User:Sydney Marshall/Notebook/Biology 210 at AU

February 16, 2014

Lab 3: Understanding The Characteristics Of Bacteria By Observing Antibiotic Resistance And Understanding How DNA Sequences Are Used To Identify Species

Introduction
This lab is titled Understanding The Characteristics Of Bacteria By Observing Antibiotic Resistance And Understanding How DNA Sequences Are Used To Identify Species. The purpose of this lab is to identify the morphology of the bacterial colonies and how these are affected by the antibiotic tetracycline. In order to understand the characteristics of bacteria colonies, wet mounts were prepared to be observed under a microscope, gram-staining of bacteria was done, and PCR preparation of DNA was done, which was a precursor next week’s lab which to purify these products of the PCR reaction. The hypothesis fro today’s experiment was that out bacteria will test negative for gram stating.
Materials and Methods

Procedure I: Observing and Counting Colony Growth on Nutrient Agar and Nutrient Agar + Tetracycline Plates

• Observe changes (such as smell or appearance) in the Hay Infusion Culture.
• Count the amount of colonies on each bacterial plate, record in Table 1 and multiply by their conversion factors to accurately determine how many colonies there are per mL.

Procedure II: Antibiotic Resistance

• Note differences between colonies in nutrient agar and nutrient agar + tetracycline plates
• Infer the effects of tetracycline on total number of bacteria and fungi.
• Determine if there are any species of bacteria that were unaffected by tetracycline.

Procedure III: Observations of Bacterial Cell Morphology and Gram-Staining

• Observe a prepared slide using 100x oil immersion and its three stained areas containing samples of bacillus, coccus, and spirillum.
• Take 4 samples from 4 seemingly distinct colonies from both the nutrient agar plates and nutrient agar + tetracycline plates.
• Prepare 2 wet mounts for each bacteria sample by putting a drop of water on a slide and scraping the bacteria on the sample and covering one group with a cover slip and another without.
  • Examine the covered slide under a microscope and identify the bacteria.
  • Gram Staining
    • Heat the slips on a Bunsen burner three times.
    • Cover the slips with crystal violet for 1 minute then rinse off with water.
    • Cover slips with Gram’s iodine for 1 minute then rinse off with water.
    • Immerse slips with 95% alcohol for 10-20 seconds and rinse gently with water.
    • Cover slips with safranin stain for 20-30 seconds and rinse off with water.
    • Dry slips with a paper towel and air dry.
    • Observe slips under a microscope and record observations.

Procedure IV: PCR Preparation for DNA sequence identification

• Transfer on colony of bacteria into a 100uL of water in a sterile tube.
• Incubate at 100 degrees Celsius and place in a centrifuge.
  • Place 5uL of the supernatant of the centrifuged liquid in the PCR reaction.

Observations and Data
Procedure I Description of Hay Infusion Culture

• Stronger smell
• Loss of water

Table 1: 100-fold Serial Dilutions

Dilution (Plate Label)	Agar	Colonies Counted	Conversion Factor	Colonies/mL
10^-3	nutrient	5000+	X10^3	5000000
10^-5	nutrient	240	X10^5	24000000
10^-7	nutrient	129	X10^7	1290000000
10^-9	nutrient	17	X10^9	1.7 X 10^10
10^-3	nutrient + tet	960	X10^3	960000
10^-5	nutrient + tet	268	X10^5	26800000
10^-7	nutrient + tet	29	X10^7	2900000000

Procedure 2-3
Description of Colonies

• Tetracycline plates
  • orange color
  • Bigger
  • uniform in size
• Nutrient Agar
  • Combination of cream and orange colored colonies
  • less uniform in size

Table 2:Description of Sampled Bacteria Colonies

Colony Label	Tet or NA	Colony Description, color, shape, texture, etc.	Cell Description, motility, shape, arrangement	Gram pos or neg
-5	NA	light yellow, smooth, small, clumped together	grouped together, circular, blue	negative
-7	Tet	orange, circular, smooth like saturated fat	rod and cocci observed, grouped together	negative
-9	NA	white, yellow, round	motile, not grouped together, round	negative

