User:Zachary Thorogood/Notebook/Biology 210 at AU

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Lab 3.
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'''Objectives:'''
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To understand the characteristics of bacteria.
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To observe antibiotic resistances.
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To understand how DNA sequences are used to identify species.
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 +
'''Procedure I: Quantifying and Observing Microorganisms:'''
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1. Make one more check on your Hay Infusion Culture and describe any changes.
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2. Count the total number of colonies on a plate.
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3. Record the data.
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'''Procedure II: Antibiotic Resistance:'''
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1. Observe the difference between the nutrient plate vs. the nutrient + tetracycline.
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2. Record the data.
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'''Procedure III: Bacteria Cell Morphology Observations:'''
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1. Choose four colonies, out of the eight, that best represent bacterial growth and microorganisms.
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2. Try and get two colonies from the nutrient agar and two from the nutrient + tetracycline plate.
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3. Prepare a wet mount and gram stain for each colony.
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4. Record the data.
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Procedure IV: Stat PCR Preparation for DNA Sequence Identification:
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1. Select one plate from the two colonies that has the best characterization.
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2. Isolate DNA from bacteria in the colonies and use two primer sequences (27F and 519R) to amplify the 16S rRNA gene.
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3. Transfer a single colony of bacteria to 100ul of water in a sterile tube.
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4. Incubate at 100C for ten minutes and centrifuge.
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5. Use 5ul of the supernatant in the PCR reaction.
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'''Data:'''
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Hay Infusion - Lots of evaporation occurred. Plant matter sunk to the bottom of the jar and the water has turned to mirky/amberish colour.
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Colony            Nutrient with/without Tetracycline            Colony Description                          Cell Description                                              Gram Positive or Negative
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2                                  Nutrient                            Mainly small sizes clumped together          Small rod and circular cells, motile.             
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                                                                            to form a spread out form. Variation          Some Bacilli arrangement. One clear                            NEGATIVE
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                                                                            in colour from dull orange to white/            Staphyloccocus.
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                                                                            grey.
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4                                Nutrient                            Blocks of colonies mainly white in colour    Small "black" circular cells, slow
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                                                                            that form longer, but less farms. Size          moving, difficult to make definitive                                NEGATIVE
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                                                                            ranges from small to large colonies.          conclusion on arrangement of cells.
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 +
                                                                             
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T3                              Nutrient + Tetracycline    Bright orange colonies mainly medium        Small "black" circular cells, fast 
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                                                                          size cells, some fungi growth.                      moving, difficult to make definitive                                POSITIVE
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                                                                                                                                              conclusion on arrangement of cells.
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Dilution            Agar          Colonies Counted          Conversion Factor          Colonies/mL
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10-3              nutrient                  300                            x10^3                        300000
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10-5              nutrient                  50                              x10^5                        5000000
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10-7              nutrient                  6                                x10^7                        60000000
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10-9              nutrient                  1                                x10^9                        1000000000
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 +
10-3            nutrient + tet          90                              x10^3                          90000
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10-5            nutrient + tet          0                                x10^5                          0
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10-7            nutrient + tet          0                                x10^7                          0
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______________________________________________
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Lab 2.
Lab 2.
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18. Take 100 microlitres of the the mixture in test tube 8 and place into both agar plates.  
18. Take 100 microlitres of the the mixture in test tube 8 and place into both agar plates.  
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Data:
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'''Data:'''
Organisms with Dichotomous Key: Very few organisms were found and those that were we could not definitively classify.   
Organisms with Dichotomous Key: Very few organisms were found and those that were we could not definitively classify.   
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'''Procedure 1: The Volvicine Line.'''
'''Procedure 1: The Volvicine Line.'''
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1. Prepare a slide of living '''Chlamydomonas''' and examine it microscopically, adding protoslo will slow motile creatures down and make them easier to observe.
1. Prepare a slide of living '''Chlamydomonas''' and examine it microscopically, adding protoslo will slow motile creatures down and make them easier to observe.
2. Observe the living culture of ''Gonium'' on a slide. It is a progressively more complex species consisting of a colony of 4, 8, 16, or 32 cells. They are held together in a gelatinous matrix and each cell can form a new colony.  
2. Observe the living culture of ''Gonium'' on a slide. It is a progressively more complex species consisting of a colony of 4, 8, 16, or 32 cells. They are held together in a gelatinous matrix and each cell can form a new colony.  

Revision as of 15:56, 16 February 2014

Lab 3.

Objectives:

To understand the characteristics of bacteria. To observe antibiotic resistances. To understand how DNA sequences are used to identify species.

Procedure I: Quantifying and Observing Microorganisms:

1. Make one more check on your Hay Infusion Culture and describe any changes. 2. Count the total number of colonies on a plate. 3. Record the data.

Procedure II: Antibiotic Resistance:

1. Observe the difference between the nutrient plate vs. the nutrient + tetracycline. 2. Record the data.

Procedure III: Bacteria Cell Morphology Observations:

1. Choose four colonies, out of the eight, that best represent bacterial growth and microorganisms. 2. Try and get two colonies from the nutrient agar and two from the nutrient + tetracycline plate. 3. Prepare a wet mount and gram stain for each colony. 4. Record the data.

Procedure IV: Stat PCR Preparation for DNA Sequence Identification:

1. Select one plate from the two colonies that has the best characterization. 2. Isolate DNA from bacteria in the colonies and use two primer sequences (27F and 519R) to amplify the 16S rRNA gene. 3. Transfer a single colony of bacteria to 100ul of water in a sterile tube. 4. Incubate at 100C for ten minutes and centrifuge. 5. Use 5ul of the supernatant in the PCR reaction.


