Bacteriology 102:
Some Photos and Notes on
the Enrichment & Isolation

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Notes on Exps. 9.3, 10.2 & 11
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Note: These would be Experiments 11.1, 11.2, 11.3, 10.2 and 9.3 in the lab manual that was in use at the time the Bacteriology 102 Website was "retired" (Fall Semester, 2006). The information on this and related pages has been updated as necessary.
Do not heed any pages dated around 2000 that were put up on a .edu website, as those pages are truly outdated and totally unauthorized.

Experiment 9.3 – Isolation of Bacteriophages

LEFTPlate inoculated with 0.5 ml of sewage (which had been passed through a filter to trap bacteria and larger particles) and E. coli strain B. Note the bacteriophage plaques appearing on the bacterial lawn. Different phages can produce different-appearing plaques (large vs. small, cloudy vs. clear, etc.). Individual plaques can be inoculated into growing broth cultures of E. coli B (as one would inoculate bacterial colonies into sterile media); thus one can propagate "cultures" of bacteriophage isolates which will lyse the bacterial cells as they replicate. The resulting suspensions ("lysates") can be filtered or chloroformed to remove any remaining viable bacterial cells.
RIGHTThree bacteriophage isolates (I, II and III) and their activity or inactivity on six strains of E. coli (B, A, etc.) streaked on petri plates. Note the large plaque wherever a particular phage can infect a certain strain of E. coli.

Experiment 10.2 – Isolation of Streptomyces

LEFTTypical dry, circular, opaque, convex colonies of Streptomyces predominate on this plate of Actinomycete Isolation Agar which had been inoculated with a dilution of soil. Such colonies are picked for future study. Shiny or filamentous colonies (some seen here) are not Streptomyces and are therefore avoided.
RIGHTThe test to determine antibiotic production. After the Streptomyces isolate (vertical streak) has grown for several days, several test organisms are streaked at right angles right up to the Streptomyces growth. Inability of a test organism to grow in the presence of Streptomyces suggests antibiotic production by the latter.

Experiment 11.1 – Enrichment & Isolation of Purple Non-Sulfur Photosynthetic Bacteria

Click here for a more detailed overview.

Experiment 11.2 – Isolation of Bacillus

Click here for a more detailed overview.

Experiment 11.3 – Enrichment & Isolation of Nitrogen-Fixing Bacteria

THE NITROGEN CYCLE: This diagram is based on that which has been found in T. D. Brock's textbooks. Other explanations and representations of the nitrogen cycle which are especially noteworthy for their completeness and organization can be found on the web here and here.

In the diagram of the Nitrogen Cycle on the right, each of the conversions in the outside circle from one type of nitrogen compound to another is carried out by certain bacteria, some of which are suited only for that particular step – such as those autotrophic organisms which oxidize ammonium to nitrite and another autotrophic group which oxidizes nitrite to nitrate. Details of the organisms involved in each step are given on our "cycles page."

Most of the "anaerobic" N2 shown on this diagram actually comes from the atmosphere.

In Experiment 7, we see how some of our cultures utlize nitrate as an electron acceptor in the process of anaerobic respiration and reduce it to nitrite and even to nitrogen gas. At various times we have seen an alkaline reaction in some media which, in most cases, is caused by ammonium released in the deamination of amino acids. See pages 17 and 143 in the manual.

In Exp. 11.3, we are concerned with free-living organisms which can take atmospheric nitrogen and convert it to usable nitrogenous compounds, and we are able to isolate aerobic and facultatively anaerobic N2-fixers on our aerobically-incubated plates. Many isolates may be recognizable as Azotobacter (oval-shaped cells in pairs) and some as Bacillus (rod-shaped cells with endospores), but others would need some extended testing to determine probable genus, an example being Klebsiella. Do not be overly concerned if you cannot identify any of your isolates, as we really did not do that many identification tests and observations! (Note the discussion here and the recommendations in the latter part of Exp. 11.3 in the manual regarding identification.) The following diagram is an overview of the Exp. 11.3 procedure.

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Page last modified on 5/1/08 at 9:15 AM, CDT.

John Lindquist, Department of Bacteriology

University of Wisconsin – Madison