BISC209/S11: Lab6 - Revision history

Tucker Crum: /* Cultured Bacterial Isolate Characterization: Example of Strategy for Dealing with Competition for Resources in a Soil Community */

2011-03-15T14:13:58Z

Cultured Bacterial Isolate Characterization: Example of Strategy for Dealing with Competition for Resources in a Soil Community

←Older revision		Revision as of 14:13, 15 March 2011
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	'''Fig: 5C-1.''' Testing of multiple isolates in one plate can be accomplished by dividing a plate into 4 (OR MORE) sections. Be sure the inoculum is placed in the center of each section and that you check the plate for growth regularly. <BR><BR>		'''Fig: 5C-1.''' Testing of multiple isolates in one plate can be accomplished by dividing a plate into 4 (OR MORE) sections. Be sure the inoculum is placed in the center of each section and that you check the plate for growth regularly. <BR><BR>

-	==Cultured Bacterial ~~Isolate Characterization~~: ~~Example~~ of ~~Strategy for Dealing with~~ Competition ~~for Resources~~ in a Soil Community==	+	==Cultured Bacterial Isolates: Examples of Competition and Co-operation in a Soil Community==

	'''Testing for Antibiotic Production'''		'''Testing for Antibiotic Production'''

Tucker Crum at 13:34, 2 March 2011

2011-03-02T13:34:07Z

←Older revision		Revision as of 13:34, 2 March 2011
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	The sequences should come back in a week or two.		The sequences should come back in a week or two.

-	=='''Culture-Dependent Isolate Characterizations: ~~Examples of Competition and Co-operation in Selected Soil Community Bacteria~~'''==	+	=='''Culture-Dependent Isolate Characterizations:'''==

	==Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain ==		==Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain ==
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	'''Fig: 5C-1.''' Testing of multiple isolates in one plate can be accomplished by dividing a plate into 4 (OR MORE) sections. Be sure the inoculum is placed in the center of each section and that you check the plate for growth regularly. <BR><BR>		'''Fig: 5C-1.''' Testing of multiple isolates in one plate can be accomplished by dividing a plate into 4 (OR MORE) sections. Be sure the inoculum is placed in the center of each section and that you check the plate for growth regularly. <BR><BR>

-	==Cultured Bacterial Isolate Characterization ~~by Metabolic and Physical Tests~~==	+	==Cultured Bacterial Isolate Characterization: Example of Strategy for Dealing with Competition for Resources in a Soil Community==

	'''Testing for Antibiotic Production'''		'''Testing for Antibiotic Production'''

Tucker Crum: /* Cultured Bacterial Isolate Characterization by Metabolic and Physical Tests */

2011-03-02T13:29:46Z

Cultured Bacterial Isolate Characterization by Metabolic and Physical Tests

←Older revision		Revision as of 13:29, 2 March 2011
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	1) To begin the antibiotic testing protocol, you will use the fresh broth culture of each of your isolates that you prepared prior to lab. <BR>		1) To begin the antibiotic testing protocol, you will use the fresh broth culture of each of your isolates that you prepared prior to lab. <BR>

-	2) Pipet one ml of the broth culture into a sterile glass tube and adjust the concentration of the cells by matching by eye the "cloudiness" (turbidity) of the cells in the glass tube to a standard provided by your instructor. We will use a McFarland 0.5 standard~~(Remember that cell concentration can be measured as optical density at 600nm in a spectrophotometer)~~; the 0.5 refers to the approximate concentration of organisms in solutions which is 1.5X10<sup>8</sup> cfu/mL for the 0.5 standard. Other common standards are shown in the table below.<BR>	+	2) Although cell density can be measured more accurately using a spectrophotometer to measure optical density (OD) at 600nm, we will use a quicker method that will work well enough for our purposes. Pipet one ml of the broth culture into a sterile glass tube and adjust the concentration of the cells by matching by eye the "cloudiness" (turbidity) of the cells in the glass tube to a standard provided by your instructor. We will use a McFarland 0.5 standard; the 0.5 refers to the approximate concentration of organisms in solutions which is 1.5X10<sup>8</sup> cfu/mL for the 0.5 standard. Other common standards are shown in the table below.<BR>

