Some notes for the General Public:
- The growth of cultures from natural sources (soil, water, human body, etc.) can be a dangerous undertaking and should only be done in an appropriate laboratory environment. 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 invite e-mail (to firstname.lastname@example.org) from those in environmental microbiology courses who may like to inquire about and perhaps add to the discussions on this and associated pages.
- A fun experiment (not yet discussed herein) involving enriching and isolating bioluminescent bacteria often leads to success. It is presently mentioned on my home page.
The following discussion expands on the material given in the introduction to Experiment 11 in the Bacteriology 102 lab 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.
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). Some of these organisms are among the examples given throughout this web page. In a broader sense, the isolation principles can also apply to the individual components of our occasional mixed unknowns (Exps. 7.2, 14.1 and 17).
Considering all of the examples (real and theoretical) that we could give and all of the similar experiments one could read about in journals, realize that each enrichment/isolation situation is unique. Never expect to replicate anyone else's results or any supposed "typical" result! For any of our experiments, we may isolate representatives of several genera from one sample and several species of one genus from another. Habitats and samples will vary considerably from each other, and will vary themselves over time. We may spot a "trend" and we may isolate something entirely new; these are things that are fun to follow up on.
Sooner or later in your lecture course you will learn about lithotrophic organisms such as the iron-oxidizing bacteria which we really should be considering in our lab. A few views of iron bacteria are shown here. Similarly we could do more with the symbiotic nitrogen-fixers; some relevant material we have will be put on the web later.
We are hopefully not "giving everything away" in the lectures and written material. As for many of our experiments, questions that could be raised can be answered by your own observations! The material in this web page is not necessarily restricted to our course but can be made generally applicable. (That's for our friends in similar microbiology courses out there in the real world who might have stumbled in here.)
Here are some items to consider as you collect, analyze and present your data and observations:
- Taking notes in lab of various observations can be greatly facilitated by the use of tables which are also valuable in reports and posters. Also, flow charts can summarize procedures in a general way and can be used to great advantage in posters and in day-to-day preparation for lab. The set-up of flow charts and tables is discussed on the Bact. 102 Homesite; click on "flow charts and tables."
- During the course of the various isolation experiments, ask yourself "what am I doing and why?" Besides the questions posed below, look over the old quiz questions in Appendix X which apply to these experiments. They are formulated to help you think about these things in an organized fashion!
- The blank table found here can be a valuable study guide once it is filled in.
- Be sure to go over the requirements for reports and posters associated with these experiments.
I. GENERAL OVERVIEW OF ENRICHMENT AND ISOLATION
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. 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.
A generalized flow chart for the enrichment, isolation and identification of microorganisms is shown here.
To help us detect and isolate a certain kind of organism and minimize interference by other organisms, the following considerations are made:
- Choice of suitable source material likely to contain the desired organism.
- Whether or not any special treatment of the source material can be helpful. Some examples: Drying of a soil sample can assist in the isolation of dessication-resistant organisms such as Bacillus and Streptomyces both of which would have produced spores as a response to nutrients becoming less available as the soil dries out. In the isolation of the endospore-former Bacillus, we can heat a suspension of soil to kill off vegetative cells and reproductive spores of a wide variety of organisms, leaving only endospores to serve as possible colony-forming units. The more "selection" that is done at this initial stage of the isolation process, the less selective the subsequent isolation medium needs to be. Also consider the filtration procedure in the isolation of bacteriophages.
- Should we plate the sample directly or begin with an enrichment?
- Often the source material is inoculated directly into a broth medium which will encourage the proliferation of the desired organism; this is called an enrichment. When an enrichment is formulated to suppress the growth of undesired competitors, it is then a selective enrichment. A selective enrichment (such as what is utilized in Experiments 11.1 and 11.3) increases the probability that colonies of the desired organism will be isolated upon subsequent streak-plating and not crowded out by others. Selective enrichment media are useful in recovering organisms which are in very low numbers, and they are often formulated like their corresponding isolation media.
- When plate counts are to be performed and/or when the desired organism is relatively abundant, no enrichment is made and the source material is plated directly (as in Experiments 10.2 and 11.2).
- Water samples can be passed through a filter which is then placed on the appropriate selective plating medium. This method is useful in the direct plating of samples containing low numbers of the desired organism and is discussed further and illustrated here.
- Suitable formulation of the isolation medium. By "isolation medium" we mean the plating medium upon which you obtain isolated colonies, having practiced this numerous times! Usually a selective medium is employed "selection" being achieved by either (1) adding a selective agent to "poison" undesired organisms (as in MacConkey Agar) or (2) making the medium restrictive by including a nutrient only certain organisms can use, or by leaving something out. More considerations about media are in Section II, below, and a general discussion is given in Appendix D of the manual (reproduced here).
- Utilization of suitable incubation conditions i.e., consideration of the following:
- Temperature: How high or low can we go without inhibiting or killing the desired organism?
- Oxygen or no oxygen?
- Does the desired organism need an increased-CO2 atmosphere?
- Is light necessary?
- We should enhance the detection of desired organisms as early in the process as possible. Some microbial groups have recognizable cultural and/or morphological characteristics which aid in their detection. There is more about this in Section III, below.
