Exercise 1: The Compound Microscope I.

Stage- a horizontal platform upon which the specimen

Types of Microscopes
1. Light microscope- uses light waves and lenses that are
associated with the light microscope
2. Electron microscope- employs electrons beam and
magnetic fields to produce image.
Microscope parts
to be examined is placed.
J. Stage clips- holds the slide in place on the stage
K1. Condenser- concentrates the light rays received
from the mirror and sends them to the objectives.
K2. Iris diaphragm- regulates the amount of light
entering the condenser
L. Base- keeps the microscope steady at any position of
the stage
M. Mirror- reflects the light into the condenser
N. Low power objective- 10x

Important features of the objectives:

a. Focal length(mm)- distance from the center of the
lense to the point where parallel rays entering the lens
are brought to the focus
b. Resolving power- property to recognize features of a
specimen that are close to each other as separate and
distinct.
c. Numerical aperture- measure of the resolving power
of an objective.
d. Working distance- the distance from the front lens
element of the objective to the closest surface of the
coverslip when the specimen is in sharp focus
e. limit of resolution- the capacity of an optical system
A. eyepiece- The lens the viewer looks through to see
to resolve point objects as separate images
the specimen.
B. Coarse focus knob- Brings the specimen into general
focus Characteristics of the objectives:
C. Draw tube- connect the eyepieces to the objective LPO HPO OIO
lenses
Focal Length (mm) 16 4 1.8
D. Fine focus knob- A knob used to fine-tune the focus
of a specimen in conjunction with the coarse focus. Linear magnification (x) 10 40 100
E. Revolving nosepiece- A nosepiece with multiple
objectives that revolves in order to enable the viewer to Numerical Aperture (N.A.) 0.25 0.65 1.25
use
Diameter of front lens (mm) 2.0 0.4 0.2
F. High power objective- 40x
Working distance (mm) 4.8 0.5 0.1
G. Arm- fastened to the base through the inclination
joint, permits the adjustment of the stage to a desired Color band yellow blue white
angle
H. Oil immersion objective- 100x
 Oil is used in oil immersion objective to have a b. Wet mount- a common type of preparation for the
high refractive index. Having a high refractive index examination of living microorganisms.
means high numerical aperture and hig resolving power.
Non-motile organisms or tiny particles in
Calibration of the Ocular Micrometer: suspension also exhibit movement characterized by an
oscillating or quivering motion known as Brownian
+ Stage micrometer- a glass slide with graduations of
movement.
known intervals. One small division is 0.01 mm or 10µm
Examination of specimens
+ Ocular micrometer- a glass disc with mounted scale.
It is inserted into the eyepiece and must be calibrated 1. Hanging drop
before measurements are made
a. Carmen dye - Brownian movement
Calibration Factor SM division value of one SM
(Value of one Ocular = subtended by OM div X division (µm) b. Protozoa (in the hay infusion)
Micrometer division) OM divisions subtended by SM division - true motility
- circular in shape
Determination of the Area of the Field of Vision
c. Micrococcus luteus
Area= πr2 - Brownian movement
ACCESSORIES - spherical in shape

Camera lucida- a prism attachment which fits on top of d. Bacillus megaterium

the eyepiece - true motility
- rod shape
Demonstration eyepiece- attached to the draw tub of
the microscope so that two persons can look at the same 2. Wet mount
specimen at the same time. a. Saccharomyces cerevisiae
Projection eyepiece- permits a projection of the image - undergoing budding
on a screen some distance - spherical in shape
b. Shizosaccharomyces cerevisiae
Exercise 2: Microscopic Observation of Living - undergoing binary fission
Microorganisms - cylindrical rod-shaped
c. Aspergillus sp.
Techniques allow for observation of living
- exhibits both asexual and
microorganisms organisms
sexual reprodcution
a. Hanging drop method- preferred if a prolonged
study of living organisms is to be made and is especially
useful for continuous observation of motility or for
monitoring the growth of individual cells in a
microculture.