	[[Image:mcfarlandtable.jpg]]<BR>		[[Image:mcfarlandtable.jpg]]<BR>

Tucker Crum: /* Cultured Bacterial Isolate Characterization by Metabolic and Physical Tests */

2011-03-02T13:27:03Z

Cultured Bacterial Isolate Characterization by Metabolic and Physical Tests

←Older revision		Revision as of 13:27, 2 March 2011
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	Start the [[BISC209/S11: Antibiotics\| Testing for Antibiotic Production]].		Start the [[BISC209/S11: Antibiotics\| Testing for Antibiotic Production]].
	<BR><BR>		<BR><BR>
-	Many microbes secrete antimicrobial compounds to help them compete with other microorganisms for habitat niche. Common antibiotic producers are the ''Actinomycetes'' (including ''Streptomycetes'' species)and many of the ''Bacillus'' species; although ~~they~~ are just a few among many, many antibiotic producing bacteria. <BR>	+	Many microbes secrete antimicrobial compounds to help them compete with other microorganisms for habitat niche. Common antibiotic producers are the ''Actinomycetes'' (including ''Streptomycetes'' species)and many of the ''Bacillus'' species; although these are just a few among many, many antibiotic producing bacteria. <BR>

	(This testing will take 3 weeks.) <BR>		(This testing will take 3 weeks.) <BR>

Tucker Crum: /* The Gram Stain */

2011-03-02T13:24:40Z

The Gram Stain

←Older revision		Revision as of 13:24, 2 March 2011
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	'''To Gram stain the previously prepared heat fixed, bacterial smear slides, perform the staining protocol from start to finish on one slide at a time. You must be careful to apply the staining reagents liberally so all the smears are evenly and completely covered and you must be sure to expose each smear to each reagent for the same amount of time.''' <BR><BR>		'''To Gram stain the previously prepared heat fixed, bacterial smear slides, perform the staining protocol from start to finish on one slide at a time. You must be careful to apply the staining reagents liberally so all the smears are evenly and completely covered and you must be sure to expose each smear to each reagent for the same amount of time.''' <BR><BR>

-	1. Place a heat-fixed bacterial smear slide on the staining tray. It is important that the slide is level during staining; therefore you should use paper towels under the tray to level the ~~slides~~. If you do this, it is much easier ~~to be sure~~ that your smears will be covered evenly with each reagent (a crucial aspect of proper staining results).<BR><BR>	+	1. Place a heat-fixed bacterial smear slide on the staining tray. It is important that the slide is level during staining; therefore, you should use paper towels under the tray to level the slide. If you do this, it is much easier ensure that your smears will be covered evenly with each reagent (a crucial aspect of proper staining results).<BR><BR>

	2. Dispense just enough Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) to completely cover each smear and stain for 1 minute. (Crystal violet is the primary stain.) <BR>		2. Dispense just enough Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) to completely cover each smear and stain for 1 minute. (Crystal violet is the primary stain.) <BR>

Tucker Crum: /* The Gram Stain */

2011-03-02T13:23:53Z

The Gram Stain

←Older revision		Revision as of 13:23, 2 March 2011
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	'''To Gram stain the previously prepared heat fixed, bacterial smear slides, perform the staining protocol from start to finish on one slide at a time. You must be careful to apply the staining reagents liberally so all the smears are evenly and completely covered and you must be sure to expose each smear to each reagent for the same amount of time.''' <BR><BR>		'''To Gram stain the previously prepared heat fixed, bacterial smear slides, perform the staining protocol from start to finish on one slide at a time. You must be careful to apply the staining reagents liberally so all the smears are evenly and completely covered and you must be sure to expose each smear to each reagent for the same amount of time.''' <BR><BR>

