Bacteriology 102:
Isolation of Bacillus

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Bacillus Isolation
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Relevant to the Bacillus isolation experiment in the
UW-Madison Bacteriology 102 Course
and not intended
to be authoritative for applied or research purposes.
Experiment numbers relate to our old lab manual.
Text and photos by John Lindquist.
Copies of this page found elsewhere are not authorized.


I. Organisms in Soil and the Nature of Their Colony-Forming Units.

A wide variety of microorganisms can be isolated from soil. In rich, moist soil, where many nutrients are available, vegetative cells of many genera of bacteria and fungi can flourish. Bacteria such as Bacillus, Streptomyces, Pseudomonas, Micrococcus, coliforms, lactic acid bacteria and (in the anaerobic pockets) Clostridium can actively metabolize decomposing plant and animal matter and various inorganic nutrients. Fungi such as molds and yeasts are also active in such an environment. The basic metabolizing "organismal unit" of any species of unicellular or filamentous bacteria is the individual, undifferentiated vegetative cell, and this basically holds true for molds and yeasts as well. These cells can replicate (usually by binary fission), giving rise to visible growth of their population. We measure such population growth in a bacteriological medium for our growth curve experiment.

As nutrients become depleted or are made less available by the drying out of the soil, vegetative cells of some organisms such as Streptomyces and molds can undergo a special type of cell division to produce many reproductive spores which can withstand a considerable degree of dryness and be carried in wind and water currents to new habitats. Microscopically, one sees the vegetative cells of Streptomyces and molds as filamentous and branching, and the round spores arise from the sides or ends of the filaments, often in clusters or chains. The spores are highly efficient in dispersing their species all over the environment, and this fact is reflected in the term "reproductive."

Vegetative cells of certain other organisms, notably of the genera Bacillus and Clostridium, can undergo a special, unequal type of binary fission where – instead of one cell dividing into two separate cells – one of the cells forms inside the other cell. The outer cell has been generally called the "mother" or "parent" cell, but this is a misnomer as the two cells are actually siblings. The new, special kind of cell is called an endospore, and it possesses several resistant outer layers and the internal features of a vegetative cell except for being almost completely devoid of water. When the enclosing cell lyses, the endospore becomes "free" and can remain viable for extended periods of time.

It can be mentioned at this point that – for purposes of convenience in our course – we have been generalizing our treatment of endospore-forming organisms as the genera Bacillus and Clostridium. As we get into the 21st Century, more and more species are being moved from these genera into newly-defined genera with varying degrees of relatedness. This situation is reflected in Bergey's Manual of Systematic Bacteriology (2nd Edition, Volume 3, 2009). So, our course continues to be slow in adapting to this new convention, and (for now) we treat the strictly aerobic and facultatively anaerobic rod-shaped endosporeformers as simply Bacillus, and those that are strict anaerobes as Clostridium.

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Endospores are metabolically inert and can withstand a wider variety of deleterious conditions than reproductive spores – such as radiation, abrasion, extremes of heat and cold, and lack of nutrients and water. Like a reproductive spore, an endospore will germinate and form a vegetative cell when conditions return for the growth of these cells, and generations of vegetative cells will again thrive as long as the appropriate nutrients and environmental conditions are present. When the nutrients begin to run out, endospores are again produced; this can be seen to happen on artificial media as well as in the normal habitat.

This diagram illustrates the life cycle of endospore-forming bacteria including the sporulation, germination and outgrowth events. The colors shown here are the differential colors one sees upon using the endospore staining procedure on smears made from our colonies. Going along with this diagram is the important fact that the vegetative cell can replicate itself and thus increase the population; subsequently, when the nutrients begin to run out, only one endospore can be produced per vegetative cell.

The following table summarizes the discussion above regarding the types of cells that may be found in a sample of soil such as what we plate out in our Bacillus isolation experiment. A vegetative cell, reproductive spore and endospore can each constitute a colony-forming unit. Any spore would have to germinate and become a vegetative cell before population growth (colony formation) could take place, as spores themselves cannot replicate to form more spores.

ORGANISMS IN SOIL possible types of cells (CFUs)
vegetative
cells
reproductive
spores
endospores
Bacillus strictly aerobic species + +
facultatively anaerobic species + +
Clostridium (strictly anaerobic) + +
Streptomyces and molds + +
Pseudomonas and others +

For a "heat-shocked" sample of soil, we expect the only colony-forming units to be endospores, the others having been killed off by the heat treatment. (Naturally we could not expect endospores to be produced during the heat-shocking treatment.) Subsequent aerobic or anaerobic incubation will determine what species of Bacillus will form colonies on the plates and whether or not we can expect colonies of Clostridium to come up. The variety of different types of colonies which arise during incubation represents different species of soil bacteria whose populations were "saved from extinction" by the fact that endospores had been produced in those populations. Accordingly, we would expect a greater variety of colony types on plates inoculated with a non-heat-shocked sample of the same soil.

