User:Amanda Zimmerman/Notebook/Biology 210 at AU

Name: Amanda Zimmerman, Group 5, The Prairie

February 3rd, 2014 Date observed: January 30th, 2014

Goals/objectives: To understand the characteristics of bacteria, to observe antibiotic resistance, and to understand how DNA sequences are used to identify species

Procedures:

• -Procedure 1: Quantifying and Observing Microorganisms
• 1) The Hay infusion culture was obtained and noted in terms of differences in smell or appearance.
• 2) A handout was obtained regarding how to characterize colony morphology
• 3) The total number of colonies on each plate were counted and recorded
• -Procedure 2: Antibiotic Resistance
• 1) Observe the colonies that grew on the tetracycline plates
• -Procedure 3: Bacteria Cell Morphology Observations
• 1) A prepared slide containing different types and shapes of bacteria were obtained and observed in order to get a base idea of the difference morphologies with both the 4x, 10x, and 40x
• 2) A small drop of oil was placed on the slide, then the 100x oil immersion lens was used to observe the slide
• 3) Two samples from the nutrient agar plate and one from the nutrient agar plus get plate were isolated to be used in a wet mount and gram stain
• 4) Each sample was scraped onto a slide with a drop of water then sterilized over an open flame then covered with a cover slip and observed under the microscope with a 10x and 40x objective lens and the cell shapes and motility noted
• 5) A Gram stain was prepared for each sample as follows:
• Each slide was labeled
• The slide was heat-fixed over a bunsen burner with the bacterial side up
• Crystal violet dye was used to cover the stain for 1 minute
• The crystal violet stain was rinsed off with water
• Gram's iodine mordant was used to cover the stain for 1 minute then rinsed off gently
• 95% alcohol was used to decolorize the slides for 10-20 seconds
• The slides were rinsed gently until the slides were completely decolorized
• Safranin stain was used to cover the stain for 20-30 seconds
• The slides were rinsed gently
• Excess water was blotted carefully with a wet paper towel
• 6) The slides were observed under a 4x, then 10x, then 40x, and finally the 100x oil immersion lens without a cover slip
• -Procedure 4: Start PCR Preparation for DNA Sequencing Identification
• 1) DNA was isolated from the bacterial colonies on the same plates as used earlier in the experiment
• 2) Each colony was transferred into separate 100 microliters of water in a sterile tube
• 3) Each tube was incubated at 100 degrees Celsius for 10 minutes
• 4) Each tube was centrifuged for 1 minute
• 5) 5 micro liters of the supernatant for each tube was placed in its own tube consisting of 27F and 519R primer sequences to locate the 16S rRNA gene presence
• 6) The tubes were saved for a PCR reaction to be done in one week

Observations:

• -Observations of quantifying and observing microorganisms:
• I don't think that archaea would have grown on the nutrient agar plates or the nutrient agar plus tet plates because the living conditions of archaea do not match those of the agar plates' environments
• The smell and change of the Hay infusion may have changed due to the lack of sunlight and over abundance of water, causing deterioration of the grass, flower, species growing off of the flower, and film on top. The deterioration may have released chemicals as it reacted, inducing an odor
• Table 1: 100-fold Serial dilutions results:

(ENTER TABLE 1 RESULTS)

• -Observations of antibiotic resistance:
• There are obvious, apparent differences in the plates with and without tetracycline, the antibiotic used in this procedure
• In the tetracycline plates, the colonies are more distinct. Additionally, the tetracycline had several golden-orange colonies that were never found in the plates without tetracycline. This resembles a different type of bacteria presence. To the naked eye, without any specialized technology, there appears to be two types of bacteria present on the tet plates- the golden-orange colonies and white-grey colonies. On the other hand, there is only one on type of bacteria present- the grey-white colonies- present on the nutrient agar plates. However, this determination was made based on our perception of the color and shape of bacteria. I think the presence of this extra golden-orange bacteria presence is an immune-type response of the bacteria. There seemed to be no fungi present on any of the plates, but it is possible that fungi were mistaken to be bacteria on both the nutrient agar and nutrient agar plus tet plates. However, overall, the get plates had a fewer number of bacterial colonies, due to the fact that get is an antibiotic and is used to kill bacteria.
• Only the 10^-3 and 10^-5 plates of nutrient agar plus tet had bacterial growth present. I believe this is because these are the more concentrated bacterial plates, so there bacteria were greater in number and strength, thus allowing them to flourish.
• According to http://mmbr.asm.org/content/65/2/232.full, tetracyclines prevent the association of aminoacyl-tRNAs with bacterial ribosomes. This association is an incredibly crucial step of protein synthesis, hence tetracycline is used to prevent bacteria from creating proteins, which are needed for the bacteria to survive and reproduce. Tetracycline has been shown to kill many bacteria, a few of which include chlamydia, Rickettsia, and Mycoplasma.
• -Observations of bacterial cell morphology:
• Table 2: Information regarding the colonies' description, amount, cell description, and gram identification

