More About the Job.
Beginning bacteriology lab courses generally confine themselves to the relatively small universe of conveniently easy-to-grow representatives of the chemoorganotrophic bacteria. Much can be learned – and applied to other living things – from this microbial group which does happen to include a lot of important pathogens, food spoilage organisms, general contaminants, and participants in various aspects of simple biodegradation – basically the "in our face" organisms. As in our previous "Bacteriology 102" course, the new, partly-virtualized Microbiology 102 course encompasses a lot of good things to know about (including anaerobic respiration) and continues to venture beyond chemotrophy into the wonderful world of anoxygenic phototrophy. Indeed, we find that the purple non-sulfur photosynthetic bacteria are a refreshing change of pace and are actually quite easy to isolate, even from hailstones!
A beginning lab course (such as ours) should really be spending some time in the fascinating worlds of cyanobacteria and chemolithotrophs. That these organisms tend to be "inconvenient" to work with is no reason to ignore them. Think about where we would be without them. So why just pay lip service to microbial diversity? In the 21st Century we may discover presently unimaginable metabolic processes that energize and fabricate life forms on Mars and elsewhere, and putting more emphasis right now on lithotrophic organisms in general microbiology teaching labs would not be a bad idea. Such has always been highly relevant to microbiology. The late P. W. Wilson made the study of the nitrogen cycle (including nitrification – an important chemolithotrophic process in the soil) a significant part of his introductory micro lab when he was here through the early 1970s.
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Certainly microbial consortia are a practical consideration for demonstration and discussion. In my explorations of sandy areas up north, I frequently come across classic examples of cryptobiotic soil – those places where mixtures of chemotrophic and phototrophic microorganisms turn the inorganic into organic and initiate substrates for higher forms of life. These organisms are Nature's Terraformers – important in the early stages of the process wherein sand develops into topsoil. They can appear as clumps of steel wool and as velvety pincushions, and cutting one open reveals a core of sand. Click on the two nearby photos which were taken while visiting the Apostle Islands where there are some restricted areas containing cryptobiotic soil that are marked off as delicate ecosystems. Microscopically one can readily see fungi and also filamentous and unicellular algae and cyanobacteria. I have yet to check out what bacteria may be present. Aside from these "soil lichens," iron bacteria are another mixed group of organisms that I have been treating superficially (so far) on the web.
Of the (at least) five general ways that organisms can generate their energy, we delve into all but oxygenic phototrophy in Microbiology 102. Along the way we realize that testing and describing organisms regarding their use of any one (or more) of these five catabolic methods is certainly more instructive than getting bogged down with the old-school method (with a thioglycollate and glucose-containing solid medium) of determining "oxygen relationships" which can only be applied productively to the characterization of those aforementioned easy-to-grow chemoorganotrophs and is often a pain for students and instructors to interpret. Students get the false idea that all organisms can be classified by this method. It is an expensive, redundant and generally ill-taught test which is better used to demonstrate the special growth patterns of microaerophiles and the relative efficiencies of fermentation vs. aerobic respiration as shown for those named in this test system as "facultative anaerobes."
How is this test redundant? Easy, when you consider the actual reasons for the growth patterns in Thioglycollate Medium and what other tests can indicate, namely whether the organism can (1) tolerate a normal aerobic environment, (2) respire aerobically and (3) ferment. The catalase test correlates with aerobic respiration, and the glucose fermentation test correlates with anaerobic growth. Then you find most species of bacteria in this world can't even grow in Thioglycollate Medium and may have other reasons to grow aerobically or anaerobically.
To be truly instructive, E. coli should be thought of as an organism that does aerobic respiration, anaerobic respiration (with nitrate) and fermentation and does not perform oxygenic or anoxygenic phototrophy – not as one that simply can grow aerobically and anaerobically (and "likes oxygen") with nothing more said.
Even though this test should have died out with the 20th Century or at least with the demolition of Fred Hall, I actually have a suprisingly objective treatment of the traditional "oxygen relationship test" which explains its limited usefulness here. I miss the real good old days back in 5th grade science class when "oxygen relationships" were simply described as whether or not an organism had the capability (for whatever reason) of growing aerobically and/or anaerobically – with the simple use of the terms "aerobe," "anaerobe" and "facultative" [sic].
