EXERCISE CHAPTER. Marine Bacteria INTRODUCTION. Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION. & Bartlett Learning,

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1 EXERCISE CHAPTER 4 Marine Bacteria INTRODUCTION Jones & Bartlett To most of Learning, us, the ecological LLC roles of large divisions or Jones phyla of & life Bartlett forms seem Learning, rather obvious. LLC Plants, whether NOT FOR SALE simple OR algae DISTRIBUTION or complex terrestrial forms, are primary NOT producers FOR SALE, converting OR DISTRIBUTION inorganic molecules into organic ones with the help of energy from the sun. Animals are consumers, unable to manufacture all their own organic molecules without first disassembling those produced originally by plants. And so it goes throughout each kingdom of living things, until one reaches the simplest, single-celled prokaryotic forms (those cells lacking a nuclear membrane separating their DNA from the rest of the cell s interior, among other attributes). Within this Jones group & of Bartlett prokaryotic Learning, life forms are LLC those that are photosynthetic Jones primary & producers, Bartlett Learning, LL chemoautotrophic NOT bacteria FOR that SALE can OR manufacture DISTRIBUTION all their organic molecules using NOT energy FOR released SALE from OR DISTRIBUT inorganic chemical sources, and disease-causing bacteria that play the ecological role of consumers by parasitizing other organisms. Finally, there are those that play important roles as decomposers, breaking down complex organic molecules into simple inorganic molecules that may eventually be incorporated into the Jones next generation & Bartlett of producers. Learning, LLC NOT The FOR prokaryotes SALE are OR usually DISTRIBUTION divided into Archaea and Bacteria (sometimes NOT FOR called SALE Eubacteria, OR DISTRIBUTION meaning true Bacteria ). Some members of the Archaea live in some of the world s most extreme environments: hot vent systems on the ocean floors, freezing desert rocks, and very high-saline or high-sulfur-content marine environments. We are most familiar with the true Bacteria. They include the Cyanobacteria (sometimes called blue-green algae), which are important photosynthetic forms found in both freshwater and marine environments. The rest NOT FOR SALE are the OR forms DISTRIBUTION that cause disease or occupy roles as NOT decomposers. FOR SALE OR DISTRIBUTION In any ecosystem, the importance of the availability of nutrients to the primary producers is well established. The release into the environment of these nutrients, which have been locked in the tissues of dead organisms and the waste products of living organisms, is the role assumed by decomposers. Decomposers are bacteria and fungi. The study of marine NOT FOR decomposers SALE OR is only DISTRIBUTION a few decades old. This is quite understandable NOT FOR when SALE one OR DISTRIBUT considers the lack of economic incentive to study the relatively small diversity of species of marine bacteria and fungi. Of the estimated 10,000,000 to 1,000,000,000 or more species of bacteria, only a fragment are considered to be marine. Many of the bacteria found in the marine environment are found only in a resistant spore Jones stage & in marine Bartlett sediments. Learning, They LLC grow when cultured in the laboratory, Jones but & Bartlett it is not known Learning, whether LLC they are NOT truly FOR marine SALE or whether OR DISTRIBUTION they were simply transported from terrestrial NOT FOR or freshwater SALE sources. OR DISTRIBUTION Some terrestrial bacterial and fungal spores can live for many years in this state of dormancy. The diversity of the marine fungi is even less marked than that of marine bacteria. Of the approximately 75,000 species of fungi in the world, less than 0.5% are found in the sea, so only the marine bacteria will be studied in this exercise. Jones & Bartlett The variety Learning, and abundance LLC of marine bacteria in Jones coastal environments & Bartlett Learning, can be appreciated LLC best by isolating and identifying a few of them. Bacteria are quite abundant in the open ocean, but the sampling.. 35

