Chapter 5 - Selected Aspects of Nutrition and Metabolism and Other Characteristics

5 - 1 Nutrition and Metabolism

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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5 - 2 Oxygen relationships

Oxygen has a tendency to form very reactive by-products (H₂O₂ and O^2-(superoxide)) inside a cell. These by-products create havoc by reacting with protein and DNA, thus inactivating them. Cells that are able to live in the presence of oxygen have evolved enzymes to cope with H₂O₂ and O^2- and thus are not inhibited by O₂. Also many anaerobes have oxygen labile Fe-S centers and no cellular machinery to protect them from the oxidizing power of oxygen. Organisms that cannot deal with the problems presented by oxygen cannot survive in air and are killed.

On the basis of oxygen tolerance, microorganisms can be placed into four classes. Strict aerobes cannot survive in the absence of oxygen and produce energy only by oxidative phosphorylation. Strict anaerobes, in many cases, generate energy by fermentation or by anaerobic respiration and are killed in the presence of oxygen. Aerotolerant anaerobes generate ATP only by fermentation, but have mechanisms to protect themselves from oxygen. Facultative anaerobes prefer to grow in the presence of oxygen, using oxidative phosphorylation, but can grow in an anaerobic environment using fermentation.

Oxygen utilization is a primary diagnostic tool when identifying microorganisms. Special media has been developed for the purposes of determining the oxygen relationship and method of metabolism (fermentation vs. respiration) of microorganisms. One such medium, Thioglycollate Agar is useful for determining the oxygen relationship of a microorganism. The medium contains thioglycollic acid, cystine and 0.35% agar, among other things. The thioglycollic acid and agar prevent oxygen from entering the entire medium. A dye, resazurin, is used as an indicator of the amount of oxygen in the medium. Resazurin is red in the presence of oxygen and turns colorless under anaerobic conditions. The medium is steamed just before use, which removes all oxygen from the tubes. After inoculation and incubation, oxygen is able to diffuse into the top part of the medium and support growth aerobically, while the bottom half of the medium remains devoid of oxygen.

A second medium used to investigate the general type of metabolism used by a microorganism is glucose O/F medium. This is a rich medium that contains glucose as primary carbon source. A pH indicator dye, brom thymol blue, is added and is green/blue under alkaline-Oxidative conditions or yellow under acidic-Fermentative conditions. Each test organism is inoculated into two tubes of glucose O/F medium. One tube is overlaid with mineral oil and the other is not. The mineral oil serves as a barrier to oxygen, which helps to create an anaerobic environment.

In this experiment you will first investigate the reactions of several known microorganisms having different types of metabolism. You will determine the characteristic reactions of thioglycollate medium and glucose O/F medium. You will then use this information to determine the oxygen relationships and catabolism type of your two unknown isolates.

Protocol for testing oxygen relationships

Period 1

Materials

8 tubes of Glucose O/F Medium

4 tubes of sterile mineral oil

4 tubes of Thioglycollate Agar (melted, in 50 °C water bath)

Cultures of

Pseudomonas fluorescens

Clostridium sporogenes

Enterococcus faecalis

Escherichia coli

2 plates of Brain Heart Infusion Agar

Anaerobe jar

In this exercise we will be first testing the oxygen relationships of some known organisms in Glucose O/F medium and Thioglycollate Agar. This will give you a sense of inoculating test media and allow you to observe their characteristic reactions.

1. Get four tubes of thioglycollate agar from the 50 °C water bath. The thioglycollate agar has been steamed for several minutes to drive off any oxygen. Keep the agar melted by incubating the tubes in a container containing 50 °C water. Label the tubes with the culture names.
2. Inoculate each culture into one tube of Thioglycollate agar. Mix the tubes by placing the palm of your hand over the top of the tube and moving the bottom of the tube in a circular fashion.
3. Incubate the tubes at 30 °C for 2-5 days.
4. Inoculate each culture into two tubes of glucose O/F medium by stabbing the medium the full length of the tube with the inoculating needle.
5. Overlay one tube of each culture of Glucose O/F medium with 2-3 cm of mineral oil. What is the purpose of the mineral oil? Incubate at 30 °C for 2-5 days.
6. Divide each plate into four sectors. Label one plate aerobic and the other anaerobic. Streak each culture onto one sector on each of the plates.
7. Incubate one plate aerobically at 30 °C. Place the other plate in the anaerobe jar. The air will be evacuated and replaced with H₂ + CO₂ atmosphere. The jar will also be incubated at 30 °C.

