CHAPTER 2

TOOLS IN MICROBIOLOGY
Microscope
- An instrument that gives an enlarged image of an object or substance that is minute
(tiny) or not visible with the naked eye
o Simple microscope – contains one magnifying lens
o Compound microscope – it is the common instrument used in the laboratory. It
contains two magnifying lens (the eyepiece and objectives). This type of
microscope is using a visible light (sunlight or built-in light bulb) as a source of
illumination. Hence the name “Compound light microscope.

Key characteristics of a reliable microscope are:

A. Magnification – ability to enlarge objects
o Magnification in most microscopes results from interaction between visible light
waves and curvature of the lens.
▪ angle of light passing through convex surface of glass changes –
refraction
▪ Depending on the size and curvature of the lens, the image appears
enlarged.
▪ extent of enlargement – magnification
o Magnification occurs in two phases –
▪ The objective lens forms the magnified real image. The real image is
projected to the ocular where it is magnified again to form the virtual
image.
o Linear Magnification
▪ Total magnification of the final image is a product of the separate
magnifying powers of the two lenses.
• Power of objective X Power of ocular (eyepiece) = total
magnification
▪ The eyepiece lens has a power or magnification (10x)
▪ The objective lens of the compound microscope has its own power or
magnification. Scanner (4x), Low Power Objective (10x); High Power
Objective (40x); Oil immersion (100x).

B. Resolving power – ability to show detail

o Resolution defines the capacity to distinguish or separate two adjacent objects –
resolving power function of wavelength of light that forms the image along with
characteristics of objective

Oil Immersion Objective

- A drop of immersion oil (cedar oil) must first be placed between the specimen and the
objective; the immersion oil reduces the scattering of light and ensures that the light
will enter the oil immersion lens.

Type of Light Microscope

A. Bright Field Microscope

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 - Most widely used; specimen is darker than surrounding field (objects are observed
against a bright background); live and preserved stained specimens

B. Dark Field Microscope

- Brightly illuminated specimens surrounded by dark field(objects are seen against a dark
background) live and unstained specimens

C. Phase Contrast Microscope

- Transforms subtle changes in light waves passing through the specimen into differences
in light intensity, best for observing intracellular structures

D. Fluorescence Microscope
- Contain a built-in ultraviolet (UV) light source. When UV light strikes certain dyes and
pigments, these substances emit a longer wavelength light, causing them to glow
against a dark background. Fluorescence microscopy is often used in immunology
laboratories to demonstrate that antibodies stained with a fluorescent dye have
combined with specific antigens; this is a type of immunodiagnostic procedure and
diagnosis of infection.

Electron Microscope
• Forms an image with a beam of electrons that can be made to travel in wavelike
patterns when accelerated to high speeds
• Electron waves are 100,000 times shorter than the waves of visible light.
• Electrons have tremendous power to resolve minute structures because resolving
power is a function of wavelength.
• Magnification between 5,000X and 1,000,000X
• Type of Electron Microscope
• Transmission electron microscopes (TEM) – transmit electrons through the
specimen. Darker areas represent thicker, denser parts and lighter areas
indicate more transparent, less dense parts.
• Scanning electron microscopes (SEM) – provide detailed three-dimensional
view. SEM bombards surface of a whole, metal-coated specimen with electrons
while scanning back and forth over it.

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Techniques in Studying Microorganism

A. Slide Preparation for Microscope

- Wet mounts and hanging drop mounts – allow examination of characteristics of live
cells: motility, shape, and arrangement
- Fixed mounts are made by drying and heating a film of specimen. This smear is stained
using dyes to permit visualization of cells or cell parts.
o Fixation is the process by which the internal and external structures of cells and
microorganisms are preserved and fixed in position. A microorganism usually is
killed and attached firmly to the microscope slide during fixation. There are two
fundamentally different types of fixation.
1. Heat-fix bacterial smears – by gently flame heating an air-dried film of
bacteria. This adequately preserves overall morphology but not
structures within cells.
2. Chemical fixation – by adding chemicals (fixatives) to smear. It is used to
protect fine cellular substructure and the morphology of larger, more
delicate microorganisms.

- Staining Techniques
o Dyes create contrast by imparting a color to cells or cell parts.
• Basic dyes - cationic, with positive charges on the chromophore
• Basic dyes bind to negatively charged molecules like nucleic acids
and many proteins. Because the surfaces of bacterial cells also are
negatively charged, basic dyes are most often used in bacteriology.

• Acidic dyes - anionic, with negative charges on the chromophore

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 • Acid dyes, because of their negative charge, bind to positively
charged cell structures.