An endospore is "activated" – i.e., ready to germinate – after an extended period of dormancy, such as those present in many of our soil samples that we keep around for months to decades. A newly-formed endospore can be activated by a short exposure to a high temperature.

II. Isolation of Bacillus.

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LEFT

Plate of Nutrient Agar inoculated (by the spread-plate method) with 0.1 ml of a heated (80°C for 15 min) dilution of soil and incubated aerobically. We would expect the CFUs (colony-forming units) to have been just endospores, as vegetative cells and reproductive spores of microorganisms would have been killed by the heat treatment. (An exception would be vegetative cells of thermophilic bacteria, but these organisms would not be growing and producing colonies under our 30°C incubation conditions.) Because of the heat treatment of the inoculum and the aerobic incubation of the plates, virtually all colonies on this plate would be those of various species of Bacillus. The spreading, filamentous growth is that of Bacillus mycoides, one of the few bacterial species which are identifiable by colony appearance. The "filaments" of B. mycoides always curve counterclockwise upon the surface they are growing.

RIGHT

Here is a plate inoculated by the streak-plate method (not utilized by us in the initial Bacillus isolation process) with a non-heat-treated soil suspension. A wider variety and greater overall number of various organisms would be expected, as the CFUs could have been not only endospores but also vegetative cells and reproductive spores of a wide variety of bacteria. This difference can be demonstrated in Experiment 11.2 when we do quantitative platings of our soil suspension before and after the heat treatment. Easily noted on this particular plate is a red-pigmented strain of Streptomyces which, being a non-endospore-former, would not show up on the plates inocluated from the heat-treated suspension.

III. Characterization of Isolates in Experiment 11.2.

Back in Experiment 7, we studied three species of Bacillus, and we could infer the oxygen relationship of each from their reactions in the catalase and glucose fermentation tests, having already noted their ability to grow well under aerobic conditions. Remember that any given species of Bacillus can be a strict aerobe or a facultative anaerobe. We do those tests again here, but – realizing that there could be dozens of different species of Bacillus on our isolation plates – we would have to run a large number of tests for species identification which we are not prepared to do. Check a recent edition of Bergey's Manual for more information.

We also run the amylase test in this isolation experiment. Is this a definitive test for the identification of the genus Bacillus? Do you or any of your neighbors get a negative reaction for any isolate?

IV. Genotypic Comparison of Eight Species of Bacillus.

When one compares the genome (specifically that part which codes for 16S rRNA) of Bacillus anthracis with those of the three species it most closely resembles phenotypically, one finds that these four species are virtually identical. At one time, B. anthracis, B. mycoides and B. thuringiensis were considered "varieties" of B. cereus, and technically (based on genetic analysis) they may still be thought of as belonging the same species. "Variety" was a taxonomic term lower in rank than species (similar to subspecies or biotype) to which strains having one or more notable phenotypic properties were assigned – for example, B. cereus var. anthracis.

These four species are compared with four more-distantly related species below. The degree of similarity may be noted to some extent when one scrolls back and forth. If you do not have an IFRAME-capable browser, click here. Programs are available into which one can feed sequence data and come up with a "phylogenetic tree" as discussed on this page (which links to a similar comparison). The sequences reproduced below are from GenBank's Nucleotide data which can be accessed from here.

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Bacillus anthracis (Ames)------------
Bacillus cereus (AH 527)-------------
Bacillus mycoides (10206)------------
Bacillus thuringiensis (ATCC 33679)--
Bacillus subtilis (AB065370)---------
Bacillus stearothermophilus (DSM 22T)
Bacillus polymyxa (DSM 36T)----------
Bacillus alvei (DSM 29T)-------------
Scroll right and left.

Primarily on account of comparative nucleic acid studies, a number of species in the genus Bacillus have been transferred to new, genetically and phenotypically-distinct genera which are not yet recognized in our Bacteriology/Microbiology 102 course which (as mentioned at the start of this page) is still mired in 20th Century taxonomy. One now finds three of the eight species considered here reassigned as follows: Geobacillus stearothermophilus, Paenibacillus polymyxa and Paenibacillus alvei. Other new genera containing species formerly in Bacillus include Alicyclobacillus, Aneurinibacillus, Brevibacillus, Halobacillus and Virgibacillus – all of which one can find detailed here.

V. More to come.

Being one of the several pages on the "retired and archived" Bacteriology 102 site (as of Fall, 2006) that is still updated as a general reference page, there is more that can be added. In the meantime, one can find out more about Bacillus by performing a search; click here and enter "Bacillus" or "endospores." A reliable modern textbook such as Brock's can be consulted to understand the finer details of endospore formation and structure and the variety of endospore-forming bacteria.


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Page content was last modified on 12/14/12 at 10:00 AM, CST.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison