Overview of microscopy in microbiology including differential and special stains Overview of common bacterial cell shapes

1. An Overview of Microscopy
By BugLady
Unless otherwise stated, all micrographs and diagrams
are the author’s own work.
Jul-14
1

2. Microscopy
Resolution and magnification
Different types of light microscopy:
principle and use
Electron microscope: principle
Stains
Recommended activity:
o Microscope tutorial
Jul-14 2

3. Principles of Light Microscopy
Light passes through specimen and through a
series of magnifying lenses
Important factors in light microscopy include
Magnification
Resolution
Contrast
Jul-14 3

4. Principles of Light Microscopy:
Magnification
Compound Microscope : microscope has two
magnifying lenses
 Lenses include ocular lens and objective lens
Lenses combine to enlarge objects
 Magnification is equal to the product of the ocular lens x
the objective lens: 10x X 100x = 1,000x
Jul-14 4

5. Principles of Light Microscopy: Resolution
Resolving power is defined as
the minimum distance existing
between two points where they
still appear as separate
Resolving power determines
how much detail can be seen
Naked eye ≈ 0.1mm
Light microscope ≈ 0.2μm
Electron microscope ≈ 2.5nm
Jul-14 5Courtesy of the CDC
Electron micrograph of Bacillus anthracis:
vegetative cell (A) and endospore (B)
Light micrograph of Bacillus anthracis

6. Principles of Light Microscopy: Resolution
Resolution
o Resolution depends on the quality of lenses
and wavelength of illuminating light
 How much light is released from the lens
o Maximum resolving power of most brightfield
microscopes is 0.2 μm (1x10-6)
 This is sufficient to see most bacterial structures
 Too low to see viruses
Jul-14 6

7. Principles of Light Microscopy Contrast
Reflects the number of visible shades in a
specimen
Higher contrast achieved for microscopy
through specimen staining
Jul-14 7

8. Microscopy: The Instruments
Two lenses in the compound microscope
o Ocular lens (x10)
o Objective lens (x4, x10, x40, x100)
Resolution and contrast are controlled
by the condenser lens and iris
diaphragm
o Close iris increases contrast (low mag)
o Open iris increases resolution (high mag)
Bacteria are transparent and must be
stained for bright-field microscopy.
Jul-14 8

9. Label the Microscope
Arm
Base
Clamp
Condenser
Coarse
adjustment
Diaphragm
Fine adjustment
Light control
Jul-14 9
Light source
Nose piece
Objective lens
Ocular lens
Power switch
Slide
positioning
Stage

10. Microscopy: The Instruments
Refractive index is the light-bending ability of a
medium.
The light may bend in air so much that it misses the
small high-magnification lens.
The refractive indexes of oil and glass are similar.
Immersion oil is used to keep light from bending.
Jul-14 10

11. Principles of Light Microscopy
Dark-Field Microscope
o Reverse image
 Specimen appears bright
on a dark background
Like a photographic
negative
o Achieves image
through a modified
condenser
Jul-14 11
Dark field microscopy of Treponema
pallidum, agent of syphilis
Courtesy of CDC

12. Electron Microscopy
Resolution is a function of wavelength:
the shorter the wavelength the higher
the resolution.
Uses electrons instead of visible light.
The shorter wavelength of electron beam
gives greater resolution.
Jul-14 12

13. Principles of Electron Microscopy
Uses electromagnetic lenses, electrons and
fluorescent screen to produce image
Resolution increased 1,000 fold over
brightfield microscope
o To about 0.3 nm (1x10-9)
Magnification increased to 100,000x
Two types of electron microscopes
o Transmission
o Scanning
Jul-14 13

14. Disadvantages of Electron Microscope
No true color
Artifacts
Large depth of field
Destroys sample
Jul-14 14

15. Staining
Live or unstained cells have little contrast with
the surrounding medium.
Cells are stained with dyes to make them
visible.
Unstained specimens are used to observe cell
behavior: motility.
Jul-14 15

16. Preparation of Specimens for
Light Microscopy
A smear is a thin film of a microbial liquid
suspension on a slide.
A smear is usually fixed by passing the
slide over a flame to attach the sample to
the slide.
Most organisms are killed by heat fixing.
Slides must be treated as potential
biohazards.
Jul-14 16

17. Preparing Smears for Staining
Stains consist of a positive ion and
negative ion.
In a basic dye, the chromophore is a
cation (Chr+).
In an acidic dye, the chromophore is an
anion (Chr -).
Staining the background instead of the
cell is called negative staining.
Jul-14 17

18. Simple Stains
Bacteria cell surfaces are slightly negatively charged
and basic dyes are used as stains.
Use of a single basic dye is called a simple stain.
Common basic dyes include
o Methylene blue
o Crystal violet
o Safranin
o Malachite green
Jul-14 18

19. Differential Stains: Gram Stain
The Gram stain classifies bacteria into Gram-positive and
Gram-negative bacteria.
The Gram stain is the most frequently used procedure to
stain bacteria.
There are as many variations on the Gram stain procedure as
there are labs.
Mostly the timing of each step and the decolorizer
composition differ.
Jul-14 19
Gram positive cocci and Gram negative rods, x1,000

20. Differential Stains: Gram Stain
Jul-14 20
Color of
Gram + cells
Color of
Gram – cells
Primary stain: Crystal violet Purple Purple
Mordant: Iodine Purple Purple
Decolorizing agent:
Alcohol-acetone
Purple Colorless
Counterstain:
Safranin/Carboxyl fuchsin
Purple Pink or Red

21. Gram Stain Procedure Animation
This animation shows all the steps involved
in the Gram stain.
http://www.medschool.lsuhsc.edu/microbiology/Flash/g
stainN.htm
Jul-14 21

22. Differential Stain: Acid-fast Stain
Used to stain organisms that
resist conventional staining
Used to stain Mycobacterium
tuberculosis
o High lipid concentration in cell
wall prevents uptake of dye
o Once stained difficult to
decolorize
Jul-14 22
Mycobacterium smegmatis

23. Special Stains
Stain specific structures in the bacterial
cell
Endospore stain
o Staining enhances endospore
o Uses heat to facilitate staining
Capsule stain
o Allows capsule to stand out around organism
Flagella stain
o Staining increases diameter of flagella to make
it visible
Jul-14 23
Klebsiella pneumoniae, x1,000
Bacillus subtilis, x1,000

24. Morphology of Prokaryotic Cells
Prokaryotes exhibit a
variety of shapes
o Most common
 Coccus
Spherical
 Bacillus
Rod or cylinder shaped
Cell shape not to be
confused with Bacillus
genus
Jul-14 24
Neisseria sicca
Bacillus megaterium

25. Morphology of Prokaryotic Cells
Prokaryotes exhibit a variety
of shapes
o Coccobacillus: Short round rod
o Vibrio: Curved rod
o Spirillum: Spiral shaped
o Spirochete: Helical shape
o Pleomorphic: Various shapes
Clubs, Chinese letters, palisade
Jul-14 25
Treponema pallidum, a spirochete
Courtesy of CDC
Corynebacterium diphtheriae
Courtesy of the CDC

26. Morphology of Prokaryotic Cells
Division along a single plane may result in pairs or
chains of cells
Pairs = diplococci
o Example: Neisseria gonorrhoeae
Chains = streptococci
o Example: species of Streptococcus
Jul-14 26
Streptococcus salivarius

27. Morphology of Prokaryotic Cells
Division along two or three
perpendicular planes form
cubical packets
Example: Sarcina genus
Division along several random
planes form clusters Example:
species of Staphylococcus
Jul-14 27
Micrococcus luteus
Staphylococcus epidermidis