Exercise 1 - The Microscope: Principles, Parts, Use and Care

(lambda) is the wavelength of light illuminating

Light Compound Microsope
the microscope can be altered using special filters.
- most familiar laboratory insrtument to Wavelength of visible white light is 550nm (indirect
microbiologists sunlight). Other electric light sources are tungsten
- optical system consisting of aligned lenses that work (light bulb), halogen lamp and fluorescent lamp.
together to magnify or enlarge the image of the
specimen under observation (eta) is the refractive index of the medium
o Objective lens - first lens that magnify the image between objective lens and glass slide.
of the specimen
Values of :
o Ocular second lens that further magnifies the
image Air 1.00
Water 1.33
Visible light of the electromagnetic spectrum ranging Linseed Oil 1.48
from 400nm to 700nm is used to illuminate this type Cedar Wood Oil 1.50
of microscope. Thus, its name Light Compound Canada Balsam 1.52
Microscope.
(theta) is the half the value of the angular
Microscope
aperture. The Angular Aperture is also known as the
- can be simple or compound cone angle; it is the cone angle that enters the
o Simple Microscope invented and used by objective lens.
Anton van Leeuwenhoek and has only one The closer the condenser to the specimen; the bigger
lens. is the cone angle.
Anton van Leeuwenhoek was the first to see
bacteria, yeasts, protists, sperm and blood cells in Cone Angle or
1673 using a 300x simple microscope. Objective Angular (theta)
Aperture
o Compound Microscope invented by Zacharias 45x dry high
Janssen in 1595 and has more than one lens. power 64 32
objective
Robert Hooke was used a compound microscope and
100x oil
was the first one to introduce the word cell in
immersion 116 58
scientific literature 1665 describing the numerous
objective
chambers (remnants of plant cells) in slices of cork.

Human eye
Another datum that is usually engraved adjacent to
- also an optical system having a resolving power of the Tube Length is the recommended thickness of
0.1mm. the coverslip to minimize spherical and chromatic
aberrations.
Resolving Power or Limit of Resolution of a microscope Coverslips have thickness ranging from 0.11mm to
0.23mm. 0.17mm is the standard recommended
- defined as its ability to gather fine details of the
thickness of the coverslip in mm.
specimen observed
o Working Distance distance between the
- more important than the magnification in evaluating
objective lens and the coverslip/slide when
the usefulness of the microscope
specimen is in focus. The longest is under the LPO
- defined by Abbes (Ernst Abbe) Equation:
and the shortest is under the OIO.
0.61 For the OIO, 1.00 is the objective magnification, 1.25
= is the numerical aperture, 160 is the tube length and

0.17 is the recommended thickness of the coverslip.
0.61 o Tube Length distance from the nosepiece-
= objective junction to the observation tube where
. . (
the ocular is inserted. It has a standard value of
- 0.61 is Abbes constant derived from light coherence 160mm for most microscopes as set by the Royal
and visibility Microscopical Society of London.
 o Xylene solvent of choice to clean hardened oil f. Stage platform under the objectives where you
sticking from the Oil Immersion Objective. place the specimen to be viewed.
g. stage aperture the opening at the stage where
Light intensity
light passes through from the condenser to the
Build-in light bulbs for microscopes can be: objectives.
h. condenser the lens located below the stage
- Tungsten (Incandescent) collects light and delivers it to the objectives in the
Incandescent like the common household light bulb is form of a cone of light whose angle is known as the
used for less expensive microscopes; it warms up angular aperture
easily and produces less intense yellowish light. i. iris diaphragm the mechanical part below the
- Fluorescent condenser which can be closed or opened with its
Fluorescent produces more intense white and cooler lever to regulate the amount of light that gets to the
light. condenser.
- Halogen Some old microscopes have the annular type,
Halogen provides brilliant light, uniform illumination different sizes of hole under the microscope.
and longer life-span. j. mirror the part that reflects light from the outside
to illuminate the field of vision
Arc lamps:
k. mirror fork the adjustable part that holds the
- mercury vapor mirror
- xenon l. base the rectangular of U-shaped part that
- zirconium supports the body of the microscope
m. arm the C-shaped part where the microscope is
grasped when it is moved or transported
Parts of the Light Compound Microscope n. fine adjustment - the knob at the body tube used to
focus the specimen viewed under the LPO
o. coarse adjustment the knob at the body tube used
to focus the specimen viewed under the LPO
p. condenser adjustment the knob at the pillar of the
microscope used to adjust the contrasting level of
the condenser
q. pillar the part connecting the body of the
microscope to the base
r. inclination joint the joint at the pillar used to tilt
the microscope for convenient observation

Exercise 2: Micrometry and the Microbial World

a. Eye piece or ocular lens where you look through in
a microscope The microbial world largest assembly of a wide variety of
b. draw tube tube where the eyepiece is attached organisms occurring nearly everywhere in nature. Major
c. dust shield the mechanical part where the group of organisms include:
revolving nosepiece is attached; protects the
Bacteria
objectives from dust Prokaryotes Cyanobacteria (blue-green
d. revolving nosepiece mechanical part where the algae)
objectives are attached; can be rotated to position Yeasts, molds, protozoa,
the objective in use. Also called Turret Eukaryotes
algae
e. Objectives the lenses above the stage attached to Viruses
the revolving nosepiece are
Scanner 4X with numerical aperture of 0.1
Viruses
Low Power Objective (LPO) 10x with
numerical aperture of 0.25 - non-living DNA or RNA nucleoprotein complex
High Power Objective (HPO) marked 40x exhibiting an obligatory intracellular parasitic nature
and sometimes called the Dry High Power
Oil Immersion Objective marked 100x
 In the classification of living organisms, there are Endospore
currently 6 proposed kingdoms and 3 domains. Metachromatic granule

