14: Larrea Tridentata Preliminary Results

Me getting my silly faces out before the presentation

Preliminary Results: Leaf Microbiome of Larrea tridentata in Urban Phoenix Versus Rural Phoenix

This week, I focused on preparing to present my preliminary results to the S-STEM Team. During the presentation, I focused on summarizing the project focus and initial results. A summary of the results can be found below:

A total of 25 colony samples were isolated, however only 24 colony samples were analyzed due to a testing error with the colony sample from the Phoenix urban area (See Table 1). Initial results showed that roughly one third of the unknown samples have a Bacillus cell morphology, 29.2% have Cocci cell morphology, and 37.5% of the samples have a Coccobacilli cell morphology. Roughly 90% of the samples tested positive for endospores. Seventy-one percent of the samples tested positive for acid production in glucose, 54.2 % of the samples tested positive for acid production in Triple Sugar Iron Agar media, and over half of the samples tested positive for acid production in Mannitol Salt Agar media.. Approximately 75.0% percent of the samples were able to grow in 42 degrees Celsius. All the samples tested positive for gram stains and negative for Simmons Citrate. Nearly half of the samples tested positive for aerobic growth. 

13: Larrea Tridentata Metabolic Test Completion

Metabolic Tests

Larrea Tridentata Metabolic Test Completion

I feel like the last two weeks have been a race against time. My goal is to finish testing and analyzing data for the remaining culture samples before our final paper is due. I finally completed all the metabolic and morphological tests on all 25 culture samples. It's definitely been a challenge to try and finish this goal, because for each culture sample, 13 different tests were needed to be completed. In total, that was 325 tests that had to be completed. I used an excel spreadsheet to keep track of all the tests ran and the results. I will focus on presenting the information collected thus far on the 25 culture samples. Next semester, I will focus on identifying the bacteria species of the 25 culture samples. 

12: Larrea Tridentata Research Paper in the Works!

Larrea Tridentata Research Paper in the Works!

This week I focused primarily on writing my rough draft of the research paper which focuses on the Larrea Tridentata leaf microbiome (see Blog 8). Later in the week, I ran an Endosphore Stain test on nine culture samples from the leaflet that was taken from the White Tank Mountains. I plan to analyze the Endospore Stains tomorrow. Additionally, I ran the Fluid Thioglycollate Media tests and they are currently in the incubator.  Once those results are recorded, I plan to start running the morphological and metabolic tests on the other 14 culture samples. My goal is to finish testing and analyzing data for the remaining culture samples before our final paper is due.

11: Creosote Bacteria Identification for White Tank Mountain Samples Continued

Gram positive result from Plate 1 Colony 6

Continued:Creosote Bacteria Identification for "White Tank Mountain Samples"

The hunt for the unknown bacteria identification continues! This week I came closer to identifying a portion of the 23 unknown bacteria samples. I am currently focusing on Plate 1 (Colonies 1-7) and am performing a range of tests. I was able to identify the cell morphologies and cell wall types. Of the samples tested, they all are gram positive. I then compared the gram stain results with results from the MacConkey tests I performed on the "Plate 1" samples. The MacConkey tests confirmed that the samples are gram positive. Additionally, I ran Glucose Fermentation tests, Trypone Tests, and Sims Citrate tests on the samples and will document the results when they come out of the room temperature incubator after the weekend. I plan to continue to run the remainder of tests and indicate the results on an excel file that I have created.

10: Larrea Tridentata (Creosote) Project Background

http://www.sci.sdsu.edu/plants/sdpls/plants/Larrea_tridentata.html

Leaf Microbiome of Larrea tridentata Grown in Urban Phoenix Versus Grown in Rural Phoenix

