, it will float, displacing a mass of the solution equal to the mass of the object, whereas an object denser than the solution will sink, displacing a volume of the solution equal to the volume of the object (Hein & Arena, 2011). Therefore, the eggs were denser than both tap water and vinegar, and less dense than the syrup solution. After being submerged for 24 hours, the weight of eggs placed in tap water and vinegar solutions increased from 116.45g to 120.80g and 109.53g to 117.00g, respectively, whereas the weight of the egg placed in syrup solution decreased from 109.17g to 102.25g (Table 4). The increase in weight of the eggs submerged in tap water and vinegar solution is comparable to the swelling of cells placed in a hypotonic solution, meaning both the tap water and the vinegar solution have lesser solute concentrations compared to the internal environment of the egg. On the other hand, the decrease in the weight of the egg placed in syrup solution is similar to the shrinking of  the cells placed in hypertonic solutions. Since, the sugar molecules in the syrup solution are large molecules which cannot diffuse across semipermeable membranes, such as the egg membrane, the greater solute concentration of  the syrup solution compared to the internal environment of the egg causes the shrinking of the egg membrane (Fosbery & McLean, 1996). Thus, water moves from an area of higher concentration (tap water, vinegar, and internal environment of the egg, respectively) to an area of lower concentration. The membrane of the egg placed in vinegar was no longer transparent and was a pale orange-white compared to the membrane of the egg placed in water which was less transparent and a slightly darker orange-white. While the membrane of the egg placed in syrup solution became dark brown. The egg membrane is an interwoven network of protein polysaccharide fibers (Belitz, Grosch, & Schieberle, 2009). The electrostatic and hydrogen bonds of polar groups of proteins found in the egg membrane would uncoil from the change in pH caused by acids and a lkalies, thereby denaturing proteins (Das, 2010). Vinegar is a diluted aqueous solution of acetic acid whereas most tap water is treated and is acid forming (Myers, 2003; Ferré, C. 2009). Therefore, the proteins of the membrane of the egg placed in vinegar were more denatured compared to that of the egg placed in water. On the other hand, the membrane of the egg placed in syrup solution became dark-brown due to the continuous bombardment of the egg membrane by the brown solute molecules which stained it dark brown (Sherwood, 2010). Lastly, all the eggs sank to the bottom of the plastic cups when placed in distilled water, therefore, the eggs were denser than distilled water (Figure 2. Day 1). Upon submerging the eggs in distilled water for another 24 hours (Figure 2. Day 2), the weight of the eggs increased (tap water: 122.45, vinegar: 118.15, syrup: 103.35). The increase in the weights of all the eggs indicate that distilled water is a hypotonic solution, thereby causing a greater influx of water into the egg cell membrane. Distilled water, compared to tap water is free of dissolved minerals. Pure distilled wa ter would have tested neutral, but it is not easily obtained because carbon dioxide in the air mixes or dissolves in water, making it somewhat acidic. As such, the membrane of the eggs initially placed in vinegar and water became pale-white and pale orange-white, respectively, compared to the previous day. On the other hand, the membrane of the egg initially placed in syrup became less dark brown due to the stretching of the membrane as water goes into the egg membrane.

D.

Osmotic Pressure in RBC

Blood tissue (or simply blood) is a connective tissue with a liquid extracellular matrix and formed elements. The extracellular matrix, blood plasma is a pale yellow fluid, mostly of water with a wide variety