Period 2

1. Observe the thioglycollate tubes for the known cultures. Notice the growth pattern of each organism. Is growth seen throughout the tube? Is there more growth at the top of the tube? What does that mean? Did any of the cultures exhibit growth only in the bottom of the tube?
2. Make drawings of each test tube culture in your lab notebook. From looking at the tubes can you infer which organisms are strict aerobes, facultative anaerobes, aerotolerant anaerobes or strict anaerobes? Record this information in your lab notebook.
3. Observe the Glucose O/F medium. Look for growth in the tubes and production of acid (yellow color). Bacteria that grow only in the tube without mineral oil have a respiratory form of metabolism. Bacteria that grow in both tubes and produce acid under anaerobic conditions can also use fermentation. What would be the reaction of a strict aerobe in this medium? Record the results in your notebook. Do the results obtained here agree with the results from the thioglycollate experiment?
4. Observe the BHI plates. Record anaerobic and aerobic growth as + or -. Do the results here agree with that which was observed for the other media in this experiment.
5. Catalase test. Add several drops of (H₂O₂) to each area of growth on the plates incubated aerobically. Observe through the top lid of the closed plates so you don't cause an aerosol of live cells to spread from a positive reaction! A positive reaction is indicated by the constant evolution of bubbles. Figure 5-15 is a movie of the catalase test for these six microbes. However, C. butyricum cannot be tested, why?

Figure 6.15 the catalase test

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5 - 3 Investigating bacterial motility by flagella

Motility in bacteria is achieved by any of several mechanisms. The most widespread mechanism is flagellar movement which 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 move-ment 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 which possess flagella are motile. Flagellation is a genetically-stable morphological trait of these cells. Certain environmental and nutritional conditions favor flagellar movement which 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 microtextbook on surface structures

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. 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.

Period 1

Materials

Young broth cultures (12-15 hours) of Enterobacter aerogenes (or other motile organism) and Staphylococcus epidermidis. These will serve as positive and negative controls (respectively) for the unknown.

Young broth culture of an unknown organism

3 tubes of Motility Medium

Demonstration (phase microscope) of Rhodospirillum, a motile, spiral-shaped bacterium.

1. Note the demonstration of Rhodospirillum set up under the phase-contrast microscope (Figure 5-2).
2. For each broth culture, prepare a wet mount as previously done. It is best to trap some air bubbles under the cover slip, as respiring organisms (such as the facultative anaerobes we are using) generally show increased motility where oxygen is available.
3. If you use the regular light microscope, focus initially with the 10X objective, switching to the 40X objective and then (but only if needed) the 100X, oil-immersion objective. Adjust the light with the iris diaphragm; optimum results are achieved with a relatively low light intensity. (Alternately, excellent results can be had by the use the phase microscope with the 40X (middle) objective lens in place. Do not use oil with this lens!)
4. Look for true motility which should be evident for the E. aerogenes culture. Do not be misled by Brownian motion or currents (see introduction). Tabulate results below.
5. Discard the wet mount (without disassembling it!) into the disinfectant.
6. Inoculate each broth culture into a separate tube of the semisolid Motility Medium. Use the needle and carefully stab-inoculate the medium about half-way down through the center. Incubate at 30 °C.

Period 2

1. Observe the tubes of Motility Medium for growth away from the line of inoculation and the subsequent cloudiness throughout the medium as discussed in the introduction. In a well-lit room, hold all of the tubes together against a darker part of the ceiling (such as the space between the fluorescent light units) so that degrees of growth can be discerned easily. Ignore all surface growth and any growth that might be creeping down from the surface along the inner wall of the tube. Tabulate your results

reaction of motile and non-motile microbes in motility medium

Figure 6-2 is a video of motile and non-motile bacteria in the microscope.

Above is a video of bacterial motility by flagella as view microscopically.

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5 - 4 Example of a growth factor requirement and protocol

Iron is required by virtually all organisms. Many microorganisms, both eukaryotes and prokaryotes, synthesize specific iron-chelating compounds called siderophores (also known as ironophores) which are extracellular (i.e., exported outside of the cell) and are involved in the solubilization and transport of iron compounds into the cell. (Siderophores may perform additional functions in the metabolism of microorganisms.) Microbially-produced siderophores act as growth factors (see Appendix D) for organisms which cannot synthesize them. Thus, in the mixed microbial populations of natural environments, siderophores essential to the growth of certain organisms are supplied by the excessive secretions of these substances by other organisms which can synthesize them. This experiment will do nothing more than demonstrate the requirement of an exogenously-supplied siderophore for growth of a siderophore auxotroph and the ability of the auxotroph to be supplied the siderophore artificially and by certain other microorganisms.

