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Chp. 3 Microscopy and Staining

I. Principles

Microscopy is the technology of making very small things visible to the human eye. Most
microbes are so small that they are measured in micrometers or nanometers. A typical bacterial
cell is about 1 um while a virus is more in the range of 10-100 nm.
Remember, Leeuwenhoek was probably the 1st to see microorganisms in the 1600’s with his invention
of his simple microscopes.

Resolution is the ability to see two objects as separate, discreet entities….kind of like the ability
to see railroad tracks as being separate tracks….GOOD resolution is being able to distinguish
the two tracks as separate…..once the two tracks merge into one, the resolution is poor!!!

Refraction is the bending of light as it passes from one medium to another of different density.
Immersion oil, which has the same index of refraction as glass, is used to replace air and to
prevent refraction at a glass-air interface. An example would be when one looks at objects just
below the surface of water in a pond or other body of water…..the objects become refracted or
“distorted” from the true image.

The total magnification of a light microscope is calculated by multiplying the magnifying power
of the objective lens by the magnifying power of the ocular lens. Increased magnification is of no
value unless good resolution can also be maintained.

Scanning (3X) x (10X) = 30X total

Low power (10X) x (10X) = 100X total
High “dry” (40X) x (10X) = 400X total
Oil immersion (100X) x (10X) = 1000X total

Most microscopes are designed so that when the microscopist increases or decreases the
magnification by changing from one objective lens to another, the specimen will remain very
nearly in focus. Such microscopes are said to be parfocal (par means equal).

Types of microscopes:

Compound Light*** (this is what we will use…know the parts and functions….we will
spend more time on this in the lab.

Dark-Field-

Phase-Contrast-
Fluorescence-

Transmission-

Scanning Electron-

II. Techniques of Light Microscopy

A. Preparation of specimens

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Wet mounts are used to view living organisms. The hanging drop technique is a
special type of wet mount, often used to determine whether organisms are motile.

Smears of appropriate thickness are allowed to air-dry completely and are then
passed through an open flame. This process, called heat fixation, kills the
organisms, causing them to adhere to the slide and more readily accept stains.

B. Principles of Staining

A stain, or dye, is a molecule that can bind to a structure and give it color.

Most microbial stains are cationic (positively charged), or basic dyes, such as
methylene blue, crystal violet, or safrannin. Some are anionic dyes (negatively
charged), or acidic dyes, such as nigrosin or India ink.

MOST bacterial surfaces are negatively charged so they will attract the basic dyes.

Simple stains use one dye and reveal basic cell shapes and arrangements.
Differential stains use two or more dyes and distinguish various properties or
organisms. The Gram stain, spore stain, and acid-fast stain are examples.

Negative stains color the background around cells and their parts, which resist
taking up the stain. (acidic dyes will “stick” to the glass slide since glass has a +
charge).

Imagine a magnet when thinking of basic and acidic dyes…..basic dyes (+) will
attract to bacteria due to their (-) parts but will be repelled by the glass because of its
(+) charge!! Acidic dyes, on the other hand, will attract to the (+) glass but be
repelled by the (-) bacterial parts!!

We will also be covering these in great detail in the laboratory.

Go to Chp. Reference

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