of dissolved substances such as nutrients, wastes, enzymes, plasma proteins, hormones, respiratory gases, and ions. The formed elements consist of red blood cells (erythrocytes)  – that transport oxygen from the lungs to body cells and remove some carbon dioxide from them, white blood cells (leukocytes)  – that are involved in phagocytosis, immunity, and allergic reactions; and platelets (thrombocytes)  – that participate in blood clotting. Consequently, an increase or decrease in the concentration of the substances in the blood ECF results into difference in the diffusion of water across the cell membrane, thereby changing the integrity of erythrocytes (Tortora & Derrickson, 2012; Guyton & Hall, 2006) Water is present within and around the cells of the body, including the blood vessels. The fluid present in blood as well as in the spaces surrounding cells is called extracellular fluid. Only about 20% of the blood is fluid  – the plasma where the blood cells are suspended. The remaining 80% of the extracellular f luid that lie between cells is called interstitial fluid. As blood flows through the blood vessels of the body, the plasma exchanges oxygen, nutrients, wastes, and other metabolic products with the interstitial fluid. These exchanges are responsible for the similarity of the concentration of dissolved substances found in the plasma and interstitial fluid, except for protein concentration. There is higher protein concentration in the plasma than in the interstitial fluid. In contrast, the composition of the extracellular fluid varies greatly from that of the intracellular fluid inside the cells. It is important, therefore, to maintain the differences in fluid composition across the cell membrane for cells to regulate their activity ( Widmaier, Raff, & Strang, 2003).

Osmosis, the diffusion of water from an area of higher concentration to an area of lower concentration across a semipermeable membrane, that is permeable to water but is not permeable to certain solutes, does not continue until no water remained on the other side of the membrane (the side initially with higher concentration). This is due to the pressure exerted by a liquid, known as hydrostatic pressure, which forces water molecules to move back into the initial side. Moreover, the solution with impermeable solutes would also exert a force called, the osmotic pressure, which is proportional to the concentration of the solute particles that cannot cross the membrane. Therefore, the higher the solute concentration, the higher is the osmotic pressure of the solution, which prevents the net influx of water (Tortora & Derrickson, 2012).

TYPES OF SOLUTIONS When the concentration inside the cell membrane is equal to that outside, there is no net movement of  water across the membrane, and solution is said to be an isotonic solution. This type of solution is suitable for the red blood cells (RBC) to maintain their normal shape and volume inside the body. On the other hand, when high concentration of solutes is observed outside the membrane, water moves towards outside the cell causing the red blood cell to shrink. This type of tonicity is referred as hypertonic solution. Lastly, when high concentration of  solutes is observed inside the cell, water rushes towards inside the cell causing the cell to swell and if the threshold of the membrane is attained, the cell will undergo hemolysis (breakdown of the cell membrane  – bursting of RBC) (Tortora & Derrickson, 2012; Guyton & Hall, 2006). To determine the effect of different salt (KCl, CaCl 2, Na2SO4) concentrations on the integrity of the red blood cell, an isotonic saline blood (ISB) suspension was prepared.

NaCl

dH20

Figure 3. Hemolysis of isotonic saline blood solution in 0.9% NaCl and distilled water (dH 2O). Figure 3 shows that when the isotonic saline blood suspension was mixed with 0.9 % NaCl, the solution was turbid, due to the dispersed RBCs in the solution. Meanwhile, when the ISB suspension was mixed with distilled water, the RBCs were hemolyzed, wherein the cellular components from the hemolyzed RBC settled at the bottom of the test tube via gravity, resulting to a clear solution. Solute inside the RBC was higher in concentration compared to water thus, water would tend to move towards inside the cell, thereby causing the cell to swell and rupture the cell membrane. On the other hand, in 0.9% NaCl, an isotonic is a solution, is the normal saline solution of the human blood, which means that water molecules would enter and exit the RBCs at the same rate due to the tonicity of 0.9% NaCl, allowing RBCs to maintain their normal shape and volume. The RBC plasma membrane + permits the water to move back and forth, but it behaves as though it is impermeable to Na and Cl ions (which enter the cell through channels or transporter but are immediately moved back out by active transport). (Tortora & Derrickson, 2012).