Project Background
Research has shown rising levels of CO2 can alter the chemical composition of leaves in Larrea tridentata. Additionally, the nutrients in the soil can be altered (Wang 2011). Research has also indicated that environmental conditions and changes can influence adaptive traits of plant function over time (Kimball et. al 2012). It is imperative to learn more about the differences in the leaf microbiome of Larrea tridentata in urban and rural areas to gain understanding of how increasing urbanization can impact microbiome development and sustainability over time.
It is imperative to expand on current research of the Larrea tridentata leaves due to continuous changes in environmental conditions and the need to further understand adaptive traits of the leaf microbiome. In the study, "Urbanization alters spatiotemporal patterns of ecosystem primary production: A case study of the Phoenix metropolitan region, USA," research showed that urbanization affects the function of an ecosystem and its surrounding environments. The study also revealed that urbanization affects the net primary production of chemical energy (Buyantuyev and Wu 2009). Additionally, the study "Temporal patterns in near-surface CO2 concentrations over contrasting vegetation types in the Phoenix metropolitan area" addresses the need to study surface levels of CO2 in urban areas and its effect on plant life (Day, et al 2002).
Furthermore, some research suggests that Larrea tridentata possesses antibacterial properties in the aerial parts of the plant which can combat the increase in bacterial infections that are drug-resistant. Currently, Larrea tridentata has antibacterial properties in its leaves and understanding differences in leaf microbiomes in urban and rural areas may provide insight on antibacterial agents and their property types in different environments (urban and rural). In the recent study, "Antibacterial activity of crude methanolic extract and fractions obtained from Larrea tridentata leaves," researchers found that phytochemicals from L. tridentata leaves may aid in the development of natural antibacterial treatment (Martins 2013). The study also indicated the need to further research the hypothesis. By testing the differences in the leaf microbiome of the Larrea tridentata in urban and rural environments, it can aid in a future understanding of how urban and rural environment affects antibacterial properties of the Larrea tridentata aerial parts.

Key Terms
Aerial parts- parts of a plant that are exposed to air (i.e. leafs, stems, roots).
Net primary production- amount of chemical energy created by produces over a period of time.

Research question
Is there a difference in the leaf microbiome of the Larrea tridentata species in the Phoenix urban area and surrounding desert rural area?

Hypothesis
Hypothesis 1: The leaf microbiome of Larrea tridentata found in the urban Phoenix area is detectably different than the leaf microbiome of Larrea tridentata found in the Phoenix rural areas.
Hypothesis 2: As a result of urban area heat islands and a greater concentration of CO2 emitting vehicles, it is hypothesized that elevated CO2 and pollution will result in different microbial communities of Larrea tridentata growing in urban areas.

References:

A. Buyantuyev and J. Wu. "Urbanization alters spatiotemporal patterns of ecosystem primary production: A case study of the Phoenix metropolitan region, USA." Journal of Arid Environments, vol. 73, 2009, p. 512-520. Retrieved from https://ac.els-cdn.com/S0140196308003649/1-s2.0-S0140196308003649-main.pdf?_tid=98c8d5e0-c052-11e7-97a5-00000aab0f01&acdnat=1509684844_197eb558cc325b63cd9a6d7f80089f02

Kimball, Sarah, et al. "Fitness and physiology in a variable environment." Oecologia, vol. 169, no. 2, 2012, p. 319+. Academic OneFile,  go.galegroup.com/ps/i.do?p=AONE&sw=w&u=mcc_main&v=2.1&id=GALE%7CA356445949&it=r&asid=76cf9b7c721d99e2e39e481da1419f8d. Accessed 20 Oct. 2017.  

K.L. Neil et al. / Journal of Arid Environments 74 (2010) 440–444. https://ac.els-cdn.com/S0140196309003231/1-s2.0-S0140196309003231-main.pdf?_tid=ec4d747e-b54d-11e7-80e7-00000aab0f6c&acdnat=1508473374_2658d691676b7ce943e2833f5216b961.

Maki Jitsuno and Yoshihiro Mimaki. "Triterpene glycosides from the aerial parts of Larrea tridentata." Phytochemistry, vol. 71 2010, p. 2157–2167. Retrieved from https://ac.els-cdn.com/S0031942210003687/1-s2.0-S0031942210003687-main.pdf?_tid=82d1fcb8-c060-11e7-96af-00000aacb361&acdnat=1509690820_e82d8b4df86e7520365baba41e5ebc45

Martins,Silvia et al. "Antibacterial activity of crude methanolic extract and fractions obtained from Larrea tridentata leaves." Industrial Crops and Products, vol. 41, 2013, Pages 306-311. Retrieved from https://doi.org/10.1016/j.indcrop.2012.04.037.

Schultz, J. C., & Floyd, T. (1999, February). Desert Survivor. Natural History, 108(1), 24. Retrieved from http://link.galegroup.com.libproxy.maricopa.edu/apps/doc/A53682802/WHIC?u=mcc_main&xid=8b7d7592.

Shen, W., Wu, J., Grimm, N.B. et al. Ecosystems (2008) 11: 138. https://doi.org/10.1007/s10021-007-9085-0.

