Chapter 3: Observing Microbes through a Microscope
This chapter is pretty straight-forward, it focuses on the basic mechanics of laboratory microscopy. Given that this is a microbiology class it is inevitable we will use microscopes quite a bit in the lab. The types of microscopes are sometimes confusing, but really you need to focus on the "big-picture" differences. The majority of types we discussed were just variations on the simple light microscope, in other words many ways to focus light through a lens. While there is subtle variations and uses for each, the principle is the same. The other main type is the electron microscopes. The principle of the electron microscope is similar to the light microscope it just uses electrons instead of photons.
The end of the chapter brings up the topic of staining bacteria. You can't see them if you don't stain them, and we discuss two main types of stains: Simple and Differential. The simple stain is what its name implies, simply staining all material so that it can be seen under the microscope. Differential stains are used to produce a difference between types of cells, the most common being the Gram Stain. While the special stains are important, you must remember the difference between the simple stains and the differential stains.
I. Units of Measurement – Metric System utilized in class
A. The standard unit of measure for length is a meter (m)
B. The most common measures in microbiology are the micrometer (µm, 1/1,000,000 m) and the nanometer (nm, 1/1,000,000,000 m)
II. Microscopy: The Instruments
A. Light Microscopy – Any microscope that uses visible light to observe a specimen.
1. Compound Light Microscope
A. The most common type of microscope in general microbiology.
B. It utilizes a series of lens to directly magnify the image present on the slide.
C. Typically staining is required to visualize bacteria.
D. Highest Effective Resolution (the ability of the device to separate fine detail) is 0.2 µm (2000X)
2. Darkfield Microscopy
A. Used for microbes that are hard to stain, or whose morphology would be disrupted by common stains. (i.e. Treponema pallidum)
B. Utilize only the reflected light from indirect illumination.
C. The samples appear light against a black background.
D. Effective Resolution similar to compound light
3. Phase-Contrast Microscopy
A. Utilizes a combination of direct and reflected light
B. Effective Resolution is slightly higher than compound light, though more sharply defined.
4. Differential Interference Contrast Microscopy
A. Similar to Phase Contrast but utilizes two beams of light.
B. Higher Resolution than phase contrast with an almost three-dimensional appearance.
5. Fluorescence Microscopy
A. Samples are stained with specific chemical called fluorochromes, which fluorescence when struck by ultraviolet light.
B. Can allow specific microbes to be picked out of a high background
6. Confocal Microscopy
A. Samples are stained with multiple fluorochromes and excited by a precisely targeted laser. Individual planes are imaged and a computer puts together three-dimensional models of from the stacked images.
B. Utilized to image biological processes in their three-dimensional perspective.
B. Scanning Acoustic Microscopy (OPTIONAL-NOT ON TEST)
C. Electron Microscopy
1. Transmission Electron Microscopy
A. Electrons are accelerated through a thin slice of material. The electrons that pass through are imaged on a photographic or fluorescent plate, producing a negative image of what they passed through.
B. Can produce very high resolution images
C. Require extensive and highly skilled prep work, which destroys the sample.
2. Scanning Electron Microscopy
A. Electrons are accelerated at a prepared material. The electrons that bounce off are collected on a photographic plate or fluorescent screen, producing a three-dimensional image of the surface of the target.
B. High resolution images of the surface of a material.
C. Highly skilled prep that destroys the sample.
D. Scanned-Probe Microscopy (OPTIONAL-NOT ON TEST)
1. Scanning Tunneling Microscopy
2. Atomic Force Microscopy
III. Preparation of Specimens for Light Microscopy
A. Preparing Smears for Staining
1. Most microbes are nearly colorless when observed in a light microscope. To counteract this they are typically stained in some fashion.
2. Staining means to color bacteria with a chemical dye.
3. Prior to staining, microbes must be fixed, attached, to a glass slide. This is accomplished with either a quick application of heat or a quick immersion in alcohol.
4. Acidic dye – a stain with a dye that is in a negative ionic state.
5. Basic dye – a stain with a dye that is in a positive ionic state. Since bacteria are slightly negatively charged at pH 7, basic dyes tend to work better.
B. Simple Stains
1. A basic dye is used to color all bacteria present, and highlight overall morphologies or simple structures.
2. Sometimes a mordant is utilized to increase the intensity of the dye. The mordant is an additive that increases the effect of the dye in some way.
C. Differential Stains – stains that react differently to different types of bacteria and can be used to distinguish between them.
1. Gram Stain
a. The most common and important differential stain utilized in modern microbiology.
b. Developed in 1884 by Hans Gram, it roughly divides the bacterial world into two camps: Gram-positive and Gram-negative.
d. Four step procedure
i. The smear is covered with a purple basic dye (crystal violet) for one minute; this imparts a purple color to all bacteria (primary stain)
ii. The crystal violet is washed off and iodine is added to the slide as a mordant. This step imparts a dark purple color to all bacteria.
iii. The iodine is washed off with an alcohol solution (decolorizing agent). This removes the purple color from Gram-negative bacteria, but not Gram-positive bacteria.
iv. The alcohol is removed and a basic red dye, safranin, is added to color the decolorized bacteria red (counterstain).
v. Gram-positive bacteria are purple and Gram-negative bacteria are red.
e. Some bacteria do not readily stain with the Gram stain.
2. Acid-Fast Stain
a. Utilized to distinguish bacteria that have a waxy outer layer (i.e. tuberculosis and leprosy bacteria).
b. Procedure
i. The red dye carbol-fuschin is applied to a smear and the slide is gently heated for several minutes.
ii. The slide is cooled and rinsed.
iii. Decolorization is done with an acid alcohol mixture which decolorizes all but bacteria with a waxy outer layer (acid-fast bacteria).
iv. The slide is counterstained with methylene blue.
v. Acid-fast bacteria are red and all other material is blue.
D. Special Stains
1. Negative Staining for Capsules
a. This does not stain the bacteria, but rather stains the background a dye such as India ink.
b. The dark background highlights the structure of cells and also allows visualization of external capsules which though colorless can exclude the background dye.
2. Endospore Staining
a. Endospores are highly resilient forms of dormant bacteria.
b. Special dyes, particularly Malachite green, are used to visualize endospores by forcing the dye into the structure through heat.
c. The samples are rinsed and counterstained with safranin.
3. Flagella Staining - A tedious and difficult stain used to visualize the flagella on bacteria.