Table 5. Hemolyzing concentrations and isotonic coefficient

Solutions KCl CaCl2 Na2SO4 Sucrose

[Hemolyzing] 0.02 0.02 0.02 0.04

i  of different solutions.

i 

0.1 0.1 0.1

 –

After determining and establishing the hemolyzing solution, the hemolyzing concentration and isotonic coefficient of different solutions were determined (Table 5). Organic compounds contain covalently bonded carbon and hydrogen atoms. In th e human body there are four major groups of organic compounds: carbohydrates, lipids, proteins, and nucleic acids. Sucrose is disaccharide (a carbohydrate) made up of one glucose and one fructose, and is considered to be a non-electrolyte because it is an organic biomolecule which dissolves in water but does not ionize when in solution. Additionally, most organic

compounds are non-electrolytes (Scanlon & Sanders, 2007). The sucrose hemolysis test is a blood test that determines the strength of the membrane of the cell. It determines the swelling of the cell under very low concentration of solute (Scott, 1993). When 0.2 M of sucrose was introduced into the isotonic saline blood suspension, the RBCs did not hemolyzed easily, because sucrose is a non-permeable molecule. It could not pass through easily into the selectively permeable membrane of the cells (Tortora & Derrickson, 2012). Additionally, the extracellular 0.2M sucrose did not result to hemolysis because the osmotic pressure due to the impermeable solutes inside the RBCs prevented the influx of water into the cell. However, dilution made the solution more hypotonic, until hemolysis was observed at 0.04 M sucrose. The osmotic gradient overcame the osmotic pressure within the cell, thereby resulting to the swelling of the cells and eventually, bursting.

On the other hand, electrolytes are chemicals that dissolve in water and dissociate into their positive and negative ions. Most electrolytes are the inorganic salts, acids, and bases found in all body fluids which help regulate the distribution of water in the cell. In the experiment, identical hemolyzing concentrations and isotonic coefficients of  KCl, CaCl2, and Na 2SO4 solutions were obtained at 0.02 M and 2.0, respectively. Compared to the electrolytes, organic compounds are generally present inside the cell, therefore they will have higher hemolyzing concentrations. A nonpenetrating nonelectrolyte solution causes hemolysis at almost the same molar concentration because it has the same number of molecules per liter. Therefore nonpenetrating nonelectrolyte solutions (e.g., glucose, sucrose, etc.) of the same molar concentrations would exhibit the same osmotic pressure (Abramoff and Thomson, 1982). Theoretically, since the intracellular abundance of the +

electrolytes is as follows: K . >so4>sodium > cl & calcium, the order of the hemolyzing concentration of the solutions should be: sucrose>KCl> Na 2SO4 > CaCl2. This order follows the principle of finding the haemolytic concentration of sucrose  – as the intracellular concentration of the electrolytes/nonelectrolytes decrease, the hemolytic concentration of the solutions containing these electrolytes/ nonelectrolytes also decrease because a hypotonic solution is necessary to produce hemolysis (Scanlon & Sanders, 2007). Additionally, the solutions of  nonpenetrating electrolytes (e.g. KCl, CaCl 2, and Na 2SO4) cause hemolysis at lower molecular concentrations than nonpenetrating nonelectrolytes because the electrolytes dissociate into two ions and every ion in the dissociated solution would exert the same osmotic pressure as is produced by an entire molecule. Therefore, at the same molar concentrations, there would be more molecules per liter in an electrolyte solution than in a nonelectrolyte solution such that the solutions would demonstrate different osmotic pressures. Also, the degree of dissociation of  an electrolyte in the solvent ca n vary the osmotic pressure exerted by different electrolyte solutions (Abramoff and Thomson, 1982). Isotonic coefficient (i) is defined as the relationship of non-permeable non-electrolytes to the non-permeable electrolytes in terms of osmotic activities (Scott, 1993). Based on the formula: i = [non-electrolyte]/[electrolyte] ,the order of the isotonic coefficients of the solutions would therefore be the opposite of the decreasing order of  their hemolyzing concentrations: CaCl2. > Na 2SO4 >KCl> sucrose (Scanlon & Sanders, 2007). Further, the computed isotonic coefficient of the different solutions (Table 5), 2.0 , would mean that 100 molecules of the electrolyte exert as much osmotic pressure as 200 molecules of sucrose (Abramoff and Thomson, 1982).

