FAR EASTERN UNIVERSITY

INSTITUTE OF HEALTH SCIENCES AND NURSING-2026

MICROBIOLOGY & PARASITOLOGY LEC- BIO1127
DEAN SALMORIN
ADAPTED FROM: POWERPOINT/LECTURE/BOOK

CHAPTER 2: TOOLS OF THE LABORATORY Isolation Technique

1. Five I’s of Microbiology  Streak plate
- Involves the streaking of a sample bacteria onto
2. Types of Media the surface of a solid agar.
- Typically done using an inoculating loop
3. Microbial size - Streak pattern allows gradual dilution where the
bacteria is gradually thinned out, for the
4. Microscopy separation of cells and colonies.
5. Specimens  Pour plate
- Involves the mixing of a volume of sample
6. Stains bacteria with a liquified medium at a temperature
that does not harm the bacteria
- Allows growth of colonies on both on the surface
of the agar and within the medium.
 Spread plate
1. FIVE I’S OF MICROBIOLOGY - Involves the spreading of a small volume of
1.1 INOCULATION sample bacteria onto the surface of a solid agar.
- Involves using a L-shaped spreader or “L-rods”
 Culture: is to grow microorganisms
 Medium (“media” if plural): is the nutrients for 1.4 & 1.5 INSPECTION AND IDENTIFICATION
microbial growth
 Inoculum: a small sample of microbes Microbes can be identified through:
 Inoculation: the “process” of introducing an inoculum  Microscopic appearance
into media to culture microbes  Characterization of cellular metabolism
 Clinical specimens are obtained from body fluids,  Determination of nutrient requirements, products given
discharges, anatomical sites, or diseased tissue off during growth, presence of enzymes, and
mechanisms for deriving energy
1.2 INCUBATION  Genetic and immunologic characteristics
 Incubator: a temperature-controlled chamber to


encourage the multiplication of microbes
Temperatures used in laboratory propagation of
2. TYPES OF MEDIA
microorganisms: 2.1 PHYSICAL STATE
Three Physical States
 20 to 45°C
 Atmospheric gases such as oxygen or carbon dioxide  Liquid
may be required for the growth of certain microbes  Semisolid
 During the incubation period, the microbe multiplies  Solid (Liquifiable)
and produces growth that is observable  Solid (Non-liquifiable)
macroscopically

1.3 ISOLATION
 Based on the concept that if an individual bacterial cell
is separated from other cells on a nutrient surface, it
will produce a discrete mound of cells called a colony
The “GROWTH” itself

 Colony: a macroscopic cluster of cells appearing on a Agar

solid medium arising from the multiplication of a single
cell
 Isolation requires the following:
 A medium with a firm surface
 A Petri dish
 An inoculating loop (streak plate method)
 Complex polysaccharide isolated from Gelidium
 Solid at room temperature
 Liquefies at 100°C; solidifies at 42°C
 Flexible and moldable
 Not a digestible nutrient for most microorganisms