d. Chlorella- spherical in shape

- green color
 e. Rhizopus oligosporus
- exhibits both asexual and
sexual reproduction
Shape and Arrangements
1. cocci- sphere or oval
a. diplococcus- cocci arranged in pairs
b. streptococcus- cocci arranged in chains
c. tetrad- arranged in squares of 4
d. sarcina- arranged in cubes of 8
e. staphylococcus- cocci arranged in irregular,
often grape-like clusters
2. bacilli- rod-shaped
a. bacillus- single bacilli
b. streptobacillus- bacilli arranged in chains
c. coccobacillus- oval and similar to a coccus
3. spirilli- spirals
a. vibrio- curved or comma- shaped rod
b. spirillum- a thick, rigid spiral
c. spirochete- a thin, flexible spiral
Exercise 3: Staining Microorganisms for Microscopic
Observation
Dye- stains that are used to make microorganisms more
easily see, to show certain cell structure, or to reveal
their chemical nature.
Kinds of Stains
1. Acidic Dye (negatively charged)
- used to stain the background (e.g. nigrosine,
india ink, carbol fuchsin)
2. Basic Dye (positively charged)
- e.g. crystal violet, malachite green, methylene
blue, safranin
Types of Staining
1. Simple Staining
a. Positive staining/ Direct- stains the cell
b. Negative staining/ Indirect- stains the
background
2. Differential Staining- more than one stain is used
- distinguished different groups of bacteria based
on certain stained cell components
a. gram staining- gram positive bacteria are
differentiated from gram negative bacteria
b. Acid fast staining- used to identify acid-fast
bacteria
3. Structural Staining- used to observe specific cell
structures such as endospores, flagella and
capsules
a. Endospore staining- resistant, dormant spore
formed within a cell, cannot be easily penetrated
by stains
- can be stained by malachite green under steam
b. Flagella staining- mordant is used as to
increase apparent diameter of the flagella
c. Capsule staining- uses acidic and basic dyes
to stain the background and bacterial cells
respectively so that the presence of capsule is
easily visualized.
Microscopic Examination and Observation
1. Positive Staining
a. Saccharomyces cerevisiae
- used methylene blue
- circular shape and blue in color
2. Negative Staining
a. Saccharomyces cerevisiae
- luminous circular cell in black
background
3. Differential Staining
a. Cheek cells
- pink squamous epithelial cell. It is flat
and has irregular shape with blue
bacteria—diplococcus salivarius that is
spherical in shape
b. Bacillus megaterium
- a gram-positive bacteria
- rod-shaped and violet in color
c. Escherichia coli
- a gram-negative bacteria
- rod-shaped and red in color
4. Structural Staining 2. Based on Consistency
a. Flagella (Salmonella tyhpi)
Consistency Agar (%) Purpose
b. Spores (Bacillus thuringiensis) For enumeration
Solid 1.5-2.0
c. Capsules (Paenbacillus velaci) Useful for
cultivation of
STEPS IN STAINING 0.5-0.7 microaerophilic
Semi-solid
1. Smear Preparation- thin, dry film of microorganism bacteria or
2. Fixation- kill microorganisms, fix or bond cells to the determination of
bacteria motility
slide so they will not be washed away during staining For observation
Liquid 0
a. Heat- pass slide through flame (3 sec) of motility
b. Chemical- flood the smear with 95% Ethanol
3. Application of stain/dye 3. Based on Purpose/ Use
a. General Purpose- allows growth of most
organisms (e.g. NA, NB, PCA)
Exercise 4: Culture Media b. Selective- suppress growth of unwanted
microorganisms and encourage growth of
Culture media desired microorganisms with inhibitors
- any nutrient material prepared for the c. Differential- allows differentiation of
growth and cultivation of microorganisms
microorganisms. d. Enrichment- increase number of desired
- must contain essential elements needed microorganisms
by microorganisms e. Characterization- test for metabolic
- C,H,O,N,P,S—basic elements in the cell for microorganisms
biosynthesis and energy production
Media Preparations—titration (pH adjustment)
The chemical compounds may be grouped as: pH 4 — bromcresol green (green to blue)
a. Nitrogen sources pH 7 — bromthymol blue (yellow to green)
b. Carbon sources pH 9 — phenolphthalein (colorless to pink)
c. Mineral
d. Growth sources Media Preparation—Determine the total volume
e. Energy sources
Glassware Approximate volume