CONCLUSION In determining the effect of the agar concentration on the diffusion of substances, the average diffusion rate of all dyes were faster in the 2% agar concentration than in the 5%, 0.113

cm/hr and 0.103 cm/hr,

respectively. Increasing the concentration of the agar decreases the pore size, thereby decreasing membrane permeability. On the other hand, it was observed that the average diffusion rate of all dyes were faster in room o

o

temperature of 28-30 at 0.113 cm/hr than in cold temperature of 11-15 C at 0.068 cm/hr due to the higher kinetic energy of molecules at high temperatures resulting to rapid movement of molecules, thereby increasing the chance of a molecule to pass through the pores. Lastly, in determining the effect of the type of dye used on the diffusion of substances, it was observed that among the three different dyes, methyl orange have the fastest rate of diffusion followed by methylene blue and congo red at 0.116 cm/hr , 0.089 cm/hr , and 0.067 cm/hr, respectively.Theoretically, the smaller the molecular weight and size of a particle, the faster is the rate of diffusion. Additionally, the colloidal property of  each dye must be considered since colloids would appear homogenous but consists of large particles dispersed throughout another substance. Therefore, non-colloids would diffuse faster than colloids thus, methylene blue should have the fastest rate of diffusion followed by methyl orange, and Congo red, with molecular weights: 319.85 g/mol , 327.33 g/mol, and 696.66 g/mol, respectively. Inconsistency of the data can be accounted for by contamination of the dye used. In determining the effect of fixatives and heat to plasma membrane permeability, it was observed that more yeast cells were stained when heated until boiling point compared to when treated with formalin. The crosslinking of membrane components (e.g. amines, imines, etc.) during treatment with formalin decreased the permeability of the cell membrane  – thus too little or none at all of the dye diffused into the cell. Meanwhile, heating the solution increased the rate of di ffusion  – increase in the kinetic energy of molecules (collisions with the cell membrane), consequently, increased the chance for the dye molecules to pass through the membrane. Additionally, heating altered the physiology of the cell  – the occurrence of metabolic changes that constitute the heat shock response of the cell, as well as the denaturation of membrane proteins or melting of membrane lipids. Therefore, more yeast cells were stained when heated due to the increase in membrane fluidity and permeability as well as the high k inetic energy of molecules. The effect of solutions having different solute concentrations (syrup, vinegar, and tap water) on osmosis was also observed. Initially, the shell-less eggs have semi-transparent white-yellowish outer membranes. The eggs sunk to the bottom of the plastic cups containing vinegar and water, whereas the egg in the syrup solution floated because the eggs were denser than both tap water and vinegar, and less dense than the syrup solution. As water moves through osmosis across a semipermeable membrane, the shape and volume of cells change  – increases or decreases. The ability of a solution to change the volume of cells through altering their water content is known as tonicity. There are three types of solutions based on their tonicity: isotonic, hypotonic, and hypertonic. Given a cell, an isotonic solution contains solute concentrations that cannot cross the plasma membrane and are equal to that inside the cell (isosmotic cell). The water molecules enter and exit at the same rate, therefore the cell keeps its normal shape and volume. On the other hand, a hypotonic solution has lower solute concentrations compared to that inside the cell (hyperosmotic cell), resulting to a greater influx of water into the cell. The cell would then swell and eventually burst. Oppositely, a hypertonic solution has higher solute concentrations compared to that inside the cell (hypo-osmotic cell), thus the water molecules move out of the cells faster than they enter such that the cells would shrink ( Tortora & Derrickson, 2012).

