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| Adapted from Schallenberg, M., Jacob, F. and Joseph B. R. (1989) Solutions to Problems in Enumerating Sediment Bacteria by direct counts. Applied & Environmental Microbiology. p. 1214-1219.<BR><BR> | | Adapted from Schallenberg, M., Jacob, F. and Joseph B. R. (1989) Solutions to Problems in Enumerating Sediment Bacteria by direct counts. Applied & Environmental Microbiology. p. 1214-1219.<BR><BR> |
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| =='''RICHNESS OF A MICROBIAL SOIL COMMUNITY: METABOLIC DIVERSITY: <BR>Isolation & Characterization of Cultured Bacteria from a Soil Habitat'''== | | =='''RICHNESS OF A MICROBIAL SOIL COMMUNITY: METABOLIC DIVERSITY: <BR>Structural Diversity in Cultured Bacteria from a Soil Community''== |
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| <font size="+1">Using General Purpose & Enrichment/Selective Media for Isolation and Identification of Soil Bacteria in a Mixed Population</font size="+1"><BR> | | <font size="+1">Using General Purpose Media for Isolation of Soil Bacteria in a Mixed Population</font size="+1"><BR> |
| Please watch the YouTube video on streaking for isolation and pay attention to your instructor's demonstration: http://www.youtube.com/watch?v=eyoW18Fzb3o<BR> | | Please watch the YouTube video on streaking for isolation and pay attention to your instructor's demonstration: http://www.youtube.com/watch?v=eyoW18Fzb3o<BR> |
| Directions for [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] are found in the [[BISC209/F13:Protocols| Protocols]] section of this wiki. <BR><BR> | | Directions for [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] are found in the [[BISC209/F13:Protocols| Protocols]] section of this wiki. <BR><BR> |
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| '''Streaking for Isolation from a Dried Soil Extract to select for endospore forming bacteria and possible antibiotic producers:'''<BR> | | '''Streaking for Isolation from a Soil Extract:'''<BR> |
| Each '''team''' of students will '''make a soil extract''' from one gram of their '''oven dried''' soil sample. | | Each '''team''' of students will '''make a soil extract''' from one gram of their soil sample. |
| <li>After recording the weight of the individual aluminum weigh boats, combine the dry soil into one weighing boat, mix gently, and then weigh out 1 gram of the mixed oven dried soil on waxed weighing paper. | | <li>After recording the weight of the individual aluminum weigh boats, combine the dry soil into one weighing boat, mix gently, and then weigh out 1 gram of the mixed oven dried soil on waxed weighing paper. |
| <li>Add the 1g of mixed dried soil to 100ml of sterile water. This is the equivalent to a 1/100 dilution. | | <li>Add the 1g of mixed dried soil to 100ml of sterile water. This is the equivalent to a 1/100 dilution. |
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| <li>We will select colonies of other potential spore-forming bacteria in lab 3. </ul> | | <li>We will select colonies of other potential spore-forming bacteria in lab 3. </ul> |
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| '''Isolation of Spore-formers by Spread Technique:'''<BR> | | '''Isolation of Bacteria by Spread Technique:'''<BR> |
| <ul> | | <ul> |
| <Li>Transfer 100 µL of soil extract onto a pre-labeled plate of glycerol yeast medium (GYEA). | | <Li>Transfer 100 µL of soil extract onto a pre-labeled plate of medium. |
| <LI>Using a sterile disposable spreader cover the surface of the plate with the inoculum. | | <LI>Using a sterile disposable spreader cover the surface of the plate with the inoculum. |
| <LI>Wait one minute to allow the inoculum to absorb into the agar. | | <LI>Wait one minute to allow the inoculum to absorb into the agar. |
| <li>Invert, and incubate the plate at RT. | | <li>Invert, and incubate the plate at RT. |
| <li>Check your plate for colonies daily and record your descriptions of the texture and shape of the colonies that appear. If any colonies arise that look tough and leathery or appear as "little, powdered-sugar volcanos", use the tip of a sterile toothpick to pick up a small but visible amount of growth (being careful not to touch anything but the tip of the colony) and "spread" the bacterial growth onto section 1 of a new glycerol yeast plate. (Inoculate one colony/plate.) Use your loop to isolation streak sections 2-4. Don't forget to flame your loop between sections! | | <li>Check your plate for colonies daily and record your descriptions of the texture and shape of the colonies that appear. If any colonies arise that look tough and leathery or appear as "little, powdered-sugar volcanos", use the tip of a sterile toothpick to pick up a small but visible amount of growth (being careful not to touch anything but the tip of the colony) and "spread" the bacterial growth onto section 1 of a new plate. (Inoculate one colony/plate.) Use your loop to isolation streak sections 2-4. Don't forget to flame your loop between sections! |