2 procedures for obtaining pelagic bacteria are complex, so they are not included here. A small sample of bay mud or beach sand collected during field studies and placed in sterile containers will provide an abundance of Jones autotrophs & Bartlett and Learning, decomposers LLCfor examination and identification. Jones After & Bartlett they are Learning, isolated on the LLC proper growth NOT FOR medium, SALE they OR will DISTRIBUTION require about 1 week to grow. Another NOT laboratory FOR SALE period OR is then DISTRIBUTION necessary for examination and identification of the isolated types. Parasitic or mutualistic bacteria will be examined from the digestive tract of bivalves. I. Types of Bacteria The majority of bacteria, either parasitic or free living, are one of three types: Myxobacteria, Eubacteria, or Spirochaeta. A. Myxobacteria The Myxobacteria Jones & are Bartlett single-celled, Learning, rod-shaped LLC organisms, generally with pointed Jones ends and & Bartlett the capacity Learning, for gliding over LLC solid substrates. NOT FOR When SALE grown OR in laboratories DISTRIBUTION on solid agar growth media, the edges NOT of FOR the colonies SALE often OR have DISTRIBUTION a lacy, flameshaped form. The colony will show a marked capacity for covering the surface of the medium in a relatively short time, although the method by which these organisms spread is unknown. B. Eubacteria NOT FOR The SALE Eubacteria OR are DISTRIBUTION by far the most common and important NOT bacteria FOR in all environments. SALE OR DISTRIBUTION Differences in the chemical composition of the cell walls cause the cells to be stained differentially when properly treated. This staining technique, called the Gram stain, was developed in the late 1800s by a Danish physician, Christian Gram. When treated, cells retaining a violet color are called gram-positive and those with a pinkish-red color are called gram-negative. Three basic shapes are evident among these bacteria: cocci, round spherical forms; bacilli, cylindrical, rod-shaped forms; and spirilla, helical, or bent-rod, forms (Figure 4.1). Jones & Bartlett Learning, LLC (a) Bacillus (rod) (b) Coccus (sphere) (c) Spiral (d) Other shapes Diplococcus (pair) Jones & Bartlett Learning, Single LLC Single Vibrio Square (comma-shaped) Appendaged Tetrad (group of 4) Staphylococcus (cluster) NOT FOR SALE Streptobacillus OR DISTRIBUTION (chain) Streptococcus (chain) Spirillum Spirochete Star-shaped Filamentous Figure 4.1 Eubacteria generally exist as one of these three basic types: (a) bacilli, (b) cocci, or (c) spirilla. (d) Miscellaneous other shapes also exist. 1. Cocci (singular coccus). Almost all of the cocci are gram-positive and, in the marine environment, usually are found in irregularly shaped clusters or in groups of eight. In other environments, they may form long chains or groups of two or four. None of the cocci are capable of locomotion. 2. Bacilli Jones (singular & Bartlett bacillus). Bacilli, Learning, or rods LLC as they are often called, occur in Jones both gram-positive & Bartlett and Learning, gram-negative LLC forms. Some move by using flagella. Some gram-positive rods produce heat-resistant spores (such as those causing the diseases botulism, tetanus, and gangrene) and are found in marine as well as terrestrial environments. The most common marine bacteria, members of the genus Pseudomonas, are flagellated gram-negative rods. Members of the genus Photobacterium are luminescent, exclusively marine forms. They also are gram-negative rods. Some are free-swimming, and many occur symbiotically with fish. Jones & 3. Bartlett Spirilla (singular Learning, spirillum). LLCThese forms are all gram-negative Jones flagellated & Bartlett organisms. Learning, LLC 36 Excercise 4 Marine Bacteria..