(ENTER TABLE 2 RESULTS) (DESCRIBE TABLE 2 WITH WORDS)

Conclusions: There was bacterial growth only on the 10^-3 and 10^-5 plates of both the nutrient agar and nutrient agar plus tet. Any lower dilution did not produce visible bacterial growth. This, I believe, is because the nutrient dilution was too low to allow the bacteria to strengthen and reproduce. Additionally, the bacteria couldn't survive on the 10^-7 nutrient agar plus tet plate because the get was too strong for the bacteria to fight. I believe that both our 10^-3 plates of nutrient agar and nutrient agar plus tet were infected with a Gram positive bacteria that colonizes in the form of streptobacilli. We think our 10^-5 plate of nutrient agar, on the other hand, were infected by Gram negative streptococcus. I'm not sure how the results varied between plates; I suspect an error was made in either spreading the bacteria or some kind of cross-contamination happened. Therefore, there are bacteria present that colonize in the form of streptobacilli and are resistant to tetracycline. (FINISH)

January 25th, 2014 Date observed: January 23rd, 2014

Goals/objectives: To understand how to use a dichotomous key and to understand the characteristics of Algae and Protists

Procedures:

• -Procedure 1: Hay infusion culture continued
• 1) The culture, without being disturbed, was brought to our group's lab space so observations of the physical appearance could be made
• 2) A sample from the bottom, middle, and top layer were all drawn and made into a wet slide to be observed under a microscope. It is important to note where the organisms are found in order to help identify their nature. Some may be near or far from the plant matter based on how much they need the plant's nutrients
• 3) The organisms found under the microscope were then described in terms of color, mobility, size, etc. and characterized with the help of a Dichotomous key
• -Procedure 2: Preparing and Plating Serial Dilutions
• 1) Four tubes of 10 mL sterile broth were obtained and labeled 10^-2, 10^-4, 10^-6, and 10^-8. A 100 mL micropipeter set and tips were also obtained
• 2) 4 nutrient agar plates were obtained and labeled 10^-3, 10^-5, 10^-7, and 10^-9 with the date and group initials
• 3) 3 agar plus tetracycline plates were obtained (labeled "T") and labeled 10^-3, 10^-5, and 10^-7 with the date and group initials
• 4) The top was then placed on the Hay infusion culture and mixed until the solution was homogenous
• 5) 100 micro liters of the mix were taken from the Hay infusion culture and added to the 10^-2 tube, then inverted several times
• 6) 100 micro liters of the mix were taken from the 10^-2 tube and added into the 10^-4 tube, then inverted several times
• 7) This was then repeated into the 10^-6 then 10^-8 tubes to make the respective dilutions
• 8) 100 micro liters of the mix from the 10^-2 tube were added to the 10^-3 plates of both the nutrient agar and agar plus tetracycline
• 9) The sample was then spread around the plate
• 10) Steps 8 and 9 were then repeated with the 10^-4 tube onto the 10^-5 plates of both the nutrient agar and agar plus tetracycline, and the 10^-6 tube onto the 10^-7 plates of both the nutrient agar and agar plus tetracycline
• 11) Steps 8 and 9 were then repeated with the 10^-8 tube onto the remaining 10^-9 plate of nutrient agar only
• 12) The plates were then incubated at room temperature for 1 week

Observations:

• -Observations of the transect: The transect is covered in several inches of snow from the storm on January 21st-22nd. There are several tracks of foot prints, both human and animal, throughout the transect.
• -Observations of last week's Hay infusion culture:
• Top layer: Brown leaf is floating on top, part of the flower bud is seen in the top layer, the top has turned into a gray-brown opaque film, there are few areas of transparency within the film, no apparent life is on the top unless the film is some sort of fungus
• Middle layer: Composed of an amber-brown transparent liquid, most of the flower bud is in this layer, the piece of grass is submerged in this layer
• Bottom later:Composed of a solid state, soil has all sunk down to the bottom, the wood chip is also on the bottom, dark brown in color
• Other observations: The piece of grass is still green with no apparent loss of pigment, the flower bud has a white spiderweb-like growth hanging off of it, and there is no strong abhorrent smell present.
• If the Hay infusion culture had sat undisturbed for another two months, I would expect the piece of grass to be completely dead, due to the lack of nutrients needed for photosynthesis. Additionally, I would expect the flower bud to have disintegrated, due to the harsh conditions of the culture. I would expect the whole culture to be a musty brown-black concoction, too harsh for anything to survive in.
• The selective pressures of the Hay infusion culture include both the soil's nutrients for growth and survival and also the competition between light and water in overwhelming or saving the plant life. These things affected the composition and livelihood of our organisms.
• -Observations of observed organisms under the microscope of the top layer with possible characterizations:

1) Algae

• Large, green masses
• Multicellular
• Immobile

2) Paramecium Voritcella

• 2.5 ocular spaces on 10x = 25 micrometers
• Clear/colorless
• Very small
• Fast swimmer
• Elongated body
• Moves in a corkscrew fashion

3) Chlamydomonas

• 1 ocular space on 10x= 10 micrometers
• Appears to be a slight green pigment
• Exhibits strange motions
• Appears to be moved by flagella
• Single cell
• Oval shaped

4) Colpidium sp.

• 20 ocular spaces on 40 x= 50 micrometers
• Clear/colorless
• Relatively small
• Oval shaped
• Fast swimmer
• Entirely covered with cilia
• Makes fast/rapid motions

Calculations:

• -Microscope calculations:
• 4x calculations: # ocular spaces x 25 = # of micrometers (not used in this lab)
• 10x calculations: # of ocular spaces x 10 = # of micrometers (ex: Paramecium vorticella= 2.5 ocular spaces on 10x--> 2.5 x 10= 25 micrometers)
• 40x calculations: # of ocular spaces x 2.5 = # of micrometers (ex: Colpidium sp.= 20 ocular spaces on 40x--> 20 x 2.5= 50 micrometers)
• -Serial dilution calculations:
• Started with 100 microliters of Hay infusion in 10 mLs of sterile broth= 1/100= 10^-2
• Took 100 microliters of 10^-2 broth and put in 10 mLs of sterile broth= .01/10 = 10^-4
• Repeated this procedure to make the 10^-6 and 10^-8 solutions
• Took 10^-2 broth and plated on a nutrient agar and nutrient agar plus tet plate with 100 microliters already placed= .01/1= 10^-3 plates
• Repeated with 10^-4 to make 10^-5; repeated with 10^-6 to make 10^-7; repeated with 10^-8 to make 10^-9 dilutions

Extra notes:

• -One of the species that demonstrates the needs of life is the paramecium:
• 1) Energy: Paramecium consume bacteria and algae for food, which it then converts into energy to survive
• 2) Cells: Paramecium are composed of membrane-bound cells
• 3) Information: Paramecium have DNA, the information that encodes its life
• 4) Replication: Paramecium reproduce asexually during binary fission, in which they replicate their DNA and pass it into their genetically-idential offspring
• 5) Evolution: Paramecium evolved from their first seen structure in order to feed and protect themselves
• All information from: http://en.wikipedia.org/wiki/Paramecium

Photos: To come of both the jar and diagram of the Hay infusion culture dilutions

January 16th, 2014 Date observed: January 16th, 2014

Goal/objective: Find the designated transect and observe it. List the physical description, biotic, and abiotic factors of the transect. Also, create a Hay infusion culture to be evaluated in one week.

• Biotic factors: Grass, leaves, soil, flowers, and bushes
• Abiotic factors: Fertilizer, concrete bench, metal lining separating the soil from the walkway, "Freidheim Quadrangle" sign, and walkway stones
• Description of area: This 20' by 20' square is located on the Quad in front of Hurst Hall. It's composed primarily of a spacious grass area. There is an arc of soil, including shrubbery and a portion of the "Freidheim quadrangle" sign. Between the soil area and the walkway is a metal lining. There are very few leaves or flowers left on the shrubbery, due to the extreme wintery conditions. Within that arc is another, smaller arc consisting of a stone walkway and concrete bench.

Procedure: Hay infusion culture

• 1) 10-12 grams of our sample was weighed and placed in a labeled plastic jar with 500 mLs of Deerpark water
• 2) 0.1 gram of dried milk was added and gently mixed up for about 10 seconds
• 3) The top of the jar was then removed and the rest of the jar was placed on a lab table with other jar

Conclusions: There isn't anything that I would change for "next time" per se, but I think it would be interesting if we made two Hay infusions and put them in different environments with varying light exposure, temperature, or nutrients and compared later. The abiotic and biotic factors will play more of a role when the weather gets nicer. I expect to see the leaves and flowers budding within a few months. Also, I'm interested in seeing how the nicer weather attracts people to the transect and how they will play a role on the transect's being.

Photos: (To come)