S

Biochem Lab
We share roughly 90 % of our
we
Activity 1 Recitation Discussion genomic content

yeast cells
... What makes saccharomyces cerevisiat cells
as an excellent model for us to observe what as why not directly extract our own blood ?

we need to observe .
I t is unethical ,
we shouldn't be subjected to biological material

unless otherwise the experiment needs it .

But if there is an alternative ,

· Because :

we will go for that .

They have the capability to metabolize

-
as well
> We are exposed to risks : Blood-borne diseases -

Hepa Th , HIV
,

as well as transport .

potentially hazardous
·
It is capable of undergoing the same processes that Biosafety
safety concerns .

almost all cells particularly cells

we see on ,
man .

>Aside from the things that was mentioned

·
they manifest cell transport processes ,
such as Diffusion ,

since we share genetic similarity with yeast cells,

·
It is eukaryotic/unicellular .
Eukaryotic who is our type of cell
we also share the same composition :
nucleus ,
mitochondria etc .

and has the ability to reproduce through a sexual repro .

Majority would be vacuoles .

It shows us the capacity of cell to "pro-create" a sexually or sexually > the composition of typical cell the lowest level limage) table
Analyzing at ,
.

via certain processes (mitosis/ meiosis) 04 . What is depicted /What makes up cells

.
It's availability Financially-wise as compared to human cells .

Red major elemental components

:

of the cell are made up of these

Easy maintenance
.
.

atoms
Followed by the Blue ones
It can survive and has the capacity

to undergo cellular physto logic processes

o . What do you think would be the reason why

human cell has the similarity w/ Yeast cells

the that they

in terms of function :
processes
undergo What .
is present in these
05 What makes these atoms different from each other What makes
.

,
diff .

cells that made them act in the same way

from nitrogen ,
0 CH ?
Because (eukaryotic)
>Presence of Nucleus -> PNA2 genetic materials Chemical level organization of the cell , we have atoms
-

lidentity ->
integral proteins /aquaporins) Qo. What do you think will be responsible for the diff
-

·All functionalities are governed by genes present in both characteristic of Catoms as compared to that of

Yeast and Human cells /but not every) patums

gene Hexokinase that

glucose and make glucose

allows the yeast cell to

G-phospate
break down

But
> No .

specifically
of Protons

in
,

subatomic
elections and neutrons

level , which among those mentioned

-
Metabolism
that's the same enzyme for
humans
Glycolisis
are responsible for the Activity/ characteristic of the atom ?
Ast enzymes
They have gene coding for cell walls -> nexokinase >
Protons (6+1 becomes N +
1 +
0

Iyeast a human)
mulapi- Electrons
mulasik -

Protons Dipole -

Dipole moments (charges on molecule residues)

Protons tells us the identity of the Atom ,
ex .

enzyme action
e

Carbon should always have G atoms if ,

we change substrate enzyme interaction

that by 1 it's Nitrogen If reduced it's a Boron .

antigen-antibody interaction (neutralize)

,

Elections can change but it does not change the identity of Why is this crucial ?
-

the atom ,
it simply changes it into its cationic or anionic > It allows polymerization

form . Meaning it simply lost

,
or gain its electron but not its -facilitated by interactions that are intermolecular

identity .
It also helps in forming a larger molecule .

forces of attraction blw small units forming

larger units .

Assembly of Atoms to
form large molecules .

Q .
Discuss whats in the slide .

&= (Image) major (2) types of Intramolecular forces of attraction .

>molecules via intramolecular FA molecules

electronegativity difference .

interacting to form a larger molecule wic may

>Part that lost the electrons has tions or may not be building blocks of the biomolecules ↑

0 <Protons
e-
and the one that received has -ions .

monosaccharides -

made up of hydroxyl group &H oneutrons

The force that will make the electron to share

<Fatty acids E
in
or
fully transfer one of the ious will be difference of ↳ Cholesterol

electronegativity (image) one of the ions or atoms

cyclopentano perhydropenathrane
-
.

-
A
would have very strong pull towards a particular electron
complex structure

so the electrons will be forced to transfer to covalence 5 carbon atoms , Yet-

con

of the acceptor ion leaving ion ↳Hydrogen

area ,
one participating
to totally lose the electron .
> Amino acids

electronegative diff (1 7 the electron will not totally ↳ glycine

.

more over the next shell .

It will get stuck midway blu 2 FG -

RCH :carbs e

participating atoms - ↳ capable of accommodating in its protonated

form
No difference
-

non-polar covalent bonding ↳ NAy earn a positive charge

>diff but not great pull-polar covalent bond ,

one
of the e- Neuclotides

will have a partial dipole positive charge , the other ↳ Adenine

Purine
partial negative dipole moment -
Intramolecular FA ↳ FG >

08 :

Intermolecular forces of Attraction of 2 or more >Biomolecules

· One of the atums must have a partial charge .

-They interact to form components of the cell

O is more electronegative than A

>Lipid bilayer
↓ N cell walls
exhibits partial
-

glycolipids -

partially -
proteins -

londy water chanels

negative charge positive sodium
-
can electrostatically -transporter
proteins
attract
<solute particles)
 nucleus How does cholesterol aid in maintaining fluidity of

>chromosomes the cell membrane

>Histone proteins
>Affect the capacity of the cell to remain

static
,
because of its structure and placement

chemical composition of cells in between the Hydrophobic Cy hydrophilic

(PicS > It also impacts the permeability
allowing entry of larger molecules
-
loosen up ,

-or v V
.

molecules
Generally , non-polar readily permeate

be
,
the membrane itself is made up of layer

Hydrophobic/philic .

Diffuse .

permeases
I
transporters , channels
-

<- Polar permeation

of materials

Facilitate Flow of materials into and out

of the cell

what forces the pic above to confirm the arrangement

making it a bilayer
> The Hydrophobicity of the fails can force the tail ends

of the bilayer to come

together .

What makes it fluid ?

< is dy trans isomers

unsaturated double bonds kinks

:

-
->
-

↳capable of molecular stacking

giving the molecules the capacity to more along

> the more stacking fluid

+ other component : Cholesterol

how does it maintain membrane fluidity

 Diffusion (Review) molecule transport
-

Representation (pics) chanels

muscle contraction

Difference between the amount of solutes ↑ concentration

,
reside

to the lower concentration order to

and movement in
synaptic
reach equilibrium .

Ion calcium

ligand-gated
movement of solutes ion transport system

↳ solute concentration to attain equilibrium >resicles a micelles

2 layer I layer
, applicable if molecules are
non-electrolytes - -

non-polar Do
or .
not carry charge nor dipole moments

But in the presence of formal charges of these molecules <non-polar compounds can easily pass through the cell

or
presence of electrolytes as solutes/ionic solutes
,
it will membrane through simple diffusion .

not be enough to attain equilibrium/equal distribution of

the concentration .
We have to consider their charges > most common molecules that is transported
-> concentration gradient G driving forces of diffusion glucose -

glucose transporters
I has to be transported
on all parts)
-
electrical gradient out of the cells
-

glycolysis -

phosphorylation
Predominant charge (Imbalance)
-

I amino acids -
distribution to blood stream
-
GLUTC (small intestines)

↳ attain equi via neutralization of +h-until there > lysosomes requires active entry of protons into the

is an equal no .

of
t
ly-charges on each side lysosomal lumen (digest enzymes) these enzymes

require ↓ PH (acidic)

pyruvate dative transport chain

-

>

trying to permeate is polar/large

mitochondria
IF the molecule
it needs a gateway/ something should facilitate its water-transporter aquaporins
-

movement (Facilitative diffusion/Passive transport) Where does reabsorption of Fluids takes place ?

