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.

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:

• 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 here.
  
  
• The blank table found here can be a valuable study guide once it is filled in.
  
  
• During the course of the various isolation experiments, ask yourself some questions. Many of these questions follow the general theme of "what am I doing and why?" We hope to see a good understanding of these things on quizzes, tests, reports and posters!
  
  • In the highlighted box above, we show a six-part multiple-true/false question. Be sure you understand why each of the six statements is false.
    
  • Questions are posed below in Sections III and V concerning the setup of the various experiments and what we hope to find out about the cultures we isolate.
    
  • Also, look over the relevant quiz questions in Appendix X of the lab manual. They test basic understanding of the general concept of isolating bacteria as well as major points about the specific kinds of organisms we are looking for.
    
  • Be sure to go over the requirements for reports and posters associated with these experiments. Some questions to think about are found on this page.

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:

• The Prokaryotes, a multi-volume set. The updated on-line edition is found here and is the primary source of reliable information I recommend when questions keep pouring in about how to isolate whatever. As stated above, I do not claim to be an expert on such things – or a general consultant, for that matter. But The Prokaryotes is where the experts on specific kinds of bacteria hang out. As an example of how one can begin to use The Prokaryotes on-line, type "Edwardsiella AND isolation" in the search box (without the quotes), and you might find a surprise.
  
  
• Bergey's Manual of Systematic Bacteriology, a multi-volume set. The first volume of the new, 2nd edition is out now and includes photosynthetic bacteria. The second volume – to appear as three separate "sub-volumes" – should begin to appear in June, 2005.
  
  
• The latest (9th) edition of the one-volume Bergey's Manual of Determinative Bacteriology is mainly for identification and has become very outdated in that respect.
  
  
• The Manual of Clinical Microbiology and the Bacteriological Analytical Manual ("BAM") also include isolation procedures; those in the latter work are intended to standardize procedures in the food industry.
  

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:

• Choice of suitable source material likely to contain the desired organism. Also, one can consider where the desired organism may occur as a significant contaminant. As mentioned elsewhere, purple non-sulfur photosynthetic bacteria have been found in samples of snow, ice and rain – surely not to be considered as habitats for microorganisms.
  
  
• 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. Selective enrichments can also be used for certain organisms that are not necessarily in low numbers; the "most probable number" method of quantitation involves their use as we shall find out in the water analysis experiment (Exp. 15).
    
  • 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 III (below) and a general discussion is given in Appendix D of the manual which is 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 IV, below.

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):

• 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, photosynthetic bacteria utilize light as their ultimate source of energy, and any compounds that can serve as energy sources for other types of organisms can be left out of the medium.
  
• Compounds which can be used as growth factors (vitamins, amino acids, nucleic acid bases, 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? Why is it beneficial to re-streak our initial colonies on an all-purpose medium?
  
• 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.
  
  

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.

• 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. Specifically, Rhodospirillum and Rhodomicrobium are especially recognizable among isolates of purple non-sulfur photosynthetic bacteria, and Azotobacter and Bacillus can be identified by their microscopic appearance in the nitrogen-fixer experiment.
  
  
• 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 colony types.)
  
  
• Plaques produced by bacteriophages infecting a suitable host culture of bacteria.
  
  
• When we get to the enrichment procedure for coliforms in Experiment 15, consider how gas production in appropriately-selective, lactose-containing broth media serves to signal their presence.
  
  

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:

• Exp. 10.2 (Streptomyces): How did we recognize probable Streptomyces colonies, and what do the cells look like microscopically? Should we expect all Streptomyces isolates to be able to produce an antibiotic? What is our official definition of antibiotic?
  
  
• 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 also to test phototaxis) and inoculated two tubes of melted Succinate Agar in the manner that we inoculated Thioglycollate Medium in Exp. 5.1 to determine oxygen relationships. (Keep in mind that the only reason for anaerobic growth in Thioglycollate Medium is fermentation which does not apply in Exp. 11.1, and the only reason for anaerobic growth in a tube of Succinate Agar is due to anoxygenic phototrophy. See the summary of what is associated with an organism's ability to grow anaerobically here.)
  
  • As we incubated one tube of Succinate Agar 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 for any isolate, what might you do with the isolation plates in order to see endospores eventually? (It's hinted at in the lab manual!)
    
  • As we have 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 what we did in Experiment 7 to show the oxygen relationships of the twelve known cultures without the use of Thioglycollate Medium.
    
  • 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.
  
  • Do not be concerned if you did not isolate anything resembling Azotobacter. Other nitrogen-fixers are possible, but they would be a bit harder to identify with what little we did in lab. A non-motile gram-negative rod might suggest Klebsiella. Sometimes Bacillus (identifiable if endospores are apparent) is isolated. Why would we not expect Clostridium on our plates? (Think about the incubation conditions of the plates.) Just characterize each isolate the best you can, and do not go beyond the recommendations and precautions mentioned in the latter part of Experiment 11.3, although some semesters we have media available to see whether or not any non-motile gram-negative rod is a Klebsiella. If you can't identify anything to genus based on the observations made, don't go sleepless about it! At least you can indicate whether or not each one was a nitrogen-fixer or a non-nitrogen-fixer.
    
  • As for the slants we inoculate our isolates onto: Would you expect nitrogen-fixers to grow on the all-purpose medium? (Why not?) Would you expect a non-nitrogen-fixer to grow by itself on the N-free medium? (Why?) For there to be any growth on the N-free medium, the growth has to be initiated by a nitrogen-fixer, so a pure culture inoculated onto such a medium which grows (without any "help" from another organism) is considered by us to be a confirmed nitrogen-fixer. We always strive to inoculate any of our test media with a pure culture and this test is no exception. Furthermore, if a mixed/contaminated culture were to be inoculated onto an N-free Agar slant and it subsequently grew, there must have been a nitrogen-fixer included in the mixed inoculum. But at this point in the semester, let's not expect to be rampant contaminators any more – what with our aseptic technique skills which had better not be deteriorating!

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.