Biol 106 Unit III

OBSERVING MICROBES
Through
MICROSCOPES
PREPARED BY: BRENT JOY H. HERNANDO 1

MICROSCOPY
the science that studies
microscope techniques
Latin word: Micro- small
Greek word: Skopos- to look at
Units of Measurement
Image taken from:

Tortora et al., 2016. Microbiology: An Introduction 12th Edition

MICROSCOPY:
THE INSTRUMENTS
“Windows in
an invisible
Realm”
Retrieved from: http://pinterest.com

MICROSCOPY:
THE INSTRUMENTS
“Leeuwenhoek’s
single lens
microscope could
magnify microbe up
to 300X”

MICROSCOPY:
The Instruments: Simple vs Compound
Simple- contains a single magnifying lens
Compound- (two-lens), additional lens

- a lamp in the base to give off visible light and illuminate
the specimen.
- a special lens called the condenser that converges or
focuses the rays of light to a single

LIGHT MICROSCOPY
refers to the use of any kind

of microscope that uses
visible light to observe
specimens.
PRINCIPLES OF LIGHT MICROSCOPY:
Compound Light Microscopy
Magnification- the ability to make objects appear
enlarged.
Resolving Power- (Resolution) the ability to show

detail.

Magnification
Magnification in most microscopes
results from interaction between
visible light waves and curvature of
the lens
Total magnification of the final image is

a product of the separate magnifying
powers of the two lenses.

Magnification
Image taken from:

Talaro, K.P.& Chess, B., 2015. Foundations in Microbiology 9th Edition

Resolution
Resolution refers to the ability of the
lenses to distinguish two points that
are a specified distance apart
A general principle of microscopy is that
the shorter the wavelength of light used in
the instrument, the greater the resolution
The white light used in a compound light microscope has a relatively long
wavelength and cannot resolve structures smaller than about 0.2 μm.

Resolution
Visible light wavelength in a spectrum is 400 nm–750 nm
Violet-blue portion- shortest (400nm)
Red portion- longest (750nm)
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Resolution
Shorter wavelength and

larger numerical aperture
will provide better
resolution
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Resolution: Refractive index
a measure of the
light-bending ability
of a medium.
Resolution
Oil immersion lens

requires the use of oil
to prevent refractive
loss of light
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MICROSCOPY RANGES

LIGHT MICROSCOPY:
Compound Light Microscopy
Under usual operating

conditions, the field of vision in a
compound light microscope is
brightly illuminated. By focusing
the light, the condenser
produces a brightfield
illumination
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TYPES OF MICROSCOPY:
Brightfield vs Darkfield vs Phase-contrast
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VARIOUS TYPES OF MICROSCOPY:
Light Microscopy
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Light Microscopy
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Confocal & Two-Photon Microscopy
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Scanning Acoustic Microscopy
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Electron Microscopy
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Electron Microscopy
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Scanned-Probed Microscopy
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PREPARATION OF SPECIMEN FOR
LIGHT MICROSCOPY
Most microorganisms appear

almost colorless when viewed
through with brightfield
microscopy
PREPARATION OF SPECIMEN FOR
LIGHT MICROSCOPY
One way is to
stain
the specimen Retrieved from: http://www.istockphoto.com/photos/paint-splash

PREPARATION OF SPECIMEN :
Preparing Smears for staining
Staining simply means

coloring the microorganisms
with a dye that emphasizes
certain structures.
Smear is a thin film of material

containing the microorganisms
is spread over the surface of
the slide after heat fixation
Retrieved from: http://microbeonline.com/wp-content/uploads/2016/05/Simple-Staining.jpg

Stains are salts composed of a
positive and a negative ion,
one of which is colored and is
known as the chromophore.

Basic Dyes Acidic Dyes
Cation (+) Anion (-)
➢ Crystal violet ➢ Eosin

➢ Methylene blue ➢ Acid fuchsin
➢ Malachite green ➢ Nigrosin
➢ Safranin

Types of staining: Positive vs Negative
(-)
(+)
Colorless
specimen
Actual
against a
specimen is
colored
stained
background

Kinds of staining
Image taken from:

Simple staining
A simple stain is an
aqueous or alcohol
solution of a single basic
dye.
Simple staining
a chemical is added to the

solution to intensify
the stain; such an additive is
called a mordant.
Differential staining
differentiate bacteria
according to their reactions
to the stains.
Differential staining: Gram Staining
developed in 1884 by the Danish bacteriologist Hans Christian Gram
Image taken from:


Differential staining: Acid-fast staining
developed in 1882 by Paul Ehrlich
Image taken from:

https://microbiologyinfo.com/category/staining-techniques/

Special Staining: Capsule, Endospore, Flagella
Image taken from:

https://microbiologyinfo.com/category/staining-techniques/

REFERENCES:
E-BOOKS:
1. Tortora, G.J.; Funke, B. R.; Case. C. L., 2016. Microbiology : an introduction 12th Edition. Pearson Education Inc., United States
of America. ISBN 10: 0-321-92915-2; ISBN 13: 978-0-321-92915-0 (Student edition)
2. Talaro,K. P.; Chess, B., 2015. Foundations in microbiology 9th Edition -Pasadena City College, Barry Chess, Pasadena City
College. ISBN 978–0–07–352260–9
WEBSITES:
1. https://microbiologyinfo.com/category/staining-techniques/
2. http://microbeonline.com/wp-content/uploads/2016/05/Simple-Staining.jpg
3. http://pinterest.com

END
THANK YOU.

Biol 106 Unit III

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OBSERVING MICROBES

PREPARED BY: BRENT JOY H. HERNANDO 1

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Compound- (two-lens), additional lens

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refers to the use of any kind

Resolving Power- (Resolution) the ability to show

PREPARED BY: BRENT JOY H. HERNANDO 8

Total magnification of the final image is

PREPARED BY: BRENT JOY H. HERNANDO 9

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Shorter wavelength and

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Oil immersion lens

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Under usual operating

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Most microorganisms appear

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Staining simply means

Smear is a thin film of material

Retrieved from: http://microbeonline.com/wp-content/uploads/2016/05/Simple-Staining.jpg

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➢ Crystal violet ➢ Eosin

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a chemical is added to the

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