Period 1

Materials

Saline suspension of cells of the siderophore auxotroph, Arthrobacter flavescens (strain

Broth cultures of Bacillus subtilis, Streptomyces griseus and Rhodotorula rosei (a yeast)

3 plates of Brain Heart Infusion (BHI) Agar plates 3 sterile swabs

Sterile paper disc saturated with a solution of Deferrioxamine B (5µg/ml), a commercially- available siderophore obtained from a species of Streptomyces and normally used in the treatment of chronic iron storage disease.

Procedure

1. Inoculate each plate of BHI Agar with the Arthrobacter flavescens suspension by means of the sterile swabs. Make sure each plate is completely and evenly covered with the inoculum. Discard the swabs into the disinfectant.
2. At the edge of one of the plates, aseptically (with flame-sterilized forceps) place a Deferrioxamine-impregnated disc. At the opposite edge of the same plate, spot-inoculate (just a dab with your loop - not a streak) the Bacillus subtilis culture.
3. On the second plate, at opposite edges, spot-inoculate Streptomyces griseus and Rhodotorula rosei.
4. Leave the third plate as is, with no further treatment. This is the control plate which will show how poorly the A. flavescens grows (if it grows at all) on a medium not supplemented with the required growth factor (i.e., the siderophore).
5. Incubate the plates at 30 °C for 3 or more days.

Period 2

Procedure

1. Observe the plates for the presence or absence of satellite growth of A. flavescens around the inoculation spots and the Deferrioxamine disc. Which organisms can provide the siderophore? For the organism which does not appear to provide a siderophore which the A. flavescens can utilize, would you expect that organism to be producing one anyway?

Example of phenotypic variation

Genotype is defined as the entire array of genes possessed by a cell, i.e., the sum of the genetic constitution of the organism, a blueprint in code. The characteristics of an organism which are based on the genotype but expressed within a given environment make up the phenotype of the organism. Thus, the genotype represents the potential of the organism, and the phenotype describes what the organism actually is and does. For example, in some species of bacteria, the production of a pigment is a phenotypic manifestation of the genotype, and the degree of actual pigment production may be influenced by temperature or nutritional variations. As another example, the ability to produce a siderophore is a genetically-controlled characteristic, but it may or may not be expressed depending on the amount of iron in the environment. When iron is in low levels, the organism will produce much more of the siderophore in order to scavenge the iron.

In this experiment, we will utilize an organism, Pseudomonas fluorescens, which produces a pigmented siderophore (fluorescein) which is easy to detect with the naked eye if it is produced in sufficient quantity. It also glows (fluoresces) when illuminated with ultraviolet light. When inoculated onto two media which differ in the concentration of iron compounds, we will see how the same organism appears differently on each medium, an example of phenotypic variation, a difference in fluorescein synthesis.

Since phenotypic characteristics are generally used to differentiate between microorganisms, it is important that all tests be done in a standardized set of conditions (medium, temperature, time, etc.).

SPECIAL SAFETY PRECAUTION: BE CAREFUL WHEN USING THE ULTRAVIOLET LIGHT. USE THE SAFETY GLASSES PROVIDED. Ultraviolet light damages the retina of the eye, and prolonged exposure to the light reflecting off the plates can also cause damage.

Period 1

Materials

Broth culture of Pseudomonas fluorescens

1 plate each of Nutrient Agar and Pseudomonas Agar-F

Procedure

1. With a single streak with the loop, inoculate the culture onto each of the plates.
2. Incubate the plates at 30 °C for 2 or more days.

Period 2

Materials

Ultraviolet lights set up in a dark room

Procedure

1. Observe the plates for production of fluorescein by the culture. This usually appears as a yellowish-green color diffused into the medium.
2. To observe the fluorescence of the pigment (hence its name), take the plates to the dark room.

Remove the covers and expose the cultures to the ultraviolet lamp. SEE THE SAFETY PRE-CAUTION ABOVE REGARDING ULTRAVIOLET LIGHT. The glowing, yellow-green fluorescence of the pigment is not itself ultraviolet light, but light within the normal, visible range. Which of the two media apparently contains more iron? (Note that iron is a trace element in each medium; this was also the case in the BHI Agar used in Experiment 2.

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