CHAPTER 3
OBSERVING MICROORGANISMS THROUGH A MICROSCOPE
Dr. ReitanoSUFFOLK COUNTY COMMUNITY COLLEGE
Microorganisms were first observed by Antonie van ____________, using a ________ microscope. A _____________microscope has only one lens.
Cowan “Microbiology”
RELATIVE SIZE as a TOOL in ________________
Cowan “Microbiology”
_____ System – International System of Units
SOME TYPES of MICROSCOPES• ________
– Bright field• __________ LIGHT
MICROSCOPE
– _____________
• ELECTRON– Transmission– Scanning
________________MICROSCOPY• DISTINGUISHING FEATURES
– Visible light passes through a series of ________ which magnify the specimen and allows fine detail to be observed (___________)
– Specimen appears _______ – Field appears lighter
• PRINCIPLE USES– Common, multi-
purpose microscope– Used to observe ____
specimens and preserved, stained
(___-living) specimens– Provides fair cellular
detail
_________________ MICROSCOPE
Figure 3.1b
___________ Light Microscopy
• In a compound microscope, the image from the ____________ lens is magnified again by the __________ lens
• Total magnification =objective lens ocular lens
PATHWAY of _____
Properties of a Compound Light Microscope: 1. _____________________
Magnification of _________
LensX
Magnification of Ocular
Lens
= Total
Magnification
Low Power ___X 10X 100X
High Dry ___X 10X 400X
Oil Immersion
___X 10X 1000X
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)
Figure 3.3
_________ 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 in the 100x objective lens
•____________ is used to keep light from bending
_______________ MICROSCOPY• DISTINGUISHING
FEATURES– Specimens are stained
with ____________dyes– ultraviolet light is used
which causes ___________ molecules in a specimen to emit light
PRINCIPLE USES Rapid detection and
identification of organisms in tissues
Excellent _____________ tool
ELECTRON MICROSCOPETYPES:
________________________________________________
__________ ELECTRON MICROSCOPY
• DISTINGUISHING FEATURES• Beam of __________(not
light) are reflected from the specimen
• __________dimensional image produced
• Magnification 1,000 to 10,000x
• Principle Uses• Observing _______
details of cells and viruses
___________ ELECTRON MICROSCOPY
• DISTINGUISHING FEATURES– Beam of __________ (not
light) pass through the specimen
– ___ dimensional image is produced
– Magnification 10,000 to 100,000x
PRINCIPLE USESPRINCIPLE USES Examination of _______ _____________ of cells
COMPARISON of _____ MICROSCOPES and ____________ MICROSCOPES
Cowan and Talaro
MICROSCOPIC OBSERVATION• TWO TYPES of SPECIMENS:
– 1. ____________• Wet Preps, Wet Mounts.• Living organisms suspended in fluid-organisms have
________________with surrounding fluid.• Used to study: size, shape, arrangement of cells,
(morphology), __________ , 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 motility.
PREPARATION of _______ SMEAR
_________ STAINING
Nester et al.
SMEAR: A ___ film of a solution of ________ on a slide A smear is usually ____ to ______ the microbes to the slide and to ______ the microbes. ______: Coloring the microbe with a ____ that emphasizes certain structures
VARIOUS STAINING CATEGORIESACIDIC STAINS
• __________ charge
(negative stain)• _________ by cells• _________ is stained• Ex.: India ink ________
stain
BASIC STAINS• ________ charge (positive stain)• ________ to cells (cells
have a _______ charge)• Cells are stained• Ex.: Methylene Blue, Crystal Violet, Safranin, Malachite Green
VARIOUS STAINING CATEGORIES cntd.SIMPLE
_____ dye Simple procedure Ex.:
• Methylene Blue
• Crystal Violet
• Safranin• Malachite
Green
DIFFERENTIAL Two dyes
__________ __________
Contrast 2 cell types or parts
Complex procedure
Ex.: _____ Stain ___________
Stain
SPECIAL Targets
_______ cell _______ Such as:
capsules flagella
spores Ex.:
India Ink Flagella Stain Spore Stain
_______________ STAIN_______________ STAINGRAMGRAM STAINSTAIN
• Developed by Dr. Hans Christian Gram in 1884• Most widely used procedure for staining ___________ • Classifies bacteria into two groups
– Based on differences in _________ STRUCTURE
Gram positive Gram negative
Basic _____ of Most Bacterial Cell _____
determine _______provide structural _________
Basic _______ of Bacterial Cell Walls:
COMPARISON of GRAM POSITIVE and GRAM NEGATIVE BACTERIA
GRAM ___________ BACTERIA
Ex.: Staphylococcus aureus Streptococcus
pyogenes
GRAM _________ BACTERIA
Ex.: Escherichia coli Klebsiella pneumoniae
Nester
STEPS:
Color of Gram-positive bacterial cells
Color ofGram-negative bacterial cells
1. ________ stain: Crystal Violet
Purple Purple
2. ____________: Iodine
Purple Purple
3. _____________ agent:** Acetone Alcohol
Purple _________
4. Counterstain: _____________
___________ ________
GRAM STAIN PROCEDURE
_______ STAINING PROCEDURE
Tortora
________________ STAINAcid Fast Stain
• Used for bacteria with _______ material in cell wall
• Several procedures*• Ex.:
– Mycobacteria species– Nocardia species
_____bacteria species
Nester
DIFFERENTIAL STAIN________________
• Used for bacteria with waxy, lipid (_____ ______) material in cell wall
• Several procedures*1. Primary stain: Carbolfuchsin 2. Decolorizer: _____ Alcohol3. Counter stain: Methylene blue
• Ex.:– Mycobacteria species– Nocardia species
Mycobacteria species
Nester
STEPS:
Color of ___________
Bacteria(Mycobacteria sp.)
Color ofNon–Acid-
fastBacteria
1. Primary stain: __________
Red Red
2.__________ agent:** _____-alcohol
Red ___________
3. Counter-stain: _____________
____ ______
___________ STAIN PROCEDURE
ACID FAST STAIN Acid-fast staining of a patient’s
________ 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?
SPECIAL STAINSUsed to distinguish _____ of cells
• CAPSULE
______________
ENDOSPORE
Tortora
COMPARISON of STAINS
Cowan et al.
SOME IMPORTANT STAINS USED in MICROSCOPY
________
- Methylene Blue
- Carbolfuchsin
- Crystal Violet
- Safranin
DIFFERENTIAL
- Gram
- Acid-Fast
SPECIAL
- __________
(Negative,
Acidic)
- Endospore
- Flagella
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