-	1. Place a heat-fixed bacterial smear slide on the staining tray. It is important that the slide be level during staining so use paper towels under the tray to ~~get it leveled~~. If you do, it is much easier to be sure that your smears will be covered evenly with each reagent.<BR><BR>	+	1. Place a heat-fixed bacterial smear slide on the staining tray. It is important that the slide is level during staining; therefore you should use paper towels under the tray to level the slides. If you do this, it is much easier to be sure that your smears will be covered evenly with each reagent (a crucial aspect of proper staining results).<BR><BR>

	2. Dispense just enough Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) to completely cover each smear and stain for 1 minute. (Crystal violet is the primary stain.) <BR>		2. Dispense just enough Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) to completely cover each smear and stain for 1 minute. (Crystal violet is the primary stain.) <BR>

Tucker Crum: /* The Gram Stain */

2011-03-02T13:22:01Z

The Gram Stain

←Older revision		Revision as of 13:22, 2 March 2011
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	'''Activity: Preparing a Gram Stain'''<BR><BR>		'''Activity: Preparing a Gram Stain'''<BR><BR>
-	The Gram stain is a standard staining technique useful for the identification of culturable bacterial organisms and you will perform it now.<BR>	+	The Gram stain is a standard DIFFERENTIAL staining technique useful for the identification of culturable bacterial organisms and you will perform it now. Remember that a differential stain is one in which different bacteria are subjected to the same stain procedure but they respond differently and show a visible differentiation. In this case, that visible difference is pink vs purple color and it is based on cell wall composition differences.<BR>
	Use the smear slides you have prepared of your isolates and the controls and follow the Gram Stain Protocol found below. This protocol can also be found in [[BISC209/S11: Stains]] in the protocol section of this wiki. <BR>		Use the smear slides you have prepared of your isolates and the controls and follow the Gram Stain Protocol found below. This protocol can also be found in [[BISC209/S11: Stains]] in the protocol section of this wiki. <BR>

Tucker Crum: /* Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain */

2011-03-02T13:18:27Z

Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain

←Older revision		Revision as of 13:18, 2 March 2011
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	3. Place the loop with the bacterial growth into the drop of water. Use a circular motion with the loop to make a smooth suspension of the bacteria in the water. Stop when you have spread out the circle of emulsified bacteria as much as possible, while making sure to leave room to make two other smears on the slide. Reflame your loop and repeat the process of inoculating the''E. coli'' control into both the middle drop of water and the drop on the far right. Mix the middle drop of ''E. coli'' emulsion as you did for the ''Staph'' smear. Flame your loop and cool it for a few seconds. Touch your loop again to a ''Staphylococcus'' colony (just a TINY bit) and inoculate the drop of water on the far right, to mix the ''Staph'' with the ''E. coli'' in the last smear. Spread the bacteria in the mixed smear evenly.<BR><BR>		3. Place the loop with the bacterial growth into the drop of water. Use a circular motion with the loop to make a smooth suspension of the bacteria in the water. Stop when you have spread out the circle of emulsified bacteria as much as possible, while making sure to leave room to make two other smears on the slide. Reflame your loop and repeat the process of inoculating the''E. coli'' control into both the middle drop of water and the drop on the far right. Mix the middle drop of ''E. coli'' emulsion as you did for the ''Staph'' smear. Flame your loop and cool it for a few seconds. Touch your loop again to a ''Staphylococcus'' colony (just a TINY bit) and inoculate the drop of water on the far right, to mix the ''Staph'' with the ''E. coli'' in the last smear. Spread the bacteria in the mixed smear evenly.<BR><BR>