3 Name Section Date Jones & Bartlett C. Spirochaeta Learning, LLC The spirochetes are very long, slender helical cells with thin cell walls. They are quite common in marine muds, where their rapid spiral movements are easily seen under a microscope. They apparently swim by contracting bundles of fibers, known as axial filaments. These filaments are made of protein threads and lie just beneath the cell wall. Most spirochetes are anaerobes (organisms that live in regions devoid of dissolved O 2 ), either free-living or parasitic. The best-known marine Jones member & Bartlett of this group Learning, is the genus LLC Cristispira, found in the digestive Jones tract of & clams Bartlett and other Learning, LL mollusks. II. Isolation and Culture Techniques for Marine Bacteria Use NOT sterile FOR techniques SALE OR and DISTRIBUTION common sense when handling these bacteria, NOT FOR because SALE some OR pathogenic DISTRIBUTION forms may be present. Sterile techniques are absolutely necessary when dealing with bacteria. It is quite common for airborne spores of terrestrial bacteria and fungi to contaminate and grow on marine growth media. To avoid contamination, it is necessary to use Jones & Bartlett a Bunsen Learning, burner to sterilize LLCsome equipment. Other equipment Jones is & sterilized Bartlett in a Learning, high-pressure, LLC high-temperature device NOT FOR SALE called OR an autoclave, DISTRIBUTION which is simply a large, elaborate pressure NOT FOR cooker. SALE OR DISTRIBUTION A. Bacterial Isolation from Marine Sediments Marine decomposers normally occur together in a confusing abundance of species. Before you can recognize and identify these organisms, it is necessary Jones & to isolate Bartlett them Learning, from each other LLC and from the sediment in which Jones they live. & Bartlett Learning, LL Part of this isolation procedure is purely mechanical. The organisms are suspended in water, and then diluted and spread out. They are then placed on plates of growth media that contain agar (a gelatin-like material made from the red alga Gelidium) and a mixture of nutrients known to be effective for growing specific types of organisms. After a few days, each bacterial cell, if it is capable of growing on the medium provided, will multiply and produce a colony containing millions of cells, all of one type. The cells of different colonies can be separated from each other and used in subsequent Jones procedures. & Bartlett Learning, LLC Procedure 1. Take two fresh sediment samples, one from a bay or estuary and one from an open-coast sandy beach. Using a sterile spatula, fill two stoppered, sterile test tubes one-third full with each of the sediment samples. 2. Add approximately one-third of a test tube of sterile seawater to both of the sediment samples and replace the Jones & Bartlett stoppers. Learning, LLC NOT FOR SALE 3. Agitate OR DISTRIBUTION the plugged samples thoroughly, and then allow NOT them FOR to stand SALE until OR the sediment DISTRIBUTION settles. These solutions will be used as your source of bacteria and fungi for subsequent experiments. Because you will be using it to inoculate various growth media, we will call it the inoculum. 4. Take three petri plates containing bacteria growth media and, using a marking crayon, label each dish with your name, the word bay, and the temperature at which each plate is to be incubated. A typical deep-sea temperature of 5 C (refrigerator), an oceanic Jones surface-water & Bartlett temperature Learning, of 15 C, and LLC 37 C (human body temperature) Jones are recommended & Bartlett incubation temperatures. NOT If normal FOR SALE environmental OR DISTRIBUTION temperatures cannot be maintained in the NOT laboratory, FOR then SALE a temperature OR DISTRIBUT Learning, LL of 20 C (room temperature) may be substituted. 5. Remove the stopper and flame the mouth of the test tube containing the inoculum (Figure 4.2a). 6. Dip a sterile cotton swab into the bay inoculum and streak the 37 C plate. Use a fresh swab each time and repeat for the other two temperatures following the streak technique shown in Figure 4.2b f. 7. Jones Repeat steps & Bartlett 4 6 using Learning, the beach inoculum. LLC Be sure to label the plates with Jones the term & Bartlett beach. Learning, LLC 8. NOT Before FOR incubating, SALE invert OR DISTRIBUTION the inoculated petri plates so that the half NOT containing FOR the SALE medium OR is on DISTRIBUTION top. Inversion prevents any moisture that may condense from dropping on the growing culture. 9. Seal plates with Parafilm or plastic sandwich bags to prevent desiccation. 10. Place the six petri plates in their proper incubation sites in the dark and allow the bacteria to grow for 1 week. 11. To complete the examination of these bacteria to determine their cellular type, arrangement, and motility, follow the Jones & Bartlett procedures Learning, outlined LLC in Part III of this exercise. II. Isolation and Culture Techniques for Marine Bacteria.. 37