| <li>We will select colonies of other potential spore-forming bacteria in lab 3. </ul><BR><BR> | | <li>We will select colonies again in lab 3. </ul><BR><BR> |
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| Over the next few weeks you will continue to sub-culture onto new plates, using your best isolation streak technique. Your goal is to continue to streak out ONE CFU until you are sure that all the bacterial growth in a colony comes from a single mother cell (pure culture). In subsequent labs you will make a bacterial smear and do a Gram stain of these genetically identical bacteria and you will perform other tests from freshly pure cultures to explore the physical and metabolic characteristics of this isolate. <BR><BR> | | Over the next few weeks you will continue to sub-culture onto new plates, using your best isolation streak technique. Your goal is to continue to streak out ONE CFU until you are sure that all the bacterial growth in a colony comes from a single mother cell (pure culture). In subsequent labs you will make a bacterial smear and do a Gram stain of these genetically identical bacteria and you will perform other tests from freshly pure cultures to explore the physical and metabolic characteristics of this isolate. <BR><BR> |
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| ''Streptomycetes'' and other ''Actinomyces'' bacteria often form tough leathery colonies, so transfer of these bacteria to new media to start a sub-culture is sometimes difficult. The powdery area may be spores. Take a sample from this area, if possible. In any case, try to "break off" a piece of the colony with your sterile loop or with a sterile toothpick and transfer that whole piece of a colony onto zone one of the new plate. Then use your loop for streaking out the other zones. The tiny spores on the surface of the colony are likely to transfer to the next plate or tube when you work with it. (That's a good thing this time.) <BR><BR>
| | Some bacteria often form tough leathery colonies, so transfer of these bacteria to new media to start a sub-culture is sometimes difficult. The powdery area may be spores which would be interesting to visualize later. To isolate them try to "break off" a piece of the colony with your sterile loop or with a sterile toothpick and transfer that whole piece of a colony onto zone one of the new plate. Then use your loop for streaking out the other zones. The tiny spores on the surface of the colony are likely to transfer to the next plate or tube when you work with it. (That's a good thing this time.) <BR><BR> |
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| Your team will obtain additional Streaking for Isolation practice by following the directions for SECONDARY ENRICHMENT and ISOLATION using the broth culture and the spread plates that you inoculated with your soil extract last week. This process will continue your attempt to find a diverse group of nitrogen cycling and other interesting bacteria in your soil community. You and your teammates should divide up the work described below and in the [[BISC209/F13: Culture Media| Enrichment Media for the Isolation of Soil Bacteria in a Mixed Population]].<BR><BR> | | Your team will obtain additional Streaking for Isolation practice by following the directions for ISOLATION using the spread plates that you inoculated with your soil extract last week. This process will continue your attempt to find a diverse group of interesting bacteria in your soil community. <BR><BR> |
| The directions for [[BISC209/F13: Aseptic Transfer| Aseptic transfer: Broth to Broth and Broth to Plate]] are found in the [[BISC209/F13:Protocols| Protocols]] section of the wiki. <BR> | | The directions for [[BISC209/F13: Aseptic Transfer| Aseptic transfer: Broth to Broth and Broth to Plate]] are found in the [[BISC209/F13:Protocols| Protocols]] section of the wiki. <BR> |
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| <font color="purple">'''Finding Nitrogen Cycling Bacteria'''<BR>
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| '''Nitrogen Fixers: Using ''Azotobacter'' Nitrogen Free Medium: </font color="purple">
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| <BR>0.08%K<sub>2</sub>HPO<sub>4</sub>; 0.02%KH<sub>2</sub>PO<sub>4</sub>, 0.02% MgSO<sub>4</sub> 0.01% CaSO<sub>4</sub>; 0.0015% FeSO<sub>4</sub>/7H<sub>2</sub>O; 0.00025% g MoO<sub>3</sub>; 0.5% sucrose (to select for nitrogen fixers).