-	4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add a tiny amount of bacteria from a fresh culture growing on solid medium of one your isolates (other than ''Hyphomicrobia''). Create the smear as you did for the controls moving from left to right and spacing the smears so that 3 isolates can fit on one slide. Repeat for the other isolates (except Hyphomicrobia) ~~on the slide~~. For your Hyphomicrobia, use your fresh broth culture of ~~your~~ isolate. Remove the culture tube top (NOT placing it on the bench) and flame the lip of the culture tube. Dip your loop into the broth culture and place a VERY small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly. <BR><BR>	+	4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add a tiny amount of bacteria from a fresh culture growing on solid medium of one your isolates (other than ''Hyphomicrobia''). Create the smear as you did for the controls moving from left to right and spacing the smears so that 3 isolates can fit on one slide. Repeat for the other isolates (except Hyphomicrobia). For your ''Hyphomicrobia'', use your fresh broth culture of this isolate. Remove the culture tube top (NOT placing it on the bench) and flame the lip of the culture tube. Dip your loop into the broth culture and place a VERY small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly. <BR><BR>

	5. Allow the slides to air dry completely! Be sure ''all'' the water on the slide has evaporated before proceeding to heat fixation!!! This drying step is crucially important. If you are impatient, you will "explode" the cells in the next step . <br><BR>		5. Allow the slides to air dry completely! Be sure ''all'' the water on the slide has evaporated before proceeding to heat fixation!!! This drying step is crucially important. If you are impatient, you will "explode" the cells in the next step . <br><BR>

Tucker Crum: /* Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain */

2011-03-02T13:17:13Z

Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain

←Older revision		Revision as of 13:17, 2 March 2011
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	3. Place the loop with the bacterial growth into the drop of water. Use a circular motion with the loop to make a smooth suspension of the bacteria in the water. Stop when you have spread out the circle of emulsified bacteria as much as possible, while making sure to leave room to make two other smears on the slide. Reflame your loop and repeat the process of inoculating the''E. coli'' control into both the middle drop of water and the drop on the far right. Mix the middle drop of ''E. coli'' emulsion as you did for the ''Staph'' smear. Flame your loop and cool it for a few seconds. Touch your loop again to a ''Staphylococcus'' colony (just a TINY bit) and inoculate the drop of water on the far right, to mix the ''Staph'' with the ''E. coli'' in the last smear. Spread the bacteria in the mixed smear evenly.<BR><BR>		3. Place the loop with the bacterial growth into the drop of water. Use a circular motion with the loop to make a smooth suspension of the bacteria in the water. Stop when you have spread out the circle of emulsified bacteria as much as possible, while making sure to leave room to make two other smears on the slide. Reflame your loop and repeat the process of inoculating the''E. coli'' control into both the middle drop of water and the drop on the far right. Mix the middle drop of ''E. coli'' emulsion as you did for the ''Staph'' smear. Flame your loop and cool it for a few seconds. Touch your loop again to a ''Staphylococcus'' colony (just a TINY bit) and inoculate the drop of water on the far right, to mix the ''Staph'' with the ''E. coli'' in the last smear. Spread the bacteria in the mixed smear evenly.<BR><BR>

-	4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add ~~1/8th of~~ a ~~1/8th~~ of ~~a pure colony~~ from a fresh culture on solid medium of your isolates other than Hyphomicrobia. Create the smear as you did for the controls moving from left to right and spacing so that 3 isolates can fit on one slide. Repeat for the other isolates (except Hyphomicrobia) on the slide. For your Hyphomicrobia, use your fresh broth culture of your isolate. Remove the culture tube top (NOT placing it on the bench) and flame the lip of the culture tube. Dip your loop into the broth culture and place a VERY small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly. <BR><BR>	+	4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add a tiny amount of bacteria from a fresh culture growing on solid medium of one your isolates (other than ''Hyphomicrobia''). Create the smear as you did for the controls moving from left to right and spacing the smears so that 3 isolates can fit on one slide. Repeat for the other isolates (except Hyphomicrobia) on the slide. For your Hyphomicrobia, use your fresh broth culture of your isolate. Remove the culture tube top (NOT placing it on the bench) and flame the lip of the culture tube. Dip your loop into the broth culture and place a VERY small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly. <BR><BR>