4 (a) Remove the stopper and flame (b) Dip the sterile cotton swab into (c) Remove the swab and flame the mouth of the test tube. Do the inoculum. Do not touch the the mouth of the test tube. not set the stopper down. sand on the bottom of the tube. (d) Replace the stopper in the tube. (e) Remove the top cover of the petri (f) dish and streak with the inoculum. Stroke the cotton swab in a zigzag fashion across the agar. Figure 4.2 Petri plate inoculation techniques. B. Bacterial Isolation of Coliform Bacteria from the Mussel Mytilus Use sterile techniques and common sense when handling these bacteria, because some pathogenic forms may be present. Filter feeders, such as oysters and mussels, concentrate bacteria from sewage wastes along with other suspended organic material to be used as food (see Exercise 9, Marine Mollusks, Part II). The bacteria that live in the digestive tract of humans and other mammals Jones do not & ordinarily Bartlett live Learning, long outside LLC the body. These coliform bacteria do not form resistant spore stages, although they NOT can FOR be found SALE in a dormant OR DISTRIBUTION state in seawater. It is possible to stimulate NOT their growth FOR by SALE providing OR DISTRIBUT a suitable growth medium. The presence of fecal coliforms may also be an indication that pathogenic (disease-causing) organisms such as those that cause salmonella, hepatitis, and a variety of staphylococcus and streptococcus infections are also in the water. It is therefore important that sterile techniques be utilized when handling these samples. The techniques used to isolate and grow coliform bacteria are similar to those used in Part A for marine sediments. Procedure 1. Using sterile instruments, such as a pair of needles, remove the visceral mass from a fresh mussel and macerate it. 2. With a sterile spatula, add the macerated visceral mass to a sterile, stoppered test tube. 3. Add approximately one-third of a test tube of sterile distilled water and replace the stopper. Jones & 4. Bartlett Agitate the Learning, stoppered test LLC tube thoroughly, and then allow Jones it to stand & until Bartlett the sediment Learning, settles. LLC This solution will be NOT FOR SALE the inoculum. OR DISTRIBUTION 38 Excercise 4 Marine Bacteria..

5 Name Section Date Jones & Bartlett 5. Take Learning, three petri plates LLCcontaining coliform bacteria Jones growth media & Bartlett (either Hektoen Learning, Enteric LLC Agar or Levine Eosin NOT FOR SALE Methylene OR DISTRIBUTION Blue Agar) and label them with your name. 6. Remove the stopper and flame the mouth of the test tube containing the inoculum (Figure 4.2a). 7. Dip a sterile cotton swab into the inoculum and streak the plate (Figure 4.2b f). Use a fresh swab for each of the other petri plates. Dispose of the swabs in a sterile manner. 8. Before incubating, invert the inoculated petri plates so that the half containing the growth medium is on top. 9. Seal the petri plates Jones with Parafilm & Bartlett or plastic Learning, sandwich bags LLC to prevent desiccation. 10. Place the three NOT petri plates FOR in SALE a 37 C incubator OR DISTRIBUTION and allow the bacteria to grow for 48 hours. NOT The FOR plates SALE may be removed OR DISTRIBUT from the incubator at that time and refrigerated for several weeks before examining. 11. If coliform bacteria are present, particularly Escherichia coli, the dominant species found in mammals, the colonies growing on the Hektoen Enteric Agar will be bright orange. When grown on Levine Eosin Methylene Blue Agar, E. coli will be a shiny, dark green colony. 12. Jones To complete & Bartlett the examination Learning, of these LLC coliform bacteria, follow the procedures Jones outlined & Bartlett in Part Learning, III to determine LLC their NOT cellular FOR type, SALE arrangement, OR DISTRIBUTION and motility. III. Bacterial Identification Jones & Bartlett Use sterile Learning, techniques LLC and common sense when handling Jones these & Bartlett bacteria, because Learning, some LLC pathogenic forms may NOT FOR SALE be present. OR DISTRIBUTION A. Cellular Arrangement and Motility To determine the cellular arrangement of bacteria (e.g., single; irregularly clumped; groups of two, four, or eight) and whether the bacteria are Jones motile, & you Bartlett must examine Learning, the live culture LLC in an aqueous preparation. The best way to NOT observe FOR the living SALE bacteria OR is DISTRIBUTION by means of a hanging drop slide. This technique NOT FOR ensures SALE that the OR organisms will not dry out before you have the opportunity to observe them under the microscope. Motility, if present, will DISTRIBUT be quite apparent as motile organisms swim actively through the drop. This active motility must be distinguished from Brownian movement, which is motion caused by the collision of molecules of water and dissolved ions with the bacteria. Brownian movement will make the bacteria appear to vibrate. Hanging NOT FOR Drop SALE Slide OR DISTRIBUTION Preparation Procedure For each colony of bacteria to be observed, prepare a freshly washed (in soap), single-concavity depression slide. 