After being submerged for 24 hours, the weight of eggs placed in tap water and vinegar solutions increased from 116.45g to 120.80g and 109.53g to 117.00g, respectively, while the weight of the egg placed in syrup solution decreased from 109.17g to 102.25 g . Therefore, tap water and vinegar are hy potonic solutions since they have lower solute concentrations compared to that inside the egg (hyperosmotic egg), which resulted to the greater influx of water into the egg membrane  – increase in weight. On the other hand, the syrup solution is a hypertonic solution since it has higher solute concentrations compared to that inside the egg (hypo-osmotic egg), which resulted to the greater efflux of water out of the egg membrane  – decrease in weight. Water would therefore move from an area of higher concentration to an area of lower concentration. Additionally, the membrane of the egg placed in vinegar became opaque and pale orange-white whereas the membrane of the egg placed in water became less transparent and slightly darker orange-white. Since the egg membrane is an interwoven network of protein polysaccharide fibers, placing the egg shell-less eggs in a diluted aqueous solution of acetic acid (vinegar) and acid forming tap water (due to treatment) uncoiled the electrostatic and hydrogen bonds of the egg membrane proteins. Therefore, the egg membrane proteins of the egg placed in vinegar were more denatured than that of the egg placed in tap water (less acidic). Meanwhile, the membrane of the egg placed in syrup solution became dark-brown from the continuous bombardment of the egg membrane by the brown solute molecules (sugar) which stained it dark brown. Lastly, all the eggs sank to the bottom of the plastic cups as they are denser than distilled water. Submerging the eggs in distilled water for another 24 hours increased the weight of the eggs (tap water: 122.45, vinegar: 118.15, syrup: 103.35). Therefore, distilled water is a hypotonic solution which caused greater influx of water into the egg cell membrane  – hence the increase in weight. Additionally, the membrane of the eggs initially placed in vinegar and water became pale-white and pale orange-white, respectively, while the membrane of the egg initially placed in syrup became less dark brown due to membrane stretching caused by the influx of  water as well as protein denaturation by the somewhat acidic distilled water  – carbon dioxide in the air mixed or dissolved in water. Osmosis, the diffusion of water from an area of higher concentration to an area of lower concentration across a semipermeable membrane, that is permeable to water but is not permeable to certain solutes, does not continue until no water remained on the other side o f the membrane (the side initially with higher concentration). This is due to the pressure exerted by a liquid, known as hydrostatic pressure, which forces water molecules to move back into the initial side. Moreover, the solution with impermeable solutes would also exert a force called, the osmotic pressure, which is proportional to the concentration of the solute particles that cannot cross the membrane. Therefore, the higher the solute concentration, the higher is the osmotic pressure of the solution, which prevents the net influx of water Lastly, in the observation of osmotic pressure, the isotonic saline blood (ISB) suspension mixed with 0.9 % NaCl, resulted to a turbid solution, due to the dispersed RBCs in the solution while the ISB suspension mixed with distilled water, resulted to hemolysis, such that the cellular components of the hemolyzed RBC settled at the bottom of the test tube via gravity, and became a clear solution. Hemolysis occurred because the osmotic gradient of the distilled water (hypotonic solution) was able to overcome the osmotic pressure  – exerted by impermeable solutes  – inside the cell. In determining the hemolyzing concentration of sucrose, KCl, CaCl 2, and Na 2SO4 solutions, dilution made the solutions increase in hypotonicity. The established hemolyzing concentration of sucrose was 0.04 M and 0.02 M for the KCl, CaCl 2, and Na 2SO4 solutions. Theoretically, the order of the hemolyzing concentration of the solutions should be: sucrose>KCl> Na 2SO4 > CaCl2, given that the intracellular abundance of  +

the electrolytes is as follows: K . >so4>sodium > cl & calcium and that sucrose, a nonelectrolyte, is nonpenetrating solute. The solutions of nonpenetrating electrolytes (e.g. KCl, CaCl 2, and Na 2SO4) cause hemolysis at lower

molecular concentrations than nonpenetrating nonelectrolytes since electrolytes dissociate into two ions and every ion in the dissociated solution exerts the same osmotic pressure as produced by an entire molecule. Therefore, at same molar concentrations, more molecules per liter is found in an electrolyte solution than in a nonelectrolyte solution  – the solutions would demonstrate different osmotic pressures. Meanwhile, the order of  the isotonic coefficients of the solutions would be the opposite of the order of their hemolyzing concentrations:

CaCl2. > Na 2SO4 >KCl> sucrose . Given the computed isotonic coefficient of the different solutions, 2.0, mean that 100 molecules of the electrolyte exert as much osmotic pressure as 200 molecules of sucrose.