AtP How do you get proximal renal tubules

Active requires energy From Atp ?
- -

> metabolism
-

lungs to maintain liquid in our lungs

>Hydrolysis of Al -
releases Free gibbs energy - bond
Nalk pume

Formation From Atp+Phosphate

transformation of chemical energy into stored potential

energy in the form of phosphodiesther bond

 Osmosis

a major forces

to be exerted onto the osmotic system

I amount of pressure

by the cell

2 :

Direct relationship

↑ concentrate osmotic or v V
.

to offset

> tonicity
-

Heating in its liquid form -

vapor pressure

↑conc . ↑ U .

p .

gas diffusion

> non-electrolyte solutes

-

commentration & electrical factor

> semi-permeable membrane ,

movement of

molecules imparts osmotic force

I lose integrity

extracellular
Hypertonic which domain has solute = .

a
-

is a solute and
water will follow where there

get out -> age

Hypotonic-

Rich
-
 PIPETTING TECHNIQUE THE PIPETTING CYCLE (FORWARD PIPETTING)

Accuracy Precision
 Accuracy is the  Precision is the
-
ability of a pipette amount by which the numerical assessment
toprovide adispersee mean value of a large of the variability of
as indicated .

set of replicate liquid several replicate

measurements measurements,
deviates from the expressed as standard
nominal volume deviation or coefficient
shown on the display Typical
of variation.
specification
>11 , of the accuracy
of the pipette. 4
-
-

specification
10% and below
↑ 35 settings at
I
can be as
the specification
much as 3x less accurate

Steps
causeu al
,
career
1.
2.
Adjust Volume I feet away from you
Put the tip on the tip of the pipette
BASIC TECHNIQUES FOR MINIMIZING ERRORS 3. Press the first stop
 Optimizing volume range 4. Immerse/Put to 2-3 millimeters
 Setting micrometer 5. Slowly release the plunger
 Tip immersion angle 6. Support the plunger while aspirating (it should
pipetting to failed experiments errors lead

 Tip immersion depth -underestimated have no air bubbles on the tip).

titer leads to over-inoculation
-

I both failed
7. Position it above the vessel

-

Tip immersion time -overestimated

-
fiter leads to under-inoculation

 Aspiration rate 8. Press until the first stop

9. Press again until the second stop (blow-out)
 Dispensing technique
10. Eject the tip to the waste vessel.
 Pre-rinsing pipette techniques
 Hand-warming effects 0 5 %
Note:
.

Turn micrometer 1/3 revolution

 Correct pipette technology for challenging liquids
 Variable volume pipettes above desired the volume

Errors from poor techniques can range from 0.1%-5% or - This pipette comes with a specific minimum and
more. maximum volume range. The volume of the liquid
to be aspirated or dispensed can be adjusted
(within the instrument's volume range) depending
PARTS OF PIPETTE on the user's requirement.
 Fixed volume pipettes
- This pipette is where the volume of liquid
aspirated or dispensed remains fixed. These
micropipettes are used when the same fluid
volume is dispensed multiple times.
 Clockwise – decrease
 Counterclockwise – increase
 You can reuse the tip a maximum of ten (10) times or
reuse it only if measuring the same solution but with
a different volume.
 There is a pointer where it should be centrally
positioned.

1|Page|CAYABYAB
 Keep the pipette vertical
 Avoid hand-warming
 Microliters
APPROPRIATE VOLUME
Specified volume range
 10-100% of volume
Recommended volume
 Typically 35%-100% of volume
 Below 35% is highly technique-dependent.

Even without splashing, inconsistent aspiration can affect

accuracy by 1-5%

Pipetting down to 10% can affect accuracy by more than
3%.
TIP IMMERSION ANGLE
 When aspirating liquid, try to keep it vertical, and the
inclination angle should not exceed 20°.
PAUSE TIME FOR MACRO-VOLUME PIPETTES
 Wait a minimum of 1 second with the tip immersed
after aspiration.
 Withdraw the tip slowly and smoothly from the liquid
 Important for large-volume samples and vicious
samples

PRE-RINSING PIPETTE TIPS

 Aspiration
sample into the
tip then
dispenses back
into reservoir or
waste.
Correct pipetting angle enhances performance by up to
2.5% microvolume pipettes  Pre-rinsing
provides
identical contact
TIP IMMERSION DEPTH surfaces for all
aliquots.
 Pre-rinse the tip
with the same
liquid that is
being dispensed.

Two pre-rinses provide up to 0.2% greater accuracy with

aqueous liquids.
ASPIRATION RATE EFFECTS
Aspirating too quickly:
 Liquid splash-up into shaft damaging piston and deal.
 Introduces aerosols and sample cross-contamination

2|Page|CAYABYAB
CONSISTENCY REVERSE PIPETTING
Use consistent
 Pipetting rhythm
 Pressure on plunger
 Speed and smoothness
For best consistency
 Use an electronic pipette
- Provides optimum consistency
- Requires less user technique approve accuracy
-

AIR DISPLACEMENT PROS AND CONS

PROS CONS  Reverse pipetting begins by depressing the
 Accurate  More training is plunger all the way down to the second stop,
with needed for thus including an extra “residual” volume in
aqueous accuracy and the aspiration cycle, which is not included in
solution at precision, the final dispense.
standard lab particularly at  Reverse pipetting was developed for work with
temperature low volumes blood and other dense/viscous material.
and pressure  The pipette can  Every researcher should determine empirically
 Manual and be damaged or whether reverse pipetting provides better
electronic contaminated if results for their particular liquid.
pipettes the sample
 The contacts the SUMMARY OF PIPETTING ERRORS
pipetting piston
technique  Inaccurate when:
can minimize - Sample and
inaccuracy lab
 Disposable temperatures
tips are less differ
expensive - Sample
 Lower viscosity or
ergonomic density varies
forces from water
 Multichannel - Sample
options volatility is
high
- Humidity is
low/vapor
pressure is
high.

3|Page|CAYABYAB
 pipetting Errors Lead to Failed Experiments ! The pipetting Cycle (Forward pipetting)
Goal : Infect mice with correct dose ((150) ·

Tip must be secured

Critical issue is not to over- or under-infect mice .

Depress the plunger

virus stocks must be titered using serial dilution Ad Hold

rjection
·
.

pipetting errors accumulate Aspirate ·

Aspirate
Under-estimated titer leads to over-inoculation .
Dispense and Blow out
Dispense &
fiter Tip
·
Over-estimated leads to under-inoculation .

Blowout
·

Ejection
Accuracy Us . Precision

-
-
Accuracy Appropriate Volum

the amount by which the mean value of a large set of Specified Volum Range
replicate liquid measurements deviates from the nominal 10-100 % of volume

volume shown on the display of the pipette .

Recommended Volume

mision ·

Typically 35-100 % of volum

the numerical assessment of the

variability of a number ·Below 35 % is
highly technique -

dependent
of replicate measurements -

expressed as standard deviation Setting the Micrometer

or coefficient of variation . Turn micrometer Its revolution above desired volume

Basic Techniques for minimizing errors Dial down to volum setting clock wise
-

· Optimizing volum range ·Aspirasion rate ·

Approach each volum in the sam direction each fir .

setting the micrometer ·

Dispensing techniques Aspiration rate effects

Tip immersion angle Pre-rinsing pipette tips Aspiration too quickly :

Tip immersion depth ·

Hand-warming effects Liquid splash-up into shaft damaging piston and seal

Tip immersion time correct pipette technology for Introduces aerosols and sample-cross contamination

challenging liquids . Eg .
Bubbles

⑰ Errors from poor technique can range from 0 1 .