1. With a toothpick, place a thin ring of petroleum jelly around the outside edge of a new glass coverslip, as shown in Figure 4.3a. Jones & Bartlett 2. With Learning, a sharp crayon, LLC mark a ring in the center of the coverslip. Jones & Bartlett Learning, LLC 3. Using an inoculating loop, place 2 loopfuls of sterile seawater in the middle of the coverslip, as shown in Figure 4.3b. 4. With an inoculating needle, transfer a minute portion of the colony to be inspected into the sterile seawater on the coverslip and stir (Figure 4.3c). Do not apply the inoculum generously, or you will not be able to see the individual bacteria. 5. Invert the depression slide and press it onto the coverslip. Be sure the drop on the coverslip fits into the center of the concavity. If the drop Jones is too & large, Bartlett the suspension Learning, will adhere LLCto the sides of the concavity, and Jones the slide & Bartlett must be placed Learning, LL in the disinfectant and a new slide made (Figure 4.3d). 6. Invert the slide quickly so that the drop does not adhere to the sides of the depression slide (Figure 4.3e). 7. Repeat the procedure for each colony to be observed. Examination Techniques Place the hanging drop slide on the stage of your microscope and observe the crayon mark under low power. After focusing on the crayon mark to get the proper focal length, adjust the slide so that NOT you observe FOR the SALE edge of OR the hanging DISTRIBUTION drop. It may be necessary to reduce the light entering the stage by closing down the microscope diaphragm located under the stage. Bright light will prevent you from seeing these very small, transparent organisms clearly. Once the edge of a drop is in focus with low power, rotate to the high, dry objective lens. Record your observations in Table 4.1. If desired, you can perform an oil-immersion examination at this time (see Appendix B)... III. Bacterial Identification 39

6 (a) (b) (c) (d) (e) Figure 4.3 Table 4.1 Hanging drop slide technique. Motility and Cellular Arrangement of Bacteria Motility (describe) Colony Jones 1 & Bartlett Learning, LLC Colony NOT 2 FOR SALE OR DISTRIBUTION Colony 3 Cell Arrangement B. Staining Before the bacterial cell wall can be stained, it is necessary to dry and fix the bacteria onto the slide. If this is not done before staining, the sample will wash off the slide. Smear Preparation Procedure 1. Wash (using soap) a plain glass slide and allow it to dry. 2. Using a loop, put 2 loopfuls of sterile seawater slightly off-center on the slide (Figure 4.4a). 3. Using an inoculating needle, touch the colony to be inspected and transfer some of it to the slide. Mix the transferred organisms into the sterile seawater and spread over a large area (Figure 4.4b). 4. Allow the slide to dry at room temperature (Figure 4.4c). 5. Use a wooden clothespin as a slide holder, and pass the slide through the flame of a Bunsen burner six times, bacteria side up. The slide should become too hot to touch but not hot enough to incinerate the bacteria (Figure 4.4d). 40 Excercise 4 Marine Bacteria.. Simple Staining Procedure The bacteria may be stained using dyes such as methylene blue, crystal violet, or basic fuchsin. Staining will enable you to see more clearly the shape and arrangement of cells.

7 Name Section Date (a) Use a flamed loop to place two loopfuls of sterile seawater on the slide. Place the two drops slightly off-center. (b) Transfer a minute amount of the bacterial colony to the seawater drop and stir. (c) Allow to air dry or waft high above the Bunsen burner flame. Do not allow the water to boil. Figure 4.4 Smear preparation technique. (d) Pass the slide through the Bunsen burner six times, organism side up. The slide should become too hot to touch but not so hot as to incinerate the organisms. 1. Place the slides on the glass tubing suspended over a drip pan, and add dye to the smear. Allow to sit for 60 seconds Jones & Bartlett (Figure Learning, 4.5a). LLC 2. Rinse with distilled water (Figure 4.5b). 3. Carefully blot dry with absorbent paper (do not rub the smear). Gram Staining A more specific bacteriological analysis may require Gram staining of the cell walls. See Appendix D for the details of this technique. C. Analysis of Community Variations in Bacterial Decomposers 1. Describe the types of bacteria (according to shape, motility, and colony color) that appear to be most common in a Jones sample from & Bartlett a local beach. Learning, LLC III. Bacterial Identification 41..

