Culturing Bacteria: Isolation and Identification

Biology Activity
Culturing Bacteria
Isolation and Identification

Introduction

This lab is actually two labs. To make this page easier to navigate, it has been divided into sections. This page contains the introduction, while the two lab activities are given their own pages, and a separate section is devoted to the Appendix, where supplemental information is found.
Culturing Bacteria Menu
Introduction
Activity #1: Bacterial Enrichment
Using a Carrot Medium
Activity #2: Enrichment of Microbes
Using a Milk Medium
Appendix
General Safety Guidelines
Microbiology Safety Guidelines

Robert and Emmy Koch
in traditional Japanese
clothing during a visit
to Japan in 1903.

In the following activities, we'll investigate the cultivation of bacteria, as well as isolation and identification of those bacteria using stains or dyes. Most of the techniques we'll be using were developed in the mid- to late 1800s by Robert Koch, Paul Ehrlich, and Hans Christian Gram. The investigations of diseases and disease-producing organisms was first developed into a science by Louis Pasteur (1822-1895). A French chemist, Pasteur was extremely adept in the laboratory as well as out in the field. (We mean “in the field” quite literally, as Pasteur studied the diseases of sheep, cattle, and other farm animals as well as human illnesses.) But a technique to distinguish one kind of bacteria from another was perfected by Robert Koch (1843-1910). The design of techniques to grow pure cultures of bacteria raised bacteriology to a regular science. In the process, Koch formalized his approach to proving that a particular microbe was the cause of any one disease in what are known as Koch's Postulates (1882):

The agent of an infectious disease must be present in every case of the disease.

The agent must be isolated from the host and grown in vitro (pure culture) for several generations.

The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host.

The same agent must be recovered once again from the experimentally infected host.

The thinking behind these postulates supported the idea that a disease is caused by one specific microbe. If the microbe could be isolated, then it might be possible to control the disease. Pasteur did isolate some bacterial strains and cultivate them in a liquid culture medium (a broth similar to bouillon). However, bacteria in a liquid appeared distorted when using better microscopes. A solid medium for bacterial growth was developed by Koch. First he used potato slices, and later he solidified broth by adding gelatin. (Beef-flavored jello...mmm!) Unfortunately, gelatin can liquefy at body temperature (37ºC), the temperature at which bacteria are often cultured. Soon scientists learned that adding an extract of Japanese seaweed called agar-agar to the culture medium would keep the medium solid. Eventually, all this was done in a special glass dish devised by Richard Julius Petri (1852-1921). You will most likely use a petri dish in your laboratory work that follows.

In 1877, Koch published a scientific paper in which he described the new technique for dry-fixing thin films of bacteria culture on glass slides, for staining them with aniline dyes, and for recording the microscopic images on film, using photographic equipment built to his own specifications. This dry-fixing technique is still a standard procedure that you also will utilize in identifying the various bacterial cultures in the activities that follow.

The idea of staining bacteria was a major step forward in the techniques for identifying specific bacteria. Although stains were used as early as 1770 in the study of the structure of wood, it was not until 1839 that Christian Gottfried Ehrenberg (1795-1876) used them to study microbes. Staining techniques depend upon the following properties:

The substance must be chromogenic—its molecules must contain groups of atoms that are color-forming.

The stain must break up (dissociate) into positively and negatively charged ions. The positive (+) ions are called cations and the negative (–) ions are called anions. An example of this is the common dye, methylene blue. When the dye molecules are added to water to make a solution, the following occurs:

When methylene blue dissociates, the positive ion (cation) is the larger ion, so methylene blue is called a cationic dye. The opposite case is illustrated with the anionic dye, eosin. Its dissociation in water is as follows:

Scientists think that the proteins of cells react with the stain to produce a colored product viewable under the microscope. One explanation is that anionic dyes like eosin (also called “acid dyes”) may interact with the cationic (+) portions of the protein, attracted by their opposite charges. The reverse would occur between cationic dyes and the anionic portions of a protein.