% -
5% or more .
 pause tim for Macro-volume pipettes ⑪ ⑧
Avoid Hand Warming
wait a minimum of I second with tip immered
Air displacement pros and cons

after aspiration PROS CONS

withdraw tip slowly and smoothy from liquid ~ Accurate with ageous solutions X More training medeo

Important for large volum samples and viscous samples .

at standard lab
temp/pressure for accuracy and precision
~ particularly at low volumes
Dispensing Techniques Manual and electronic pipettes

Thre techniques : ~ pipatting technique can minimize X Pipette can be

damaged or

contaminated if sample
·Along side-wall (touch off) accuracy

·Above liquid surface ~ Disposable tips are less expensive contacts piston .

V X Inaccurate when :
·
Into liquid Cour ergonomic forces

considerations ~ Multichannel options -sample and lab

key :

Pause before blowout with viscous liquids temperatures differ

If dispensing in liquid , keep piston at second stop until What is reverse pipetting ? -
Sample viscosity or

tip is removed to prevent unintentional aspiration .

density differs from water

Dispense
Pre-rinsing pipette tips
Tip ·

and -sample volatility is high

Ejection
Blawan
Aspirate sample into tip , and then dispense back into reservoir
-
Humidity lowl vapor

or to waste Blowondual old

pressure is High

pr-rinsing provides identical contact surfaces for all aliquots

Mistsantere e Aspirate
ample
pre-vince tip with sam liquid that is being dispensed .

by
Maintaining Consistency Reverse P .

begins depressing plunger all the way

Use Consistent For best consistency down to the 2nd Stop thus including an extra
- -

pipetting rhythm -
use an electronic pipette residual volume" which is not included in the

-pressure or
plunger ·

provides optimum consistency final dispension .

requires less user technique It is developed for work wy blood and other
-speed and smoothness
·

dense viscous material .

 I ↳mi /
Saccharomyces cerevisiae
rearranging of cells-tails hydrophobic -
a bilayer of membrane

Baker's teast head hydrophilicity

Wht yeast cells instead of human cells

?
presence of kink-unsaturation

Hal Aspect cholesterol -

fluidity

within
Reproduction/life cycle hyper compressibility
stal-
is continuous hours of immers on

Budding concentration gradient - down ↓

-
Fermentation Electrochemical gradient -
charge of molecules

↳stic( organisms Net charge should be zero (0)

I
-

I
Mouse Genous (Xenophous) Nucleotid / Sugar Transporters

Bacterium (E . Coli) Musculus Transporters -

Enzymes Na , K permease

cerevie ee Aquaporins facilitate massive water through cells

accharomyc
Chimpanzee -
movement of amounts of

mars
Lam
renal tubule-water re-absorption
(
- I
impacts #20 movement on kidneys
-

cell division
Westine
Absorption site -
z

DNA- gene expression Transportation between difusion and Osmosis

Simulation of biochemical studies vapor pressure lowering

-

colligative property

Law
90 % -

CHON Carbon Hydrogen Oxygen Nitrogen -Raoult's

Hydride L Solvent above solution is equal to the mole fraction of solvent in the

Carbide soln times the vapor pressure of pure solvent .

weak non-covalent interaction Vant Hoff Factor

small organic building blocks large organic molecules

Exposing cells to high concentration of solution

sugars polysaccharides , glycogen , starch (plants) New Osmotic theory Dialysis: Graham's law

Colloidal

w
fatty acids fats and membrane lipids Implications to living cells Molecules

amino acids proteins isotonicity -

Equilibrium /Balance
pumping of
inorganic ions
-

hypertonicity -

Shrink
-
Expulsion of water

fatty Acids nucleic acids

of inorganic ious hypotonicity - Swell
-Importation
 1 .
A 20 2 -
Dye 3 7 2 3

A O 200 A O 200 -

B 10 190 B 20 -

C 20 180 C48 -

D 30 170 D 60 -

E 40 160 Ego u

F 50 150 F 100

G 60 140 G 120

H 70 130 H 140

ML
 AN INTRODUCTION TO BIOCHEMISTRY
SEPTEMBER 2023| PRELIMS
BIOCHEM LAB

POST-LAB ACTIVITY 01 1. Nuclear Pore

● form the natural channels for exchange of components
INFERRING THE CHARACTERISTICS OF IN VIVO SYSTEMS
between the nucleus and cytosol, whereby export and
import pathways can be distinguished.
I. Major components of Saccharomyces cerevisiae ● Regulates what comes in and out of cell
● Major Components and Organelles
A. Cell Envelope 2. Nucleolus
B. Cytoplasm and Cytoskeleton ● locates the rRNA genes, and is the site for the synthesis and
C. Nucleus processing of rRNA.
D. Organelle Compartment ● It is also involved in the assembly of the ribosomal subunits
and in pre-mRNA processing
A. Cell Envelope
● Comprises 15% of the cell 3. Chromosomes
● Primary contribution : permeability and osmolarity ● genetic element
● Cytoplasm : characterized as acidic 5.2 uph; cellular
activity is observed D. Organelle Compartment
● Cytoskeleton : mechanical barrier w/c provides structure of ● Modification, stability, and transport of proteins and then ready to be
the cell composition; involved in movements of mitosis and transported to golgi apparatus
meiosis process; mobility, stability ○ Golgi apparatus - the primary process of rRNA/ protein
● Nucleus : all about primary activity; genetic material synthesis
houses chromosomes 1. Endoplasmic Reticulum
● Organelle compartment (vacuoles, peroxisomes, ER, GA): ● key organelle for all processes controlling the stability,
purpose is for modification and trafficking of proteins modification, and transport of proteins.
1. Cell Wall
a. Initial Characteristics 2. Golgi Apparatus
● yeast cell aggregation - asexual reproduction process ● processing and sorting events include synthesis and
● Agglutination - due to sexual reproduction process processing of complex biomolecules.
● Clumping - due to specific proteins, glycoprotein turned
into flucillin responsible for adhesion to one yeast cell to 3. Vacuoles
another ● involved in intracellular protein trafficking and nonspecific
intracellular proteolysis
2. Plasma membrane ● vacuole is a “drain.”
The primary functions of the yeast plasma membrane are:
● Physical protection of the cell. Functions of Vacuoles;
● Control of osmotic stability. ● storage compartments
● Control of cell wall biosynthesis. ● Osmoregulation, cell structure
● Anchor for cytoskeletal compounds. ● homeostatic regulation of cytosolic ion concentration and
● Selective permeability barrier controlling compounds that enter or that pH
leave the cell. Of prime importance in active transport of solutes is the
activity of the plasma membrane proton-pumping ATPase 4. Mitochondria
● Transport-related functions in endocytosis and exocytosis. ● respiratory-competent organelles
● Location of the components of signal transduction pathways. ● will adopt different morphologies, depending on the
● Sites of cell-cell recognition and cell-cell adhesion conditions.
3. Periplasmic space ● Metabolism process - glycolysis to fermentation
● comprises mainly secreted proteins that are unable to ● Powerhouse of the cell - production of ATP and
permeate the cell wall, such as invertase & phosphatase fermentation
(responsible for interaction; catabolize the substrates that 5. Peroxisomes
are trying to permeate the cell) ● also called microbodies
B. Cytoplasm and Cytoskeleton ● Catalytic enzymes for observations of metabolic process
and participating in regulation of cell structure
1. Microtubules ● endowed with several metabolic functions that are of
● are conserved cytoskeletal elements outstanding importance for cell viability