8 (a) (b) Figure 4.5 Staining apparatus technique. Connect two glass rods with surgical rubber tubing to form a slide rack. Suspend the rack over a pan to collect waste solutions. (a) Place stains directly on the dried smear. (b) Use a squeeze bottle to rinse with distilled water. 2. Describe the types of bacteria (according to shape, motility, and colony color) that appear to be most common in the bay sample. 3. Which of the & twobartlett samples seems to containllc the greater number of bacteria? number of colonies that grew Jones Learning, (Estimate Jones the & Bartlett Learning, LLC on the petri plates.) 4. Form a hypothesis to explain this variation. 5. What effect does temperature have on the growth rates of these bacteria? 42 Excercise 4 Marine Bacteria _CH04_PASS02.indd 42 10/10/16 2:20 pm

9 Name Section Date Jones & Bartlett 6. Abyssal Learning, bottom temperatures LLC commonly range from 0 C Jones to 5 C. & Assume Bartlett that Learning, a particular species LLCof bacteria found in NOT FOR SALE warm OR coastal DISTRIBUTION waters is also common in abyssal environments. NOT FOR Formulate SALE a OR hypothesis DISTRIBUTION to estimate the relative turnover rates of organic material by these bacteria in the two different habitats. D. Analysis of Bacteria Found in Mytilus 1. NOT Were FOR coliform SALE bacteria OR such DISTRIBUTION as E. coli present in the visceral mass of the NOT mussel? FOR SALE OR DISTRIBUTION 2. Describe the types of bacteria (according to shape, motility, Gram stain, and colony color) that appeared on the growth media. 3. If coliform bacteria were present, what was their likely source? IV. Autotrophic Bacteria Not all autotrophic organisms use chlorophyll to manufacture their food, although those that do so are dominant by far. Bacteria also are capable of utilizing inorganic compounds to produce carbohydrates, proteins, lipids (fats), and nucleic acids. These bacteria fit Jones into one & of Bartlett two groups: Learning, chemoautotrophic LLC (chemosynthetic) or photosynthetic. Jones & Because Bartlett the synthesis of food by chemoautotrophic NOT FOR SALE bacteria OR does DISTRIBUTION not need light to proceed, these organisms NOT can be FOR found SALE throughout OR the DISTRIBUT Learning, LL water column. In contrast, photosynthetic bacteria must have light energy to survive. Consequently, all photosynthetic bacteria must occupy the photic zone. A. Purple and Green Photosynthetic Bacteria Photosynthesis and Pigments All photosynthetic bacteria use a pigment system, as do green plants and photosynthetic protistans, to utilize certain wavelengths of light energy. The photosynthetic bacteria use red and infrared wavelengths. These pigment systems do not contain the same pigments found in other photosynthetic life forms, but instead have their own complement of pigments called bacteriochlorophylls... IV. Autotrophic Bacteria 43

10 It is these pigments, together with carotenoids, that divide the photosynthetic bacteria into two groups, the purple and the green bacteria. Besides the color differences, there are some other basic distinctions between chlorophyll-bearing life Jones forms & Bartlett and photosynthetic Learning, bacteria. LLC Chlorophyll-bearing life forms Jones in terrestrial & Bartlett and marine Learning, environments LLC must grow in NOT FOR an SALE area where OR free DISTRIBUTION O 2 is available for respiration. They are dependent NOT FOR on the SALE energy OR released DISTRIBUTION from the breaking up of H 2 O molecules during photosynthesis to reduce CO 2. The disruption of H 2 O molecules also causes a release of free O 2. Photosynthetic bacteria, in contrast, often will not grow in an area that has free O 2. Instead of releasing O 2, these anaerobic bacteria use H 2 S (hydrogen sulfide) to reduce CO 2 and, in the process, release sulfur. This sulfur may be excreted to the environment, forming sulfur crystals outside the walls of the bacteria, or it may crystallize internally. The following chemical formula illustrates Jones the synthesis & Bartlett