• Positive staining – surfaces of microbes are negatively charged and attract

basic dyes
• Negative staining – microbe repels dye, the dye stains the background (ex.
Capsule staining)

o Other Staining Techniques

• Simple stains – one dye is used; reveals shape, size, and morphological
arrangement of cell
• Differential stains - divide bacteria into separate groups based on staining
properties.
• use a primary stain and a counterstain to distinguish cell types or
parts (examples: gram stain, acid-fast stain)
• Gram Staining – this stains the cell wall of the bacteria specifically
the peptidoglycan
Stain Gram Positive Gram Negative
Primary Stain (Crystal Violet) Blue - Purple Blue – Purple
Mordant (Grams Iodine) Blue – Purple Blue – Purple
Decolorizer (Ethanol or Blue – Purple Colorless
Acetone)
Counter Stain (Safranin) Blue – Purple Red – Pink

• Acid-Fast Staining – This method for differential staining of bacteria

is based on the high lipid content of acid-fast cell wall known as
mycolic acid. A few species, particularly those in the genus
Mycobacterium reacts with this type of stain
Stain Acid-Fast Positive Acid-Fast Negative
Primary stain (Carbol Red Red
Fuchsin)
Mordant (Heat) Red Red
Decolorizing Agent (Acid Red Colorless
alcohol)
Counterstain (Methylene Red Blue
blue)

• Special stains – reveal certain cell parts not revealed by conventional

methods: capsule and flagellar stains and endospore stain.
• Capsule Staining – Capsule composed of carbohydrate or
glycoprotein that contributes to virulence by preventing phagocytic
cells. There are few bacteria species posses this structure such as
Streptococcus pneumoniae and Klebsiella pneumoniae.
• Flagella Staining – Flagella are tiny hair-like structure of locomotion.
• Endospore Staining – Endopsore is a body formed within the
vegetative cells of bacteria. This is a specialized structure for
survival and protection against changes of environment such as
intense heat. There are two genera that are commonly known to
have endospore namely: Bacillus and Clostridium. Endospore former
are difficult to destroy, thus autoclave and special sporocidal
disinfectants are used to kill such microorganism.
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B. Culturing Microorganism
1. Inoculation – introduction of a sample into a container of media to produce a culture of
observable growth
2. Isolation –separating one species from another
3. Incubation – under conditions that allow growth – temperature-controlled chamber at
appropriate temperature and atmosphere; microbe multiplies and produces
macroscopically observable growth
4. Inspection – observation; macroscopic and microscopic
– pure culture – grows only single known species of microorganisms
– mixed cultures – hold two or more identified species or microorganisms
– contaminated culture – once pure or mixed culture that has unwanted microbes
growing
5. Identification – macroscopic and microscopic appearance, biochemical tests, genetic
characteristics, immunological testing

Isolation Technique
• If an individual bacterial cell is separated from other cells and has space on a nutrient
surface, it will grow into a mound of cells - a colony.
• A colony consists of one species.
• Isolation techniques include:
• Streak plate technique – A culture produced by lightly stroking an inoculating
needle or loop with inoculum over the surface of a solid medium. The microbial
mixture is transferred to the edge of an agar plate with an inoculating loop or
swab and then streaked out over the surface in one of several patterns. The
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 principle behind streak plate is separating species of microorganism from the
other species and will multiply and give rise to one specific colony.

• Pour plate technique - The original sample is diluted several times to reduce the
microbial population sufficiently to obtain separate colonies when plating. Then
small volumes of several diluted samples are mixed with liquid agar that has
been cooled to about 45°C, and the mixtures are poured immediately into
sterile culture dishes.

• Spread plate technique - A small volume of dilute microbial mixture containing

around 30 to 300 cells is transferred to the center of an agar plate and spread
evenly over the surface with a sterile bent-glass rod. The dispersed cells develop
into isolated colonies.

Culture Media
- Media can be classified according to three properties:
1. Physical state – liquid, semisolid and solid
o Liquid – broth; does not solidify
o Semisolid – clot-like consistency; contains solidifying agent (agar or gelatin)
o Solid – firm surface for colony formation; contains solidifying agent; liquefiable
and non-liquefiable
o Most commonly used solidifying agent is agar. The following are the
characteristic of agar:
▪ A complex polysaccharide isolated from red algae
▪ solid at room temp, liquefies at boiling (100oC), does not resolidify until
it cools to 42oC
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 ▪ provides framework to hold moisture and nutrients
▪ not digestible for most microbes
o Most commonly used media:
▪ nutrient broth – liquid medium containing beef extract and peptone
▪ nutrient agar – solid media containing beef extract, peptone and agar

2. Chemical composition – synthetic (chemically defined) and nonsynthetic (complex)

o Synthetic – contains pure organic and inorganic compounds in an exact chemical
formula
o Complex or nonsynthetic – contains at least one ingredient that is not
chemically definable

3. Functional type – general purpose, enriched, selective, differential, anaerobic, transport,

assay, enumeration
o General purpose media- grows a broad range of microbes, usually nonsynthetic
o Enriched media- contains complex organic substances such as blood, serum,
hemoglobin or special growth factors required by fastidious microbes
o Selective media- contains one or more agents that inhibit growth of some
microbes and encourage growth of the desired microbes. It favors growth of
particular bacteria.
o Differential media – allows growth of several types of microbes and displays
visible differences among desired and undesired microbes
▪ Media that distinguish between different groups of bacteria and even
permit tentative identification of microorganisms based on their
biological characteristics. Blood agar is both a differential medium and
an enriched one.

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