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This experiment is virtual and a prelude to Experiment 7

Of all the physical factors that can affect microbial growth, the most important are temperature, pH (hydrogen ion concentration) and oxygen tension (oxidation-reduction potential).  Here we will demonstrate the relationship between “oxygen relationships” and certain physiological reactions as they apply to typical chemoheterotrophic organisms.  Be sure you have read the Introductory Material and the Appendix D as a prelude to the following.

I.  METHOD OF ENERGY GENERATION

A chemotrophic organism uses a substrate to extract energy, in what is known as a catabolic “pathway”. In essence the organisms are removing high-energy electrons, or oxidizing the substrate, and also in the process produce various end (“waste”) products.  As the substrate is broken down in this pathway, the energy that is released is trapped in ATP, the energy currency.  For a phototrophic organism (such as what we will encounter in Experiment 11), light is the ultimate source of energy.

The actual generation of ATP, incorporating the released energy, is called phosphorylation of which there are three kinds:  oxidative, substrate-level and photophosphorylation.  The path that a particular organism takes is a fundamental part of their genetic make-up. We will leave the intricate details of these processes to your lecture course and textbook, although there is a general overview of catabolism that may be helpful in Appendix D in this manual.

In this course, we will be dealing almost exclusively with chemotrophic bacteria, specifically the chemoheterotrophs.  (In Experiment 11B, we will be dealing with photoheterotrophs.)  Depending on the abilities of the specific organism and the environment in which it is found, the catabolic pathways of chemotrophs are associated with either oxidative or substrate-level phosphorylation.  If the former, the organism is undergoing respiration; if the latter, the process is fermentation.  Relative comparisons are made between respiration and fermentation below.

1.Respiration:

a.   There is a wide variety of potential substrates (amino acids, sugars, even inorganic compounds, etc.). The substrate is more completely broken down than it is during fermentation processes.

b.   A relatively smaller variety of end products is produced. A small amount of acid is produced

when sugars are respired.

c.   ATP is generated by oxidative phosphorylation wherein relatively more ATP is generated

than by substrate-level phosphorylation, and oxygen is often utilized as the terminal electron acceptor.  (Certain organisms can use an alternate terminal electron acceptor such as nitrate under anaerobic conditions; this situation is termed anaerobic respiration and will not be considered or tested for until we get to Experiment 7.)

d.   Respirers tend to grow “better” than organisms relying on fermentation processes.

2.   Fermentation:

a.   A smaller variety of substrates can be fermented, some microbes ferment sugars, some may ferment amino acids. The substrate is not completely broken down, we call this incomplete oxidation.

b.   Relatively larger variety of end products is produced.  Much acid (and possibly gas) is pro-

duced when sugars are fermented.

c.   ATP is generated by substrate-level phosphorylation wherein relatively less ATP is

generated than by oxidative phosphorylation.  Oxygen is not involved in the process.

d.   Microbes fermenting are growing less efficiently.

 II. the test for “OXYGEN RELATIONSHIP” WITH the USE OF THIOGLYCOLLATE MEDIUM

Oxygen relationship is generally defined as the growth response of an organism while in the presence or absence of oxygen.  To a laboratory bacteriologist, whether or not growth occurs aerobically or anaerobically is dependant on the tolerance of the organism to oxygen and the mode of chemotrophic energy generation used by the organism – specifically (1) whether or not the organism can perform aerobic respiration and (2) whether or not the organism can ferment.

The following categories of oxygen relationships are based on those found in Bergey’s

Manual of Determinative Bacteriology (9th ed., 1994) but they are only applied to chemotrophic organisms.  We will refer to these terms often this semester, especially when we attempt to identify microorganisms.  We often determine the oxygen relationship of a microbe in this process.

1.   Strict aerobes (also called obligate aerobes) possess only a respiratory type of metabolism

and use oxygen as the terminal electron acceptor.  They can grow in a normal air atmosphere.  A related group is Strict Respirers, they often use oxygen as the terminal electron acceptor but can grow anaerobically if they are able to use an “oxygen substitute” such as nitrate.  (Alternate electron acceptors are not included in Thioglycollate Medium.)

2.   Microaerophiles require oxygen in their metabolism, usually for respiration, but they cannot

grow under normal atmospheric O2 levels.  Optimal growth is achieved at a much lower O2 con-centration.  We consider microaerophiles very little in this course.

3.   Facultative anaerobes are as described for strict aerobes but they have the additional ability

to obtain energy by fermentation.  Anaerobic growth occurs when a fermentable compound is present; most facultative anaerobes can ferment glucose and certain other sugars.  As respiration is a more efficient means of energy generation than fermentation, growth is heavier in the presence of O2.