2. Actin
● direct polarized cell growth and to segregate organelles
prior to cell division.

3. Motor Proteins
● provide the energy necessary for motility.

● Myosin & Kinesins

○ are proteins that are able to bind to polarized cytoskeletal
filaments and use the energy derived from repeated cycles
of ATP hydrolysis to move along them.
● Dynein
○ largest motor protein active in the movement of the mitotic
spindle that must move into the narrow neck between the
mother cell and the bud in order to segregate duplicated
chromosomes accurately

C. Nucleus
Page 1 of 8
BIOCHEMISTRY LABORATORY - Ma’am Julie De Guzman

III. Fermentation
● Mechanism of Fermentation
○ Saccharomyces cerevisiae kinuha si glucose undergoes
glycolysis. Glucose to pyruvate to acetaldehyde to by
products, ethanol, carbon dioxide (cause inflation)
■ Presences of real oxygen - will have different by
products, it can use oxygen to produce CO2 and
H2O

○ “Pasteur effect”
○ The ATP yield from glycolysis under anaerobic conditions
(2 ATP per molecule of glucose) is much smaller than that
from the complete oxidation of glucose to CO2 under
aerobic conditions (30 or 32 ATP per glucose)

II. Characteristics of Cells

● Suspension A - it has almost 2 layers of cell well (cell envelope and
cell membrane)
● Suspension B - temperature destroy the membrane/ cytoskeleton due
○ Increasing tempt- lumuluwag and can’t be regulated by
cholesterol, mabubuwag and;
○ Destruction of enzyme protein kaya walang clear structure
● After staining : alive still has regulating enzyme because it reduces the
stain
● Methylene Blue
○ cationic stain - basic pH; cell structure is acidic
■ So there’s attraction
○ Determine cell mortality
○ Light blue - Reduction reaction due to removal of hydrogen
and went not P found in cell
○ Methylene blue interact to cell tries to permeate, enzyme
will take in action (lactate dehydrogenase)
Uses of Methylene Blue *viability of the cell is its target IV. Osmosis
1. For determining cell damage ● Osmosis - all about movement of solvent
-

2. Identification of Microorganisms - some of them are highly acidic ● Diffusion- all about movement of solute
-

3. Identifying Nucleic Acids ● Concept of Osmosis

4. Identifying RNA Sequences ○ The passage of solvent through a semipermeable membrane
5. Calculating viable cells in yeast sample separating a dilute solution from a more concentrated
6. Identifying distinctions between bacterial, viral and fungal diseases solution
7. As diagnostic agent ● Osmotic Pressure
○ must be applied to prevent the net flow of solvent through a
semipermeable membrane from a lower concentration to a
higher concentration to still attain equilibrium
○ Colligative properties - movements of solvents
● Thermodynamic drive
○ Provide equilibrium for water molecules for concentration
to what comes in and out of the membrane
○ There is a thermodynamic drive for water molecules to
achieve an equivalent concentration across a permeable
membrane

Page 2 of 8
BIOCHEMISTRY LABORATORY - Ma’am Julie De Guzman
○ Net Effect: Reduction of entropy - when heat is actually
removed, water molecules entropy decrease ; when added
interaction increases entropy
● Raoult’s Law
○ All about concentration of vapor pressure (property:
colligative)
○ States that a solvent’s vapor pressure in a solution (or
mixture) is equal or identical to the vapor pressure of the
pure solvent multiplied by its mole fraction in the solution
○ related to vapor pressure estimation
○ Solute-blocking provides a kinetic explanation for why
Raoult’s Law and the other colligative properties depend on
the mole fraction (not the size) of the solute particles
○ Note: Raoult’s Law estimates vapor pressure lowering
Note: Aquaporins and its Roles and/or Location
V. Law of Osmotic Pressure
● Van’t Hoff’s Theory of Osmotic Pressure
○ a substance in solution behaves exactly like a gas, and the
osmotic pressure of a dilute solution is equal to the pressure
which the solute would exert if it were a gas at the same
temperature occupying the same volume as the solution
● New Osmotic Pressure Formula
○ Osmotic Force Equilibrium Formula
○ atmospheric pressure ∙ osmotic effective area on the solvent
side = (atmospheric pressure + osmotic pressure) ∙ osmotic
effective area on the solution side
○ osmotic pressure as π
○ atmospheric pressure as p
○ molar concentration of solution as [Ci ]
○ osmotic effective area on the solution 1 − k[Ci ])
○ temperature at melting point of solvent T/ T0
VI. Solute Movement across Cell Membranes
● Mechanism 1: Non-specific Permeation (Diffusion)
● Mechanism 2: Protein-mediated Transport
○ Facilitated diffusion
○ Active Transport
● Aquaporins
○ Doesn’t need ATP
Page 3 of 8
○ Ion dipole & hydrogen bonding
○ “Water channels”
○ Description:
■ Small
■ Very hydrophobic
■ Intrinsic membrane
■ Proteins on the cell membrane
○ Function:
■ Water permeability and exclude the passage of
other solutes
■ Transcellular water transport
○ Aqua-glyceroporins can also conduct glycerol, carbon
dioxide, ammonia, and urea across the membrane
○ In a span of seconds, the possible changes are observed in
the cell, attributed to transport mechanism
■ The transport mechanism, such as aquaporins, is
a protein membrane
■ It does not need ATP. It does not need energy.
■ Aquaporins is one of the factors that helped in
fast uptake in cell structure
○ Aquaporins are in different parts/organ that is actually
distributed throughout the body
○ Alka-helical proteins help in stability of the structure and its
regulation, but most importantly, the middle structure has
positive charge
○ It contains the positive charge, so that whatever water
molecule will pass through, they will not be aggregated or
uniform
○ It should gatekeep the needed molecules to not release
them, it should be in order and maintained concentration of
the molecules, and lets the water molecules to go
inside-outside vice versa
● Glucose Transporter (GLUT)
○ In glycolysis process, we were able to observe that the
fermentation reaction process
○ In GLUT, there are two mechanisms in which we are able to
observed, all of which is influences insulin
○ Insulin
○ Glucose could also be transported hand in hand together
with sodium-potassium
○ We have sodium-glucose transporter, similar to the
transport mechanism of sodium-potassium pump but with
the presence of glucose
● Sodium-Potassium ATPase
○ For you to observe the sodium-potassium pump, what do
you need to activate the transporter? Remove phosphate
group in ATP, because the phosphate group will bound in
the transport membrane, thus it will allow the uptake of the
3 molecules of sodium
○ Upon the uptake of sodium, the potassium will enter. When
the phosphate group is released, then potassium will also be
released.
VII. Dialysis
● Concept of Dialysis
○ process in which a semipermeable membrane allows the
passage of solvent, dissolved ions and small molecules but
BIOCHEMISTRY LABORATORY - Ma’am Julie De Guzman
blocks the passage of colloidal- sized particles and large ○ Sucrose is disaccharides, made up of glucose and fructose
molecules ○ If sucrose is subjected to heat, the positive result is
○ Process which separates molecules based on diffusion caramelization
○ A semipermeable membrane allows the movement of ● Monosaccharides, glucose and
certain molecules based on size fructose
○ Applications: desalting, removal of buffer from a ● Three byproducts: acetic acid, furan,
biomacromolecule (e.g. protein) and maltol
○ Dialysis is all about the process of separating the molecules ■ The degradation of the sucrose that is actually
based on the idea of diffusion, it is actually losing the will observe glycosidic bonding, and further
semipermeable membrane for you to be able to determine degradation will result to burnt smell
the specific molecules will pass through
○ Physiologic application: hemodialysis with kidney failure
patients

