LAB LONG TEST 1

Exercise 1: Microscopy: Cell Size Measurements and Counting Cells

Microscopy
microscope is instrumental in the discovery of the cell and is an essential tool in the study of the structure of
organisms at the histological and cellular levels
Magnification = visually enlarging or enhancing a thing that is to be viewed
degree of visual enlargement of the object being observed
determined through the product of the magnifying power of the objective and the magnifying power
of the ocular
Micrometry
technique used to measure objects
more accurate measurements
involves the calibration of the ocular micrometer using the stage micrometer
Ocular Micrometer —> glass disk with 100 equal divisions on it but is arbitrary and has no absolute value
placed on the ocular of the microscope
used to measure the size of a specimen
Stage Micrometer —> looks like a microscope slide but has a standard scale etched into it
used to calibrate the ocular micrometer
smallest divisions are 0.01 mm in length
like a tiny rules
one space is equal to 0.01 mm or 10 mm
Why do we need to calibrate?
each objective lens has different magnifications, with numerical values that hold true only for that
specific objective lens, therefore as the objectives change, the ocular spaces with it changes as well, so by
calibrating the microscope, despite theses differences we will be assured accurate measurements
How to calibrate?
1. fit an ocular micrometer into the eyepiece of the microscope
2. place slide micrometer and focus under LPO
3. adjust the stage micrometer so that the 0 line of the ocular micrometer is exactly superimposed on the 0 line
of the stage micrometer
4. find another point at the extreme right where the other two lines superimposed
5. count the number of division lines on the ocular micrometer between the 0 lines and the superimposed lines
6. count the number of 0.1 mm division lines between the 0 line and the second superimposed line on the
stage micrometer
7. divide the number of stage micrometer (OM) lines by the number of ocular micrometer (OM) lines —>
calibration constant (value of one OM)
8. convert millimeters to microns —> 1 mm = 1 microns (μm)
if you change microscopes, the calibration process must be done again for each objective lens because the
magnification is different on different microscopes
Counting Cells
Hemocytometer = apparatus used for the visual counting of cells in a blood or fluid sample
glass device with 4 corner squares made up of 16 smaller wherein the cells are counted per square
commonly used for blood or sperm counts; to determine cell size, and process cells for culturing
used to calculate the density of cells in suspension
Viable cells = have the ability to grow; can take up the stain
Nonviable cells = incapable of further growth; cannot take up the stain
Calculations
Mean no. of cells/0.1 mm3 = (total no. of cells in four corner squares)/4
Total no. of cells per mL = (mean no. of cells/0.1 mm3) x 104 x dilution factor
Total no. of cells = Total no. of cells per mL x Volume of solution (mL)
Viable cells (%) = Total number of viable cells / Total number of cells (viable + nonviable)

Exercise 2: Analysis of Human Blood Proteins Using SDS-PAGE

Blood —> specialized body fluids with different components

Red blood cells —> contains hemoglobin
hemoglobin = carries oxygen from the lungs to the tissues in the body
 four global chains with an iron-containing heme group
two alpha polypeptide chains and two beta polypeptide chains
White blood cells —> responsible for fighting infections and killing parasites
Platelets —> helps in the clotting process
Plasma —> liquid in which the three other components are suspended
mostly water
functions in defense, clotting and molecular transport
SDS-PAGE
procedure to separate proteins and determine their molecular weights
molecular weight marker (kilo Dalton) used as size standard
PAGE = polyacrylamide gel electrophoresis
used to separate and identify blood proteins according to their molecular weight
used to identify hemoglobin
electrophoresis = applying an electrical field to a protein solution to enable molecules to migrate
migration rate is dependent on molecular size or charge
SDS = sodium dodecyl sulfate
anionic detergent (negatively charged) used to denature the proteins by binding to the hydrophobic
regions
non-covalent bonds will be disrupted
masks the charge of proteins so that their migration is solely based on molecular weight
coats proteins with an over-all negative charge —> this allows the molecules to move towards the
positive electrode
typically composed of two gel layers
Stacking gel —> top most layer; low pH and low percentage of acrylamide
Separating or resolving gel —> lower layer; high pH and high percentage acrylamide
the difference in pH and acrylamide concentration provides better resolution and sharper bands in
the separating gel
gels are polymerized between two glass plates in a gel caster with a comb inserted at the top to
create the sample wells
Ammonium persulfate = initiator for gel formation
TEMED (tetramethylethylenediamine) = improves polymerization
Advantages
can withstand high voltage gradients
amendable to staining and destaining procedures
digested to extract separated fractions of proteins
dried for permanent recording
tank buffer stabilizes the pH within the gel
buffer should be unreactive and not modify or react with most proteins
Staining: Coomassie brilliant blue
stained to detect the proteins present
after staining different biomolecules appear as distinct bands within the gel
run a molecular weight size marker of known molecular in separate lane to determine
the approximate molecular mass of unknown biomolecules