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| Note that the ingredients of this medium are not extracts of undefined sources such as beef digest but rather defined chemicals. The medium contains no nitrogen and only one carbon source-- one could use any sugar, but we will use sucrose. Microorganisms that can't use sucrose for all of their carbon needs and that can't fix nitrogen from the atmosphere to synthesize proteins and other required nitrogen containing compounds will not grow on this medium. Those that can, such as the ''Azotobacter'' group will be selected.<BR><BR>
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| <font color="black">
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| '''Secondary Enrichment and Isolation for Nitrogen Fixers''':<ul>
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| <li>Secondary enrichment:<BR><ul>
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| Each pair should take a loopful of the slime from the pellicle (media surface) or from the side of the flask and place it into 1 ml of sterile water in a small tube. Cap the small tube. Vortex the tube to disperse the sample. (Vortexing in this way helps break up the other microbes that will be embedded in the slimy material. The other microbes are taking advantage of the by-products of Nitrogen compounds excreted by the N<sub>2</sub> fixers. )
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| OR use a sterile swab to remove some of the slime and a little liquid from the flask of Azoto medium and inoculate the first zone of an isolation streak plate directly.
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| <LI>''Each Pair'' should streak onto two plates of ''Azotobacteria'' agar medium suspension, using your best isolation streak technique. (Follow the protocol in [[BISC209/F13: Streaking for Isolation | Streaking for Isolation ]]).
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| <LI> Incubate one streak plate at room temp and the other at 30 °C. Use the shelf in the 30°C walk in chamber that is designated for your 209 lab section.
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| </LI></UL></ul>
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| '''Isolation:'''<BR>
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| Watch for the appearance of isolated, slimy colonies on either plate.
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| Continue to streak to make sub-cultures onto fresh ''Azotobacter'' medium or on NA until you are sure that you have a pure isolate. Note that your bacteria will probably grow better on NA and it is fine to switch to nutrient agar after the initial selection. <BR><BR>
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| '''Characterization:'''<BR>
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| In a later lab you will make a bacterial smear and do a Gram stain and other tests to explore the physical and metabolic characteristics of your nitrogen fixing bacteria.
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| <font color="purple">'''Finding Nitrogen Cycling Bacteria: Bacteria that can Extract Nitrogen from Ammonium Compounds'''<BR>
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| '''Ammonium & Citrate Users: Using Simmons Citrate Medium''': </font color="purple"><BR>
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| 0.02% MgSO<sub>4</sub>(Magnesium Sulfate), 0.1% NH<sub>4</sub>H<sub>2</sub>PO<sub>4</sub>(monoammonium phosphate), 0.1% K<sub>2</sub>HPO<sub>4</sub>(dipotassium phosphate), 0.2% C<sub>6</sub>H<sub>5</sub>Na<sub>3</sub>O<sub>7</sub>(sodium citrate), 0.5% NaCl(sodium chloride), 2.5% agar, 0.008% C<sub>27</sub>H2<sub>7</sub>Br<sub>2</sub>O<sub>5</sub>SNa(bromothymol blue) at pH6.9
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| <BR><BR>
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| Simmons Citrate Medium selects for microorganisms that can utilize citrate as their sole source of carbon in a medium containing inorganic ammonium salts as its only source of nitrogen. In 1923 an investigator named Koser invented a broth medium in which ammonium phosphate supplied the only source of nitrogen and each organic acid was added individually, allowing the introduction of carbon utilization as a diagnostic aid. Later, an investigator named Simmons converted Koser’s liquid medium to a solid by the addition of agar and added an indicator system by incorporating bromthymol blue. The exact nature of the alkaline reaction produced by the organisms that are able to use citrate as their sole source of carbon is poorly understood. It appears that the alkaline reaction that gives the color change characteristic of a positive Simmons-Citrate test most likely occurs when excess CO<sub>2</sub> is generated when citrate is cleaved to form oxaloacetate. This by
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| product is then decarboxylated to pyruvic acid and CO<sub>2</sub>. The excess CO<sub>2</sub> combines with sodium and water to form sodium carbonate. Note that bacteria that utilize citrate in this way are able to extract nitrogen from ammonium phosphate in the medium, resulting in the production of ammonia which combines with water to form NH<sub>4</sub>OH. These reactions in combination produce an alkaline pH (greater than 7.6), resulting in a color change in the indicator from green to blue. Some members of the genera''Klebsiella''in the ''Entrobacteriacea'' family are Simmons Citrate positive.<BR><BR>
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| <font color="black">
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| '''Isolation of Ammonium & Citrate Users from Simmons Citrate Selective Medium''':<BR>
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| Compare the relative number of colonies that grow on NA to the number observed on Simmons Citrate at each dilution plated from the 1% soil extract. There should be fewer colonies on the Citrate plate (A). Why? What are some important characteristics of the bacteria that are growing on this medium? <ul>
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| <LI>Count the CFU on a plate with 30-300 colonies and using a Sharpie circle and number a few of the different appearing ones on the bottom of the plate or give them code names (your initial and a number or a more creative coding scheme).