Thomas A Day, et al. "Temporal patterns in near-surface CO2 concentrations over contrasting vegetation types in the Phoenix metropolitan area." Agricultural and Forest Meteorology, vol 110, Issue 3, 2002, p. 229-245. Retrieved from http://www.sciencedirect.com.libproxy.maricopa.edu/science/article/pii/S016819230100288X#aep-section-id24

Wang, D. (2011). A meta-analysis of plant physiological and growth responses to temperature and elevated CO2. Oecologia, 169(1), 1-13. Retrieved October 19, 2017, from http://hh2wl8hm4u.search.serialssolutions.com/?ctx_ver=Z39.88-2004&ctx_enc=info%3Aofi%2Fenc%3AUTF-8&rfr_id=info%3Asid%2Fsummon.serialssolutions.com&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=A meta-analysis of plant physiological and growth responses to temperature and elevated CO2&rft.jtitle=Oecologia&rft.au=Wang%2C Dan&rft.au=Heckathorn%2C Scott A&rft.au=Wang%2C Xianzhong&rft.au=Philpott%2C Stacy M&rft.date=2012-05-01&rft.pub=Springer-Verlag&rft.issn=0029-8549&rft.eissn=1432-1939&rft.volume=169&rft.issue=1&rft.spage=1&rft.epage=13&rft_id=info:doi/10.1007%2Fs00442-011-2172-0&rft.externalDBID=n%2Fa&rft.externalDocID=2012_442_169_1_2172mdict=en-US.

Weijun Shen, et al. "Effects of Urbanization-Induced Environmental Changes on Ecosystem Functioning in the Phoenix Metropolitan Region, USA." Ecosystems (2008) vol. 11, 2008, p. 138–155. Retrieved from http://leml.la.asu.edu/jingle/Wu-Publications-PDFs/2008/Shen_etal-2008-Ecosystems.pdf

9: Creosote Bacteria Identification Continued

Continued:Creosote Bacteria Identification for "PC Sample" and "White Tank Mountain Samples"

This week I continued to perform two more tests on the creosote sample that was taken on the Phoenix College campus. The first test I performed was  Mannitol Salt Agar (MSA) which is used to identify if a bacteria can tolerate a high saline concentration. The second test performed this week was called  MacConkey Agar (MAC) which is selective for gram-negative bacteria. The inoculated plates were placed in the incubator at 37 degrees Celsius and results will be recorded by the end of the school week.
Additionally, the creosote plate samples from the White Tank Mountain area were continued to be further isolated with the goal of obtaining pure culture samples.

8: Larrea Tridentata (Creosote) Research Proposal

Retrieved from: Sonora-desert-detective

Leaf Microbiome of Larrea tridentata Grown in Urban Phoenix Versus 
Grown in Rural Phoenix

Background Information

Larrea tridentata is a species of plant common to the Mojave, Sonoran, and Chihuahan deserts. Larrea Tridentata’s unique properties allow for its shrubs not only to grow deep roots but spread out across more surface area in the which allows the shrub to dominate absorption of nutrients in the soil. Due to the chemical durability of the Larrea Tridentata species, many other organisms have adopted the Larrea Tridentata as their host environment (Schultz 1999). However, research has shown rising levels of CO2 can alter the chemical composition of leaves in Larrea tridentata. Additionally,the nutrients in the soil can be altered. (Wang 2011). Research has also indicated that environmental conditions and changes can influence adaptive traits of plant function over time (Kimball et. al 2012). It is imperative to learn more about the differences in the leaf microbiome of Larrea tridentata in urban and rural areas to gain understanding of how increasing urbanization can impact microbiome development and sustainability over time.

Research question

Is there a difference in the leaf microbiome of the Larrea tridentata species in the Phoenix urban area and surrounding desert rural area?

Hypotheses

Hypothesis 1:

The leaf microbiome of Larrea tridentata found in the urban Phoenix area is detectably different than the leaf microbiome of Larrea tridentata found in the Phoenix rural areas.

Hypothesis 2:

As a result of urban area heat islands and a greater concentration of CO2 emitting vehicles, it is hypothesized that elevated CO2 and pollution will result in different microbial communities of Larrea tridentata growing in urban areas.

7: Creosote Bacteria Identification from White Tank Mountain Area

Creosote Bacteria Identification from White Tank Mountain Area

This week was focused on completing more bacteria-identification tests on the leaf sample collected from a creosote bush here on Phoenix College's campus. The second focus of this week was to isolate bacteria samples taken from two creosote bushes located in the White Tank Mountain Area (Sample Extraction Coordinates:33⁰ 34’ 31’’ N, 112⁰ 34’ 44’’ W.

