Chapter 3

Observing Organisms Through a Microscope

Table 3.1

Units of Measurement Used n Microbiology

Figure 3.2

mm

Figure 3.1 - Overview

Compound Light microscopy

• Have ocular and objective lenses

• Calculate total magnification by:Objective x ocular = total magnification

Objective = 5x (scanning), 10x (low), 40x (high), and

100x (oil)

Ocular = 10x

So, 10x (ocular) with 100x (objective) = 1,000x

• Resolution = ability to distinguish between two points

White light = 0.2um resolution

• Field of vision decreases as objective power increases

• 100x objective requires the use of immersion oil

Brightfield used for stained smears

Darkfield or Phase-contrast used for unstained.

Example: Trepomena pallidum

Compound Light microscopy

Figure 3.3

Figure 3.4 - Overview

Figure 3.4a (1 of 2)

Figure 3.4a (2 of 2)

Figure 3.4b (1 of 2)

Figure 3.4b (2 of 2)

Figure 3.4c (1 of 2)

Figure 3.4c (2 of 2)

Figure 3.5

Table 3.2 (1 of 7)

Table 3.2 (2 of 7)

Table 3.2 (3 of 7)

Table 3.2 (4 of 7)

Table 3.5 (5 of 7)

Table 3.2 (6 of 7)

Table 3.2 (7 of 7)

Fluorescence Microscopy

• Principle – substances absorb short

wavelengths (ultraviolet UV) and give off light at

a longer visable wavelength.

• Need a microscope with special filters.

• Good for screening where there are few

organisms.

– Example: Auramine O – a fluorochrome for

Mycobacterium tuberculosis

Figure 3.6 - Overview

Figure 3.6a

Figure 3.6b

Figure 3.7

Figure 3.8

Electron Microscopy

• Uses a beam of electron instead of light

• Get better resolution and can see

organisms 100,000 times smaller than with

visible light.

– Have to use to see viruses

Figure 3.9 - Overview

Figure 3.9a (1 of 2)

Figure 3.9a (2 of 2)

Figure 3.9b (1 of 2)

Figure 3.9b (2 of 2)

Figure 3.10 - Overview

Figure 3.10a

Figure 3.10b

Preparing Smears for Staining

• Fix specimen to slide

• Have to ‘fix’ specimen to slide. We will use heat in our lab. Can use methanol to get better preservation.

• Stains are salts composed of positive and negative ions of which one is colored.

• Basic dyes= positive colored ion.

• Acidic dyes = negative colored ion.

• Bacteria have a slightly negative charge. So, basic dyes are attracted to bacteria. These are crystal violet (purple) and safranin (red) in the gram stain.

• Apply stain and then view under the microscope

Simple Stain

• Solution of single basic dye

• Mordant may be added (to intensify stain)

– Typical lab simple stain = Methylene blue

Differential Stains

• Acts differently with different bacteria and used to distinguish different bacteria.

• Gram Stain - most important differential stain in microbiology

• How does it work? Gram positive bacteria retain the purple/blue stain (crystal violet + mordant (iodine)) after the decolorization step; gram negative bacteria do not and thus appear pink/red from the counterstain (safranin).

• Why? Due to the structural differences in the cell wall: more or less peptidoglycan.

– Gram positive = purple color

– Gram negative = pink color

Figure 3.11 - Overview

Figure 3.11b

Acid-Fast Staining

• Called AFB stain for acid fast bacilli.

• Binds strongly to waxy cell wall material in some bacteria (Mycobacterium tuberculosis)

• Acid-fast organisms do not stain by normal Gram methods

• Mycobacterium tuberculosis, M. avium, other AFB, and Nocardia sp. Retain carbolfuchsin after acid-alcohol decolorization and appear red; non acid-fast organisms take up methylene blue counterstain and appear blue.

Figure 3.12

Special Stains

• Stains used to show structures such as capsules, flagella, and endospores.

• Capsule stain = colloidal solution of dark particles .

• Example: India ink for Crytococcus neoformans (stain everything but the capsule).

• Endospore = malachite green as primary and safranin as secondary to detect special resistant, dormant structure that protects bacterium from adverse conditions. Example Clostidium tetani.

• Flagella = very thin, difficult to see, so use mordant to increase diameter so can see with light microscope.

Figure 3.13 - Overview

Figure 3.13a

Figure 3.13b

Figure 3.13c

Table 3.3

GRAM STAIN PROCEDURE

1.The slide should be heat fixed on the heat block, or methanol fixed,

prior to staining.

2.Methanol fixation is accomplished by flooding the slide with 95%

methanol. Let the slide sit for 2 minutes, drain off the excess

methanol, and allow to air dry.

3.Flood the slide with crystal violet for 30 seconds. Rinse with water.

4.Flood the slide with iodine for 60 seconds. Rinse with water.

5.Flood the slide with decolorizer for about ten seconds – decolorization

is complete when the solution runs clear from the slide. Rinse with

water.

6.Flood the slide with safranin for 30 seconds. Rinse with water.

7.Blot the slide dry with absorbent paper and examine the slide under an

oil immersion lens.

INTERPRETATION

gram negative organisms = pink color

gram positive organisms = blue color

Slide Show

Examples of staining techniques