ACTIVITY 2

CELL
Raymond Godfrey Dagwasi, RPh
wet mount slide
most common type of slide
preparation for microscope work
used to view living organisms,
as well as liquid substances of
all kinds
used for any sort of specimen
that needs to be kept moist
Microscopy
practice that involves the use of a
microscope for the purposes of
observing small scale structures
that cannot be viewed using the
naked eye and often cell staining
is necessary as structures are
difficult to discern due to
insufficient contrast
Cell staining
technique used for the main
purpose of increasing
contrast through changing
the color of some of the
parts of the structure being
observed thus allowing for a
clearer view

Cell staining
technique that can be used to
better visualize cells and cell
components under a microscope.
By using different stains, one can
preferentially stain certain cell
components, such as a nucleus or
a cell wall, or the entire cell.
Cell staining preparation
Permeabilization - treatment of
cells, generally with a mild
surfactant, which dissolves cell
membranes in order to allow
larger dye molecules to enter
inside the cell.
Cell staining preparation
Fixation - serves to "fix" or preserve
cell or tissue morphology through
the preparation process. This
process may involve several steps,
but most fixation procedures involve
adding a chemical fixative that
creates chemical bonds between
proteins to increase their rigidity.
Cell staining preparation
Mounting - involves attaching
samples to a glass microscope
slide for observation and analysis.
Cells may either be grown directly
to the slide or loose cells can be
applied to a slide using a sterile
technique.
Cell staining preparation
Staining - application of stain to a
sample to color cells, tissues,
components, or metabolic processes.

This process may involve immersing
the sample (before or after fixation
or mounting) in a dye solution and
then rinsing and observing the
sample under a microscope.
Methylene blue
homologue of Toluidine Blue O

stains animal cells to make nuclei more
visible

Result: the intracellular metachromatic
granules stain a ruby-red to black color;
with the remainder of the cell staining a
pale blue color.
Section through an animal cell showing the major components visible
by electron microscopy. To simplify, a few important components,
including intermediate filaments, have been omitted.
Paramecium, HPO (640x)
The Membrane Proteins
Integral Proteins: span the plasma
membrane exposed on one surface or both
surfaces of the plasma membrane.
: exposed on both surfaces of the
plasma membrane are called
Transmembrane Proteins

Peripheral Proteins: attached to integral
proteins or the phospholipids heads on the
cytoplasmic face of the plasma membrane
Some functions of membrane proteins, p. 68.
Transport
(a) A protein that spans the membrane
may provide a hydrophilic channel
across the membrane that is selective
for a particular solute. (b) Some
transport proteins hydrolyze ATP as an
energy source to actively pump
substances across the membrane.
Enzymatic activity
A protein built into the membrane may
be an enzyme with its active site
exposed to substances in the adjacent
solution. In some cases, several
enzymes in a membrane act as a team
that catalyzes sequential steps of a
metabolic pathway as indicated
(right to left) here.
Receptors for signal transduction
A membrane protein exposed to the
outside of the cell may have a binding
site with a specific shape that fits the
shape of a chemical messenger, such
as a hormone. The external signal
may cause a conformational change
in the protein that initiates a chain of
chemical reactions in the cell.
Intercellular joining
Membrane proteins of adjacent cells
may be hooked together in various
kinds of intercellular junctions.
Some membrane proteins (CAMs)
of this group provide temporary
binding sites that guide cell
migration and other cell-to-cell
interactions.
Cell-cell recognition
Some glycoproteins (proteins
bonded to short chains of sugars)
serve as identification tags that
are specifically recognized by
other cells.
Attachment to the
cytoskeleton
and extracellular matrix (ECM)
Elements of the cytoskeleton (cells internal
supports) and the extracellular matrix (ECM)
may be anchored to membrane proteins,
which help maintain cell shape and fix the
location of certain membrane proteins.
Others play a role in cell movement or bind
adjacent cells together.
(a) (b)
ATP
Cell junctions, p. 70.
Linker
proteins
Plaque
Intermediate
filament
Intercellular
space
Intercellular space
Interlocking
junctional proteins
Microvilli
Tight junction
Plasma membranes
of adjacent cells
Gap junction
Underlying
basement membrane
Extracellular
space between
cells
Desmosome
Intercellular
space
Channel
between cells
(connexon)
(b) Desmosome
(a) Tight junction
(c) Gap junction
Membrane Junctions
Tight Junction fusion of integral proteins in
plasma membrane of adjacent cells forming an
impermeable junction
Desmosome linker proteins extending from
plaques on the cytoplasmic surface of the plasma
membrane of adjacent cells interdigitate to hold the
cells together and prevent their separation; also
known as anchoring junction
Gap Junction formed by hollow cylinder called
connexons; it allows for the rapid transfer of ions
between cells; also known as communicating
junction
MEMBRANE TRANSPORT the
plasma membrane is a selective barrier
1. Passive processes substances cross the plasma
membrane without any energy input
2 main types: Diffusion and Filtration
Diffusion 3 subtypes:
Simple diffusion
Facilitated diffusion
Osmosis
2. Active processes the cell provides energy required to
move substances across the plasma membrane
2 main types: Active transport ( = solute pumping)
and Vesicular transport
Vesicular transport Exocytosis and Endocytosis
Exocytosis movement of substances out of the cell
Endocytosis movement of substances into the cell
Phagocytois
Pinocytosis
Receptor-mediated endocytosis
Diffusion through the plasma membrane,
p. 72.
Extracellular fluid
Cytoplasm
Lipid-
soluble
solutes
Lipid
bilayer
Lipid-insoluble
solutes
Water
molecules
Small lipid-
insoluble
solutes
(a) Simple diffusion
directly through the
phospholipid bilayer
(c) Channel-mediated
facilitated diffusion
through a channel
protein; mostly ions
selected on basis of
size and charge
(b) Carrier-mediated facilitated
diffusion via protein carrier
specific for one chemical;
binding of substrate causes
shape change in transport
protein
(d) Osmosis, diffusion
through a specific
channel protein
(aquaporin) or
through the lipid
bilayer
FILTRATION
A passive process no energy input