The samples obtained included taking swabs from a creosote bush's leaves and streaking them onto three TSA plates (Sample ID Label = Plate 1 C1-C7; Plate 2 C1-C6; and Plate 3 C1-C8). From an additional creosote bush, three vials of leaf samples were collected and are in the process of being streaked on TSA plates. 
Plates 1-3 were taken and observed for different colony types. Next isolation streaks on TSA were performed for each colony type observed. A total of 21 colonies were observed and streaked on TSA. After being incubated at 37 degrees Celsius, the TSA plate samples were observed for colony growth, and it was determined that the TSA plate samples for the 21 colonies should be further isolated with the goal of obtaining pure cultures. Currently, the second round of isolating the colonies is in process. Below are pictures of the three plate samples collected from the one of the creosote bushes in the White Tank Mountain area. 
     

6: Cold Case of the Flu

Cold Case of the Flu
This past week, the identification of the unknown bacteria found on the Creosote sample 1 "PC Sample" was put on hold because I came down with the Flu. Three reasons why I would have rather contracted some other virus or sprained a body part in lieu of getting the Flu:
1. Vomit makes me cry (there was a lot of vomit associated with my Flu).
2. I had the worst headache of my life, I thought I was going to die.
3. It took me like nine days to start to feel better.
It's been nine days since I came down with the flu, I still feel nauseous and have an upset stomach. Nonetheless, I am glad to be back at school and working on my S-Stem project.

5: Creosote Bacteria Identification Continued

                                                                  

Gelatin Test (Pre-incubation)

Creosote Bacteria Identification Continued
This week was focused on completing a serious of tests to gather information which will aid in the identification of the unknown bacteria sample collected from a creosote bush here on Phoenix College's campus. The test performed this week: Simmon's Citrate, Growth at 42 degrees Celsius, TSI, Gelatin, FTM, Glucose Fermentation, and Triptone.  The tests completed were the Glucose Fermentation Test and the Growth at 42 degrees Celsius. The unknown sample was negative for glucose fermentation and positive for growth at 42 degrees Celsius. I have created an excel spreadsheet to keep track of tests that were completed and their results (See table below). 

4: Adventure of the Creosote

Positive gram stain and Cocci dipole cell morphology

Adventure of the Creosote (Larrea tridentata)
Fun fact: In Mexican culture, the Creosote is known as "The Governess."Another fun fact: The Creosote is not the most visually appealing plant to look at, but it is partially responsible for contributing to that wonderful "desert rain smell."
For the remainder of the semester, I will be working on a project that focuses on learning about the various types of bacteria present on Creosote bushes (Larrea tridentata) in urban and non-urban environments; convenience sampling was used as a result of limited access to creosote bushes. After a leaf sample was collected from a Creosote bush on the Phoenix College campus, the process of identifying microbes had begun. First, the leaf sample was mixed with Peptone water and vortexed for 20 seconds. After, I used the aseptic technique to inoculate a TSA plate with an isolation streak. Next, the TSA sample incubated for approximately twenty hours. Individual off-white, punctiform colonies were present and a Gram Stain test was performed; the bacteria tested positive for the gram stain and consisted of a cocci dipole cell morphology. The Catalase test was also positive for the catalase enzyme. After, a Glucose Fermentation test was also performed on the sample and is currently in the incubator.  Next steps for the sample are recording the results for the Glucose Fermentation test and following the next tests outlined in the identification guide.

3: Endospore Stain, Glucose Fermentation, and MSA Tests of Unknown Bacteria

Endospore Stain, Glucose Fermentation, and MSA Tests of Unknown Bacteria
Throughout my experimental process of identifying an unknown bacteria, I have so far observed that my unknown bacteria has a positive cell wall and bacillus cell morphology. To further test my unknown bacteria in hopes of identifying the Genus species, I performed an Endospore Stain, followed by a Glucose Fermentation test when the unknown bacteria tested positive for endospores. After the Glucose Fermentation test revealed that the unknown bacteria had a pH level< 7, I performed a MSA (Mannitol Salt Agar) test. The MSA test was positive. Using the Identification Flow Chart, I was able to identify the unknown bacteria as Bacillus subtilis. See below for Experimental Procedures and Results.






