1. The Study of Microbial Structure: Microscopy and Specimen Preparation
Chapter 2
The Study of Microbial Structure: Microscopy and Specimen Preparation

2. Measurement of microbes – units known as micrometers.
1000 micrometers = 1 millimeter
Length of bacteria – 2 um to 7 um
Diameter 0.2 um to 2 um
Bright field microscope
Total magnification = magnification by the objective lens X magnification by the ocular lens
Resolving power (resolution) clarity/sharpness of the image

4. The Light Microscope many types bright-field microscope
dark-field microscope
phase-contrast microscope
fluorescence microscope

5. The Bright-Field Microscope
produces a dark image against a brighter background
has several objective lenses
parfocal microscopes remain in focus when objectives are changed
Compound microscope- ocular lens and objective lens
total magnification
product of the magnifications of the ocular lenses and the objective lenses

6. Figure 2.3

7. Microscope Resolution
ability of a lens to separate or distinguish small objects that are close together
Resolving power of the microscope in our lab 0.2 um
1000 um = 1 mm
1 um = 1/1000 mm

8. The Dark-Field Microscope
produces a bright image of the object against a dark background
used to observe living, unstained preparations
For eucaryotes has been used to observe internal structures
For procaryotes has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis

9. Fig. 2.8a

10. The Phase-Contrast Microscope
enhances the contrast between intracellular structures
excellent way to observe living cells
Especially useful for detecting bacterial components such as endospores and inclusion bodies

11. Fig. 2.8d
endspores

12. The Fluorescence Microscope
exposes specimen to ultraviolet, violet, or blue light
specimens usually stained with fluorochromes auramine O
shows a bright image of the object resulting from the fluorescent light emitted by the specimen
Has applications in medical microbiology
Mycobacterium tuberculosis

13. Fig. 2.13b
Mycobacterium

14. Figure 2.18

15. Beam of electrons is used in place of light.
TEM - thin sections of the specimen are obtained and placed on a copper mesh grid. Magnifies the object 10,000X to 100,000X. It has the resolving power of um. This used to observe internal structures.
SEM – used to observe structures found on the surface of microbes. Magnifies the object 1000X to 10,000X.
Resolving power of 0.02 um.

16. Fig. 2.17b
TEM Rhodospirillum
100,000X

17. Fig. 2.17c
TEM T4 bacteriophage

18. Fig. 2.24a
32,000X

19. Preparation and Staining of Specimens
increases visibility of specimen
accentuates specific morphological features (shape, arrangement)
preserves specimens

20. Dyes and Simple Staining
make internal and external structures of cell more visible by increasing contrast with background
ability to bind cells (ionic bond is formed between the cell and the dye)

21. Dyes and Simple Staining
Ionizable dyes have charged groups
basic dyes have positive charges
acid dyes have negative charges
simple stains
a single stain is used
use can determine size, shape and arrangement of bacteria

22. Dyes are salts
Basic dye – positive ion has the color.
Methylene blue chloride.
Acidic dye – negative ion has the color.
Sodium eosinate
Basic dyes are used to stain the cell.
Bacterial cell is negatively charged. It is attracted to the positive ions. Ionic bond is formed between the cell and the stain.

23. Negative cell is repelled by the negative ions
Negative cell is repelled by the negative ions. They are used to stain the background.
Nigrosin is used – background is black and cells are bright. Image is similar to what is seen in the case of dark-field.
(negative staining)
Simple staining – a basic dye is used to stain the cell to determine the shape and arrangement of the cells.
Gram staining is a differential staining. It places bacteria into 2 groups.

24. Fig. 2.14a

25. Differential Staining
divides microorganisms into groups based on their staining properties
e.g., Gram stain
e.g., acid-fast stain

26. Gram staining most widely used differential staining procedure
divides bacteria into two groups based on differences in cell wall structure

27. Gram staining
Crystal violet – primary stain
Iodine – mordant
Alcohol-acetone - decolorizer
Safranin – counterstain
Gram + are purple
Gram – are pink

28. Figured 2.15

29. Gram staining is based on the cell wall structure.
Gram positive cells have thick cell walls. They hold on to the primary stain.
Gram negative cells have thin cell wall.
One or two layers of peptidoglycan. They also have an outer membrane – lipids.
Alcohol causes damage to the lipids. Primary stain leaks out.

30. Acid-fast staining
particularly useful for staining members of the genus Mycobacterium
e.g., Mycobacterium tuberculosis – causes tuberculosis
e.g., Mycobacterium leprae – causes leprosy
high lipid (mycolic acid) content in cell walls is responsible for their staining characteristics

31. Acid-fast staining Differential staining Two genera are acid-fast
Mycobacterium and Nocardia
They have a waxy substance known as mycolic acid in their cell walls.

32. Carbolfuchsin – primary stain
Acid-alcohol – decolorizer
Methylene blue – counterstain
Acid-fast – red
Nonacid-fast - blue

33. Fig. 2.14d

34. Capsule staining
A capsule is a gelatinous substance found around the cell wall.
It cannot be stained
Stain the background using nigrosin.
Stain the cell with crystal violet.
Background is black.
Capsule shows up as a clear ring around the stained cell.

36. Staining Specific Structures
endospore staining
double staining technique
bacterial endospore is one color and vegetative cell is a different color
flagella staining
mordant applied to increase thickness of flagella

37. Endospore staining
Two common genera of bacteria that make endospores are Bacillus and Clostridium.
Endospores are resistant to hostile environmental conditions.
Heat, UV light, disinfectant, desiccation.
Endospores are formed within the vegetative cell. Once the formation is complete, endospores are released into the environment.

38. Endospore Staining Malachite green - primary stain Water – decolorizer
Safranin – counter stain
Endospores – green
Vegetative cells -pink

40. Fig. 2.14g
Tannic acid – increases thickness
fuchsin

41. Beam of electrons is used in place of light.
TEM - thin sections of the specimen are obtained and placed on a copper mesh grid. Magnifies the object 10,000X to 100,000X. It has the resolving power of um. This used to observe internal structures.
SEM – used to observe structures found on the surface of microbes. Magnifies the object 1000X to 10,000X.
Resolving power of 0.02 um.