4.   Aerotolerant anaerobes:  We have separated this group from the facultative anaerobes for

their inability to respire and their indifference to the presence of O2.  Many texts and reference works (such as Bergey’s Manual) include these organisms in the facultative anaerobe category.  Aerotolerant anaerobes obtain energy only by fermentation and will show the same amount of growth under aerobic and anaerobic conditions.  Lactic acid bacteria (Experiment 12) are aerotolerant anaerobes as a rule.

5.   Strict anaerobes (also called obligate anaerobes):  Oxygen is toxic to these organisms, and

thus there is no growth under aerobic conditions.  Generally these organisms have a fermentative type of metabolismbut certain exceptional strict anaerobes not studied in this course (such as sulfate-reducers and methane producers) mainly utilize forms of anaerobic respiration.

There are two things to note about Thioglycollate Medium when interpreting the growth patterns observed – namely: (1) this medium does not allow for anaerobic growth due to other modes of catabolism, namely anaerobic respiration or anoxygenic phototrophy, and (2) there are a number of organisms that simply cannot utilize its nutrients, and these organisms include chemolithotrophs and many chemoheterotrophs.  This “oxygen relationship” concept is still useful in comparative, descriptive studies of commonly-encountered chemoheterotrophic bacteria as we will see in the upcoming lab experiments.  For example – utilizing the definitions in the above section – describing an organism as a “strict aerobe” will imply its tolerance of oxygen, the use of oxygen in metabolism (i.e., aerobic respiration) and the inability to ferment and grow anaerobically as a result.

When one considers what this medium is really testing for – as shown in the following table – one can see that the use of Thioglycollate Medium (or equivalent) becomes redundant when other routine tests are done on the organisms, namely the tests for glucose fermentation and catalase as noted in the next section.  This is good to know, as Thioglycollate Medium is quite expensive.

Corresponding tube number above	1	2	3	4
OXYGEN RELATIONSHIP	STRICT AEROBE	FACULTATIVE ANAEROBE	AEROTOLERANT ANAEROBE	STRICT ANAEROBE
	(Darker shade denotes heavier growth.)
TOLERANCE OF O2	+	+	+	–
AEROBIC RESPIRATION and the Catalase Reaction	+	+	–	–
FERMENTATION and Anaerobic Growth	–	+	+	+
EXAMPLES	Bacillus (some) Micrococcus Pseudomonas Alcaligenes	Bacillus (some) Staphylococcus Escherichia Enterobacter	Lactobacillus Streptococcus Enterococcus Lactococcus	Clostridium

III.  CORRELATION OF CERTAIN TESTS WITH OXYGEN RELATIONSHIPS

           

Glucose Fermentation Broth is a differential medium designed to detect acid (and gas) produced from the fermentation of glucose.  We would not expect strict aerobes to ferment any sugar.  Also, if an organism is going to ferment, it will generally be able to ferment at least glucose.  This medium is explained in Appendix G and also on the following web page:

http://www.jlindquist.net/generalmicro/dfnewgfbpage.html

Glucose O/F Medium is a differential medium designed to detect acid produced from the fermentation or respiration of glucose.  Like a “fermentation broth,” Glucose O/F Medium consists of a sugar (in this case, glucose), peptone and a pH indicator.  If the inoculated organism cannot catabolize glucose, it may still derive energy by respiring one or more of the amino acids in the peptone.  Unlike fermentation broth, we will not be using this medium in the lab but will only observe it as a demonstration in Experiment 7, as it has limited application that is noted in the following explanatory web page.

http://www.jlindquist.net/generalmicro/dfnewglucoseofpage.html

The Catalase Test:  Those organisms that respire tend to produce hydrogen peroxide (H2O2) continuously in a side reaction while growing in the presence of oxygen.  As H2O2 is toxic, these organisms possess an intracellular enzyme called catalase that detoxifies H2O2 with this reaction:

2H2O2  –>  2H2O  +  O2

A positive result with the catalase test is seen by the evolution of O2 bubbles upon application of H2O2 and correlates with an organism which is able to respire (i.e., strict aerobes and facultative anaerobes).  As we shall see in Experiment 7, knowledge of the catalase reaction and whether or not glucose is fermented can assist in determining oxygen relationship.

Tolerance to Azide:  Those organisms that respire tend to be inhibited by respiratory poisons such as sodium azide.  How sodium azide can be used as a selective agent to select for “aerotolerant anaerobes” is explained in Experiment 12.

IV. –BACTERIAL MOTILITY

Some (many) bacteria actively locomote in their environments. We are addressing this topic along with the metabolic and growth characteristics of the organism because it is probably related to how they “make their livings”, or how they gain energy. Perhaps this is simplistic but it seems like a good place to study this aspect of a microbe. Motility in bacteria is achieved by any of several mechanisms. The most widespread mechanism is flagellar movement that allows travel in a liquid medium and is mediated by special threadlike organelles extending from the cell surface called flagella. Most rods and spirilla are motile by means of flagella; cocci are usually non-motile. A somewhat modified version of the bacterial flagellum is responsible for the movement of the bacteria known as spirochetes. These organisms possess an axial filament, consisting of two sets of flagella-like fibrils anchored at the two poles of the cell. Another type of movement observed for bacteria is known as gliding motility. It is the sole method of movement for certain of the cyanobacteria and myxobacteria. These organisms can move slowly over solid surfaces. Motility seen for any organism in this course will be due to flagella, the main focus of this experiment.

