Bacteriology 102:
Principles of Enrichment and
Isolation of Bacteria

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Enrichment & Isolation Principles
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This has been the bacterial enrichment & isolation web resource page for Bacteriology 102 at UW-Madison to supplement the material in the then-current lab manual. Outside visitors to this page may find a number of things that may be of value. Websites which post old (circa 2000) copies of this and related web pages perform no useful service and should be avoided; these pirated pages are not authorized and generally have broken links, missing images and outdated course information.

References herein to the writing of lab reports and posters were current through Fall Semester, 2006. Since then, report guidelines for any current semester are in the present lab manual for Microbiology 102. However, even though this website for our old Bacteriology 102 course (splammo.net/bact102) was "retired" as of the end of Fall Semester, 2006, subject matter details throughout the site are still current, and questions asked on this page are generally worth pursuing regarding isolation of microorganisms, lab reports on such experiments, and examinations.


I. GENERAL INTRODUCTION

This page on our Bacteriology 102 website expands on the material given in the introduction to Experiment 11 in the manual and also serves to summarize major points regarding the following specific isolation experiments: 11.1 (purple non-sulfur photosynthetic bacteria), 11.2 (Bacillus), 11.3 (N2-fixers), 10.2 (Streptomyces) and 9.3 (bacteriophages). Even though bacteriophages are not bacteria but, rather, viruses which infect bacteria, many of these general principles will apply.

In this course, the selective enrichment/isolation concept applies not only to the isolation of the organisms indicated in the above-named experiments, but wherever we are isolating a certain type of organism from a natural source. This includes gram-negative bacteria from hamburger (Exp. 4), lactic acid bacteria from sauerkraut (Exp. 12), Staphylococcus aureus from the body (Exp. 13.1) and coliforms from water (Exp. 15). The basic principles also apply to the isolation and identification of the mixed unknowns (Exps. 7.2, 14.1 and 17).

The generalized procedure for the isolation and identification of any particular type of bacteria can be represented in the following flow chart:

ENRICHMENT AND ISOLATION PURE CULTURE WORK
SOURCE
MATERIAL
BROTH
ENRICHMENT
PLATING FOR
ISOLATION
STOCK
CULTURES
CHARACTERIZATION & IDENTIFICATION
•Consider inoculum: what organisms may or may not be present.
•May pre-treat inoculum, e.g., by heat-shocking.
•Usually is selective.
•May be skipped altogether.
•Usually selective or selective-differential – but not always!!  
Throughout procedure, appropriate media and incubation conditions must be considered.

In these experiments, we show how this general plan is applied productively to several specific, naturally-found groups of organisms. Students who took our Bacteriology 320 course from the mid-1970s through the 1990s will recognize this approach and recall the dozen or so different kinds of bacteria we obtained from a variety of natural sources – intestinal and otherwise. Naturally the successful isolation of the small set of organisms in either course will not make us experts in the isolation of all kinds of bacteria – nor has this author ever claimed to be such an expert(!) – but we can appreciate the general plan and how samples, media and incubation conditions can be chosen appropriately for each type of organism such that the desired growth is enhanced with the inhibition of as many other kinds of organisms as possible. So, we provide the basic framework upon which we can hang the specifics of a given isolation situation, and such can be kept in mind out in the real world if the student encounters an entirely new isolation project. The author has applied this concept in the isolation of Edwardsiella (with appropriate pH-based differential media) and the purple non-sulfur photosynthetic bacteria – both of which have been great fun – and may possibly consider iron-oxidizing bacteria his third area of expertise in bacterial isolation.

I often put a multiple-true/false question like this on a quiz or final. We expect all statements to be false and sincerely hope no one teaches such heresy.

To isolate a certain kind of organism from a natural source, utilizing the enrichment and isolation principles we learned about in our experiments:

      We can examine a sample from anywhere to find any kind of organism, as bacteria do tend to get around and can be found everywhere.

      It is best to utilize an all-purpose medium to isolate as many different kinds of bacteria as possible. Then we can study all of the colonies obtained and choose which ones we want to continue with.

      We always wish to compare our results to a general key to find out if we isolated the correct strains and got the correct determination of CFUs per gram of the sample.

      We always try to duplicate the original habitat of the organisms in the laboratory as much as possible such that all organisms in any sample can continue to grow in the laboratory as they did out in their normal habitat.

      We can expect to find a pure culture growing in any selective enrichment.

      In the experiment on purple non-sulfur photosynthetic bacteria, we expect each different kind of colony on a plate to represent a different genus.