● Expected Results
○ Table salt solution = POSITIVE
○ Starch solution = NEGATIVE
○ Sugar solution = POSITIVE
○ NaOH = pH change
● The results are governed by means of the molecular size
VIII. Le Chatelier’s Principle
● In terms of molecular size, Salt ions and small molecules such as
● Not always applicable in vivo processes. Take consideration of the
glucose are able to pass through the semipermeable membrane.
limitation in applying the principle.
Molecules of starch are too big to pass through. Because it is made up
● This principle focuses on three ways in which we can change the
of monomers of glucose
conditions of a chemical reaction at equilibrium:
● The basis in dialysis is colloidal particles.
● (1) changing the concentration of one of the components of the reaction
● (2) changing the pressure on the system
● (3) changing the temperature at which the reaction is run.
● Determine: shifting
○ Provide equilibrium para if there are instances na increase
or decrease temperature, the body will shift towards or
away from the factor involved
● Example: homeostasis

● Balanced Equation
○ The helical structure of Amylose forms a charge transfer
(CT) complex with iodine, wherein iodine is present inside
the spiral or helical structure of the Amylose.
○ In the charge transfer complex of polyiodide ions and
Amylose, electrons absorb light energy and get excited to a
higher energy level.
○ The complementary color to the light energy absorbed by IX. Diffusion
the charge transfer complex is perceived as a blue-black ● Movement of solute molecules from an area of high concentration to an
color by the human eye. area of lower concentration
● More ↓ volume = ↑ surface-volume ratio = ↑ diffusion rate

● Important Factors the Influence Diffusion

1. Distance
● The shorter the distance the more quickly
○ Double-displacement reaction concentration gradients are eliminated
■ NaCl is small enough, and since it is present, it 2. Molecule size
is possible to have double-displacement reaction ● Ions and small organic molecules diffuse more
with AgNO3 rapidly than larger ones do
○ If you will be performing some test wherein you will 3. Temperature
determine possible presence of starch and polysaccharides, ● The higher the temperature, the faster the
you will utilize Iodine’s Test diffusion rate
■ To have blueish-black coloration, which is the 4. Gradient Size
positive result, Iodine will form an interaction ● The larger the concentration gradient, the faster
with one of the components of the starch. diffusion proceeds
■ Starch has two components: amylopectin and 5. Electrical Forces
amylose ● Opposite charges attract each other like charges
■ Once the iodine will interact with amylose, it repel
will form a specific charge transfer. ● ↑ Diffusion Rate
■ One IMF of attraction is observed in charge ● ↑ Kinetic Energy = ↑ Diffusion Rate
transfer is electrostatic forces of attraction to
form bluish-black color.

● Caramelization

Page 4 of 8
BIOCHEMISTRY LABORATORY - Ma’am Julie De Guzman
X. Fick’s First Law of Diffusion
● The molar flux due to diffusion is proportional to the concentration
gradient
○ Highly affected by the changes in the concentration and
distance change, and diffusion coefficient
● Negative sign means solutes go opposite to that of increasing
concentration
● Provide an idea on why we have selected particles and ions that can
diffuse in and out of the cell
● If there is an observable changes in the concentration or in diffusion
coefficient, expect that molar flux will shiftings/changes taking place

● Application in Living System of Fick’s Law

1. Diffusion of Gases
2. Cellular Dimension
● Limited by oxygen diffusion

Page 5 of 8
BIOCHEMISTRY LABORATORY NOTES
SPECTROPHOTOMETRY
DEFINITION:
- a method to measure how much a chemical
substance absorbs light by measuring the
intensity of light as a beam of light passes
through sample solution
INSTRUMENT:
• A spectrophotometer is used to measure the
light transmitted by a solution to determine the
concentration of the light-absorbing substance
in the solution.
PRINCIPLE:
Specific particles in a solution can be measured based on
their ability to absorb a passing light. So, when a beam of
incident light (𝐼𝑜 ) passes through a solution, a part of the
incident light is reflected (𝐼𝑟 ), a part is absorbed (𝐼𝑎 ) and
rest of the light is transmitted (𝐼𝑇 ).
𝐼𝑜 = 𝐼𝑟 + 𝐼𝑎 + 𝐼𝑇
𝐼𝑜 = the amount of incident light is known
𝐼𝑟 = the amount of the reflected light is negligible so it
can be removed from the equation
𝐼𝑇 = transmitted light is measured by the photodetector
𝐼𝑎 = 𝑎𝑚𝑜𝑢𝑛𝑑 𝑜𝑓 𝑎𝑏𝑠𝑜𝑟𝑏𝑒𝑑 𝑙𝑖𝑔ℎ𝑡 = 𝑐𝑜𝑚𝑝𝑢𝑡𝑒𝑑
For example: To determine glucose concentration,
serum is reacted with a reagent specific for glucose.
Afterwards, the solution is placed in a cuvette for
spectrophotometric reading. The amount of light
absorbed by the sample is measured and correlated to
the concentration.
COMPONENTS:
1. Light Source
- The light source is required to generate
lights within the spectrophotometer.
- The material is used in light sources should
be excited to high energy states by a high
voltage electric discharge (or) by electrical
heating to serve as excellent radiant energy
sources.
- incandescent tungsten or tungsten-iodide
lamp
o common light source to provide
light in visible and near-infrared
regions
- deuterium discharge lamp and the mercury
arc lamp
o common light source to provide
light in UV region
2. Collimator (Lens)
- points the light to a monochromator or
prism from the light source
3. Monochromators (Prisms/Gratings)
- used to separate the polychromatic
radiation into component wavelength (or)
bands of wavelengths
- determines polychromatic radiation into its
individual wavelengths and isolates these
wavelengths into very narrow bands.
*The white light (incident light) is from the light source. The
collimator will collect and direct the light towards the prism.
The prism receives the light and separate them into their
individual wavelengths. Based on the chosen wavelength, the
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transmitted light passes through the slit and passes through
the sample.
*prism and grating are different, but both are
monochromator. A spectrophotometer either has a prism or a
grating (more common).
*mono = one, chromo = color → white light only has many
colors. Monochromator isolates each color
4. Wavelength selector (slit):
- It selects a particular wavelength of light
from the split wavelengths and passes it
through the cuvette.
5. Sample cell
- used to hold the sample to be studied
o cuvette must be stable and not
moved during reading to ensure
proper absorbance if light
- commonly use quartz CUVETTES
o Cuvettes with scratched optical
surfaces scatter light and should be
discarded
6. Photodetectors
- convert the transmitted radiant energy into
an equivalent amount of electrical energy
- the electrical energy is measured as the
amount of transmitted light
- transmitted light is indirectly proportional to
absorbed light and directly proportional to
electrical energy
Summary of the spectrophotometry principle:
* Analogy: A box of fruit containing 100 oranges
(amount of incident light) were given to a class
(cuvette) with unknown number of students
(concentration). Each student will strictly have only
one orange (amount of absorption). After taking the -
oranges, the teacher made a 30 cups of orange
juice (amount of electrical energy) from the
remaining oranges (amount of transmitted light). If
one orange = 1 cup of orange juice, how many
students (unknown concentration) were in the
room?
*30 cups of orange juice = 30 remaining orange = 70
students since there were 100 oranges
*More concentrated samples would have higher
absorbance of light, and less transmitted light/electrical
energy.
Theory of Ultraviolet-Visible (UV-Vis)
Spectroscopy
- based on the absorption of ultraviolet light
or visible light (incident light) by chemical
compounds (sample), which results in the
production of distinct spectra (transmitted
light)
- When matter absorbs ultraviolet radiation,
the electrons present in it undergo
excitation. This causes them to jump from a
ground state (an energy state with a
relatively small amount of energy associated
with it) to an excited state (an energy state
with a relatively large amount of energy
associated with it).
- the difference in the energies of the ground
state and the excited state of the electron is
always equal to the amount of ultraviolet
radiation or visible radiation absorbed by it
*The use of ellipsis (*) indicates antibonding. σ indicates sigma
orbital, while π indicates pi orbital. N means the electrons are
not bonded – lone pairs.
σ → σ* energy transitions
- An electron in a bonding s orbital is excited
to the corresponding antibonding orbital.
- Normally absorbed at 125nm (below the
visible and UV region)
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π → π* and n → π* energy transitions