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 FAR EASTERN UNIVERSITY
INSTITUTE OF HEALTH SCIENCES AND NURSING-2026
MICROBIOLOGY & PARASITOLOGY LEC- BIO1127
DEAN SALMORIN
ADAPTED FROM: POWERPOINT/LECTURE/BOOK
2.2 CHEMICAL CONTENT OF MEDIA
Defined or synthetic
 Composition is precisely chemically defined
 Contain pure organic and inorganic compounds that
vary little from one source to another
 Molecular content specified by means of an exact
formula
Complex
 One or more components is not chemically defined
 Contains extracts of animals, plants, or yeasts
 Blood, serum, meat extracts or infusions, milk, yeast
extract, soybean digests, and peptone
Oftenly used complex components are blood, meat extracts,
and peptone.
2.3 MEDIA FOR DIFFERENT PURPOSES
General-purpose Media
 Grow as broad a spectrum of microbes as possible
 Generally complex
Enriched Media
 Contains complex organic substances such as blood,
serum, hemoglobin, or special growth factors for the
growth of fastidious microbes
 Used in the clinical laboratory to encourage growth of
pathogens present in low numbers
Blood agar is different from chocolate agar, blood agar is
uncooked and chocolate agar is cooked blood agar.
Selective Media
Contains one or more agents that inhibit the growth of
a certain microbe or microbes:
 Important in the primary isolation of a specific type of
microorganism from a sample containing dozens of
different species
 Speed up isolation by suppressing unwanted
background organisms and favoring the growth of the
desired ones
Differential Media
Allows multiple types of organisms to grow but display
visible differences in how they grow.
 Variations in colony size or color
 Media color changes
 Production of gas bubbles
 Variations often come from chemicals in the media with
which microbes react
Selective AND Differential Media
A medium can be both selective and differential:
 Example: MacConkey agar suppresses the growth of
some organisms while producing a visual distinction
between the ones that do grow
 Dyes are used as differential agents because many are
pH indicators that change color in response to the
production of an acid or a base
Miscellaneous Media
 REDUCING MEDIA
- Contains a substance that absorbs oxygen or
slows the penetration of oxygen
- Important for growing anaerobic bacteria
 TRANSPORT MEDIA
- Used to maintain and preserve specimens that
have to be held for a period of time before clinical
analysis
 CARBOHYDRATE FERMENTATION MEDIA
- Contains sugar that can be fermented with a pH
indicator to show this reaction
 ASSAY MEDIA
- Used by technologists to test the effectiveness of
antimicrobial drugs
- Used by drug manufacturers to test the
effectiveness of disinfectants, antiseptics,
cosmetics, and preservatives on microorganisms
 ENUMERATION MEDIA
- Used by industrial and environmental
microbiologists to count the numbers of organisms
in milk, water, food, soil, and other samples
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 FAR EASTERN UNIVERSITY
INSTITUTE OF HEALTH SCIENCES AND NURSING-2026
MICROBIOLOGY & PARASITOLOGY LEC- BIO1127
DEAN SALMORIN
ADAPTED FROM: POWERPOINT/LECTURE/BOOK

 Diaphragm: a part of the condenser that controls the

3. MICROBIAL SIZE
 Yeast is generally 3 to 4 µm
 The smallest bacteria measure around 200 nm; largest
around 750 µm
 Most viruses measure between 20 nm and 400 nm;
some can be as big as 800 nm or 1500 nm (as big as
cells)
amount pf light passing through the specimen.
Regulates brightness and contrast of the image..
 Light source: The light source.
 Coarse and fine adjustment knob: Usually located
on the side of the microscope, it allows you to adjust
focus of the objective lenses. Coarse adjustment
moves the stage rapidly while fine adjustment moves
the stage slowly for precise focusing.

3.1 Size of Macroscopic versus Microscopic

Organisms
 The dimensions of macroscopic organisms are given
in centimeters (cm) and meters (m)
 The dimensions of microscopic organisms range from
millimeters (mm), to micrometers (μm), to nanometers
(nm)
 Arm: the curved part of the microscope that connects
the eyepiece and the base. (Provides stability and
support)
 Base: bottom of the microscope that provides stability
and support to the entire instrument.
 Revolving nosepiece: holds the objective lenses in
place and allows you to switch between magnifications
by rotating it.

4.2 MAGNIFICATION
Magnification occurs in two phases:
4. MICROSCOPY
4.1 PARTS OF A MICROSPCOPE
 Real image: formed by the objective

 Eyepiece/Ocular lens: Lens at the top of a

 Virtual image: formed when the image is projected up
through the microscope body to the plane of the
microscope that you look through, 10x magnification.
eyepiece, the ocular lens forms a second image
 Objective lenses: A set of lenses located on a rotating
nosepiece just above the specimen stage. Provides Total Magnification:
different levels of magnification. 4x scanner, 10x LPO
(low power objective), 40x HPO (high dry objective,
and 100x oil immersion objective lens.
 Stage: a flat platform below the objective lenses where
the specimen is placed for observation
 Condenser: located beneath the stage, it focuses and
directs the light from the light source onto the
specimen. Helps enhance the illumination and clarity of
view.

