Chapter 2 Bio461

CHAPTER 2

Observing Microorganisms
Through a Microscope

2.1 Units of measurement

2.2 Specimen preparations
2.3 Staining techniques in microbiology
2.1 Units of measurement
• Microorganisms are measured in units
that are unfamiliar to many of us in
everyday life.
• The standard unit of length in the metric
system is the meter (m).
• But, microorganisms and their structural
components are measured in even
smaller units, such as a micrometers and
nanometers.
Advantages : its all related to each other by

factors of 10.
BIO461:Mic/DNA/UiTM NS_2017
• A micrometer (µm) = 0.000001 m = (10-6m).
• The prefix micro indicates that the unit following it should
be divided by 1 million, or 106.
• A nanometer (nm) = 0.000000001 m = (10-9m).
• Angstrom (Å) = 0.1nm = (10-10 m) .
Table 2.1 Metric Units of Length and US Equivalents
Metric Unit Meaning of Prefix Metric Equivalent US Equivalent
1 kilometer (km) Kilo=100 1000 m=103m 3280.84 ft or 0.62 mi
1 meter (m) Standard unit of length 39.37 in or 3.28 ft or

1.09 yd
1 decimeter (dm) Deci=1/10 0.1 m=10-1m 3.94 in
1 centimeter (cm) Centi = 1/100 0.01 m=10-2m 0.394 in; 1 in = 2.54 cm
1 milimeter (mm) Mili=1/1000 0.001 m=10-3m
1 micrometer (µm) Micro=1/1000 000 0.000001 m=10-6m
1 nanometer (nm) Nano=1/1000 000 000 0.00000000 m=10-9m
1 picometer (pm) Pico=1/1000 000 000 000 0.00000000000 m=10-12m
Microscopy : The instruments
• Microscopes are
essential for
microbiological
studies
• Light microscopes:
cellular resolution
– bright-field (stains)
– dark-field
– phase contrast
– fluorescence (stains)
• Electron
microscopes:
subcellular
resolution
a. Light Microscopy: Optics
• Refers to the use of any kind of microscope that uses visible light to
observe specimens.
• Visualization depends on magnification
(lenses) and resolution (physical
properties of light)
• The limit of resolution for a light microscope is
about 0.2 m (or 200 nm)
– Objects closer than 0.2 m cannot be resolved
• Total magnification is product of the
magnification of its ocular and its
objective lenses
• Compound LM
• Series of lenses
• Use visible light as source of illumination.
• To examine very small specimen and fine detail.
Resolution (resolving power) is the ability of the lenses to distinguish
fine detail and structure of specimens.
– Ability of lenses to distinguish 2 points a specified distance apart.

– Ex. : Microscopes with 0.4nm resolving power : it can distinguish 2
points if they are at least 0.4 nm.
– Shorter wavelength of light used, the greater the resolution.
• Refractive Index is a measure of the light bending ability of a

medium.
- We can change the RI of specimens by

staining the specimens.
- To achieve high magnification (1000x) with
good resolution, the objective lens must be
small ( do not want to lose light rays after
they have passed through the stained
specimens)  Immersion oils.
b. Phase contrast microscopy
• Permit detailed examination of
internal structures in living
microorganisms.
• Not necessary to fix (attach the
microbes to the microscope slide) or
stain the specimen- procedure that
could distort of kill the
microorganisms.
• Internal structure of cell become more
sharply defined.
c. Dark-field microscopy
• Use to examine live microorganisms that either
are invisible in the ordinary light microscope,
cannot be stained by standard methods or are so
distorted by staining that their characteristics
then cannot be identified.
d. Fluorescence Microscopy
• Takes advantage of fluorescence, the ability of
substances to absorb short wavelength of light
(ultraviolet) and give off light at a longer
wavelength (visible)
e. Differential Interfere Contrast (DIC) Microscopy
• Similar to phase-contrast microscopy but using difference refractive indexes.
• Using 2 beam of light instead of one.
• Split by prisms, adding contrasting colors to the specimen.
• Therefore, resolution of DIC microscope is HIGHER than that of a standard
phase—contrast microscope.
f. Confocal Microscope
• Technique in light microscopy used to reconstruct
3D images.
• Specimens are stained with fluorochromes so
they will emit or return light.
1
6 2
5 3
2.2 Specimens preparations
1
Because most microorganisms appear almost colorless when viewed
through a standard light microscope.
3
2
Before the microorganisms can be
stained, however they must be fixed
(attached)to the microscope slide.
• A thin film of material