In a separate category is the Gram stain, named after the Danish physician Hans Christian Gram (1853-1938) who worked out the procedural details in 1884. The first step of the process is to stain the bacteria with a dye called gentian violet. The second step is to wash the stained bacteria with an iodine solution. The third step is to wash the bacteria with ethyl alcohol. For some bacteria, the second and third steps wash away the dye, leaving them without the color of the gentian violet. These are called Gram-negative bacteria. Other bacteria remain colored even after washing with the iodine solution and with the ethyl alcohol. These bacteria are called Gram-positive.

This leaves the features of Gram-positive bacteria visible under a microscope, but what about those Gram-negative critters? The last step of the Gram-staining procedure is to stain the bacteria in a sample with a reddish-pink dye. Only the Gram-negative bacteria will be stained by this dye. When we're done, the Gram-negative bacteria will have pink structural features and the Gram-positive bacteria will have purple or purple structural features.

Whether a bacterium is Gram-negative or Gram-positive is often a good indicator of whether the bacteria can be destroyed using a given antibiotic. Many antibiotics will kill Gram-positive bacteria, but Gram-negative bacteria are often tougher to kill, resisting common antibiotics.

Gram-positive and Gram-negative bacteria have very different types of cell walls. The cell wall of a Gram-negative bacteria will dissolve in the mixtures of ethyl alcohol and acetone that are used in the third step of the procedure. When the cell wall dissolves, there is nothing to keep the Gram stain inside the cell, so the stain can easily be washed away. Meanwhile, the cell wall of a Gram-positive bacterium will not dissolve in mixtures of ethyl alcohol and acetone. The cell wall stands firm, holding the Gram stain inside the bacterial cell. What's more, the ethyl alcohol-acetone mixture absorbs water from the cell wall, causing it to contract and closing the pores in the cell wall. This means the cell wall can hold the Gram-stain inside even more effectively.

Like was said above, Gram-negative bacteria are often more resistant to antibiotics than Gram-positive bacteria. In Gram-negative bacteria the cell wall is surrounded by an extra layer made of polysaccharides, proteins, and phospholipids. We talked about this layer when we explained how Gram-staining works. This layer, though easily washed away by alcohol-acetone mixtures, blocks many antibiotics from reaching the peptidoglycan cell wall. Since b-lactam antibiotics like penicillin work by attacking the cell wall, this outer layer makes Gram-negative bacteria resistant to such antibiotics.

During the 1800s two great bacteriologists, Marinus Willem Beijerinck (1851-1931) and Sergius Winogradsky (1856-1953), made it easier to find microorganisms with given properties. They found that different culture media support different mocroorganisms. They learned how to bait microbes with the properties they wanted by selecting the right culture medium. Their system works like this: Place a sample of soil or whatnot on the culture medium, and of all the microbes in the soil, only those with a desired property will grow, if you have selected your culture medium carefully. Therefore, by using just the right culture medium, you can isolate the microbes that have antibiotic properties, for example, from all the other microbes in a soil sample.

The investigations that follow (Bacterial Enrichment Using a Carrot Medium and Enrichment of Microbes Using a Milk Medium) allow you to isolate and identify, through staining, the particular organisms produced in the different culture media. The various procedures have their historical roots in the 1800s when bacteriology became not only a respected science, but a vital one, responsible for significantly improving the health of all.
For more information, at other Web sites...

Gram Stain—an overview, part of the University of Pennsylvania Medical Center Guidelines for Antimicrobial Therapy.
Gram Stained Images of Medically Important Bacteria—from Loyola University Chicago.
New Gram Stain Atlas—a large collection of Gram-stained images, part of the University of Pennsylvania Medical Center Guidelines for Antimicrobial Therapy.

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Reference

Porter, Roy. The Greatest Benefit to Mankind: A Medical History of Humanity. New York: W.W. Norton, 1998.

Image Credits

Robert and Emmy Koch...: Courtesy National Library of Medicine.

Copyright ©2002 The Chemical Heritage Foundation