BIOL 2320 J.L. Marshall, Ph.D.

HCC-Stafford Campus

Chapter 3- Tools of the Laboratory: Methods of Studying Microorganisms*

*Lecture notes are to be used as a study guide only and do not represent the comprehensive information you will need to know for
the exams.

3.1 Methods of Microbial Investigation

There are challenges to studying microbes that are not encountered in other fields on biology. Microbes are
not visible to the naked eye and they grow in complex associations. So, to deal with these challenges
microbiologists have developed several procedures for investigating and characterizing microbes. These
techniques are called the six “I’s”: inoculation, incubation, isolation, inspection, information gathering, and
identification. (Scoping Out The Chapter, pg. 61; table 3.1). These steps may be performed in several
combinations and orders.

3.2 The Microscope: Window on an Invisible Realm

Microscopy is the science that studies microscope techniques.

Magnification and Microscope Design

Two key characteristics of a microscope are magnification, the ability to make objects appear larger (figure
3.1), and resolving power, the ability to show detail (figure 3.3). An image is visible in the microscope when
placed in the path of a spherical lens and light source. The image appears enlarged to a particular degree,
which is called its power of magnification, and denoted by the unit “X”, read as “times”. The most common
type of microscope used by the biology student is the modern compound light microscope (figure 3.2).

The magnification of an object in a microscope is accomplished by two lenses, the ocular lens and the
objective lens. To determine the total magnification (X) of an object, multiply the ocular by the objective.
Sample calculations are on page 64.

In order to attain better resolving power using the 100X objective, immersion oil is used to help prevent
refractive loss of light. (figure 3.4).

Variations on the Optical Microscope

A summary of the different types of microscopy is in table 3.2. Two are listed here.

Bright-field microscopy – most widely used, used for live, unstained material, and preserved, stained material.
(table 3.2A).

Dark-field microscopy – uses a disc called a stop in the condenser to block all light except the light that is
reflected off the sides of the specimen itself. The specimens are surrounded by a dark field. Best for observing
live, unstained specimens. (figure 3.2 A).
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BIOL 2320 J.L. Marshall, Ph.D.
HCC-Stafford Campus

Electron Microscope
The electron microscope (EM) allows visualization of the tiniest details. EMs form an image using a beam of
electrons. (figure 3.7). Resolution of an EM is high, up to 1,000,000X for biological specimens. (table 3.2 C).

Two forms of EM are transmission electron microscope (TEM) and scanning electron microscope (SEM). TEM
is useful to visualize cell and viral structures. (figure 3.7a). SEM can produce extremely detailed 3-D view of
biological organisms. (figure 3.7b).

See also Table 3.3 for comparison of light and electron microscopes.

3.3 Preparing Specimens for Optical Microscopes

Specimen preparation for optical microscopy includes using a suitable glass slide, whether the specimen is
living or preserved, the expectation of what to visualize, and the type of microscope used, such as bright-field
or dark-field.

Wet mounts are used to visualize live samples of microbes, figure pg. 71.

Fixed, stained smears are used to prepare permanent specimen for long term study. The specimen is
prepared on a glass slide by making a smear, heat-fixing the specimen to the slide, and then stained with dyes
to impart color.

A positive stain is where the dye sticks to the cell and imparts color. A negative stain does not impart color to
the specimen, but instead “stains” the background. See Table 3.4 for comparison.

A simple stain uses only one dye to stain the specimens all one color (figure 3.8a). A differential stain uses
two different color dyes to stain the specimens, and is used to distinguish between cell types or parts. The
types of differential stains are Gram stain (Gram positive or Gram negative, figure 3.8b); acid-fast stain
(figure 3.8b); and endospore stain (figure 3.8b). Structural stains emphasize cell parts that are not revealed by
conventional staining methods, such as capsule (figure 3.8c).

3.4 Additional Features of the Six “I’s”

Inoculation, Growth, and Identification of Cultures
To grow microbes, or produce a culture, one introduces an inoculum to growth medium. This process is called
an inoculation. The observable growth in the medium is called a culture. Not all microbes have the ability to
grow under laboratory conditions (3.1 Secret World of Microbes, The Uncultured).

Some Special Requirements of Culturing

It is important to practice sterile, aseptic and pure culture techniques. You want to prevent unwanted
contamination and the release of infectious agents.

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BIOL 2320 J.L. Marshall, Ph.D.
HCC-Stafford Campus

Isolation Techniques
A method to separate an individual cell from other cells, if given sufficient surface area (figure 3.9). When an
individual cell has the ability to grow on this surface, it can for a colony. There are several methods used to
separate cells: streak plate method, where the bacteria are streaked across the surface of the agar plate
(figure 3.10a, b); loop dilution / pour plate technique, the sample is diluted and mixed with cooled molten
agar, then poured into a petri plate (figure 3.10c, d); and the spread plate technique, where a volume of a
diluted sample is spread across the surface of the agar plate using a “hockey stick” (figure 3.10e, f). Once the
medium has been inoculated, it is then incubated at a temperature that will promote bacterial growth.

A pure culture is the growth of only one type of microbe in or on the growth medium. A mixed culture has
several different types of microbes. A contaminated culture has unwanted microbes growing in the growth
medium (figure 3.11).

Identification Techniques
A more efficient way to distinguish different types of bacteria is to use biochemical tests, which are based on
cellular metabolism (figure 3.12).

3.5 Media: The Foundations of Culturing

The rise of microbiology was the development of pure culture techniques in the laboratory. In the laboratory
microbes are grown using various types of media with various types of composition. Sterile technique is
essential in the microbiology laboratory.

Types of Media
Three Categories of Media summarized in table 3.5.

Physical States of Media

Liquid / broth media are water-based solutions that do not solidify at room temperature (figure 3.13). A
commonly used liquid medium is called nutrient broth.

The solidifying agent used to make growth media solid is agar. Solid media provides a firm surface on which to
produce colonies (figure 3.14 and figure 3.15).

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BIOL 2320 J.L. Marshall, Ph.D.
HCC-Stafford Campus

Chemical Content of Media

Chemically defined / synthetic media contains components where the exact composition (substance and
weight) are known (table 3.6 A).

Complex / nonsynthetic media does not have a definable composition, the exact composition (substance and
weight) are not known (table 3.6 B).

Media to Suit Every Function

Different types of media can grow different types of microbes. General-purpose media are designed to grow a
variety of microbes that do not have special growth requirements. An enriched media contains complex
organic substances such as blood, or special growth factors, that certain species must have in order to grow.
Bacteria that require growth factors and complex nutrients are called fastidious (figure 3.16). A selective
medium inhibits most bacteria, but allows one, or a few types, to grow, and is often used for primary isolation
(table 3.7; figure 3.17 b). A differential medium will show visible differences among microbes, such as colony
color (figure 3.17 c; figure 3.19; table 3.8). Miscellaneous media are those such as reducing medium, that
helps to determine oxygen usage and transport media, that are used to maintain and preserve specimens for a
period of time before clinical analysis.