 I tend to spend an awful lot of time trying to find less time-consuming ways to teach the more complicated stuff, and there is no time to get cute and condescending in presenting the subject matter. After I can overcome the frequent mental blocks and get things understandable, then the oral presentations in lab become more effectual, and the board diagrams, handouts, manual material and associated web pages write themselves.
Regarding the webworks, I am still pumping out the not-so-interactive reference material in an attempt to upload what little I know about bacteriology besides what is in the 2006 lab manual cited below. The following apples/oranges list reflects some points:
- The catabolism page (still ranking reasonably high in a Google search for "catabolism" but no longer no. 1) arose out of an attempt to summarize energy/electron/ATP generation in the Farm Microbiology Short Course (a bright spot in the school year and certainly a place to expand on soil microbiology and lithotrophy) where lecture time is very short indeed. A "whiteboard" presentation developed during a Farm Micro lecture on the subject is shown here, and going along with this are the correct definitions and differentiations of the terms lithotroph vs. organotroph and also chemotroph vs. phototroph – keeping in mind respiration and fermentation only apply to the chemotrophic organisms.
- It is always best to summarize the basic strategy of any biological process first. For catabolism, then, further details can come along as time and relevancy to the course permit and may include such things as specific intermediates and pathways, the internal electron transfers in fermentation pathways, the importance of NADP as an electron acceptor in phototrophy, cyclic vs. non-cyclic photophosphorylation, what photoorganotrophy really amounts to, and the interesting variations of anaerobic respiration presented by methanogenesis and "anammox." (Anammox should be included in any discussion of the nitrogen cycle.)
- Another web effort is the beta-galactosidase page which is hidden away here.
- More bacteriological concepts whose practical applications and interpretations tend to follow basic patterns include quantitation, media, isolation, identification, and the cycles of elements. These things – as well as proper usage of terms – are gone over in the web pages expanded upon below and also in the 2006 manual.
- The enteric bacteria are especially suited as practical examples to use in the discussions regarding differential media formulation (see references 3 and 4 below) and genotypic identification, and that is where I tend to concentrate my interest concerning these organisms. When dealing with enterics in the teaching lab over the past several decades, useless memorization of genera and species and their characteristics and habitats have long ago given way to a more practical approach. A handout associated with our enteric experiment (which includes one of our classic "thought questions") is shown here.
The above-mentioned web material is simply what I have let happen, and I have no intention of making work for myself (and thereby creating misery) by putting together something more all-inclusive. Enough qualified individuals are posting on-line textbooks, and my interests in microbiology may not be all that comprehensive. Doing the web thing works best for me when I can go at my own speed. Thank you very much.
One kind of writing course that should be required concerns something I entirely regret not getting the hang of decades ago, and that is taking lecture notes in real-time that are complete, organized, and understandable. Maybe I should have learned stenography in high school. The inability to generate a "things to do" list that is totally inspiring and adhered to has been another problem. Yet when it comes to writing in general – whether it be academic, travel-related, fiction or whatever – who cannot be inspired by the highly-talented likes of a Robert J. Sawyer or Timothy Hallinan? Charles Dickens' contemporary Ned Buntline just may be able to teach us a thing or two; one can imagine the kind of blog that might have come out of him.
Getting back to topic, here is a generality that seems to hold up well: All dilution plating problems are basically the same problem but with different variables. That same idea applies when one is interpreting the various pH-based differential media. The solutions to these things need not be made unnecessarily complicated, and each particular medium or dilution problem need not be dealt with from scratch as a special case, for crying out loud.
No lab protocol is perfect in its content or organization, and there is always a need for improving such things. Students can look at a manual protocol for an upcoming experiment and come up with a clarified flow chart to get the lab done efficiently. Also, with a good command of basic microbiology, any thoughtful instructor or student can come up with some really creative ways of doing and finding out certain things, and it is encouragement of that sort of thing that is behind the thought exercise here.
As one structures one's course over its term and expects it to be a firm foundation for further coursework and productive research, "mixing apples and oranges" and otherwise playing loose with the basic concepts and definitions will turn it into a house of cards. Graduating ignorant and spending one's professional life addicted to consultants is not part of anyone's definition of "leadership."