Data:

Hay Infusion - Lots of evaporation occurred. Plant matter sunk to the bottom of the jar and the water has turned to mirky/amberish colour.


Colony Nutrient with/without Tetracycline Colony Description Cell Description Gram Positive or Negative

2 Nutrient Mainly small sizes clumped together Small rod and circular cells, motile.

                                                                            to form a spread out form. Variation           Some Bacilli arrangement. One clear                             NEGATIVE
                                                                           in colour from dull orange to white/            Staphyloccocus. 
                                                                           grey.

4 Nutrient Blocks of colonies mainly white in colour Small "black" circular cells, slow

                                                                           that form longer, but less farms. Size          moving, difficult to make definitive                                NEGATIVE
                                                                           ranges from small to large colonies.           conclusion on arrangement of cells.


T3 Nutrient + Tetracycline Bright orange colonies mainly medium Small "black" circular cells, fast

                                                                          size cells, some fungi growth.                      moving, difficult to make definitive                                 POSITIVE
                                                                                                                                             conclusion on arrangement of cells.

Dilution Agar Colonies Counted Conversion Factor Colonies/mL 10-3 nutrient 300 x10^3 300000 10-5 nutrient 50 x10^5 5000000 10-7 nutrient 6 x10^7 60000000 10-9 nutrient 1 x10^9 1000000000

10-3 nutrient + tet 90 x10^3 90000 10-5 nutrient + tet 0 x10^5 0 10-7 nutrient + tet 0 x10^7 0

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Lab 2.

Objectives:

To understand how to use a dichotomous key. To understand the characteristics of Algae and Protists.

Procedure:

1. Obtain a dichotomous key and make a wet mount from a sample of random organisms. 2. Add Protoslo to increase chances of finding an organism. 3. Record observations.

4. Using the hay infusion, that was created from the previous lab, take random samples from varying points to see which organisms inhabit the hay infusion. 5. After gathering the sample, create a wet mount and add Protoslo to increase chances of finding any organisms. 6. Record observations.

7. Obtain four 10ml test tubes that contain sterile broth and label them 2,4,6,8. 8. Obtain eight agar plates - four nutrient agar plates and four agar + tetracycline plates. 9. Mix the hay infusion carefully, yet enough to create an adequate mixture. 10. Obtain a micropipette that is set to100 microlitres. 11. Take 100 microlitres of the mixed hay infusion and place into the 10ml test tube labelled 2. 12. Take 100 microlitres of the the mixture in test tube 2 and place into test tube 4. 13. Take 100 microlitres of the the mixture in test tube 4 and place into test tube 6. 14. Take 100 microlitres of the the mixture in test tube 6 and place into test tube 8. 15. Take 100 microlitres of the the mixture in test tube 2 and place into both agar plates. 16. Take 100 microlitres of the the mixture in test tube 4 and place into both agar plates. 17. Take 100 microlitres of the the mixture in test tube 6 and place into both agar plates. 18. Take 100 microlitres of the the mixture in test tube 8 and place into both agar plates.

Data:

Organisms with Dichotomous Key: Very few organisms were found and those that were we could not definitively classify.

Hay Infusion Observations: Water is amber brown with plant and soil matter primarily located at the bottom of the infusion.

Niche 1 in Hay Infusion: Plant and soil matter located at the bottom. Niche 2 in Hay Infusion: Plant and soil matter located in the middle/top.



Lab 1.

Objectives:

To understand natural selection. To understand the biotic and abiotic characteristics of a niche.

Procedure 1: The Volvicine Line.

1. Prepare a slide of living Chlamydomonas and examine it microscopically, adding protoslo will slow motile creatures down and make them easier to observe. 2. Observe the living culture of Gonium on a slide. It is a progressively more complex species consisting of a colony of 4, 8, 16, or 32 cells. They are held together in a gelatinous matrix and each cell can form a new colony. 3. Observe the living culture of Volvox on a slide. It represents the peak of evolutionary complexity in this line of green algae consisting of thousands of spiked cells making up a spherical Volvox colony.

Procedure 2: Defining a Niche at AU.

1. Set the 20x20 feet dimensions of the transect (either 1, 2, 3, 4, or 5) with four popsicle sticks. 2. Describe the general characteristics of the transect: Location, Topography, etc. 3. List the abiotic and biotic components of the transect. 4. Use the sterile 50ml conical tube to take a soil and ground vegetation sample. Make it is representative of the ground and what is on the surface of the ground.

Procedure 3: Make a Hay Infusion Culture.

1. Weigh 10 to 12 grams of the soil/ground sample and place in the plastic jar with 500ml of deerpark water. 2. Add 0.1 grams of dried mil and gently mix it for about 10 seconds. 3. Take the top off and place the jar in a safe place in the lab. 4. Label the jar so the group can find it.

Data:

Five Biotic Observations:

1. Two Squirrels. 2. A Bird. 3. Shrub/Trees. 4. Ground Cover Plants. 5. Tall Grass.

Five Abiotic Observations:

1. Two Metal Light Poles. 2. Loose Garbage. 3. Sidewalk. 4. Mulch. 5. Soil.

Hay Infusion Observations:

Mirky dark water with transect components mixed all around. Mass of the overall hay infusion is 11.03 grams.

Very good start. Don't rewrite protocol try to put into own words. Could include some more detail. SK

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