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| <li>Some of the colonies on this plate may floresce. In order to see this, you will need to take a marker, safety goggles, the hand-held UV light on the instructors' desk, and your SC plate culture to a completely dark location (such as the bathroom in the hall outside the lab). Put on the glasses, turn on the UV light and shut off the white lights. If you see any colonies that "light up"(B), mark them with a small dot or circle on the bottom side of the plastic plate and attempt to isolate at least one of them to pure culture. <BR>
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| <li> Make observations in your lab notebook about the #, appearance, color, consistency or florescence of any colonies growing on your Simmons Citrate plates.
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| <li> Each teammate should try to isolate a unique colony from any of the Simmons Citrate plates (based on colony color, consistency, and/or florescence).
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| <li> Obtain new sterile Nutrient Agar plates or SC plates (one for each student in your group).
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| <li> Each student should use her flame sterilized and cooled loop and touch it to the center of the colony on Simmons Citrate that you are attempting to isolate. DO NOT touch anything on this plate other than the desired colony!
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| <li>Follow the directions found in [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] in the [[BISC209/F13:Protocols| Protocols]] section of this wiki carefully. Transfer bacteria from this colony to the O zone of a new NA plate.
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| <li> Flame and cool your loop before going back into sequential, inoculated zones to transfer fewer and fewer bacteria as you streak each zone.
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| <LI>Parafilm and save any plate that contains a colony you or your teammates are attempting to isolate. Discard plates that you do not contain colonies that were subcultured.
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| <li> Check the sub-cultures frequently and sub-culture over and over to new media until you are sure that your culture is pure. Note how fast your organism grows and if the initial colony appearance and/or fluorescence is maintained.
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| <li> Your goal is to end with well isolated single colonies that grow from a single bacterium (all genetically identical) on nutrient agar (NA). It will probably take several transfers over the next few weeks to take a CFU from this original selective medium plate and grow bacteria from it in pure culture on nutrient agar.
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| </li></ul>
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| <font color="purple">'''Finding and Isolating Other interesting Bacteria to Pure Culture''':</font color="purple"> <BR>
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| NA: 0.3% Beef extract, 0.5% Peptone, 1.5% Agar; Deionized water to 1 L at pH 6.6- 7.0 at 25°C <BR>
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| NA+Starch: NA (above) plus added 2.5% starch<BR>
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| Dilute NA: NA diluted 1:10 (0.03% beef extract, 0.05% Peptone, 1.5% agar)
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| Nutrient Agar is a rich, general purpose medium. It is not used for enrichment or selection although not all microorganisms will grow on it. Those that won't are considered, with great variability, "fastidious". NA + starch is similarly non-selective and should grow the same microorganisms that grow on NA although you may find that the addition of starch encourages the growth of organisms that can use the starch effectively and/or observe interesting pigmentation to some colonies.<BR>
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| You and your partners should spend time observing and making notes in your notebook about the variety of bacterial colonies growing on these media. Observe differences and similarities between the number and variety of colonies on the dilute NA and NA plus starch plates at the same dilutions. Each of you should choose one or two different looking colonies to attempt to isolate in pure culture on NA. <ul>
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| <li>Number on the bottom of the plate or use a code for the colonies each member of your group selects.
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| <li>Parafilm and save these plates in the refrigerator in your group's rack.
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| <li>Follow the directions carefully that are found in [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] in the [[BISC209/F13:Protocols| Protocols]] section of this wiki. Transfer bacteria from one of your selected colonies to the O zone of a new NA plate.
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| <li> Flame and cool your loop before going back into sequential inoculated zones to transfer fewer and fewer bacteria as you streak each zone of the new plate.
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| <li> Your goal is to end with well isolated single colonies that grow from a single bacterium (all genetically identical) on nutrient agar (NA). It will probably take several transfers over the next few weeks to take a CFU from this original selective medium plate and grow bacteria from it in pure culture on nutrient agar.
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| </ul>
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| <BR><BR>
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| The complete procedures for Enrichment and Isolation of Selected Bacteria are described in [[BISC209/F13: Culture Media | Enrichment Media for the Isolation of Soil Bacteria in a Mixed Population]] protocols). <BR><BR>
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| For more information about culture characteristics of Azotobacter refer to on-line reference books such ''The Prokaryotes'' or ''Bergeys'' (link for access in the [[BISC209/F13:Resources|Resources]] section of the wiki. For an online example of images of colonies and stained cells of ''Azotobacter vinelandii'' [http://inst.bact.wisc.edu/inst/index.php?module=book&func=displayarticle&art_id=274] http://inst.bact.wisc.edu/inst/index.php?module=book&func=displayarticle&art_id=274
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| ==CLEAN UP== | | ==CLEAN UP== |