containing the 4
5 microorganisms is spread
over the surface of the slide.
Fixing simultaneously kills and
• This film, called a SMEAR is fixes them to the slide
allowed to air dry.
• Stains are salts composed of a positive and a
negative ion, one of which is colored and is
known as chromophore.
ACIDIC DYES BASIC DYES

• +ve ions • -ve ions
• Acidic dyes • Basic dyes
Examples : Crystal violet, Examples : Eosin, Acid
Methylene blue, fuchsin, Nigrosin
Malachite green
2.3 Staining techniques in microbiology
• Staining is used to increase contrast in bright-field
microscopy
Simple staining : an aqueous or alcohol solution of

1 a single basic dye
Differential staining : combination of dyes allows

differential staining of different populations 2
Special staining : used to color and isolate specific

3 parts of microorganisms, such as endospores,
flagella and to reveal the presence of capsules.
Simple stain
•One dye stains all cells
•Eventhough different dyes bind
specifically to different parts of cells,
the primary purpose of a simple stain is
to highlight the entire microorganism
so that cellular shapes and basic
structures are visible.
• The stain is applied to the fixed smear
for a certain length of time and then
washed off and the slide is dried and
examined.
• A chemical is added to the solution to
intensify the stain called mordant.
• Mordant function is to increase the
affinity of a stain for a biological
specimen.
•Example : methylene blue, carbol
fuchsin, crystal violet and safranin.
ANSWER ME NOW….
1. State the purpose of heat fixation in the
preparation of a bacterial stain. (3 marks) – Jun 2015
Answer :
Heat fixation kills the microbes and causes the

specimen to adhere to the glass slide so that it does
not easily wash off during staining. Fixation generally
also preserve the shape and size of the microbes.
ANSWER ME NOW….
2. Explain the relationship between contrast and
staining in microscopy. (4 marks) – Dec 2015
Answer :
Contrast refer to differences in intensity between two
objects or between an objects and its background and
important in determining resolution because microbes
are colorless.
Stain are used to make microbes and their parts more
visible because satins increase contrast between
structures and between specimen and its background.
Differential stain
• Differential stain react
differently with different kinds of
bacteria and thus can be used to
distinguish them.
•The differential stains most

frequently used for bacteria are
the Gram stain and Acid-fast
stain.
i.Gram positive cells retain the

dye and remain purple.
ii.Gram negative cells do not
retain the dye; they are colorless
until counterstained with red
dye. BIO461:Mic/DNA/UiTM NS_2017
•The Gram method is one of
the most important staining
techniques in medical
microbiology.
•Gram stain results are not

universally applicable,
because some bacterial cell
stain poorly or not at all.
•The Gram reaction is most

consistent when it is used on
young growing bacteria.
Example of Gram stain
Acid-fast stain
• The stain binds strongly only to bacteria that have
a waxy materials in their cell walls.
• The bacteria that have been stained red with an
acid-fast strain.
• Non-acid fast cells are stained with the
methylene blue counterstain.
Special stains
• Used to color and isolate specific parts of
microorganisms.
Negative staining for capsules
• It is more difficult than other types of staining
procedures because capsular material are
soluble in water and may be dislodged or remove
during rigorous washing.
Endospore staining
Endospore : a special resistant, dormant structure form within a cell
that protects a bacterium from adverse environmental conditions.
• Endospore cannot be stained by ordinary

