Download Chapter 3: Observing Microorganisms Through a Microscope

CHAPTER 3
OBSERVING MICROORGANISMS
THROUGH A MICROSCOPE
Dr. Reitano
SUFFOLK COUNTY COMMUNITY COLLEGE
Microorganisms were first observed by Antonie
van Leeuwenhoek, using a simple microscope. A simple
microscope has only ____ _____.
Cowan “Microbiology”
_________ ____ as a TOOL in IDENTIFICATION
Cowan “Microbiology”
THE __________ SYSTEM
International System of Units
Equals
Note: millimeters
are smaller than
centimeters, but
there are a larger
number of
millimeters in a
given length.
http://cassfos02.ucsd.edu/physics/ph7/Units.html
http://www.personal.psu.edu/hw7/Biology110/metricsystem.htm
SOME TYPES of MICROSCOPES
• LIGHT -uses visible light
to observe specimen
– Bright field
• COMPOUND LIGHT
MICROSCOPE
– Fluorescence
• ELECTRON-uses electrons
instead of visible light
– Transmission
– Scanning
BRIGHT FIELD
MICROSCOPY
• DISTINGUISHING FEATURES
– ______ light passes through
a series of lenses which
• PRINCIPLE USES
________ the specimen and – Common, multiallows fine detail to be
purpose microscope
observed (____________)
– Used to observe live
specimens and
– Specimen appears darker
preserved, stained
– Field appears lighter
(non-living)
specimens
– Provides fair cellular
detail
____________ LIGHT MICROSCOPE
Compound Light Microscopy
• In a compound
microscope, the
image from the
_____________ is
magnified again by
the ocular lens
• Total magnification =
objective lens 
ocular lens
PATHWAY of LIGHT
Figure 3.1b
Properties of a Compound Light Microscope:
1. __________________
Magnification of
Objective Lens
X
Magnification of
Ocular
Lens
=
Total
Magnification
Low Power
10X
10X
100X
High Dry
40X
10X
400X
100X
10X
1000X
Oil
Immersion
2. ______________
The ability of a lens system to accurately distinguish
between two separate points, that lie close to each other, as
separate and distinct.
(structures less than 0.2um cannot be resolved
with the compound light microscope)
__________ in the Compound Microscope
_________ index - a measure of
the light-bending ability of a
medium, such as air
•Air may bend the light so much
that it misses the small size of
the opening of the 100x
objective lens
•Immersion oil is used to keep
light from bending
Figure 3.3
______________ MICROSCOPY
• DISTINGUISHING
FEATURES
– Specimens are stained
with fluorescent dyes
– Ultraviolet light is used
which causes
fluorescent molecules
in a specimen to emit
light

PRINCIPLE USES
 Rapid detection and
identification of
organisms in tissues
 Excellent _________ tool
_____________ MICROSCOPE
TYPES:
_________________
TRANSMISSION
__________ ELECTRON MICROSCOPY
• DISTINGUISHING
FEATURES
• Beam of electrons (not
______) are reflected
from the specimen
• _______ dimensional
image produced
• Magnification 1,000 to
10,000x
• Principle Uses
• Observing _______
details of cells and
_________
___________ ELECTRON MICROSCOPY
• DISTINGUISHING
FEATURES
– Beam of electrons (not
light) pass through the
specimen
– ____ dimensional image is
produced
– Magnification 10,000 to
100,000x