Generally speaking, it would be unthinkable to streak out a plate of an all-purpose medium from a sample and then examine all of the resulting colonies to find what we are after. (That is the literal "looking for the needle in the haystack" approach.) Most likely, the desired type of organism would be totally overgrown. In looking for some obscure organism in the environment – for example, Edwardsiella tarda from a swimming beach – one would never be expected to have generated a list of genera or species in that environment that were encountered on the way to finding the E. tarda. You don't have to be an expert on the microbial flora in the environment – that's a separate issue for those who care to do that. Using selective procedures you would be inhibiting or killing most of them anyway in your attempt to recover (more easily) the desired organism.

Hopefully for our experiments, we will have a variety of samples from various probable habitats of these organisms. (Note the admonition in the manual to bring in your own samples!) Habitats from which the samples are taken are not homogenous and will vary considerably from each other, and they will themselves vary over time. We must never expect to replicate exactly anyone else's results or any supposed "typical" result! There is no "key" to tell us if we are "correct" when we isolate anything – that is, there is no tally of specific genera or species to check off for any given source! For any of our experiments, we may isolate representatives of several genera from one sample and several species of only one genus from another. We may spot a "trend," and we may isolate a new species; these are things that are fun to follow up on. At least we will come up with some new strains – as we define that term here.

Sooner or later in your lecture course you will learn about lithotrophic organisms such as the iron-oxidizing bacteria which we really should be including in our lab course. (A few views of a habitat in which they are found are shown here.) Similarly we could do something with the symbiotic nitrogen-fixers, but – at least – we get an appreciation of what "special" medium can help sort out free-living nitrogen-fixers from other soil organisms, and we have demonstration materials that feature the effects of Rhizobium, a genus of organisms that fix nitrogen whilst in symbiosis with leguminous plants.

Here are some items to consider as you collect, analyze and present your data and observations:

Reference material concerning the isolation of various organisms can include some professionally-produced web pages, a good textbook (such as recent editions of Brock) and also these items:


II. TAILORING THE ENRICHMENT/ISOLATION PLAN TO SPECIFIC TYPES OF ORGANISMS

Many specific kinds of microorganisms can be obtained from their natural habitats (soil, water, etc.) by the creation in the laboratory of an artificial environment which will enhance their growth over competing organisms. We would not get far by replicating exactly the habitat from which a sample was taken. Morphological and/or physiological characteristics of the desired organisms which can give them special advantages over others are exploited in the formulation of culture media, the choice of incubation conditions, and any special treatment of the original source material itself. We want to inhibit as many "undesirable" organisms as possible so they do not interfere with the isolations of the desired organisms, and we want to satisfy the nutritional requirements of the desired organisms such that they grow well.

To help us detect and isolate a certain kind of organism and minimize interference by other organisms, the following considerations are made:

Note how the blank table on this page can be filled in to summarize the above points for the organisms we are isolating in these experiments.


III. ENRICHMENT AND ISOLATION MEDIA

Essential medium components can be manipulated (these are items from Appendix D):

Here are some questions about specific medium components and procedures:


IV. DETECTION OF THE DESIRED ORGANISMS DURING THE ENRICHMENT AND ISOLATION PROCESS

The following gives a few examples of recognizable cultural and/or morphological characteristics of certain organisms which aid in their detection during enrichment and/or isolation. Click on the highlighted text for images and further explanation.


V. CHARACTERIZATION AND IDENTIFICATION OF OUR ISOLATES

We have neither the time nor the materials necessary to identify any of our bacterial isolates to the species level. Find a copy of Bergey's Manual and see what it takes to identify the dozens of species for the various genera we consider in our isolation experiments. A little more about bacterial identification is given here.

We can at least run some tests on pure cultures of our isolates to see if they follow the general pattern of what is expected for the genus or type of organism under consideration. And we can perform some "special" tests which are not essential to identify anything to the genus level, such as testing Streptomyces isolates for the ability to produce antibiotics and Bacillus isolates for the production of amylase.

For the organisms in Experiments 10.2 and 11, consider the following:


VI. A FEW GENERAL WORDS OF CAUTION
especially to those not taking Bact. 102 at UW-Madison

The growth of cultures from natural sources (soil, water, human body, etc.) can be a dangerous undertaking and should only be done in a for-real microbiology lab. One can never tell if the growth of pathogens is being enhanced. Make sure all cultures are handled with the utmost care and are completely sterilized before they are discarded!

I cannot advise about laboratory techniques (such as learning the various bacteriological manipulations) via e-mail or even the phone. Take the course in the appropriate laboratory environment at a college or university and get your techniques certified.


              GO TO:
Supplement to this page
    Selected Groups of Bacteria
    Bacteriology 102 Website
    Site Outline of related pages

Pages on this Bacteriology 102 site have copyright by John Lindquist
and found their permanent sanctuary on www.splammo.net in 2001.
Copies found elsewhere are neither authorized nor up to date.
See statement (in red) regarding internet piracy here.
This page was last modified on 5/27/05 at 4:15 PM, CDT.
Caveat on top of page expanded on 12/15/10 at 9:00 PM, CST.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison