1. CHAPTER 3 OBSERVING MICROORGANISMS THROUGH A MICROSCOPE
Dr. Reitano
SUFFOLK COUNTY COMMUNITY COLLEGE

2. Microorganisms were first observed by Antonie van Leeuwenhoek, using a simple microscope. A simple microscope has only ____ _____.
Cowan “Microbiology”

3. _________ ____ as a TOOL in IDENTIFICATION
Cowan “Microbiology”

4. 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.

5. 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

6. BRIGHT FIELD MICROSCOPY
DISTINGUISHING FEATURES
______ light passes through a series of lenses which ________ the specimen and allows fine detail to be observed (____________)
Specimen appears darker
Field appears lighter
PRINCIPLE USES
Common, multi-purpose microscope
Used to observe live specimens and preserved, stained (non-living) specimens
Provides fair cellular detail

7. ____________ LIGHT MICROSCOPE

8. Compound Light Microscopy
PATHWAY of LIGHT
In a compound microscope, the image from the _____________ is magnified again by the ocular lens
Total magnification = objective lens  ocular lens
Figure 3.1b

9. Properties of a Compound Light Microscope: 1. __________________
Magnification of Objective Lens X
Magnification of Ocular
Lens =
Total Magnification
Low Power
10X
100X
High Dry
40X
400X
Oil Immersion
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)

10. __________ 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

11. ______________ MICROSCOPY
PRINCIPLE USES
Rapid detection and identification of organisms in tissues
Excellent _________ tool
DISTINGUISHING FEATURES
Specimens are stained with fluorescent dyes
Ultraviolet light is used which causes fluorescent molecules in a specimen to emit light

12. _____________ MICROSCOPE TYPES: _________________ TRANSMISSION

13. __________ ELECTRON MICROSCOPY
Principle Uses
Observing _______ details of cells and _________
DISTINGUISHING FEATURES
Beam of electrons (not ______) are reflected from the specimen
_______ dimensional image produced
Magnification 1,000 to 10,000x

14. ___________ 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

15. ELECTRON PHOTOMICROGRAPHS
Tortora et al.

16. COMPARISON of ____ MICROSCOPES and ELECTRON MICROSCOPES
Cowan and Talaro

17. 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 _______________.

18. 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.

19. 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

20. VARIOUS STAINING CATEGORIES
_________
____ dye
Simple procedure
Ex.:
Methylene Blue
Crystal Violet
Safranin
Malachite Green
SPECIAL
Targets specific cell parts
Such as: _________ flagella
spores
Ex.:
India Ink
Flagella Stain
Spore Stain
______________
____ dyes
Primary
Counterstain
Contrast 2 cell types or parts
Complex procedure
Ex.:
_____ Stain
Acid-fast Stain

21. 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

22. Basic Structure of Most _________ Cell ______
Basic Function of Bacterial Cell Walls:
determine _____________ provide structural support

23. COMPARISON of GRAM POSITIVE and GRAM NEGATIVE BACTERIA
GRAM _________ BACTERIA
Ex.:
Staphylococcus ________
Streptococcus pyogenes
GRAM ___________ BACTERIA
Ex.:
Escherichia ______
Klebsiella pneumoniae
Nester

24. Gram-positive bacterial cells Gram-negative bacterial cells
Gram Stain PROCEDURE
STEPS:
Color of
Gram-positive bacterial cells
Gram-negative bacterial cells
1. _______ stain:
Crystal violet
Purple
__________
2. __________:
Iodine
3. Decolorizing
agent:**
Alcohol-acetone
Colorless
4. _______stain:
Safranin
______

25. GRAM STAINING PROCEDURE
Tortora

26. 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

27. Acid-Fast Stain Procedure
STEPS:
Color of
Acid-fast Bacteria
(Mycobacteria sp.)
Non–Acid-fast
Bacteria
1. Primary stain:
Carbolfuchsin
Red
2. Decolorizing agent:**
Acid-__________
____________
3. Counterstain:
Methylene _____
___________

28. 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?

29. __________ STAINS Used to distinguish _____ of cells
CAPSULE
FLAGELLA
ENDOSPORE
Tortora

30. COMPARISON of STAINS
Cowan et al.

31. Identify the Types of Stains Used2 1 4 3

32. SOME STAINS USED in MICROSCOPY
SIMPLE
- Methylene Blue
- Carbolfuchsin
- Crystal Violet
- __________
DIFFERENTIAL
- Gram
- __________
SPECIAL
- Capsule
(Negative,
Acidic)
- __________
- Flagella