1. Observing Microorganisms Through a Microscope
Microscopy
Observing Microorganisms Through a Microscope

2. Microorganisms are so small that metric prefixes may be unfamiliar
Units of Measurement
Microorganisms are so small that metric prefixes may be unfamiliar
centi = 1/ or
milli = 1/ or
micro = 1/1,000, or
nano = 1/1,000,000, or

3. The Instruments

4. Compound Light Microscope
Uses visible light
Has at least 2 sets of lenses
Can achieve maximum 2000X magnification
Resolution of objects as small as 0.2 m

5. Light Microscopy
In a light microscope visible light passes through the specimen and then through glass lenses.
The lenses refract light such that the image is magnified into the eye or a video screen.

6. Light Microscopes
Microscopes vary in magnification and resolving power.
Magnification is the ratio of an object’s image to its real size.
Resolving power is a measure of image clarity.
It is the minimum distance two points can be separated and still viewed as two separate points.
Resolution is limited by the shortest wavelength of the source, in this case light.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

7. Resolution of Light Microscopes
The minimum resolution of a light microscope is about 2 microns, the size of a small bacterium
Light microscopes can magnify effectively to about 1,000 times the size of the actual specimen.
At higher magnifications, the image blurs.
Fig. 7.1
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

8. Brightfield Illumination
Usual operations
Specimens must be stained for viewing
Best magnification and resolution with the oil immersion objective
Oil has same refractive index as glass

9. Darkfield Microscopy
Only light reflected from specimen enters objective lens
Organism appears light against a dark field
Useful for examining
Live organisms
Microorganisms which cannot be stained by standard methods
Treponema pallidum, the causative agent of syphilis

10. Phase-Contrast and Differential Interference (DIC) Microscopy
Uses wave nature of light
One set of light rays are direct and one set are reflected
Makes detailed images of internal structure of living microorganisms possible
Image in greyscale
Uses differences in refractive indices
Uses 2 beams of light
Resolution higher
Brightly colored image
Image appears nearly three-dimensional

11. Electron micrographs

12. Fluorescence Microscopy
When illuminated with short  light some dyes emit light with longer 
Enables viewing of cells located on an opaque surface such as a soil particle
When illuminated with UV or halogen light source preparations glow
Bovine pulmonary artery endothelial cells. Photometrics, Ltd.

13. Fluorescent stain of cell

14. Confocal Microscopy Preparations also stained with fluorochromes
3-D confocal microscopy of Salmonella-infected macrophage (green) with XY-slice showing bacteria (red) inside the cell
Preparations also stained with fluorochromes
Exceptionally clear two-dimensional images
Three-dimensional images obtained by computer construct

15. Electron Microscopy
Beam of electrons has shorter  so gives better resolution than visible light
Electromagnetic lenses rather than glass
Done in a vacuum
Can resolve to 0.5nm and magnify up to 100,000 times.
Specimen must be dry….dead

16. Transmission Electron Microscopy (TEM)
Resolves objects as close as 2.5nm
Magnification 10,000 to 100,000X
Ultra-thin sections
Specimens must be dehydrated
Preparation of specimen may generate artifacts
Lambda Bacteriophage DNA (TEM x153,000)

17. Scanning Electron Microscopy (SEM)
Resolves objects as close as 20 nm
Magnification between 1,000 and 10,000X
Whole specimens
3-dimensional view of specimen
Specimen dehydrated
Slime Mold Fruiting Structure, Lamproderma sp. (SEM x290)

18. Scanning Tunneling Atomic Force
Thin metal probe scans specimen
Resolving power much greater than electron microscopes
no special specimen preparation
detailed views of silicon chips & DNA molecule
Metal and diamond probe forced down along surface of specimen
3-dimensional image
no special preparation of specimen is required
views of detailed structure of biological molecules

19. Preparation of Microscopy Specimens
Microorganisms must be spread over the surface of a slide (smear)
Microorganisms must be attached to the slide (fixed)
Microorganisms must be colored (stained)

20. Making the Smear and Fixing It
Put a small amount of organism into a drop of water on a clean microscope slide & spread .
When dry pass through the flame of a Bunsen burner or flood with methyl alcohol

21. Stains Dyes Negative Stain Simple Stains Differential Stains
stains the background not the organism
Simple Stains
everything stained a single color
Differential Stains
distinguish among bacteria based on particular characteristics

22. Characteristics of Dyes
Basic Dyes
Chromophore is in the positive ion
Used to stain most bacteria
Used alone as simple stains in combination for differential stains
Acidic Dyes
Chromophore is in the negative ion
Used in negative staining & for staining nuclear material

23. Gram Stain
The Gram Stain is the single most important test in microbiology. The principal utility of the Gram Stain rests on its speed and simplicity. Most bacteria may be divided in two groups by this procedure
developed by the Danish physician Hans Christian Gram to differentiate pneumococci from Klebsiella pneumonia
difference between Gram-positive and Gram-negative bacteria is in the structure of the cell wall

24. Procedure

25. Results G+ cocci G- rods
Websites with more samples of gram stained bacteria
GRAM STAINED IMAGES OF MEDICALLY IMPORTANT BACTERIA
Loyola University Medical Center
GRAM STAIN TUTORIAL

26. Acid Fast Stain
Acid Fast Staining is used primarily for detection of organisms with a thick outer coat composed of true waxes, mycolic acids and phosphatides
Mycobacteria are not decolorized and retain the stain, appearing pink under the light microscope (hence are 'fast', like color-fast clothes).
Acid Fast Stain can also be used to identify several protozoa, such as Cryptosporidium and Isospora belli. These two coccidia have recently acquired greater clinical significance because of their widespread occurrence in immuno- compromised patients, such as those infected with HIV.

27. Special Stains Spore Stain Capsule Stain Flagella Stain
Bacillus subtilis
Streptococcus pneumoniae
Pseudomonas aeruginosa