Smear Preparation and Endospore Stain Method Procedure:
1. Prepared two slides by drawing a nickel-sized circle on each slide and labeled slides “1” and
“2”.
2. Used micropipette to place one drop of water within each circle of the slides.
3. Placed inoculating loop in bacti-cinerator for 5 seconds (used stopwatch to keep time).
4. After, used the sterilized inoculating loop and aseptic technique to obtain a sample of the
    unknown bacteria and smeared it into the water droplets of each slide.
5. Let bacteria smear dry on each slide.
6. After each bacteria smear dried, they were heat-fixed for 5 seconds each (used stopwatch to
    keep time).
7. Brought beaker full of water to a boil using hot plate.
8. Placed slide 1 over beaker of boiling water using rack tool and covered the slide with a
    paper towel.
9. Soaked the paper towel in Malachite Green solution and timed it for five minutes using
    stopwatch. Added more Malachite Green solution as needed when paper towel dried.
10. After five minutes, rinsed slide for 40seconds with water.
11. Added Safranin Stain and timed it for 75 seconds. Blotted dry with bibulous paper.
12. Repeated steps 8-11 for slide 2.
13. Prepared both slides for oil immersion under microscope.
14. Placed coverslip on slides and focus objective to 40x lens.
15. Rotated 40x lens halfway to 100x lens and placed one drop of immersion oil on each slide.
16. Viewed slides 1 and 2 through 10x and 40x lens.
17. Unknown bacteria was positive for endospores.
Experiment errors:
Initially placed wrong coverslip on slides.
Timing for second slide was 30 seconds instead of 40.
Could not focus to 100x lens.
Glucose Test Fermentation Procedure:
1. Used Aseptic Technique and took inoculation loop and retrieved sample of unknown bacteria.
2. With the inoculating tool, placed sample of unknown bacteria in test tube.
3. Incubated test tube at 37°C for approximately 16 hours.
Results: The Glucose Fermentation test revealed a yellow liquid color indicating a positive result for a pH <7.
MSA Test Procedure:
1. Used Aseptic Technique and took inoculation loop and performed isolation streak of the microbe onto the surface of the MSA plate.
4. Incubated MSA plate at 37°C.
5. Left inoculated MSA plate in incubator for approximately 16 hours.
Results: The isolation streak revealed a yellow color indicating a positive result

2: The Streak Plate and Gram Staining Processes

Lawn culture  (Left) Iso Streak (Right)
YAY SCIENCE!!!!

The Streak Plate and Gram Staining Processes
This past week was filled with lots of learning through trial and error. At the beginning of the week, I was tasked with identifying an unknown bacteria. In preparation for this challenge, I learned about the process of streak plating bacteria using two techniques, the Streak Plate method and the Lawn Culture technique. After isolating the unknown bacteria and allowing them to incubate for a few days, I learned about the Gram Staining process which would aid in the identification of the unknown bacteria. After multiple attempts of gram staining the unknown bacteria, it was identified as having a gram positive cell wall. I believe I was having challenges with the gram staining process for three possible reasons: I didn't allow my unknown bacteria to dry fully on the slide, I used too much de-colorizer, and/or my bacteria spread was too thick. With this processes I was able to figure out that my unknown bacteria is gram positive and has a rod-like cell morphology. Next week, I will continue the process of identifying my unknown bacteria by performing an Endospore stain to see if the bacteria contains any spores.

1: The Start of My S-STEM Adventure

The Start of My S-STEM Adventure
This week marked the kick-off of my S-Stem internship. I focused on learning about safety protocols in the laboratory and research ethics via online tutorials. The safety protocol videos covered topics such as: hazardous chemical storage and organization, chemical spill clean-up procedures, and the emergency evacuation process. The online ethics tutorials reviewed information over research rights and obligations; Collaboration, Communication, and Grants Management; and Intellectual Property. One new piece of information I learned this week is that elements of research can be protected as intellectual property. Additionally, I was shocked to learn that the average cost of patenting intellectual property can exceed well over $10,000. That is so expensive!!!! This week, I was also provided with the opportunity to apply to be a part of a Western Alliance To Expand Student Opportunities project. The project's focus is comparing leaf microbiome of wild Larrea tridentata versus those growing in urban areas. If granted the opportunity, I will be able to learn more about Larrea tridentata. As of now, the only information I know about Larrea tridentata is that it is a Creosote bush which contributes to the "Desert rain smell" that I love so much!

Image credit: gocomics.com