- Most absorption spectroscopy of organic

compounds is based on these transitions.
o absorption peaks for these
transitions fall in the UV-VIS region
(200-700nm) These transitions
- need an unsaturated group in the molecule
to provide the pi electrons

n → σ* energy transitions
* When a sample absorbs light of a particular color, it
- non-bonding electrons (lone pairs) are
transmits the complementary color, i.e., the color opposite
excited to the antibonding sigma orbital
the absorbed color on the color wheel. For example, if a
- range 150 - 250 nm sample absorbs purple light, the sample will appear green.
- requires less energy than σ → σ*

BEER-LAMBERT LAW
VISIBLE SPECTRUM OF COLORS
- the working principle of the
Spectrophotometer is based on this law
- states that the amount of light absorbed by
a color solution is directly proportional to
the concentration of the solution and the
length of a light path through the solution

*The visible light spectrum is at 400-700nm.

*Light falling on a colored solution is either absorbed or
transmitted. A colored solution absorbs all the colors of white
light and selectively transmits only one color. This is its own
color. Refer to the table:

A = εbC

A = Absorbance

ε = molar absorptivity (constant)

b = length of light path

C = Concentration

RELATIONSHIP:

Absorbance is INCREASED if concentration of sample and

the length or distance travelled by the light INCREASES

*since the length of the cuvette is standardized, only the

concentration of analyte affects the absorbance

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Beer’s law also states that the concentration of a How much diluent must be added per tube if each tube
substance is directly proportional to the amount of light has a dilution factor of 5 and has 150uL of solute per
absorbed or inversely proportional to the logarithm of tube?
the transmitted light
DILUTION → 1 : 5
SERIAL DILUTION

DEFINITION

- series of dilutions, with the dilution factor

SOLUTION = SOLUTE + SOLVENT
ideally staying the same for each step
Total parts (parts of the solution) = parts of solute +
DILUTIONS
parts of solvent

PARTS OF DILUENT = 5 – 1 = 4 parts

Volume of diluent:

1 part : 150uL = 4 parts diluent : x

x = 150uL (4parts) / 1part = 600uL

𝑪 𝟏 𝑽𝟏 = 𝑪 𝟐 𝑽𝟐
600uL of diluent must be added to 150uL of stock
𝑪𝟏 = 𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 solution to have a dilution of 1:5 since the volume of
𝑪𝟐 = 𝒇𝒊𝒏𝒂𝒍 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 solute to solution will be 150uL: 750uL.

𝑽𝟏 = 𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆 Shortcut: Since 1 part of solute is equal to 150uL, 4 parts

will be 4 times the solute volume. So, diluent will be
𝑽𝟐 = 𝒇𝒊𝒏𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆 150uL * 4 = 600uL.
Sample computations:
How much diluent must be added to 5mL of 5% RCS to Dilution Factor
produce a 2% RCS?
𝑽𝒇
𝑫𝑭 =
𝑪 𝟏 𝑽𝟏 = 𝑪 𝟐 𝑽𝟐 𝑽𝒊
𝟓%(𝟓𝒎𝑳) = 𝟐% 𝑽𝟐
𝟓%(𝟓𝒎𝑳) 𝑽𝒇 = 𝑭𝒊𝒏𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆 (𝑽𝟐)
= 𝑽𝟐
𝟐%
𝑽𝒊 = 𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆 (𝑽𝟏)
𝑽𝟐 = 𝟏𝟐. 𝟓𝒎𝑳
𝑫𝒊𝒍𝒖𝒆𝒏𝒕 = 𝟏𝟐. 𝟓𝒎𝑳 − 𝟓𝒎𝑳 = 𝟕𝒎𝑳
Compute for the dilution factor and the final
ANSWER: 7mL of NSS must be added to dilute the 5% concentrations of tubes 1, 3 and 5 if 1mL of 500g/mL of
RCS to 3.5% the stock solution is transferred to tube 1.
*hint: V2 is the volume of the solution after the addition of diluent,
so it is higher than the V1, while V1 is the volume of the solute or
the “stock” solution. That’s why the volume of diluent is not
directly computed from the given formula.

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 APPLICATIONS

ANTIBODY TITRATION

*serum/plasma as stock solution is diluted in a series of

tubes to determine the titer or concentration of
antibody in the serum/plasma.

Given:

𝑽𝟏 = 𝟏𝒎𝑳 (𝒗𝒐𝒍 𝒐𝒇 𝒕𝒉𝒆 𝒔𝒐𝒍′𝒏 𝒃𝒆𝒊𝒏𝒈 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒓𝒆𝒅)

𝑽𝟐 = 𝟐𝒎𝑳 (𝑽𝟏 + 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒅𝒊𝒍𝒖𝒆𝒏𝒕)
= 𝟏𝒎𝒍 + 𝟏𝒎𝒍
𝑽𝒇 𝑽𝟐 𝟐𝒎𝑳
𝑫𝒇 = = = =𝟐
𝑽𝒊 𝑽𝟏 𝟏𝒎𝑳 *RCS is added as reactant/ indicator
FINAL CONCENTRATIONS (TUBES 1 and 3) *RCS has surface antigens that is reacted upon by
antibodies
𝑪 𝟏 𝑽𝟏 = 𝑪 𝟐 𝑽𝟐
TUBE 1 (1:2) *antibodies bind with antigen linking the red cells
together leading to crosslinking which presents a
𝒈 AGGLUTINATION
(𝟓𝟎𝟎 ⁄𝒎𝒍)(𝟏𝒎𝑳)
=𝑪𝟐
𝟐𝒎𝑳 HETEROTROPHIC PLATE COUNT
𝑪 𝟐 = 𝟐𝟓𝟎𝒈/𝒎𝑳 *a bacterial suspension or broth is diluted in several
Tube 3 (1:8) dilutions to determine bacterial concentration.

𝒈 *After diluting, it is dispensed or plated in culture media

(𝟏𝟐𝟓 ⁄𝒎𝒍)(𝟏𝒎𝑳)
=𝑪𝟐 to be observed for growth after incubation. The number
𝟐𝒎𝑳 of colonies are counted afterwards.
𝑪 𝟐 = 𝟔𝟐. 𝟓𝒈/𝒎𝑳

You can also use the formula:

𝑪𝟏
𝑪𝟐 =
𝑫𝑭
Tube 1 (1:2)
𝟓𝟎𝟎𝒈/𝒎𝒍
𝑪𝟐 = = 𝟐𝟓𝟎𝒎𝒈/𝒎𝒍
𝟐
Tube 3 (1:8)
𝟏𝟐𝟓𝒈/𝒎𝒍
𝑪𝟐 = = 𝟔𝟐. 𝟓 𝒎𝒈/𝒎𝒍
𝟐

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BIOCHEMICAL SYSTEMS

IN VIVO A=Surface area

D=Diffusion coefficient
- A medical experiment or a test that is
-
performed on a living organismIunder
-
P1-P2=Partial Pressure gradient
controlled physiological condition
- Usually uses animal testing, i.e. guinea pigs, T = Thickness of membrane
mouse, rabbit
- Clinical trial of drugs
𝑨 × 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒐𝒏 𝒈𝒓𝒂𝒅𝒊𝒆𝒏𝒕 𝒅𝒊𝒇𝒇𝒆𝒓𝒆𝒏𝒄𝒆
𝑫𝑹 =
IN VITRO 𝑻
- A medical experiment or a study that is
-
performed only in laboratory dish or a test
“the rate of diffusion is proportional to both the surface
tube under controlled environmental
area and concentration difference and is inversely
conditions
proportional to the thickness of the membrane”
- Serologic tests, i.e. antigen or antibody tests
↑DR = ↑SA = ↑CG = ↓T
EX VIVO
Fick’s first law:
- A medical experiment or a study that is
-
performed in or on a tissue in an artificial
- - The amount of movement of particles (diffusion
environment outside the organism with a
-
-

flux) from high to low concentration is directly

minimum alteration of natural conditions proportional to the particle’s concentration
gradient
IN SILICO ↑DR = ↑CG
- A medical experiment or a study that is * It means the greater the difference of concentration
-
-
simulated on a computer between the high concentration region and low concentration
- Molview region makes particles diffuse faster.