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 FAR EASTERN UNIVERSITY
INSTITUTE OF HEALTH SCIENCES AND NURSING-2026
MICROBIOLOGY & PARASITOLOGY LEC- BIO1127
DEAN SALMORIN
ADAPTED FROM: POWERPOINT/LECTURE/BOOK

4.3 RESOLUTION 4.4 RESOLUTION

Also known as resolving power  Refractive index:
 The degree of bending that light undergoes as it
The capacity of an optical system to distinguish two passes from one medium to another
adjacent objects or points from one another:  The higher the difference in the refractive indexes (the
more bending of light), the sharper the contrast
 Resolving power of the human eye: 0.2 mm registered by the microscope and the eye
 Resolving power of the light microscope using the oil  Too much light can reduce contrast and burn out the
immersion lens: 0.2 μm image:
 Iris diaphragm: controls the amount of light coming into
Oil Immersion Lens Reduces Light Scatter, the condenser
Increasing Resolution  Increase contrast with special lenses (such as a phase-
contrast microscope) and by adding dyes

4.4 TYPES OF MICROSCOPY

Bright-Field Microscopy
 The most widely used type of light microscope
 Forms its image when light is transmitted through the
specimen
 The specimen, being denser and opaquer than its
surroundings, absorbs some of this light, and the rest
of the light is transmitted directly up through the ocular
 Can be used for both live, unstained material and
preserved, stained material

Importance of Resolution

Dark-Field Microscopy
 A bright-field microscope can be adapted as a dark-
field microscope by adding a special disc called a stop
to the condenser
 The stop blocks all light from entering the objective
lens, except peripheral light that is reflected off the
sides of the specimen itself
 The resulting image is a particularly striking one:
brightly illuminated specimens surrounded by a dark
(black) field
 The most effective use is to visualize living cells that
would be distorted by drying or heat or that cannot be
stained with the usual methods

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 FAR EASTERN UNIVERSITY
INSTITUTE OF HEALTH SCIENCES AND NURSING-2026
MICROBIOLOGY & PARASITOLOGY LEC- BIO1127
DEAN SALMORIN
ADAPTED FROM: POWERPOINT/LECTURE/BOOK
Phase-Contrast Microscopy
 The phase-contrast microscope has been constructed
to take advantage of the fact that cell structures differ
in density
 Contains devices that transform the subtle changes in
light waves passing through the specimen into
differences in light intensity
 The amount of internal detail visible by this method is
greater than by either bright-field or dark-field methods
 Most useful for observing intracellular structures such
as bacterial endospores, granules, and organelles, as
well as the locomotor structures of eukaryotic cells
such as cilia
Fluorescence Microscopy
 The fluorescence microscope is a specially modified
compound microscope furnished with an ultraviolet
(UV) radiation source
 The name comes from the use of certain dyes
(acridine, fluorescein) and minerals that are
fluorescence. The dyes emit visible light when
bombarded by short ultraviolet rays.
 For an image to be formed, the specimen must first be
coated or placed in contact with a source of
fluorescence
 Shining ultraviolet radiation on the specimen cause it
to give off light that will form its own image, usually an
intense red, blue, or green against a black field
 Has its most useful applications in diagnosing
infections and pinpointing particular cellular structures
Confocal Microscopy
 The scanning confocal microscope overcomes the
problem of cells or structures being too thick by using
a laser beam of light to scan various depths in the
specimen and deliver a sharp image focusing on just a
single plane
 Able to capture a highly focused view at any level,
ranging from the surface to the middle of the cell
 Most often used on fluorescently stained specimens
but it can also be used to visualize live unstained cells
and tissues
Transmission Electron Microscope (TEM)
 TEM - the method of choice for viewing the detailed
structure of cells and viruses
 Produces its image by transmitting electrons through
the specimen
 Because electrons cannot easily penetrate thick
preparations, the specimen must be sectioned into
extremely thin slices (20–100 nm thick) and stained or
coated with metals that will increase image contrast
 The darker areas of TEM micrographs represent the
thicker (denser) parts, and the lighter areas indicate the
more transparent and less dense parts
Scanning Electron Microscope (SEM)
 SEM provides some of the most dramatic and realistic
images in existence
 Designed to create an extremely detailed three-
dimensional view of all kinds of objects, from plaque on
teeth to tapeworm heads
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 FAR EASTERN UNIVERSITY
INSTITUTE OF HEALTH SCIENCES AND NURSING-2026
MICROBIOLOGY & PARASITOLOGY LEC- BIO1127
DEAN SALMORIN
ADAPTED FROM: POWERPOINT/LECTURE/BOOK