PRINCIPLE USES


Examination of viruses
____________ of cells
ELECTRON PHOTOMICROGRAPHS
Tortora et al.
COMPARISON of ____ MICROSCOPES and
ELECTRON MICROSCOPES
Cowan and Talaro
MICROSCOPIC OBSERVATION
• TWO TYPES of SPECIMENS:
– 1. ______________
• Wet Preps, Wet __________.
• Living organisms suspended in fluid-organisms have little
contrast with surrounding fluid.
• Used to study: size, shape, arrangement of cells,
(morphology) behavior , and _______________.
– 2. STAINED
• Fixed Smear Preparations.
• _____________organisms.
• Contrast is created to allow cellular characteristics to
stand out.
• Used to study size, shape, arrangement of cells
(morphology) but not _______________.
PREPARATION of ________ SMEAR
Smear: A thin film of a solution of ________ on a slide.
A smear is usually fixed to ______ the microbes to the
slide and to kill the microbes.
Staining: ________ the microbe with a dye that
emphasizes certain structures.
_________ STAINING
Nester et al.
VARIOUS STAINING CATEGORIES
_______ STAINS
• _________ charge
(negative stain)
• Repelled by cells
• _________ is stained
• Ex.: India ink capsule
stain
BASIC STAINS
• __________ charge
(positive stain)
• ________ to cells (which
have a negative charge)
• Cells are stained
• Ex.: Methylene ______,
Crystal Violet, Safranin,
Malachite Green
VARIOUS STAINING CATEGORIES
_________
 ____ dye
 Simple
procedure
 Ex.:
• Methylene
Blue
• Crystal
Violet
• Safranin
• Malachite
Green
______________
 ____ dyes
 Primary
 Counterstain
 Contrast 2 cell
types or parts
 Complex
procedure
 Ex.:
 _____ Stain
 Acid-fast
Stain


SPECIAL
Targets specific
cell parts
 Such as:
_________
flagella
spores
Ex.:
 India Ink
 Flagella
Stain
 Spore Stain
DIFFERENTIAL STAIN
_______ STAIN
• Developed by Dr. Hans Christian Gram in 1884
• Most widely used procedure for staining bacteria
• Classify bacteria into two groups
– Based on differences in CELL _____ STRUCTURE
Gram ________
Gram negative
Basic Structure of Most _________ Cell ______
Basic Function of Bacterial Cell Walls:
determine _____________
provide structural support
COMPARISON of GRAM POSITIVE and
GRAM NEGATIVE BACTERIA
GRAM _________ BACTERIA
 Ex.:
Staphylococcus ________
Streptococcus pyogenes
GRAM ___________ BACTERIA
 Ex.:
 Escherichia ______
 Klebsiella pneumoniae
Nester
GRAM STAIN PROCEDURE
STEPS:
1. _______ stain:
Crystal violet
2. __________:
Iodine
3. Decolorizing
agent:**
Alcohol-acetone
4. _______stain:
Safranin
Color of
Gram-positive
bacterial cells
Purple
Color of
Gram-negative
bacterial cells
__________
Purple
Purple
Purple
Colorless
Purple
______
GRAM STAINING PROCEDURE
Tortora
DIFFERENTIAL STAIN
______________ Stain
• Used for bacteria with waxy, lipid (mycolic
acid) material in cell wall
• Several procedures*
1. ________ stain: Carbolfuchsin
2. Decolorizer: ______ Alcohol
3. Counter stain: Methylene blue
• Ex.:
– Mycobacterium species
– Nocardia species
Mycobacterium
species
Nester
ACID-FAST STAIN PROCEDURE
STEPS:
Color of
Acid-fast
Bacteria
(Mycobacteria sp.)
Color of
Non–Acidfast
Bacteria
Red
1. Primary stain:
Carbolfuchsin
Red
2. Decolorizing
agent:**
Acid-__________
Red
___________
_
3. Counterstain:
Methylene _____
Red
___________
ACID FAST STAIN
Acid-fast staining of a patient’s
sputum is a rapid, reliable, and
inexpensive method to diagnose
___________. What is the genus
and species of this organism?
This is an acid-fast
stain of a patient’s
__________. What is
the disease
associated with this
organism?
__________ STAINS
Used to distinguish _____ of cells
• CAPSULE

FLAGELLA

ENDOSPORE
Tortora
COMPARISON of STAINS
Cowan et al.
Identify the Types of Stains Used
2
1
3
4
SOME STAINS USED in MICROSCOPY
SIMPLE
DIFFERENTIAL
- Methylene Blue
- Gram
- Carbolfuchsin
- __________
- Crystal Violet
- __________
SPECIAL
- Capsule
(Negative,
Acidic)
- __________
- Flagella