	5. Allow the slides to air dry completely! Be sure ''all'' the water on the slide has evaporated before proceeding to heat fixation!!! This drying step is crucially important. If you are impatient, you will "explode" the cells in the next step . <br><BR>		5. Allow the slides to air dry completely! Be sure ''all'' the water on the slide has evaporated before proceeding to heat fixation!!! This drying step is crucially important. If you are impatient, you will "explode" the cells in the next step . <br><BR>

Tucker Crum: /* Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain */

2011-03-02T13:15:20Z

Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain

←Older revision		Revision as of 13:15, 2 March 2011
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	2. Prepare the "control" slide first by using your loop to apply three tiny drops of deionized water to different parts of this pre-labeled slide. Flame sterilize the loop, allow it to cool for a few seconds and touch the cooled loop to a single colony of the control culture plate of ''S. epidermidis''. Pick up a VERY, VERY TINY bit of growth from a single bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony will give you plenty of bacteria. If you take a glob, the bacteria will be so thick, you will not get good stain results.<BR><BR>		2. Prepare the "control" slide first by using your loop to apply three tiny drops of deionized water to different parts of this pre-labeled slide. Flame sterilize the loop, allow it to cool for a few seconds and touch the cooled loop to a single colony of the control culture plate of ''S. epidermidis''. Pick up a VERY, VERY TINY bit of growth from a single bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony will give you plenty of bacteria. If you take a glob, the bacteria will be so thick, you will not get good stain results.<BR><BR>

-	3. Place the loop with the bacterial growth into the drop of water. Use a circular motion with the loop to make a smooth suspension of the bacteria in the water. Stop when you have spread out the circle of emulsified bacteria as much as possible, while making sure to leave room to make two other smears on the slide. Reflame your loop and repeat the process of inoculating the''E. coli'' control into both the middle drop of water and the drop on the far right. Mix the middle drop of ''E. coli'' emulsion as you did for the ''Staph'' smear. Flame your loop and cool it for a few seconds. Touch your loop again to a ''Staphylococcus'' colony (just a TINY bit) and inoculate the drop of water on the far right, to mix the ''Staph'' with the ''E. coli'' in the last smear. Spread the ~~two~~ bacteria in the mixed smear evenly.<BR><BR>	+	3. Place the loop with the bacterial growth into the drop of water. Use a circular motion with the loop to make a smooth suspension of the bacteria in the water. Stop when you have spread out the circle of emulsified bacteria as much as possible, while making sure to leave room to make two other smears on the slide. Reflame your loop and repeat the process of inoculating the''E. coli'' control into both the middle drop of water and the drop on the far right. Mix the middle drop of ''E. coli'' emulsion as you did for the ''Staph'' smear. Flame your loop and cool it for a few seconds. Touch your loop again to a ''Staphylococcus'' colony (just a TINY bit) and inoculate the drop of water on the far right, to mix the ''Staph'' with the ''E. coli'' in the last smear. Spread the bacteria in the mixed smear evenly.<BR><BR>

	4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add 1/8th of a 1/8th of a pure colony from a fresh culture on solid medium of your isolates other than Hyphomicrobia. Create the smear as you did for the controls moving from left to right and spacing so that 3 isolates can fit on one slide. Repeat for the other isolates (except Hyphomicrobia) on the slide. For your Hyphomicrobia, use your fresh broth culture of your isolate. Remove the culture tube top (NOT placing it on the bench) and flame the lip of the culture tube. Dip your loop into the broth culture and place a VERY small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly. <BR><BR>		4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add 1/8th of a 1/8th of a pure colony from a fresh culture on solid medium of your isolates other than Hyphomicrobia. Create the smear as you did for the controls moving from left to right and spacing so that 3 isolates can fit on one slide. Repeat for the other isolates (except Hyphomicrobia) on the slide. For your Hyphomicrobia, use your fresh broth culture of your isolate. Remove the culture tube top (NOT placing it on the bench) and flame the lip of the culture tube. Dip your loop into the broth culture and place a VERY small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly. <BR><BR>