Virtually all bacteria that possess flagella are motile. Flagellation is a genetically-stable morphological trait of these cells. Certain environmental and nutritional conditions favor flagellar movement that can cease with increasing age (of the culture), temperature and concentration of waste products. Thus, optimal conditions for growth of the organism should be provided when one wishes to detect motility. The presence of flagella, as well as their number and distribution on the cell, are important characteristics for purposes of identification and classification of bacteria. When one or more flagella arise only from one or both ends of a rod or spiral-shaped cell, the arrangement is termed polar. When flagella arise randomly over the entire surface of the cell, the arrangement is termed peritrichous.

Motility is important in that an organism can swim toward optimal concentrations of nutrients and away from toxic substances. As an organism moves (runs), stops (twiddles), moves off in another direction, stops, etc. – in what superficially looks like a random fashion – one finds that the longest runs are in the direction toward the nutrient or away from the toxic substance. This type of purposeful movement is called chemotaxis. Other forms of tactic response include phototaxis (movement toward optimal light concentration or wavelength) and magnetotaxis (orientation and movement along lines of magnetic force). For more information of motility, see the chapter in the Virtual Microbiology manual.

Unlike flagella of eucaryotic cells (protozoa, algae, etc.), bacterial flagella are beyond the resolving capability of our lab microscopes. Flagella stains have been developed which coat the flagella to make them visible with the microscope and much practice is necessary to get consistently good results. Were we to have access to an electron microscope, we would be able to see the flagella (if present) and their arrangement for the organisms in this and other experiments in this course.

In our lab, determining the presence or absence of flagella is done by indirect methods, as we detect whether or not motility is evident under growth conditions made as favorable as possible for the organisms. With the wet mount, an actively growing, young culture is required for observation. Motile organisms are usually easily seen as they move among each other in separate directions. One must, of course, discount Brownian motion, the movement due to bombardment of submicrosopic particles in the liquid, where the cells (alive or dead) appear to remain in one position but shake somewhat. One must also not confuse true motility with movement in a current of liquid where all cells (again, alive or dead) appear to be swept in one direction.

In lab, we will also utilize tubes of Motility Medium wherein only motile bacteria can move away from the line of inoculation in the low (0.5% or less) concentration of agar; descendants of cells which have migrated throughout the medium show up as evidenced by turbidity (cloudiness) in the medium. One advantage of using Motility Medium is that a culture of any age can be used for inoculation – as long as it is pure and viable! In observing the tubes, one must always ignore growth at the surface of the medium and also between the medium and the wall of the tube. Such growth is not necessarily evidence of actual motility. Certain motile organisms, such as many which are strict aerobes, may grow with difficulty in the depths of the medium and ultimately give a false-negative reaction. Generally, one seeks to confirm microscopically any negative reaction seen in the tube.

 

V.  DESCRIBING ORGANISMS ACCORDING TO TYPE OF CATABOLISM AND OTHER CHARACTERISTICS: Virtual Experiments

A classification of organisms based on the mode of energy generation would be more descriptive and comprehensive than one that is based on “oxygen relationship.”  When one considers the thousands of species of bacteria – only a relatively small number of which can grow on normal laboratory media including Thioglycollate Medium – one can still find ways to test whether any organism (chemotrophic or phototrophic; organotrophic or lithotrophic) can perform any one or more of the following catabolic processes:

•     aerobic respiration

•     anaerobic respiration

•     fermentation

•     anoxygenic phototrophy

•     oxygenic phototrophy

We will refer to these processes throughout the semester and consider questions such as these:

1.   Which of these processes are classified as chemotrophic?

      As phototrophic?

2.   Which are associated with anaerobic growth?

In this course – where we mainly work with the easy-to-grow, chemoheterotrophic bacteria –

we will still utilize the concept of “oxygen relationships” and the associated descriptive terms (strict aerobe, facultative anaerobe, etc.) that go along with aerobic respiration and fermentation capabilities.

(In Experiment 7, we will introduce the concept of anaerobic respiration with the utilization of nitrate as an “oxygen substitute.”  Then – in Experiment 11 – we will work with the purple non-sulfur photosynthetic bacteria that are capable of both aerobic respiration and anoxygenic phototrophy.) We will also examine and use for classification other basics traits of some microbes including production of growth factors and motility.

Go back to the table of contents for this chapter and do, Oxygen relationships, Investigating bacterial motility by flagella, and Example of a growth factor requirement and protocol.

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