methods, such as simple staining and Gram stain
because they do not penetrate the wall of the
endospore.
Flagella staining
• Flagella, a structure for locomotion
• Too small to be seen with a light microscope
without staining.
• A tedious and delicate staining procedure uses a
mordant and the stain Carbol fuchsin to build up
the diameter of the flagella until they become
visible under the light microscope.
Table 2.2 A summary of various stains and their uses
Stain Principal uses
Simple (methylene Used to highlight microorganisms to determine cellular shape and

blue, carbol fuchsin, arrangements. Aqueous of alcohol solution of a single basic dye
crystal violet, safranin. stains cell.
Differential : Used to distinguish different kind of bacteria

Gram
Acid-fast stain Used to distinguished Mycobacterium sp. And some special .
Special: Used to demonstrated of capsules

Negative
Endospore Used to detect the presence of endospore in bacteria
Flagella Used to demonstrated the presence of flagella
Microscopy: Dark Field
• Greater resolution
• Light reaches specimens
only from the side
• Only the specimen itself
is illuminated
Microscopy: Phase Contrast
• May be used to
visualize live
samples and avoid
distortion from cell
stain
• Image contrast is
derived from the
differential
refractive index of
cell structures.
Microscopy: Fluorescence
• Visualization of
autofluorescent cell structures
(e.g., chlorophyll) or
fluorescent stains
• Can greatly increase the
resolution of cells and cell
structures
• Many functional probes
available
Example for Differential Fluorescence Stain
*Psuedomonas (green) *Bacillus (orange)
Microscopy: Electron Microscopy
• Electron microscopes have far greater resolving
power than light microscopes, with limits of
resolution of about 0.2 nm
• Two major types of electron microscopes
– Transmission electron microscopy (TEM) for observing
internal cell structure down to the molecular level
– Scanning electron microscopy (SEM) for 3-D imaging and
examining surfaces
Electron Microscopy
TEM SEM
Pseudomonas Mycobacterium

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CHAPTER 2

2.1 Units of measurement

Advantages : its all related to each other by

• A nanometer (nm) = 0.000000001 m = (10-9m).

• Angstrom (Å) = 0.1nm = (10-10 m) .

Metric Unit Meaning of Prefix Metric Equivalent US Equivalent

1 kilometer (km) Kilo=100 1000 m=103m 3280.84 ft or 0.62 mi

1 meter (m) Standard unit of length 39.37 in or 3.28 ft or

1 centimeter (cm) Centi = 1/100 0.01 m=10-2m 0.394 in; 1 in = 2.54 cm

1 milimeter (mm) Mili=1/1000 0.001 m=10-3m

1 micrometer (µm) Micro=1/1000 000 0.000001 m=10-6m

1 nanometer (nm) Nano=1/1000 000 000 0.00000000 m=10-9m

1 picometer (pm) Pico=1/1000 000 000 000 0.00000000000 m=10-12m

– Ability of lenses to distinguish 2 points a specified distance apart.

• Refractive Index is a measure of the light bending ability of a

- We can change the RI of specimens by

• A thin film of material

ACIDIC DYES BASIC DYES

Simple staining : an aqueous or alcohol solution of

Differential staining : combination of dyes allows

Special staining : used to color and isolate specific

Heat fixation kills the microbes and causes the

•The differential stains most

i.Gram positive cells retain the

•Gram stain results are not

•The Gram reaction is most

• Endospore cannot be stained by ordinary

Stain Principal uses

Simple (methylene Used to highlight microorganisms to determine cellular shape and

Differential : Used to distinguish different kind of bacteria

Acid-fast stain Used to distinguished Mycobacterium sp. And some special .

Special: Used to demonstrated of capsules

Endospore Used to detect the presence of endospore in bacteria

Flagella Used to demonstrated the presence of flagella

*Psuedomonas (green) *Bacillus (orange)

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Psuedomonas (green) Bacillus (orange)