February, 9, 2014

Lab 2: Identifying Algae and Protists from a Hay Infusion Culture using a Dichotomous Key and Preparing and Plating Serial Dilutions

Introduction

The title of this lab is Identifying Algae and Protists from a Hay Infusion Culture using a Dichotomous Key and Preparing and Plating Serial Dilutions. The purpose of this lab was to understand the characteristics of Algae and Protists and utilize a dichotomous key to identify the species of such organisms. This procedure helped in determining the organisms observed from different niches in our hay infusion culture. The main hypothesis for this experiment is as follows: *If our transect consisted of a large variety of botanical the hay infusion culture is going to have a large variety of organisms.

Materials and Methods

Procedure I: Using a Dichotomous Key to Identify Organisms in Hay Culture Observations

• Carefully, bring the jar to your workstation and note its appearance and smell.
• Take 1-drop samples from two major niches in the jar (middle and bottom) using a transfer pipette and note specific locations of the samples.
• Observe the organisms on a microscope and sketch detailed pictures of what is seen in the slides while noting its size in micrometers.
• Characterize the different organism by determine the mobility, photosynthesizing ability, and similarities with the dichotomous key.

Procedure II: Using a Hay Infusion Culture to Prepare and Plate Serial Dilutions

• Take 4 tubes of 10mL sterile broth and label them 2, 4, 6, and 8.
• Obtain four nutiret agar plates and four agar plus tetracycline plates and label them 10-3, 10-5, 10-7, and 10-9.
• Using a micropipeter, obtain 100 microliters of hay infusion culture and place it in the tube 2 to make a 10^-2 dilution. Mix well.
• Obtain 100 microliters of the mix in tube 2, and place it in tube 4 to make a 10^-4 dilution. Mix well.
• Repeat for tubes 6 and 8 to make 10^-6 and 10^-8 dilutions, respectively.
• Take 100 microliters of mixed liquid in the 10^-2 tube, and carefully spread it on the surface of the nutrient agar plate labeled 10^-3.
• Repeat step 5 using tube 4 on the 10^-5 plate, tube 6 on the 10^-7 plate, and tube 8 on the 10^-9 plates.
• Incubate for a week at room temperature.

Observations and Data

Procedure I: Using a Dichotomous Key to Identify Organisms in Hay Culture Observations
Description of Hay Infusion Culture

• Smell: strong and musty
• Appearance: dirty, murky, moldy, has a layer of film on the surface of water

Figure 1: Two organisms found in middle and bottom of hay infusion culture, respectively.

Table 1: Descriptions of the two organisms found in hay infusion culture

'	(left)	(right)
Type of organism	Bacteria	Algae
Shape	Bacilli	Oval
Color	Black	Green
Size	2 micrometers	2 micrometers
Photosynthesic capabilities?	No	Yes

Figure 2: Nutrient Agar Plate Set-Up

Conclusion

In this lab, we were supposed to determine whether the diversity of botanical life corresponded to the amount to life in our hay infusion culture. I determined that since I was only able to identify two organism that my hypothesis was proved false. I interestingly found bacteria in addition to algae and thought it met all the needs of life because it is unicellular, acquires and uses energy by decomposition of organisms, processes and transfers genetic material by means of conjugation, capable of replication through colonies that will be observed next week, and is a product of evolution because it can evolve by being resistant to certain antibiotic strains.

If the hay infusion culture were to be observed for another few months, I would predict that more organisms could be easily identified. I managed to find bacteria as one of my organisms which is usually very tiny, and given time, more could have been found in the culture. Also, more mold could grow on the sides of the culture. Some selective pressures that affected the compositions of my samples were that my transect was in a very shady area surrounded by large trees. Therefore, there is a lot of moisture held in this area giving rise to diverse populations of algae. Even though I was only able to find one algae sample, it is possible that there were more types in other niches in my culture.

