Bacteriology 102
Exps. 12, 13 & 14:
Additional Notes

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Notes on Exps. 12, 13 & 14
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Modified:
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With these experiments, we begin to consider various "groups" of bacteria:

  • Exp. 12 concerns a group of gram-positive bacteria – the "lactics" – which produce lactic acid as the only acidic end-product of fermentation and were at one time considered members of the same taxonomic family.
  • Exp. 13.1 deals with several non-related genera of gram-positive and negative cocci which have clinical significance.
  • Exp. 14 concerns a group of gram-negative rods – the "enterics" – which are members of the same taxonomic family, the Enterobacteriaceae.

The table below shows the general characteristics of these organisms and is also the origin of the dichotomous key on page 150 of the lab manual (which is also reproduced here). As noted in our discussion of dichotomous keys, one does not have to consider all of the possible reactions of the organisms. For such a key, one usually begins with characteristics generally known (gram reaction and cellular shape) and continues on with whatever relevant tests or observations eventually differentiate the individual organisms.

Included on this table are tests which demonstrate some special phenotypic property and/or show differences useful in specific identification. Not only is this a good table to know, but the various tests must be understood as to their purposes and principles. Much of this can relate to our page on bacterial identification.

  gram rx.,
shape
catalase
reaction1
glucose
fermen-
tation1
oxidase
reaction
some additional tests of interest
Neisseria –, cocci +   +  
Staphylococcus +, cocci + + coagulase
Micrococcus2 +, cocci + +  
Lactic acid bacteria (the "lactics") +, cocci and rods + hot loop, hemolysis (for the "streptococci"), slime from sucrose, etc.
Enteric bacteria (the "enterics")3 –, rods + + phenylalanine, H2S, methyl red, lactose fermentation, etc.
Pseudomonas2 –, rods + +  

1  From the pattern of reactions for the catalase test and glucose fermentation (and the fact that all can grow in the presence of air), one can deduce the probable oxygen relationship. We also did this for the cultures in Exp. 7 and the various Bacillus isolates in Exp. 11.2.

  • catalase+ and glucose– : strict aerobe
  • catalase+ and glucose+ : facultative anaerobe
  • catalase– and glucose+ : aerotolerant anaerobe

2  Micrococcus and Pseudomonas are included in this table for comparison with Staphylococcus and the enterics, respectively.

3  Enteric-like organisms which are oxidase-positive include Vibrio, Aeromonasand Chromobacterium. The last-named was included among the cultures in Experiment 7.1 and showed a bright purple pigmentation.


Experiment 12: The Lactic Acid Bacteria

The lactic acid bacteria ("lactics") include the "streptococci" in the general sense – i.e., Streptococcus, Enterococcus and Lactococcus– all of which were lumped together as Streptococcusnot too long ago. Also included in the lactics are Leuconostoc, Aerococcusand Lactobacillus. As they are considered "aerotolerant anaerobes" and also "fastidious," the following will assist in the isolation of lactic acid bacteria as explained in the introduction to Experiment 11: incorporation of sodium azide into a rich plating medium (i.e., one containing lots of growth factors and also glucose for fermentation) and aerobic incubation. Sodium azide inhibits the activities of iron-porphyrin compounds such as catalase and cytochromes. As the lactics do not possess these compounds, they are not affected by sodium azide.

For the lactic acid bacteria, the hot loop test differentiates between the "homofermenters" (those that produce lactic acid as the end product of glucose fermentation) and the "heterofermenters" (those that produce equal amounts of lactic acid, ethanol and carbon dioxide from glucose fermentation). Dissolved gas that comes out of solution and forms bubbles when the hot loop is jammed into the medium (usually APT Broth) constitutes the positive reaction and indicates the presence of a heterofermenter.