Well, there goes the language. Would you believe there are still those publicly-funded individuals who teach their students that an autotroph is defined as one that "makes its own food"? No organism can do that for cryin' out loud, as each living thing processes its nutrients for catabolism and biosynthesis according to its kind. The emerging institutional redefinition of "mentor" (and "mentoring," etc.) threatens to obliterate the original meaning of the term which suggests a helpful personal and/or professional relationship that is simply allowed to happen – and for free! How can that be mandated by some special funded program? Redefinitions are running rampant. The abbreviation "i.e." is coming to mean "for example" which is an impossible stretch as explained here. "Aliquot" popularly refers to any specified volume rather than its original intended meaning which still has practical application wherein a better word cannot substitute. As for is, it is the fashion to say "media is" and "bacteria is" – as the actual singular forms of these nouns are disappearing. Self-appointed pseudo-mentors are holding sway, constantly coming up with more nonscience. Pretty soon, the Constitution will not be so. And these people are getting paid with your tax money.
Working with the teaching labs' culture collection over the several decades of my employment here has been quite educational. A few of our stock cultures are descendants of ATCC strains (many generations removed), but the majority of our cultures have been isolated by myself or students and have wound up being "archived" for one reason or another. My "ET Project" introduced a number of enterics into our teaching labs including a strain of the very rare Edwardsiella tarda Biogroup 1 whose origin was a small, warm bay of Lake Superior which harbored a lot of seagull feces and dead fish. Biogroup 1 of E. tarda is sometimes mentioned in the literature as being inherently H2S-negative – as if that statement is to be accepted by faith alone. In some media (such as TSI Agar) detection of H2S is negated by excess acid production from fermentation. Biogroup 1 shows a good positive reaction in other media such as KIA and the API-20E test; also see Figure 2 and Table 3 here.
Our spectacular Photobacterium was isolated from store-bought shrimp in the early 1980s by our "Advanced General Microbiology" lab (the late and lamented Bacteriology 320). The strains of E. coli and K. pneumoniae that we use routinely were isolated from pitcher plants, and our Rhodomicrobium came from water trapped by a bromeliad; click here for more about these plant sources.
So, what is a "strain"? It is a commonly mis-used word, and it can get so overly-used that it can easily start to sound ugly. Clarification is hopefully made here. I stand by that discussion 100%.
Even though some of our cultures come from well-known, commercial sources, we cannot put any strain number on any of our cultures but our own. Stock culture curators may not be able to prove that any particular strain of theirs has the same exact genotype and phenotype of the isolate or certified strain it was descended from – no matter how few generations have transpired. For certified cultures for research or industry, one must go to suppliers of such cultures – for example, ARS/NRRL and ATCC.
Lyophilization is probably the best method of storing cultures for indefinite periods, and it has been an ongoing process when our 1940's-era machine could be MacGyvered to work reliably. Lately I have come across a storage method that involves methylcellulose in which one does not have to put up with problems associated with lyophilization or ultra-low temperature freezing.
We have experienced the loss of some cultures while in refrigerated storage. One lesson I have learned is never to store Edwardsiella tarda on Heart Infusion Agar. (Nutrient Agar works better for extended viability under refrigeration for reasons unknown.) Also, we have seen major characteristics of a few of our refrigerator-stored strains visibly shift during serial subculturing. Some examples follow:
- The type strain of Pseudomonas aeruginosa loses its intense blue pigment upon repeated transfer.
- A Streptomyces griseus strain we used as a good "positive control" for showing filamentous growth and antibiotic production lost both properties in a few years. We now use an unspeciated student isolate that continues to be reliably spectacular on both accounts.
- An occasional mutant in our Klebsiella pneumoniae strain that produces no capsule (and consequently an atypical colony on EMB and MacConkey Agars) can show itself in great numbers after a few serial subcultures – necessitating the re-isolation of a typical mucoid colony before using the culture in class.
- A minority component (probably a mutant) in many of my Edwardsiella tarda isolates which has a delayed hydrogen sulfide reaction can "take over" in just a few generations! This has happened along with a decrease in alkaligenic activity (less amino acid deamination and/or lysine decarboxylation) – resulting in colonies on our Edwardsiella Isolation Agar (which is almost like a "hybrid" of MacConkey and XLD Agars) that give a greater net acidic reaction. Come to think of it, the greater acidity may be behind the decreased detection of hydrogen sulfide (as can happen in TSI Agar). Presently, what I can find of my original poster of my "ET Project" is reproduced here as the start of a new "ET Site" which will expand to show photos of colonies along with various aspects of isolation and identification. (See reference 3 below.)
So, what is frequently a thankless chore can also be a kind of education one cannot pick up from one's usual coursework or laboratory teaching duties.
- Robert H. Deibel and John A. Lindquist. 1981. General Food Microbiology Laboratory Manual. Pearson Education, Paramus, NJ. ISBN 0-8087-5559-5. In this lab manual, the real principles of food microbiology are dealt with in organized fashion and include: (1) detection and identification of contaminants, spoilage organisms, food-borne pathogens, and indicator organisms; (2) production of fermented foods by wild fermentation and with the aid of starter cultures; (3) respect for and practice of the concepts of aseptic technique in the laboratory and compartmentalization in food processing; and (4) some experiments involving microbial growth and the control of such growth. Regarding the last point, an understanding of the concept of "water activity" is most important. In the giddy rush to modernize courses, a lot of basic things tend to get discarded. (Throw out baby; keep bathwater.) However, these are items that should be part of any food microbiology lab course as is a discussion of the art and science of epidemiology as it relates to tracing the course of an outbreak of foodborne disease. Common sense from a real food microbiologist (R.H.D.) infuses each page of this manual, and my usual blanket (even though benign) disparagement of lab manuals does not apply here.
The bacteriological nomenclature may be a bit dated, but at least by 1981 we finally got away from using "Aerobacter." However, that genus name seems to have made a virtual comeback now that the classic "Aerobacter aerogenes" strains – which have been lately classified as Enterobacter aerogenes (motile) and Klebsiella pneumoniae (non-motile) – are closely allied again with the creation of Klebsiella mobilis for the motile strains. (Enterobacter aerogenes is now synonymous with Klebsiella mobilis in the new Bergey's Manual which has a citation below.)
- John A. Lindquist. 1975. Bacteriological and Ecological Observations on the Northern Pitcher Plant, Sarracenia purpurea. M.S. Thesis, Department of Bacteriology, University of Wisconsin, Madison, WI. This project came about when my proposal to pursue a coherent problem involving cyanophages was summarily rejected. (The question was this: Is lysogenic conversion responsible for Microcystis aeruginosa toxicity?) It would be great to get some momentum going with cyanophages again. As for pitcher plants, one expects (and does find) proteolytic and chitinolytic bacteria, but who would have thought these plants were such a great source of photosynthetic bacteria and a repository of E. coli? (Before e-mailing me about pitcher plants, please go to this page first!)
When it comes to green and leafy life-forms, I have been finding mulleins to be quite fascinating and instructive. Maybe they will find some practical use above and beyond their alleged medicinal value. Considering the abundance of cellulose in these ubiquitous plants, perhaps mulleins can contribute to the production of cellulosic ethanol? Or maybe not. When it comes to providing for our own energy needs from whatever source we may possess in abundance, we have been stalled most miserably.
- John A. Lindquist. 1991. Medium and Procedure for the Direct, Selective Isolation of Edwardsiella tarda from Environmental Water Samples. (Poster presented at ASM Meeting in Dallas on May 8, 1991.) Abstr. Annu. Meet. Am. Soc. Microbiol. 1991, C-303, p. 302. This project showed how a hypothetical selective-differential medium to isolate a certain physiological type of bacterium can work for real as it does on paper. Even in the 21st Century, this sort of thing still has relevance in the isolation and culture of pathogens and other organisms of interest from the environment. More about the "programming" of such media is here. This truly is great fun and is highly instructive – easily fitting in with lab teaching. As mentioned above, my original Edwardsiella tarda poster is reproduced here as the start of a new site devoted to isolation and identification of the species. Also, the isolation medium is briefly discussed in the Edwardsiella chapter of The Prokaryotes.
Not fun was another project involving searching for a pathogen among soil samples, but the isolation medium that came out of that made the whole ordeal worthwhile.