January 30, 2014

Lab 1: Observing the Evolutionary Specializations of Cells in the Volvocine Line and Determining the Characteristics of a Niche on Campus

Introduction

The title of this lab is Observing the Evolutionary Specializations of Cells in the Volvocine Line and Determining the Characteristics of a Niche on Campus. The purpose of this lab was to determine the evolutionary changes of cells within the Volvocine line, specifically from the Chlamydomonas, Gonium, and Volvox. The second procedure was to take a sample of our transect #2 in order to make a hay infusion culture for studying bacteria in the following week. The two hypotheses for each procedure this experiment are as follows:

• If the origin of the Volvocine line is Chlamydomonas, then the evolutionary progress of the following cells should exhibit more complexity.
• If the transect is in an area not frequently disturbed by human presence, then there will be a wide range of organisms to observe.

Materials and Methods

Procedure I: Observing the Evolutionary Changes in Cells within the Volvocine Line

• Prepare slides of Chlamydomonas, Gonium, and Volvox using a light microscope.
• Analyze the evolutionary specializations such as number of cells, colony size, relations of structure to function, and reproductive specializations.

Procedure II: Observing Characteristics of a Transect on Campus

• Go to a 20x20 foot transect near campus and describe general characteristics such as location and land profile.
• Determine the biotic and abiotic components of of the transect.
• Take a soil sample from the transect that is an accurate representation of the transect as a whole.
• Create a hay infusion culture using the retrieved soil sample.
  • Put sample in a jar with 500ml of deerpark water.
  • Add .1 grams of dried milk in the jar and label it for next week's use.

Observations and Data

Procedure I: Observing the Evolutionary Changes in Cells within the Volvocine Line

Table 1: Evolutionary Specialization of Members of the Volvocine

Characteristic	'	Chlamydomonas	Gonium	Volvox
Number of Cells		13	14	10000+
Colony Size		1μm	4μm	5μm
Describe any functional specialization of cells		motility (flagella)	form colonies with similar cells	motile flagellate cells, form spherical colonies made of glycoproteins
Describe any reproductive specialization (isogamy vs oogamy)		isogamy of motile cells fusing together to make a gamete	isogamy - cells can function as gametes and fuse together to make a zygote	oogamy - female is nonmotile and males are motile

Figure 1: Sketches of Three Organisms within the Volvocine Line

Figure 1: Sketches of Three Organisms within the Volvocine Line

Procedure II: Observing Characteristics of a Transect on Campus
General Characteristics:

• Located Behind Cassell Hall and in front of Wesley Theological Seminary
• Shaded under flowering and non-flowering organisms
• Abiotic components:
  • Holly
  • Ivy
  • American Sycamore
  • Pine Tree
• Biotic components:
  • Aluminum Foil
  • Soil

Conclusions

Relating back to the purpose of the lab, our group was to determine whether there was significant evoltionionary change in the original Chlamydomonas algae based on its predecessors, the Gonium and Volvox. According to Table 1, the number in cells in each slide increased, as the Chlamydomonas algae evolved over time. Also, the colony size increased from 1um to 5um. The most significant differences were the structures, starting as individual motile cells to spherical motile colonies made from glycoproteins. In addition, the reproductive specialization changed from isogamy, the fusion of cells to make gametes to oogamy, with the female gamete being nonmotile and the male sperm motile. Therefore, the original hypothesis is confirmed by this increase in complex progression over time. A sample sketch of what was seen in the slide can be found in Figure 1.

Based on the transect observations, it was to be determined whether or not a lack of human presence within the transect indicated a large amount of organisms. Our group found that there were a variety of biotic factors found such as Ivy, holly, pine, and sycamore. Only one unique abiotic component, other than soil, was found, which was aluminum foil, and I assumed that because of this, this transect was not commonly disturbed by human company. Although this information is not enough to determine whether the transect is a place containing a large variety of organisms, I conclude that my data does not support my hypothesis because visually, our group could not find organisms other than botanical organisms . However, I believe that in next week’s lab, I will be able to look at a microscopic view of the organisms that the transect can contain, possibly supporting or refuting my hypothesis. Because of this lack of information in the experiment, I would maintain my hypothesis for the next experiment to determine whether or not there is a large variety of bacteria and protists, In addition, it was hard to see the transect in the dark, therefore had there been an adequate amount of light, there would have been other organisms that our group could have observed.

Excellent Lab 1 entry. Very thorough, well explained and organized. SK

January 22, 2014

Test.