6 kingdoms
Calibration of the ocular micrometer with a stage
Prokaryotes that do not micrometer
Archaea have peptidoglycan in their
cell wall
Prokaryotes which have
Bacteria peptidoglycan in their cell
wall
Unicellular eukaryotes
Protista comprising the protozoans,
algae and slime molds
Multicellular eukaryotes
Animalia which takes in their food by
ingestion
Multicellular eukaryotes
Plantae
which are photosynthetic
Multicellular non-
chlorophyllous eukaryotes
which performs Calibration Factor
Fungi
extracellular digestion and
1000
then absorbs their food =
from the outside
Other units can also be used
3 Domains 1000 = 10 = 0.01
Bacteria, Archaea, Eukarya ( BAE )

The most recent form of microbiological hazard to be Exercise 3: Preparation and Sterilization of Culture Media
recognized are the small proteinaceous infectious particles and Glasswares
are called PRIONS lack nucleic acid and VIROIDS small
circularly closed RNA molecules infecting plants. Except for The study of microorganisms in the laboratory require them
these and the viruses and some disease-producing species of to be grown or cultured in an axenic (pure; presence of a
microorganisms, more species perform important and single species) state. This condition is achieved by cultivating
beneficial roles in the grand scheme of biosphere. The them in artificial chemical preparations referred to as culture
prokaryotes and the eukaryotes are, in general, viewed under media.
the compound microscope either in their living or Culture media
fixed/stained state. This process facilitates observation of
their morphological and in some, locomotory properties. - usually come in powder form containing essential
ingredients for the metabolism of microorganisms
Lantern slides (macronutirents, micronutrients and trace elements)
- rectangular glass the size of your palm containing - pH indicators for the cultivation of microorganisms
photographic images of bacteria contained in the - can be prepared in liquid form called Broth or in
microscopic field solid form called Agar
The presence of agar is therefore a solidifying agent.
Bacilli Without it the culture medium remains in liquid
Cell Shape Cocci form.
Spirals - need to be sterilized by autoclaving, heating or
Singly membrane filtration when it is prepared.
In Pairs If it is not sterilized, then ubiquitous unwanted
Groupings In Chains microorganisms called Contaminants may
Tetrads
proliferate and grow in them thereby spoiling them
Clusters
beyond use.
Capsule
Cell Structure
Flagella
Sterilization Sterilization

- the process of killing all forms of life including heat- - voiding all microorganisms
resistant bacterial endospores and viruses
Disinfection
- it is achieved mainly through moist and dry heat
- can apply to inanimate objects
The former uses steam under pressure to achieve
- kills pathogenic microorganisms
temperatures exceeding those of boiling water. This in turn
kills contaminants. Antiseptic
most common method of moist heat - living tissues
sterilization
Autoclaving
uses to sterilize both glasswares and Sanitization
culture media
- reduce germs/microorganisms
uses the principle of convection: heat
transfer via air Culture media
process takes longer since air is not an
Dry heat
effective conductor of heat compared - a nutrient material prepared for the growth of
sterilization
to steam microbes in a laboratory
only recommended for sterilizing
glasswares Inoculum
uses special filters with known pore - microbes that are introduced into a culture medium
size that will not allow the passage of
to initiate growth
Membrane contaminants
Filtration used only for heat sensitive media Culture
components such as amino acids and
sugars - microbes that grow and multiple on a culture
medium
- comprises of:
Conditions during sterilization (autoclaving): 1. Red Algae (Gracilaria and Gelidium)
1. 15 psi or 118 KPa 2. Agar
2. 121C(250F) 3. Peptone
3. 15 to 20 minutes in the autoclave 4. Trace elements (K, P, Fe, S)
5. Yeast Extract
Exercise 4: Aseptic Technique/Transfer of Cultures 6. Casein Hydrolase
7. pH indicators
Pure cultures (axenic) are an indispensable aid in the study
8. serum, whole blood(?), heated whole blood(?)
of microorganisms because in microbiology one deals with a
population of cells and all of them must be mirror image Types of media:
(clone) of one another. Hence, all attempts must be made to
prevent the inclusion of unwanted microorganisms 1. Synthetic medium/Chemically defined medium
(contaminants) which can easily come from the immediate - medium prepared in the laboratory for materials of
environment like from the air. precise or reasonably well-defined composition
2. Complex medium
As long as asepsis is observed in every transfer, no - contains reasonably familiar materials by vary
problems should arise in the maintenance and 3. Selective medium
handling of pure cultures. - encourages the growth of some organisms but
suppresses the growth of other
Aseptic technique is a must for all activities that involve
4. Differential medium
culturing and isolating desired microorganisms. It starts
- has a constituent that causes in a color change or
from:
change in pH
a. sterilization of glasswares 5. Enrichment culture
b. preparation of media - contains special nutrients that allow the growth of a
c. culturing and subculturing particular organism that might not otherwise

Media preparation Sterilization and Inoculation

 Broth 5mL
Slant 6mL
Butt-slant 6mL
Deep/Stab/Butt 5mL

Agar plate 15-20mL

Setting the tube

a. For stabs or butts: let it solidify upright

b. For slants: set tubes in a slanted position

Broth medium
Inoculating loop
Agar Plate
Stab/deep/butt agar
Inoculating needle
Butt/Slant
Slant Inoculating loop or needle

Interrupted Multiple Streaking

- Pure Culture Technique which means that it is

used to purify and ensure of pure (axenic cultures)
from mixed cultures
o Interrupted flaming of the loop before doing a
streaking process
o Multiple streaking is done in four quadrants on
the agar plate
Colony pure culture that originate from a single cell;
visible mass of billions of cells