Exercise 3: Colorimetric Analysis of Protein Concentration (Bradford Assay)

Bradford Assay Method

use of Coomassie Brilliant Blue G-250 dye to which protein binds altering the light absorbance properties of
the dye
protein concentration will be determined using absorption visible wavelengths based on protein-binding to a
dye
when the dye is prepared as an acidic solution (in 85% phosphoric acid), it maximally absorbs light with a
wavelength of 465 nm
addition of protein results in a shift of the dye’s absorption maximum to 595 nm
as protein concentration increases, the absorbance of light at 595 nm increases linearly
hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change
sensitive to about 5 to 200 micrograms protein
useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold
concentration range
 BSA protein = bovine serum albumin protein
used in order to measure and plot a standard curve of protein concentration versus absorbance at
595 nm
Bradford Reagent = made by dissolving 100 mg Coomassie Blue G-250 in 50 ml 95% ethanol, adding 100
mL 85% (w/v) phosphoric acid and diluting the mixture to 1 liter with water
Spectrophotometer
commonly used to estimate the level of an analyte (protein) in solution
proteins absorb light at a specific wavelength
can be used directly to measure the concentration of a purified protein in solution
method based on the two laws of light absorption by solutions, namely:
Lambert’s Law
Beer’s Law —> I = Io10 -gcd
Where Io = intensity of the incident radiation, I = intensity of the radiation transmitted
through a cell of thickness d (in centimeters) that contains a solution of concentration c (in
M or grams/100 mL) and g is an absorptivity constant (extinction coefficient) characteristic
to the substance being investigated
absorbance, Abs is directly related to Io and I by Abs = log10 (Io/I)
Abs = gcd, and Abs is directly proportional to concentration
DNA absorbs maximally at 260 nm because of the presence of aromatic rings in the nitrogenous bases of the
nucleotides
purine absorbance maximum is slightly below 260 nm and pyrimidine absorbance maximin is slightly above 260 nm
proteins absorbs maximally at 190-230 nm range due to the peptide bonds, and at 280 nm due to aromatic amino
acids (tyrosine, phenylalanine, and tryptophan)

Exercise 4: Enzyme Activity: Digestion of Starch by Amylase

Enzymes
proteins that act as biological catalysts in speeding up rates of reactions without being used up in the reaction
achieved by binding their active site to their specific substrate and lowering the energy of activation required
for conversion to product
rate of activity of enzymes is strongly affected by several factors, such as temperature, pH, presence of
inhibitors, and concentration of the enzyme
Saliva
contains an enzyme called salivary amylase
the substrate for amylase is starch
salivary amylase breaks down starch into maltose by hydrolysis
in the stomach salivary amylase is inactivated by the lower pH
salivary amylase act on a specific target molecule (substrate) to produce a product
establishing the presence of enzyme and substrate —> variable conditions (temperature and pH) for
enzyme activity —> diffusion of enzyme of varying concentrations
Test for starch = iodine solution
positive result —> black color
negative result —> brown color
Test for sugar = Benedict’s solution
used to detect the presence of sugar
color of the obtained precipitate gives an idea about the quantity of the sugar present in the solution
positive result ranges from green to bright red; negative result would appear blue and indicate no sugar
blue = negative
green to yellow = traces of reducing sugar
orange to red = moderate presence of sugar
brick red = large amount of reducing sugar