Experiment 14: The Enteric Bacteria

The enteric bacteria ("enterics") also have a differentiating test that divides the enteric group into two major physiological subgroups based on fermentation. The methyl red test differentiates between the "mixed acid fermenters" (those that produce a mixture of acids and ethanol from glucose fermentation) and the "butanediol fermenters" (those that produce acids, ethanol and "neutral products" – the production of which consumes some of the acid). The very low pH maintained by the "mixed acid fermenters" (after at least two days of incubation at 37°C) turns the methyl red a red color which constitutes the positive reaction and indicates the presence of a mixed acid fermenter. Click here for a graphic representation of what goes on in the medium for both types of organisms.

Ingredients in enrichment and plating media which assist in the isolation of enteric bacteria include various selective agents which inhibit the growth of gram-positive bacteria. There is no medium which supports growth of enterics exclusively. Pseudomonasis an important non-enteric to consider as it frequently "gets in the way" during our efforts to isolate enterics from samples we are testing (clinical, environmental, food, etc.). To drive this fact home, we add Pseudomonas to our mixture of enteric unknowns in Experiment 14.1, and we find we can eliminate it sooner or later during the isolation process.

The following "flow chart" is based on the main elements of bacterial isolation as taught and applied in our enrichment and isolation exercises and specifically summarizes the general enteric isolation procedure as discussed in the introduction to Experiment 14.1 in the manual.

ENRICHMENT AND ISOLATION IDENTIFICATION
SOURCE
MATERIAL
x SELECTIVE-DIFFERENTIAL
PLATING MEDIUM
FOR ISOLATION
x PRELIMINARY
SCREENING
MEDIA
x ADDITIONAL
BIOCHEMICAL
TESTS
  x   x   x   x  
  SELECTIVE-
DIFFERENTIAL
ENRICHMENT(S)
  SEROLOGICAL
TESTING
 

Realize that the intent of Experiment 14.1 is not to identify unknowns, as we assume that will come about "automatically" if basic aseptic technique is followed. With the enterics, we can learn more about differential media – even what it might take to "program" such a medium to highlight a certain physiological type of organism. This can be helpful in the isolation and identification processes.

The pH reaction one might see in many of our differential media is the net result from competing alkaline and acidic reactions shown on "Table I" on page 82 in the manual. This idea is explained in general here – and more specifically here for Kligler Iron Agar (KIA).

You can play around with the idea of programming media with the "thought questions" given here: Question #1 is virtually identical to Question 14I in Appendix X of the manual and involves the formulation of an enteric plating medium that will make colonies of "Excalibacterium" look a certain, detectable way, and other enterics look differently. Question #2 is replicated on a handout given during Period 3 of Exp. 14.1 and involves the differentiation of cultures of "Sorgobacter" from other enterics in a KIA-based medium.

Caution: Please don't waste time looking for our fictional organisms in the library or elsewhere! We often make use of them as hypothetical examples on the web and also in quizzes and exams while we wait for our new genus to be published and thus available as a true example of media programming in action!

If you are interested in reading more about Salmonella– the subject of Experiment 14.2 – you can start with our Salmonellapage which includes a photo of the slide agglutination test as done in lab. With the antisera we use, we do not get to identify our culture to any specific serotype (serovar), but rather a "serological group" to which the serotype would belong, such as "Group B" or "Group C1."

Click here for a brief discussion of genotypic identification of bacteria – something that has become useful in the definition and identification of bacterial species, genera and higher groups. A summary of how genotypic characterization of bacteria can lead to inferences about evolutionary relationships is shown here – the only place on the web where this author has mentioned our new organism by name while awaiting publication (as mentioned above).


  • The lab manual referred to herein is referenced here.
  • Our revised and simplified demonstration of enteric plating media which was applicable to the course as taught through 2006 is shown here.
  • The introductory handout for the enteric experiment (wherein is a comparison with lactic acid bacteria) is reproduced as a pdf file here. (The two media questions on this handout are also here as Questions 1 and 2.)
  • Home Page of Bacteriology 102 Web Site
  • Index of the General Bacteriology Pages

Page last modified on 3/23/07 at 2:45 PM, CDT.
John Lindquist, Department of Bacteriology
University of Wisconsin – Madison