1. Tools of the Laboratory: The Microscope
Tools of the Laboratory: The Methods for Studying Microorganisms
Microbiology: A Systems Approach
Tools of the Laboratory: The Microscope
Chapter 3
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Chapter 3, pages 55 to 79

2. Learning Goals
Name parts of the microscope and describe their function
List and describe the three elements of good microscopy: magnification, resolution, and contrast.
Identify a use for darkfield, phase-contrast, differential interference contrast (DIC), fluorescence, confocal, transmission and scanning electron microscopy, and compare each with brightfield illumination.
Explain the purpose for simple staining. Differentiate between positive and negative staining.
List the steps in preparing a Gram stain, and describe the appearance of G+ and G- cells after each step.
Compare and contrast Gram stain and acid-fast stain
Explain why each of the following is used: capsule stain, endospore stain, flagella stain.

3. Viewing Microbial Cells
Too small for the human eye
Need to magnify the image
Leeuwenhoek used simple microscopes
Hooke used an early compound microscope

4. Compound Microscope

5. Microscope provides
Magnification: creates an optical image of the object which appears enlarged.
Resolution: capacity to separate two adjacent objects.
Contrast.

6. Refraction
Refraction: bending or changing an angle of the light ray as it passes through a medium such as lens

7. Magnification of an object by a compound microscope occurs in two phases

8. Resolution
Resolution is the ability to distinguish two adjacent objects or points from one another
Also called resolving power (smaller is better)
Resolving power (RP) = Wavelength of light in nm
2 x Numerical aperture
Shorter wavelengths provide a better resolution
Numerical aperture is how well the lens gathers light

9. Effect of Wavelength on Resolution

10. The Oil Immersion Lens Immersion oil reduces refraction of light
More light is gathered
Numerical aperture increases
Resolution improved
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Objective lens
Air
Oil
Slide

11. Crystal violet stain of Escherichia coli stain of Corynebacterium
Increasing Contrast
Most microbes are…
Colorless
Very Small
Difficult to see
Staining increases contrast by increasing refractive index
Increases size sometimes
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(a)
Simple Stains
Crystal violet stain of Escherichia coli
Methylene blue
stain of Corynebacterium
a1: © Kathy Park Talaro; a2: © Harold J. Benson

12. Conclusions:
The most common microscope used in microbiology is the compound light microscope. It uses visible light.
The total magnification of an object is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens
The maximum resolution the ability to distinguish between two points) of a compound light microscope is 0.2m, maximum magnification is 2000x.
Immersion oil is used with the oil immersion lens to reduce light loss between the slide and the lens
Specimens are stained to increase the difference between the refractive indexes of the specimen and the medium

13. Light Microscopy
Bright-field
Dark-field

14. Light Microscopy
Phase contrast
DIC

15. Brightfield
Darkfield
Phase-contrast

16. Light Microscopy
Fluorescence
Confocal

17. Electron Microscopy Focused electron beam Wavelength = 6pm
Magnification100,000 X
Offers much better resolution

18. Conclusions Brightfield illumination is used for stained smears.
Unstained cells are more productively observed using dark field, phase-contrast, or DIC microscopy.
The darkfield microscope shows a light silhouette of an organism against a dark background. It is most useful for examining living organisms that are invisible in brightfield, do not stain easily, or are distorted by staining.
A phase contrast microscope brings direct and reflected light rays together to form an image. It allows detailed observation of living organisms.
The DIC microscope provides a colored, three-dimensional image of the object. It allows detailed observation of living cells.

19. Conclusions:
In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet as a light source. The organisms appear as bright objects against a dark background.
In confocal microscopy, a specimen is stained with a fluorescent dye and illuminated one plane at a time. Using computer, two- and three-dimensional images can be produced.
A beam of electrons, instead of light, used in an electron microscope. Electromagnets, instead of glass lenses, control focus, illumination, and magnification.
Thin sections of microorganisms can be observed using transmission electron microscopy (TEM). Three-dimensional of the surfaces of microorganisms can be obtained with scanning electron microscope (SEM)

20. Microbial Stains Simple staining: Positive staining Negative staining
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Simple staining:
Positive staining
Negative staining
Differential staining:
Gram staining
Acid fast staining
Spore staining
Special staining
Capsule staining
Flagella staining
Differential
Stains
(a)
Simple Stains
(b)
(c)
Special Stains
Crystal violet
stain of Escherichia
coli
Gram stain
Purple cells are G+
Red cells are G -
India ink capsule stain of
Cryptococcus neoformans
Acid-fast stain
Red cells are acid-fast.
Blue cells are non-acid-fast.
Flagellar stain of Proteus vulgaris
A basic stain was used to
build up the flagella.
Methylene blue
stain of Corynebacterium
Spore stain, showing spores (red)
and vegetative cells (blue)

22. Purple cells are gram-positive. Red cells are gram-negative.
Gram Staining
The most universal diagnostic staining technique
Differentiation of microbes as gram positive(purple) or gram negative (red)
Gram stain
Purple cells are gram-positive.
Red cells are gram-negative.

23. Gram Staining Procedure

24. Over-decolorized
Positive control
Under-decolorized

25. Conclusions:
Staining means coloring microorganisms with a dye to make some structures more visible.
Fixing uses heat or alcohol to kill and attach microorganisms to a slide.
Simple stains make cellular shapes and arrangements visible. Bacteria are negatively charged, and the colored positive ion of basic stain will stain bacterial cells. The colored negative ion of an acidic dye will stain the background of a bacterial smear producing a negative stain.
Differential stains differentiate bacteria according to their reaction to the stains.

26. Conclusions
The gram stain procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain. G+ bacteria retain the purple stain after the decolorization step; G- bacteria do not, and thus appear pink from the counterstain.
Acid-fast microbes, such as members of genera Micobacterium and Nocardia, retain carbolfuchsin after acid-alcohol decolorization and appear red; non-acid-fast microbes take up the methylene blue counterstain and appear blue.
Negative staining is used to make microbial capsules visible.
The endospore stain and flagella stain are special stains that only color certain parts of bacteria.