II. ENRICHMENT AND ISOLATION MEDIA
Essential medium components can be manipulated (these are items from Appendix D):
- Carbon source: As an example, one can leave organic compounds out of the medium to allow for the growth of autotrophs which can obtain their carbon from atmospheric CO2 which diffuses into the medium.
- Nitrogen source: For example, one can leave nitrogenous compounds out for the growth of "nitrogen-fixers" which can obtain atmospheric nitrogen (N2). The so-called "nitrogen-free media" are indeed free of nitrogenous compounds but not N2 which diffuses in from the air.
- Energy source: For example, one can leave it out for photosynthetic bacteria which can use light as their energy source.
- Compounds which can be used as growth factors (vitamins, amino acids, nucleic acids, fatty acids, etc.) and other special medium ingredients can be added as needed.
Here are some questions about specific medium components and procedures:
- Regarding purple non-sulfur photosynthetic bacteria: What is the "magic" of succinate? Why use it as the carbon source?
- What do we really mean when we say "non-sulfur" regarding the very metabolically-diverse group of photosynthetic bacteria we are working with? If we were to look for various kinds of photosynthetic bacteria that can grow autotrophically, why might one include a sulfide compound in the medium?
- Regarding Streptomyces: What kind of compound serves as the primary source of carbon and energy (and nitrogen)? What does an organism need to produce in order to utilize such a compound?
- Regarding the nitrogen-fixers: Why is there so much sugar in the enrichment and isolation media? Is it possible for non-nitrogen-fixers to grow in our enrichments and on our plates? How do we really know that we are isolating nitrogen-fixers?
- Why don't we need a selective medium when isolating Bacillus from soil?
- Why does the combination of (1) heating the initial soil suspension to 80°C and (2) aerobic incubation assure us of having virtually only Bacillus on our isolation plates? What two genera would we expect if we were to incubate the isolation plates anaerobically?
- When we get to Experiment 12 on the lactic acid bacteria, consider why the combination of (1) a "rich" plating medium with sodium azide and (2) aerobic incubation conditions assures us of having virtually only aerotolerant anaerobes on the plates.
III. 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.
- Recognizable appearance of colonies produced by Streptomyces.
- Pigmentation seen in enrichments and colonies of purple non-sulfur photosynthetic bacteria. A mass of reddish color seen in enrichments (and in the natural habitat on occasion) is called a "bloom."
- Peculiar cell shapes seen for certain genera of photosynthetic and nitrogen-fixing bacteria.
- Endospores seen microscopically in older cultures of Bacillus and other endospore-formers.
- A generally off-white appearance for colonies of Bacillus. Beyond this similarity, the many species of Bacillus tend to produce a wide variety of different types of colonies.
- Plaques produced by bacteriophages infecting a suitable host culture of bacteria.
- Also consider gas production by coliforms in lactose-containing broth media (Experiment 15).
IV. CHARACTERIZATION 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 Experiment 11, consider the following:
- Exp. 11.1 (purple non-sulfur photosynthetic bacteria): For each different type of pigmented colony which we could isolate, we observed a wet mount (for morphology and probable genus identification) and inoculated two tubes of melted Succinate Agar (like we inoculated Thioglycollate Medium in Exp. 5.1 to determine oxygen relationships).
- As we incubated one tube in the dark and one in the light, which tube shows the pigmented phototrophic growth? Where do you see it? (Aerobically or anaerobically?)
- As we expect most purple non-sulfur bacteria to be "facultative phototrophs," do you see any growth in the tube incubated in the dark? (Aerobically or anaerobically?) Is there corresponding growth in the "light" tube?
- Click here to see the appearance of a "facultative phototroph" in this test.
- Exp. 11.2 (Bacillus): For each of three isolated colonies from our plates from the "heat-shocked" soil suspension, we performed the endospore stain and set up tests for glucose fermentation, catalase and amylase.
- Why do we expect virtually any isolate from these plates to be a member of the genus Bacillus? If you don't see endospores, what might be a solution? (It's hinted at in the lab manual!)
- As we've mentioned a number of times, some species of Bacillus are strictly aerobic and the others are facultatively anaerobic. How does this relate to the tests for catalase and glucose fermentation? Recall Experiment 7.
- Must we expect all isolates of Bacillus to give positive results for the amylase test? Remember in Experiment 7 that we tested only three of the dozens of Bacillus species.
- Exp. 11.3 (nitrogen-fixers): For each of our chosen isolated colonies on N-free Agar, we made a wet mount and/or gram stain and we also inoculated a slant of N-free Agar and a slant of an all-purpose medium.
- Don't be concerned if you did not appear to have Azotobacter. Other nitrogen-fixers are possible but would be hard to identify with what little we did in lab. A non-motile gram-negative rod might suggest Klebsiella, and sometimes Bacillus (identifiable by the presence of endospores) is isolated. Others are possible; just characterize each isolate the best you can at least as either a nitrogen-fixer or a non-nitrogen-fixer.
- Would you expect nitrogen-fixers to grow on the all-purpose medium? (Why not?) Would you expect a non-nitrogen-fixer to grow on the N-free medium? Here you can see why inoculating these slants with a pure culture is absolutely necessary in order to tell whether or not a particular isolate is a nitrogen-fixer! This should hint at the probability that we did have non-nitrogen-fixers growing in our "N-free" enrichments and plates.