Movement of solution from area of higher
pressure to area of lower pressure; down a
pressure gradient
A gradient is any difference in
intensity between two sides of a
demarcation.
DIFFUSION
Movement of substances from area of higher
concentration to area of lower concentration;
substances move down their concentration gradient
Simple diffusion nonpolar/hydrophobic/lipid-
soluble substances diffuse through the plasma
membrane.
Facilitated diffusion transport of large/polar
substances mediated by carrier proteins embedded
in the plasma membrane. Facilitated diffusion
exhibits saturation, specificity
Osmosis movement of water from area of lower
solute concentration to area of higher solute
concentration through a semi-permeable membrane

Water moves through specific channels called
aquaporins

Influence of membrane permeability on diffusion and osmosis, p. 73.
Left
compartment:
Solution
with lower
osmolarity
Membrane
Solute
molecules
(sugar)
Right
compartment:
Solution
with greater
osmolarity
Both solutions have
the same osmolarity:
volume unchanged
H
2
O
Solute
Left
compartment
Membrane
Right
compartment
Both solutions have identical
osmolarity, but volume of the
solution on the right is greater
because only water is
free to move
H
2
O
(a) Membrane permeable to both solute
molecules and water
(b) Membrane impermeable to solute molecules,
permeable to water
Questions
diffusion is faster in what type of matter -
gas, liquid or solid?

Which state of matter has the highest
kinetic energy?

Which state has the most space between
the particles?
DIFFUSION in Gases
the natural rapid and random movement of the
particles in all directions means that gases readily
spread or diffuse.

The net movement of a particular gas will be in
the direction from lower concentration to a higher
concentration, down the so-called diffusion
gradient.
Diffusion continues until the concentrations are
uniform throughout the container of gases, but
ALL the particles keep moving with their ever
present kinetic energy!
Diffusion of gas occurs through the one cell layer
thick alveoli in the lungs.

The speed of diffusion is dependent on different
factors, one being the surface area and second
the size of the diffusing particles.
In the lungs there is ample surface and Oxygen
and CO2 are small particles so the rate of
diffusion is great.
Hemoglobin has a high affinity for O2 in the
lungs and a low affinity for CO2.
In the tissues the role reverses and hemoglobin
has a low affinity for O2 and a greater affinity for
CO2.
Now in the tissues since they are small
molecules O2 and CO2 readily diffuse across
the cell membranes and require no special
transport mechanisms.
This is simple diffusion where gasses are
diffusing down their concentration gradient.

Diffusion in liquids
If colored crystals of potassium manganite (VII) are
dropped into a beaker of water and covered at room
temperature.

Despite the lack of mixing due to shaking or
convection currents from a heat source the bright
purple color of the dissolving salt slowly spreads
throughout all of the liquid but it is much slower
than the gas because of the much greater density
of particles slowing the spreading due to close
proximity collisions.

BLOOD
Oxygen diffuses into red blood cells in the lungs,
while Carbon Dioxide is diffusing out of red blood
cells at the same time.

Nutrients diffuse from the intestines into the
blood.

KIDNEYS
water and other nutrients diffuse by condensing
the waste products in the urine and reabsorbs
majority of the water.
STOMACH (gastric juice)
medicine diffuses through its capsule, through the
stomach lining, and into the blood stream.
Diffusion in solids (colloid)
Diffusion is almost impossible in solids because
the particles are too closely packed and
strongly held together with no empty space for
particles to move through.

With increase in temperature, the particles
vibrate faster and more strongly as they
gain kinetic energy.
Cytoplasm : watery, gel-like material in which cell parts move
and cell activities take place
Thus,
Diffusion is faster in gases
than liquids where there is
more space for them to move
and diffusion is negligible in
solids due to the close packing
of the particles.
through a membrane
Silver nitrate will react to sodium chloride to
form a white precipitate (silver chloride)

Nitric acid will coagulate the protein albumin



OSMOSIS
the movement of water from an
area of higher water concentration,
across a selectively permeable
membrane, to an area of lower
water concentration.
from an area of lower solute concentration to
an area of higher solute concentration.
Movement is always toward equilibrium.
Dilution is toward equilibrium.
raisins absorb water and swell up

water has very little solute concentration than
the raisin, so osmosis occurs, water enters the
raisin, and raisin swells up

(It is also called end osmosis or deplasmolysis)
Dialysis
the movement of a non-water particle
across a barrier.
This movement is based on the size of
the particle in relation to the size of the
pores within the barrier.
In the living system this barrier is the
cell membrane.

Observe osmosis with
a thistle tube. This
device consists of a
hollow bell attached to
a long tube. The tube
is filled with sugar
solution, and the large
opening of the tube is
covered with dialysis
tubing and secured
with a rubber band.
The thistle tube is
placed in a beaker of
water and clamped to
a ring stand.
Tonicity
Movement of water in and out of cells can change the
shape or tone of cells.
a. Isotonic solution concentration of solution
inside and outside of the cells is the same; the
same amount of water moves in/out of the cells/
shape of cells remain unchanged
b. Hypertonic solution cells placed in solution with
a higher concentration than solution inside cells;
water moves via osmosis from the cells; cell
crenate ( shrink)
c. Hypotonic solution cells are placed in a
solution with a lower concentration than solution
inside cells; water moves via osmosis into the cells
cells swell and eventually burst
cell retain its normal size and shape since the
concentration outside the cell is same solute/water
concentrations as inside the cell
Isotonic solution
the cell take in water by osmosis because it
contains no solutes, water continues to enter
the cell until they finally burst or lyse.
Hypotonic solution
the cell loses water and shrink (crenate) since the 25%
saline contains more solutes than what is present inside
the cell
Hypertonic solution
What determines the osmotic
pressure of any given solution?
Vant Hoff 's Laws of Osmotic Pressure

Vant Hoff Boyles Law
Osmotic pressure (p) of a solution at a constant
temperature is directly proportional to its
concentration (C)

Vant Hoff Charles Law
For a solution of fixed concentration, the osmotic
pressure (p) of a solution is directly proportional to
its absolute temperature (T).

5% glucose solution is isotonic to
cellular fluid, but a 5% saline
solution is hypertonic.
D5W is unique in that it may be
categorized as both an isotonic and a
hypotonic solution. The amount of
dextrose in this solution makes its initial
tonicity similar to that of intravascular fluid,
making it an isotonic solution. But
dextrose (in this concentration) is rapidly
metabolized by the body, leaving no
osmotically active particles in the plasma.
Active Processes
Energy (ATP) is required for the movement of
substances across the plasma membrane
2 types: Active transport and Vesicular
transport
Active transport movement of solutes/ions from
area of lower solute concentration to area of higher
solute concentration (against a concentration
gradient); mediated by carrier proteins. Active
transport exhibits saturation and specificity.
Ex. sodium/potassium pump ( Na+/K+ pump)
Hence, active transport is also known as
Solute Pumping
Figure 3.10: Operation of the sodium-potassium pump, an antiport
pump (Na
+
K
+
ATPase), p. 76.
Cytoplasm
Extracellular fluid
K
+
is released
and Na
+
sites
are ready to
bind Na
+

again; the
cycle repeats.
Cell ADP
Phosphorylation
causes the
protein to
change its shape.
Concentration
The shape change
expels Na
+
to the
outside, and
extracellular K
+
binds.
Loss of phosphate
restores the original
conformation of the
pump protein.
K
+
binding triggers
release of the
phosphate group.
Binding of cytoplasmic
Na
+
to the pump protein
stimulates phosphorylation
by ATP.
Na
+
Na
+
Na
+
K
+
K
+
Na
+
Na
+
K
+
K
+
K
+
K
+
Na
+
Na
+
Na
+
ATP
P
P
Na
+
Na
+
Na
+
K
+
K
+
P
P
i
1
2
3
4
5
6

symport antiport
ATP ATP
Active Process Vesicular Transport
2 types Exocytosis and Endocytosis

Exocytosis movement of substances
enclosed in vesicles from the interior of cells
to the exterior.
Hormones, enzymes are secreted via
exocytosis

Endocytosis movement of substances
from the exterior of cells to the interior
Exocytosis, p. 78.
Extracellular
fluid
Cytoplasm
Molecules to
be secreted
Vesicle
SNARE
Plasma membrane
SNARE
Secretory vesicle
(a)
(b)
Movement of substances
from the interior of the cell
to its exterior
Clathrin-mediated endocytosis, p. 79.
Recycling of
membrane and
receptors (if present)
to plasma membrane
Cytoplasm
Extracellular
fluid
Extracellular
fluid
Plasma
membrane
Detachment
of clathrin-
coated
vesicle
Clathrin-
coated
vesicle
Uncoating
Uncoated
vesicle
Uncoated
vesicle
fusing with
endosome
To lysosome
for digestion
and release
of contents
Transcytosis
Endosome
Exocytosis
of vesicle
contents
Clathrin-
coated
pit
Plasma
membrane
Ingested
substance
Clathrin
protein
(a) Clathrin-mediated endocytosis
Endocytosis -
Movement of
substances
from the
exterior of the
cell to its
interior
Endocytosis
Movement of substances from the exterior of a cell into the
cells interior.
3 types:
i) Phagocytosis movement of solid particles from the
exterior into the cell; solid particles are enclosed in vesicles
called PHAGOSOMES
ii) Pinocytosis movement of solution into cells by
enclosing the solution in vesicles
iii) Receptor-mediated endocytosis substances bind to
specific receptors on the surface of the cell; clathrin appear
where the substances are bound; coated pits form to
transport substances into the cell.
Receptor-mediated endocytosis exhibits saturation and
specificity
Figure 3.13: Clathrin-mediated endocytosis, p. 79.
Recycling of
membrane and
receptors (if present)
to plasma membrane
Cytoplasm
Extracellular
fluid
Extracellular
fluid
Plasma
membrane
Detachment
of clathrin-
coated
vesicle
Clathrin-
coated
vesicle
Uncoating
Uncoated
vesicle
Uncoated
vesicle
fusing with
endosome
To lysosome
for digestion
and release
of contents
Transcytosis
Endosome
Exocytosis
of vesicle
contents
Clathrin-
coated
pit
Plasma
membrane
Ingested
substance
Clathrin
protein
(c) Receptor-mediated endocytosis
Extracellular
fluid
Cytoplasm
Bacterium
or other
particle
Pseudopod
Clathrin
protein
(b) Phagocytosis
Clathrin
protein
Membrane
receptor
(a) Clathrin-mediated endocytosis
1
3
2
c) Pinocytosis movement of solution into cells
Transcytosis and Vesicular trafficking
Trancytosis: Movements
of substances enclosed in
caveolae into a cell, across
the cell and released on the
opposite side of the cell via
exocytosis

Vesicular Trafficking:
Intracellular movement of
substances in coatmer-
coated vesicles from
organelle to organelle with
the cell
The role of K
+
in generating the resting membrane
potential, p. 82.
Potassium
leakage
channel
Protein anion
Cytoplasm
K
+
diffuse down their steep
concentration gradient (out
of the cell) via leakage
channels. Loss of K
+
results
in a negative charge on the
inner plasma membrane
face.
A negative membrane potential
is established when the movement
of K
+
out of the cell equals K
+
movement into the cell.
K
+
also move into the cell
because they are attracted
to the negative charge
established on the inner
plasma membrane face.
+







+
+
+ +
+
+
+
Na
+
Na
+
K
+
K
+
K
+
K
+
K
+
K
+
K
+
K
+
K
+
K
+
K
+
K
+
A

A

K
+
K
+
K
+
Cl

Cl

1 2
The Resting Membrane Potential
(RMP)
RMP of resting cells is between -50mV to -100mV
RMP is established by the partial/selective permeability of
the plasma membrane to potassium ion (K+) diffusion over
sodium ion( Na+) diffusion.
K+ concentration is higher inside the cell than outside the
cell K+ diffuses out of the cell down its electrochemical
gradient
Na+ concentration is higher outside of the cell than inside
the cell Na+ diffuses into the cell down its
electrochemical gradient
The plasma membrane is about 75 times more
permeable to K+ than Na+ = more K+ diffuses out of cell
than Na+ diffusing into the cell = more positive ions
move out of the cell making the cytoplasmic surface of
the plasma membrane negative compared to the
extracellular surface