Fick’s second law:

CELL TRANSPORT - States the relation between the change in

concentration gradient of the particles and time.
DIFFUSION

- It is an example of passive transport Other Diffusion Factors

- Movement of solute or solvent from an area of
A. Temperature and Agitation
high concentration to an area of lower
- Temperature and agitation increase kinetic
concentration (along the concentration
energy of the particles allowing them to bump
gradient)
into each other more, hence, it leads to faster
Fick’s law diffusion.

- describes the relationship between the rate of B. State of matter

diffusion and the three factors that affect
- The phase of the diffusing particle and the
diffusion.
diffusion method influence the rate of diffusion
𝐀 × 𝐃 (𝐏𝟏 − 𝐏𝟐) as well. Gas particles diffuse the fastest while
𝐃𝐑 = solid is the slowest.
𝐓
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 C. Distance in HCl allows H+ ions to diffuse through the agar making
- The farther the distance, the longer the diffusion the pH acidic, subsequently decolorizing the gelatin
as particles has to travel and be diffused longer cube.

D. Particle size
- The smaller the particle, the easier it is to travel
across barriers or surfaces.

DIFFUSION AND GAS EXCHANGE

Diffusion Rate
(𝒗𝒐𝒍 𝒐𝒇 𝒄𝒖𝒃𝒆 − 𝒗𝒐𝒍 𝒐𝒇 𝒄𝒐𝒍𝒐𝒓𝒆𝒅 𝒑𝒐𝒓𝒕𝒊𝒐𝒏)
𝑫𝑹 =
𝒗𝒐𝒍 𝒐𝒇 𝒄𝒖𝒃𝒆
× 𝟏𝟎𝟎%
*Both alveoli and capillaries are lined with simple
cuboidal cells, so their cell membrane are thin. They are
closely aligned, so the distance is small. Alveolus has a A gelatin cube with 3X3X3cm dimensions is immersed in
large surface area and small volume so it has more HCl for 3minutes. Afterwards, the length of colored part
contact with the capillaries → more diffusion area. All of is measured at 2cm. If the diffusion is equal in all sides,
these allows faster diffusion and exchange of gas. In how much of the cube has been diffused? What is the
Pneumonia, fluid infiltrate the alveolus making diffusion diffusion rate per minute?
slower, hence gas exchange is also inhibited. This is
(𝒗𝒐𝒍 𝒐𝒇 𝒄𝒖𝒃𝒆 − 𝒗𝒐𝒍 𝒐𝒇 𝒄𝒐𝒍𝒐𝒓𝒆𝒅 𝒑𝒐𝒓𝒕𝒊𝒐𝒏)
manifested by the difficulty in breathing. 𝑫𝑹 =
𝒗𝒐𝒍 𝒐𝒇 𝒄𝒖𝒃𝒆
DIFFUSION AND DECOLORIZATION × 𝟏𝟎𝟎

pH indicator: phenolphthalein

pH indicator – changes color depending in pH (𝟐𝟕𝒄𝒎𝟑 − 𝟖𝒄𝒎𝟑 )

𝑫𝑹 = × 𝟏𝟎𝟎
𝟐𝟕𝒄𝒎𝟑
𝑫𝑹 = 𝟕𝟎. 𝟑𝟕% 𝒇𝒐𝒓 𝟑𝒎𝒊𝒏𝒖𝒕𝒆𝒔 = 𝟐𝟑. 𝟒𝟔% /𝒎𝒊𝒏𝒖𝒕𝒆

𝑫𝑹 = 𝟐𝟑. 𝟒𝟔% /𝒎𝒊𝒏𝒖𝒕𝒆 𝒐𝒓 𝟔. 𝟑𝟑𝒄𝒎𝟑 /𝒎𝒊𝒏𝒖𝒕𝒆

*to get the diffusion rate per cubic volume/minute, take

the 23.46% of the total cube volume 27𝑐𝑚3 .

Another cube with same dimension was immersed in a

stronger acid. Upon computation, the diffusion of H+
Gelatin or Agar cube has phenolphthalein and NaOH
into the cube was measured at 𝟖𝒄𝒎𝟑 /𝒎𝒊𝒏𝒖𝒕𝒆. How
dyeing it pink due to the alkalinity. Immersing the cube
much time is needed to decolorize the tube completely?

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100% decolorization = 100% diffusion = 𝟐𝟕𝒄𝒎𝟑 Reverse Osmosis
𝟖𝒄𝒎𝟑 : 𝟏 𝒎𝒊𝒏𝒖𝒕𝒆 = 𝟐𝟕𝒄𝒎𝟑 : 𝒙 - Application of pressure to allow water to move
𝟑
𝟐𝟕𝒄𝒎 (𝟏𝒎𝒊𝒏) through a semipermeable membrane from an
𝒙= = 𝟑. 𝟑𝟖 𝒎𝒊𝒏𝒖𝒕𝒆𝒔 area of high solute concentration to low solute
𝟖𝒄𝒎𝟑
concentration (against the concentration
gradient)
OSMOSIS - This is applied in water purification to separate
water from large particulates.
- Passive transport
WATER TRANSPORT AND OSMOSIS IN
- Movement of SOLVENT from an area of HIGH to
LOW water potential or LOW to HIGH solute CELLS
concentration through a semipermeable
membrane to equalize solute concentration on Osmotic theory of water absorption
both sides.
- proposed by Akins and Priestly
- This theory explains the absorption of water by
the roots from the soil.
- water moves up the soil with the help of xylem
along with increasing diffusion pressure deficit
which is the suction pressure causing water to
be absorbed. Afterwards, endosmosis (water
moves in via osmosis) happens.

Water transport in cells

Osmotic pressure

- It is the additional pressure which gets exerted

on the solution due to the osmosis of pure
solvent into it through a semi permeable
*Water is able to pass through the phospholipid bilayer
membrane.
as it is small and neutral so it is not repelled. Also, the
- It is one of the colligative properties of solution phospholipid bilayer is amphipathic. Water interacts
- It always increases with the rise in temperature more with the polar head and avoid the non-polar tail.
based on vant Hoff equation π = CRT. So, the phospholipid molecules are constantly in motion,
- Also, it increases with the increase in jostling and drifting around the membrane, flexing and
atmospheric pressure because as the extending their tails. However, this is extremely slow and
atmospheric pressure increases, there is a inefficient so other transport mechanism is also used.
greater tendency of the solvent molecules to
pass into the solution through the semi-
permeable membrane thereby causing an
increase in osmotic pressure.