- John A. Lindquist and J. J. Farmer III. 1999. Isolation and Characterization of a New Genus of Edwardsiella-like Bacteria from Wisconsin Lakes. (Poster presented at ASM Meeting in Chicago on May 31, 1999.) Abstr. Annu. Meet. Am. Soc. Microbiol. 1999, R-1, p. 616. This is something brand new that tried to pass itself off as Edwardsiella tarda on the medium mentioned just above. After consulting the enteric identification tables in recent editions of the Manual of Clinical Microbiology (ISBN 1-55581-371-2), it appears that this organism is unique among the gram-negative, oxidase-negative fermenting rods in being both arginine-positive and mannitol-negative. The Enteric Identification Lab of the CDC did a complete workup of its phenotypic and genotypic characterization, and the organism has been established as CDC Enteric Group 121. GenBank has its 16S rRNA sequence on the web: Click here and search Nucleotide for AF015258. The tentative name of the organism that you will see there translates as "water unit from Hayward"! Where I found the first isolate is shown here.
- John A. Lindquist and J. J. Farmer III. 2005. Genus XXXVIII. Trabulsiella. In Brenner, Krieg and Staley (eds.), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, pp. 827-828. Springer, New York. ISBN 0-387-24144-2. No, this isn't the new genus mentioned just above. Stay tuned for that!
- John A. Lindquist. 2006. General Microbiology: A Laboratory Manual, Fourth Edition. McGraw-Hill Companies. ISBN 0-07-339101-8. This is the last edition of the manual we used through 2006 when Bacteriology 102 was an entirely hands-on lab course. As alluded to previously, one cannot judge a general laboratory course only by its manual. See no. 8 below.
- Henry S. Gibbons, Stacey M. Broomall, Lauren A. McNew, Hajnalka Daligault, Carol Chapman, David Bruce, Mark Karavis, Michael Krepps, Paul A. McGregor, Charles Hong, Kyong H. Park, Arya Akmal, Andrew Feldman, Jeffrey S. Lin, Wenling E. Chang, Brandon W. Higgs, Plamen Demirev, John Lindquist, Alvin Liem, Ed Fochler, Timothy D. Read, Roxanne Tapia, Shannon Johnson, Kimberly A. Bishop-Lilly, Chris Detter, Cliff Han, Shanmuga Shozhamannan, C. Nicole Rosenzweig, Evan W. Skowronski. 2011. Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant. PLoS ONE 6(3): e17836. doi:10.1371/journal.pone.0017836 This article is found on-line here.
- The 2006 General Microbiology lab manual listed above (no. 6) has been supplemented or complemented by my two main microbiology websites which will remain on the web in perpetuity – freely accessible to serve as supplements wherever appropriate. Handouts to go along with lectures in various courses have been derived from much of this material. Here is what's up with the two sites, and I cannot put it any more plainly:
- The "GENERAL TOPICS WEB PAGES" are on jlindquist.net and are indexed here. Most had their origins in review or remedial material provided as handouts when I taught the Bacteriology/Food Science 324 lab course way back in the last millennium. One is expected to have a good general microbiological background and understand this material fully when dealing with (1) federal and industry manuals which detail methods and regulations and are not meant to serve as textbooks, (2) reference works such as Bergey's Manual and The Prokaryotes, and (3) practicing microbiologists out there in the real world who (by the way) occasionally express serious concern about new hires who have not retained their proper aseptic technique procedures since their graduation.
- The "retired" BACTERIOLOGY 102 SITE is on splammo.net and was freshened-up for each semester that I happily taught it as a totally hands-on laboratory course. One would think that every course taught at a public-funded university should have an accessible site on the internet. The Archived Site for Fall Semester, 2006 begins at its home page, and the updates page for that semester (1) gave some focus to the absolute glut of web resources and (2) reiterated announcements given in the lab. Certain pages containing background material are still updated as necessary. Now that much of the course is taught on-line, students can access information, grades, etc. via the "Learn@UW" website. (Watch out – here comes an update!)
- An Update as of Fall Semester, 2010: As the Bacteriology 102 Site went over so well during its active tenure, and as Learn@UW can be a problem regarding instructor access, student access, and organization, I have gone back to the web to develop a new MICROBIOLOGY 102 SITE (on jlindquist.net) which will permanently make the retired/archived Bact. 102 site even more so. Analogous to "Course News" and "Content" on Learn@UW are the easier-to-access updates and links (to course-related matters) on the home page. Learn@UW can still serve to summarize grades privately.
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