pH is properly adjusted since different microorganisms Petri dish 15-20 mL
have different pH requirements: Big test tubes (18 mm diameter)
stabs/ broth 10
molds — pH 4-7 slants 6-8 mL
yeasts — pH 5-8 Small test tubes (13 mm diameter)
bacteria— pH 6.5-7 stabs/ broth 5 mL
slants 3 mL
Types of Culture Media Erlenmeyer flask not more than 60% of the
1. Based on Composition total capacity
a. Chemically defined/synthetic
- exact chemical composition Cotton is a common and efficient air filter used
in biological work. Cotton plugs permit the interchange
Nutrient Agar Amount per liter (g/L)
of gases but bot of the microorganisms in the air. The
Peptone from meat 5.0
plug should project into the test tube to a distance of 2-3
Meat extract 3.0
Agar 15.0 cm, and should protrude 2cm from the mouth to protect
the outer edge of the tube. Plugs for flasks should
b. Complex medium protrude 3 cm into the neck of the flasks and should have
- exact composition is unknown enough cotton outside to cover the lip of the flasks.
- undefined components (e.g. meat extract,
coconut water) Effect of initial pH on the gelling power of agar
- the lower the pH, the lower the gelling power
Exercise 5: Sterilization and Disinfection
Methods used to destroy and/ or control microbial
growth:
1. Sterilization- any treatment of process
that results in the death of all living organisms
and viruses in a material.
2. Disinfection- process of eliminating
nearly all pathogens.
The process said above can be achieved by
1. Physical- filtration, heat and radiation
2. Chemical Method- alcohols, chlorine
compounds, and chemotherapeutic agents to
control infectious diseases
a. cidal agents- chemicals that kill
organisms
b. static agents- inhibits growth
METHODS OF STERILIZATION
1. Physical Heat
a. Direct heat (alcohol lamp, Bunsen burner)
b. Dry Heat (mechanical convention oven)
- hot air oven at 170 °C
- 30 min and 60 min time exposure
c. Moist Heat (autoclave, pressure cooker)
- heated for 5 min and 15 min at 121 °C
2. Filtration
- filtered and unfiltered
3. Radiation
- exposed to UV light for 30 min w/ and w/out
cover
4. Disinfection
- different concentration of QUATS solution and
time exposure
Minimum inhibitory concentration (MIC) is the
lowest concentration of a chemical, usually a drug,
which prevents visible growth of a bacterium or bacteria.
Factors
1. Type of microorganism present
2. Concentration and nature of disinfectants
3. Length of treatment
4. Temperature
Exercise 6: Isolation of Bacteria into Pure Culture
Pure Culture- population of cells which
have originated from a single cell and thus, are
identical and of the same kind and species.
Methods
1. Plating- streak, pour and spread plating
2. Enrichment- use of special nutrients
(usually carbon and nitrogen source)
3. Serial dilution- makes use of series of
diluents (distilled water, buffer, saline
solution, phosphate buffered saline, broth)
4. Single cell isolation- actual and direct
separation of one cell from the other cells
5. Membrane filtration technique- employs
a membrane filter of appropriate pore
size to trap the microbial cells coming from the
sample
Plating
1. Streak Plating
1st to be added: media
2nd to be added: inoculum
Volume of inoculum: loopfull
Equipment used: wireloop. Petri dish
Purpose: for isolation
Types of colonies observed: surface colonies
Advantages: makakaisolate ng distinct colonies
Disadvantages: prone to contamination, media must be
pre-dry, no exact amount of the inoculum
2. Pour Plating
1st to be added: inoculum
2nd to be added: media
Volume of inoculum: 1 mL
Equipment used: petri dish, test tubes
Purpose: for counting
Types of colonies observed: surface and subsurface
Advantages: microaerophiles, aerobes and anaerobes can
be classified, need no spreader
Disadvantages: cannot be used in heat sensitive
microorganisms. Nakaembed sa agar yung nasa
subsurface, time consuming
3. Spread Plating
1st to be added: media
2nd to be added: inoculum
Volume of inoculum: 0.1 ml
Equipment used: spreader, test tubes, petridish
Purpose: for counting
Types of colonies observed: surface colonies
Advantages: heat-sensitive microorganisms are not
affected, easier to pick colonies, can be used for
enumeration
Disadvantages: higher tendency for the microorganisms
to be crowded, media must be pre-dry