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 Isotonic

- It is the ideal environment for cells as the

*On the other hand, water can also pass through via
facilitated diffusion either with a non-specific
transporter, a specific transporter or an aquaporin. An
aquaporin is an integral (transmembrane) protein that is
found in the cell membrane. This allows unimpeded
movement of water through the membrane. Water
moves in or out of the cells based on the osmotic
gradient.
Osmotic solutions
BALANCE
SIRINK swil
osmotic gradient in both sides is equal, hence,
movement of water in and out of the cell is also
equal. Since there is no decrease nor increase in
water in the cell, no damage to the cell is
observed.
- Examples: 0.9% normal saline solution
Hypotonic
- The cell has more solutes than its surrounding.
When this happens, water moves into the cell
until there’s equal osmotic gradient. If too much
water moves in, the cell will swell and it may
burst or lyse (hemolysis for red cells) due to
increased water pressure inside the cell.
- Red cells can lyse in extremely diluted urine.
- Example: distilled water
Factors of Osmosis
Dialysis
INSIDE
~/

- It is still a passive transport and it is a

combination of osmosis and diffusion as both
water and solute may move through a
YOUTSIDE
semipermeable (dialyzing) membrane.
- Primarily observed in the renal function filtration
stage of urine formation.

Hypertonic Selective Permeability

- The surrounding environment has more solute

than the inside of the cell. As such, water moves
out of the cell to equalize the osmotic gradient
in both sides. When too much water moves out,
cell shrinks (other cells) or crenate (red cells).
- Example: RBCs in concentrated urine sample like
in dehydrated patients, 5% dextrose solution
- High blood Na+ makes blood hypertonic causing
water to move out of tissue cells and blood cells
into the blood. This increases the blood volume. *the kidneys have many aquaporins as lots of water is
Since blood volume increased, blood pressure needed to produce urine
consequently increases as well to “push” blood Glomerulus- it is the filtering structure of the kidneys. It
towards the upper extremities. is made up of capillaries lined with a single endothelium.

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 Analyte testing
Test for NaCl

- Na and Cl ionizes in water, and each ion is small

Test for sucrose
Due to large MOLECULAR size, large proteins and blood
cells will stay in the glomerulus and in the blood. The
filtrate has to pass through the glomerulus, the blood
proteins and the Bowman’s capsule. Blood proteins are
negatively charged so it repels anions like HCO3- and Cl-.
That’s why despite the small molecular size they will not
easily enter the tubules.
enough to pass through the hotdog casing. So,
when AgNO3 was dropped to the dialysate,
white precipitate should have been observed.
This is based on the equation:
𝑁𝑎𝐶𝑙 + 𝐴𝑔𝑁𝑂3−→ 𝐴𝑔𝐶𝑙 (↓) + 𝑁𝑎𝑁𝑂3 (𝑎𝑞)
- Sucrose is a disaccharide, but it should be small
enough to pass through the hotdog casing.
Presence of sugar is tested by heating the
dialysate over low heat. Heating allows
evaporation of water, leaving sucrose molecule.
When the sugar reaches its melting point, it
starts to break down and caramelize.

Test for NaOH

- Same with NaCl, NaOH also dissociates in water.

As such, sodium and hydroxide can also pass
through the membrane. Presence of OH can be
detected qualitatively through litmus paper (red
to blue) or quantitatively though pH meter by
checking difference of pH before and after
dialysis.

Test for starch

However, HCO3- and Cl- anions are still able to enter the
tubule due to the net pressure. There are 3 pressures
that is involved in the filtration process. Glomerular
pressure pushes the blood components towards the
tubule, while osmotic and hydrostatic pressure pushes
the blood back. Normally, the glomerular pressure is
stronger so it can push anions towards the filtrate
despite the electric repulsion. If the glomerular pressure
increases too much, it may even push the proteins
towards the tubule, which may now be seen in urine.
Orthostatic hemoglobinuria may be seen with healthy
individuals who has to stand most of the time. Gravity is
stronger when you are standing, which adds to the
glomerular pressure.
- Starch is a polysaccharide. It is composed mostly
of amylose which is large and branched. If tested
inside the hotdog casing using Lugol’s iodine,
bluish-black color must be observed.
- The amylose imbibed the iodide (𝐼 − ) ions in its
coils. The 𝐼 − ions electrons move from Iodine to
Iodine allowing absorption of yellow light (iodine
color). After which, there is a change in
wavelength, so the complementary color is
observed (blue). Refer to the earlier color wheel.

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*This is the amylose. Notice the coils and branching. Inside the
coil is the iodine.
YEAST
- They are eukaryotic, single-celled
microorganisms classified as members of the
fungus kingdom
- may be pathogenic or opportunistic
o presence of yeast in biological samples
like blood and urine indicates fungal
infection
- used in the food industry (baker’s yeast)
o Saccharomyces cerevisiae
These are yeast cells in urine. Sometimes, presence of
mycelium (stem-like structure) and budding are
observed which may be used to differentiate yeast from
red cells.
*to ensure correct identification of red cell or yeast cell, acetic
acid is added to the sample to lyse red cells. If the prior
observed cells disappeared, then it was red cells. If the cells
resist the acetic acid, it is reported as yeast cells.
Reactions
Solution A Solution B
Viability of cell Alive Dead

Reaction to stain colorless colored

Budding present absent

Fermentation present absent
Osmosis Yeast cells will appear smaller in 1M
*1M NaCl (hypertonic), same in 0.1M NaCl
*0.1M NaCl (isotonic) and bigger in d-H2O
*d-H20 (hypotonic)
*solution B contains yeast cells that are heated and boiled.
Boiling kills yeast due to protein denaturation.

*Dead cells do not exhibit biochemical processes anymore like

budding and fermentation as these requires energy.

*Alive yeast cells are not colored as the stain is transported

out by the cell’s active transport system. Additionally, viable
\ cells are capable of metabolic process that produces NADH+.
This nucleotide may lose its electron to Methylene blue
These are red cells in urine. It has a similar appearance turning the dye colorless.
with yeast cells. Sometimes, red cells are crenated in
*Methylene blue is a basic stain that stains the negatively-
urine due to hypertonicity of urine, so this may
charged structures in the cell.
differentiate red cells from yeasts.

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 Then, with the presence of a catalyst – enzyme
zymase – the yeast acts on the glucose. Glucose
is then converted to a series of products till the
final products are produced. (Ethanol, CO2, H+)
o Yeast is used as a leavening agent in
baking bread as it produces CO2 (gas)
that “puffs” the bread.
- Generally, fermentation of carbohydrates
*dead cells may still exhibit passive transport like diffusion and
produces an alcohol, an acid and gas.
osmosis as it doesn’t require energy.

Reproduction of yeast

Glucose→G6P→F6P→F-1,6-
- Yeast reproduces in three forms. P→Glyceraldehyde→phosphoglycerate→
o Vegetative by fission and budding pyruvate→acetaldehyde→ethanol
▪ Fission forms 2 daughter cells
▪ Budding forms 1 daughter cell
o Asexually by sporulation
▪ Forms 4 haploid endospores
o Sexual reproduction (meiosis)
▪ Forms 4-8 ascospores
- Vegetative reproduction is under favorable
conditions, while yeast reproduce asexually or
sexually only in unfavorable conditions.

Fermentation

- S. cerevisiae produces two types of enzymes: an

extracellular invertase and an intracellular
zymase. The invertase hydrolyses cane sugar to
dextrose or invert sugar and zymase breaks
invert sugar into ethyl alcohol and carbon
dioxide.
- Sugar (sucrose) is first broken down to its basic
monosaccharide units – glucose and fructose.
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