 SEM bombards the surface of a metal-coated Hanging Drop Technique

specimen with electrons while scanning back and forth
over it
 A shower of electrons deflected from the surface and
the electron pattern is displayed as an image on a
television screen
 The color is always added afterward; the actual
microscopic image is black and white

5. SPECIMENS
5.1 PREPARING SPECIMENS FOR THE
MICROSCOPE
Specimens are generally prepared by mounting a
sample on a glass slide that sits on the stage between
the condenser and the objective
The manner in which a specimen is prepared depends
on:
 The condition of the specimen: living or dead
 The aims of the examiner: observation of overall
structure, identification, or movement
 The type of microscopy available: bright-field, dark-
field, phase-contrast, or fluorescence
5.2 FRESH, LIVING PREPARATIONS
Placed on wet mounts or in hanging drop mounts to
observe as close to the natural state as possible
6. STAINS
Unstained cells in a fixed smear are difficult to see,
regardless of magnification and resolving power
Staining
 is any procedure that applies colored chemicals (dyes)
to specimens:
 Basic dyes have a positive charge
 Acidic dyes have a negative charge
Bacteria have numerous negatively charged substances
and attract basic dyes
Acidic dyes are repelled by cells
6.1 Negative versus Positive Staining
Positive Stain
 dye sticks to the specimen and gives it color
Negative Stain
 does not stick to the specimen but settles some
distance from its outer boundary, forming a silhouette:
 Negatively charged cells repel the negatively charged
dye and remain unstained
 Smear is not heat fixed so the distortion and shrinkage
of cells is reduced

Cells are suspended in water, broth, or saline to  Also used to accentuate a capsule

maintain viability and provide a medium for locomotion  Nigrosin and India ink are used

Wet mount: 6.2 Simple versus Differential Staining

 Consists of a drop or two of culture placed on a slide

and overlaid with a cover slip Simple stains
Hanging drop:
only require a single dye and an uncomplicated
 A drop of culture is placed in a concave (depression)
procedure:
slide, Vaseline adhesive or sealant, and cover slip are
used to suspend the sample
 Cause all the cells in the smear to appear more or less
the same color, regardless of type

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 FAR EASTERN UNIVERSITY
INSTITUTE OF HEALTH SCIENCES AND NURSING-2026
MICROBIOLOGY & PARASITOLOGY LEC- BIO1127
DEAN SALMORIN
ADAPTED FROM: POWERPOINT/LECTURE/BOOK

 Reveal shape, size, and arrangement Differential Stains: Endospore Stain

 Similar to the acid-fast stain in that a dye is forced by
heat into resistant bodies called endospores
 Stain distinguishes between endospores and
vegetative cells
 Significant in identifying gram-positive, spore-forming
members of the genus Bacillus and Clostridium

Differential stains
 Use two differently colored dyes: the primary dye and
the counterstain
 Distinguish cell types or parts
 More complex and require additional chemical
reagents to produce the desired reaction Special Stains
Used to emphasize cell parts that are not revealed by
Differential Stains: The Gram Stain
conventional staining methods
Developed in 1884 by Hans Christian Gram
Capsular staining
Consists of sequential applications of:
 Used to observe the microbial capsule, an
unstructured protective layer surrounding the cells of
 Crystal violet (the primary stain) some bacteria and fungi
 Gram’s iodine (the mordant)  Negatively stained with India ink
 An alcohol rinse (decolorizer)
 A contrasting counterstain (for example Safranin) Flagellar staining
 Different results in the Gram stain are due to  Used to reveal tiny, slender filaments used by bacteria
differences in the structure of the cell wall and how it for locomotion
reacts to the series of reagents applied to the cells  Flagella are enlarged by depositing a coating on the
 Remains the universal basis for bacterial classification outside of the filament and then staining it
and identification
 A practical aid in diagnosing infection and guiding drug
treatment

Differential Stains: Acid-Fast Stain

Differentiates acid-fast bacteria (pink) from non-acid-
fast bacteria (blue)

Originated as a method to detect Mycobacterium

tuberculosis:

 These bacteria cell walls have a particularly impervious

cell wall that holds fast (tightly or tenaciously) to the
dye (Carbol fuchsin) when washed with an acid alcohol
decolorizer
Also used for other medically important bacteria, fungi,
and protozoa

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