pathway Learning, followed by these LLCbacteria: 2CO 2 + H 2 S + H 2 O Jones 2(CH 2 O) & + Bartlett H 2 SO 4 Learning, LL Diagnostic Characteristics of Purple and Green Photosynthetic Bacteria Aside from slight variations in the wavelengths utilized by purple and green bacteria, the two may be distinguished physically. All photosynthetic bacteria are gram-negative. Some are motile, some are not. The green bacteria are always bacilli, whereas the purple bacteria may be bacilli, cocci, or spirilla. Green bacteria do not form sulfur crystals within their cell wall but Jones instead deposit & Bartlett sulfur crystals Learning, the liquid LLCenvironment. Although a few Jones purple bacteria & Bartlett also deposit Learning, sulfur this LLC manner, NOT most FOR store SALE it as visible OR inclusions DISTRIBUTION in their cells. B. Chemoautotrophic Bacteria Chemosynthetic bacteria have the capacity to grow in the dark, on completely inorganic media. Some are facultative (able Jones & to utilize Bartlett a completely Learning, inorganic LLCgrowth medium if that is all that Jones is available), & Bartlett whereas Learning, others are obligative LLC (able to grow NOT FOR only SALE on an OR inorganic DISTRIBUTION medium). Most are members of the Eubacteria NOT FOR and SALE are gram-negative. OR DISTRIBUTION Some are aerobic; others are anaerobic in shallow marine environments such as bays, salt marshes, and estuaries. These bacteria synthesize food by one of the following chemical processes: Aerobic chemosynthesis: CO 2 + H 2 O + H 2 S + O 2 (CH 2 O) + H 2 SO 4 Anaerobic chemosynthesis: 2CO 2 + 6H 2 (CH 2 O) + CH 4 + 3H 2 O In the late 1970s, biotic communities were found associated with vent systems in the deep sea where crustal movement allowed seawater to come in close contact with the molten magma beneath the seafloor. These spectacular colonies of tube worms, clams, and other life forms are supported by chemoautotrophic bacteria. Both aerobic and anaerobic bacteria have been found associated with these communities. The most common chemoautotrophic forms found in shallow waters are anaerobic bacteria from the genus Desulfovibrio. Jones Members & Bartlett of this genus Learning, are motile, LLC short, curved rod forms (occasionally Jones helical). & Bartlett One of the Learning, major products LLC of their respiration is H 2 S (hydrogen sulfide), the compound utilized by most photosynthetic bacteria as an energy source. This is the gas that gives the characteristic rotten egg smell to organically rich muds. C. Analysis of the Winogradsky Column Your instructor will provide a coastal wetlands sediment sample that was treated specifically to encourage the growth of Jones & autotrophic Bartlett bacteria Learning, (see Field LLC Study 1 for the Winogradsky column Jones procedure). & Bartlett To study Learning, these autotrophic LLCbacteria, isolate NOT FOR a few SALE bacteria OR using DISTRIBUTION the following procedures. 1. Remove the cover of the cylinder. Do you detect any unusual odor? Describe it. To what compound do you attribute this odor? What kinds of microorganisms must be present to produce this compound? 44 Excercise 4 Marine Bacteria..

11 Name Section Date Jones & Bartlett 2. Collect Learning, samples of green LLC or purple photosynthetic bacteria Jones with & Bartlett a sterile spatula. Learning, Observe LLC these bacteria using the NOT FOR SALE hanging OR DISTRIBUTION drop slide technique. Describe any motility NOT you see. FOR SALE OR DISTRIBUTION 3. Prepare stained slides of each different chemosynthetic form and record the colony color and cell shape below. Jones & Bartlett 4. Collect Learning, samples of bacteria LLC from the center of the column Jones and examine & Bartlett them using Learning, a hanging LLC drop slide. Describe any NOT FOR SALE motility. OR DISTRIBUTION 5. Prepare stained slides of these bacteria and record the cell shape in the space below. Note the presence of any internal sulfur crystals. IV. Autotrophic Bacteria 45..

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