THE METHODS FOR STUDYING MICROORGANISMS

BIO 2302

Introduction

Growing up you were probably told to wash your hands so you would not become infected by
germs. You probably only did so to stay out of trouble because no matter how well you focused
on your hands, you never saw a germ on them. Today you realize that a germ is a microorganism,
one of many that surround us. Microorganisms cannot be seen with the naked eye, but you can see
them with the aid of a microscope. In this lesson you'll learn how to view microorganisms under a
microscope.

The Five I's

The Five I's is a method used to locate, grow, observe and characterize microorganisms. It is not
necessary to perform all of these steps, or to perform them exactly in this order. In some cases, one
may proceed right from the sample to inspection, and in others, only inoculation and incubation
on special media are required. The first step is the collect your specimen. Common specimens are
body fluids, foods, water or soil.

One you have your specimen collected you can perform the Five I's:

1. Inoculation: The sample is placed into a container of sterile medium that provides
microbes with the appropriate nutrients to sustain growth.
2. Incubation: An incubar can be used to adjust the proper growth conditions of a sample.

3. Isolation: The end result of inoculation and incubation is isolation of the microbe.

4. Inspection: The cultures are observed for obvious growth characteristics that could be
useful in analyzing the specimen contents.
 5. Identification: Determine the type of microbe, usually to the level of species.

The Microscope

The principal way a microbiologist studies microorganisms is by observing them through a

microscope. A microscope is a device that enlarges objects using a process called magnification.
The simplest form of a microscope is a magnifying glass consisting of a single lens. And the
simplest magnifying glass is the bottom of an empty glass. Some glasses are slightly bent at the
bottom, causing a magnifying effect if held at a certain height over an object.
The single-lens magnifying lens or glass is a thing of the past. Scientists today use a microscope
that has two sets of lenses (objective and ocular), which is called a compound light microscope.
View different slides of organisms from algae to bacteria.

A light compound micrscope is a good tool for observing many kinds of microorganisms, however,
it isn't capable of seeing the internal structure of a microorganism nor can it be used to observe a
virus. In order to view internal structures of viruses and microorganisms, microbiologists use an
electron microscope where specimens are viewed in a vacuum. The electron microscope uses
beams of electrons and magnetic lenses rather than light waves and optical lenses to view a
specimen.

Providing Nutrients in the Laboratory

In order to grow microbes out of their natural habitats and in pure form in the laboratory, certain
nutrients are required. Nutritional requirements of microbes vary, and at least 500 different types
of media are used in culturing and identifying microorganisms. Culture media are contained in test
tubes, flasks, or Petri dishes. Media are extremely varied in nutrient content and consistency and
can be specially formulated for a particular purpose. There are different levels of media such as
liquid, semisolid, and solid. The most widely used media is agar, a complex polysaccharide. The
benefits of using agar as the media include that is is solid at room temperature, and melts at the
boiling temperature of water. It is flexible and moldable and provides a basic framework to hold
moisture and nutrients, though it is not itself a digestible nutrient for most microorganisms.
Preparing Specimens

There are two ways to prepare a specimen to be observed under a light compound microscope.
These are a smear and a wet mount.

Smear

A smear is a preparation process where a specimen is spread on a slide. You prepare a smear using
the heat fixation process:

1. Use a clean glass slide.

2. Take a loop of the culture.

3. Place the live microorganism on the glass slide.

4. The slice is air dried then passed over a Bunsen burner about three times.

5. The heat causes the microorganism to adhere to the glass slide. This is known as fixing the
microorganism to the glass slide.

6. Stain the microorganism with an appropriate stain.

Wet Mount

A wet mount is a preparation process where a live specimen in culture fluid is placed on a concave
glass slide or a plain glass slide. The concave portion of the glass slide forms a cup-like shape that
is filled with a thick syrupy substance. The microorganism is free to move about within the fluid,
although the viscosity of the substance slows its movement. This makes it easier for you to observe
the microorganism. The specimen and the substance are protected from spillage and outside
contaminants by a glass cover that is placed over the concave portion of the slide.

Staining a Specimen

Not all specimens can be clearly seen under a microscope. Sometimes the specimen blends with
other objects in the background because they absorb and reflect the same light waves. You can
enhance the appearance of a specimen by using a stain. A stain is used to contrast the specimen
from the background.
A stain is a chemical that adheres to structures of the microorganism and in effect dyes the
microorganism so that it can be easily seen under a microscope. Stains used in microbiolody are
either basic or acidic.

Basic stains are cationic and have a positive charge. Common basic stains are methylene blue and
crystal violet. These are ideal for staining chromosomes and the cell membranes of many bacteria.

Acid stains are anionic and have a negative charge. Common acidic stains are eosin and picric
acid. Acid stains are used to stain cytoplasmic material and organelles or inclusions.

There are two types of stains: simple and differential. A simple stain has a single basic dye that is
used to show shapes of cells and structures within a cell.

A differential stain consists of two or more dyes and is used in the procedure to identify bacteria.
One of the most commonly used differential stains is the gram stain. Gram-positive
microorganisms stain purple. Gram-negative microorganisms stain pink. A common bacterium
that causes food poisoning, Staphylococcus aureus, is gram-positive. Escherichia coli is gram-
negative.

Review

The Five I's is a method used to locate, grow, observe and characterize microorganisms which
include inoculation, incubation, isolation, inspection and identification. The principal way a
microbiologist studies microorganisms is by observing them through a microscope, either a
compound light or electron. In order to grow microbes out of their natural habitats and in pure
form in the laboratory, certain nutrients are required. The most widely used media is agar. There
are two ways to prepare a specimen to be observed under a light compound microscope: a smear
or a wet mount. Not all specimens can be clearly seen under a microscope. You can enhance the
appearance of a specimen by using a stain.
Practice Assignment

Answer the following questions to send to your instructor:

1. _____________________ have thinner walls, containing an outer layer of

lipopolysaccharide that permits dye to escape.

2. _____________________ are long whip-like structures that enable bacteria to move

through the water.

3. Asexual reproduction is often known as _______________________.

4. During the _________________________ the bacteria become acclimated to the

environment and begin to produce enzymes to break down the "food".

5. _________________________ is sometimes known as fermentation.

6. _______________________ is the movement from a region of higher concentration to a

region of lower concentration.

7. The ______________________ regulates the flow of nutrients into the cell and removes
waste from the cell by opening and closing passages called channels.

8. _________________________ bacteria retain the original violet dye and cannot be

counterstained.

9. ___________________________ are gram negative, facultative anaerobic, rod-shaped

bacteria which live in the intestines of warm-blooded animals.

10. ___________________________ are round cells, sometimes slightly flattened when they
are adjacent to one another.