Native Instincts Kicked In

Well I can say I have a green thumb. The pea plant seeds came out with all pots successfully growing into sprouts. Figure 1 below shows the 100% success of all pots growing pea plants. Furthermore, the green tray was split with my lab made divider using yellow tape and cotton balls. I split the tray so the plants wouldn’t share the liquid of DI water and faucet water, but it turned out that both liquids different in pH both grew the plants. I think my last plants didn’t grow because the temperature was at 34 degrees Celsius; also, the soil was soaked rather than lightly moisten and the mixed soil with vermiculite.

Figure 1. The bottom left corner shows a close up of the success. There is a yellow arrow showing the yellow tape dividing the two rows of pots.

I, also, sampled two locations off campus for agrobacterium tumefaciens (A.tumefaciens). Figure 2 below show the samples in 0.1% Peptone of 45mL with the corresponding test tubes. Once the sample was chiseled out with sprayed 70% alcohol of a hammer, flat head screwdriver, and chisel, it was immediately put into a urine sample cup of Peptone of 90mL for isolation of bacteria. The piece of the crown gall was then rinsed with sterile DI water then cut using a sterile scalpel and tweezers. This whole procedure was performed in the flow hood for low risk of contamination. The pieces cut were place in two sterile test tubes of 6mL of Peptone and two sterile test tubes of 6mL of Tryptic Soy Broth (TSB). Then, the eight test tubes were stored in a cabinet for room temperature of 25 degree Celsius for proper growth of A.tumefaciens.

Figure 2. The neon green top rows of test tubes are tree "A." The next 2 bottom yellow rows are tree "B." Basically, the top row has TSB. The next row has Peptone, then the next row is TSB. The final row has Peptone. 

Interesting WEEK,

In the beginning of this week I started mixing my own soil for perfect growing conditions for my pea plants. The taxonomic name would be Pisum sativum (P. sativum). These dicot plants would be a good host for infection of the Agrobacterium tumefaciens (A. tumefaciens).  Figure 1 below show the arrangement of the seeds in the pots, and I used 70/30 of soil/vermiculite with a pH of 6.23. I was, also surprised on how to calibrate a pH meter. Furthermore, Josh told me that there was a pH meter for the soil that Matt had in his room. This meter gave a reading of 7 and the calibrated meter gave a reading of 6.23. On the other hand, the soil needed to be slightly acidic for growing conditions. Thus, the top row would have only DI water and the bottom row would have regular faucet water. I, also, trained newbie intern Alex on my procedure to prepare media and sampling technique used to obtain bacteria on the made contact plates with TSA.

Figure 1. The pots are set up just the same way as this previous pic, but there is a divider in the middle so the water I put in don't mix.

Failed Experiment

Figure 1. This is just five of the twenty randomly used for observation and statistical analysis of the CFU. The rate of this group is fair.

Figure 2. The red obscure circular shaped colony in the top middle row is very interesting. The small white of a guess would be S. epidermis and the yellow medium sized circular would be S. aureus. The rate would be poor.

Figure 3. I would say the rate for this group would be fair.

Figure 4. 

Figure 5. I would rate this group as very poor. There is a rigid circular shaped colony in the bottom row of the left plate.

Figure 6. I would rate this group as poor.

The plants that I seeded with Sonora wheat and Anasazi beans died because there was no watering due to my absence of the lab. Although I infected three Anasazi beans with the possibly A. tumefaciens, I wasn’t able to get any results because of my poor responsibilities in the lab. On the other hand, my first project with the fomites is going somewhat okay. I have sampled more than 8 locations around campus, and each location had about 20 plates used for each site. If my calculations are correct, I used about hundred and eighty plates, and trashed about a sleeve of incorrect plates when pouring. The figures above show some of the randomly obtained samples from each site that was going to be analyzed using Photoshop software. Furthermore, I would rate the group as poor, fair, or acceptable for either necessary action or non-necessary action to be handled in the caption.

Interesting Article

I found a simple formula for how to calculate the sampling sites of a location.

Although the whole article was based on the pharmaceutical industry of manufacturing healthcare products, the whole concept of using contact plates is to make the sampling easier and quicker for my project. The previous semester of using swabs and sterile tubes was a long process from swabbing to inoculating to TSA media for incubation of CFU. It would take about almost two hours just to sample an area with the swabs, but now the contact plates make it easier to take samples. In other words, the PDF article was beneficial on how to sample areas of potential contamination of microorganisms. It, also, explains the proper procedures to take when analyzing the results of the incubated plates after 24 hours. The simple equation above used in a real situation would be used like the example below.

 The room used would be the DB-Building lab room 107 of the bottom floor.

Figure 1. Shows the location of sampling to be taken.

Figure 2. Shows the idea of how to calculate the minimum number of sampling locations.

Where, N_L is the minimum number of sampling locations (rounded up to a whole number)

A is the area of the clean room or zone in meters².
Article Link

Fast Week with Progress

The plants that were seeded last week didn’t germinate this week, so the new approach was to use another type of soil known as vermiculite. Vermiculite holds more moisture for less watering for the inexperienced gardener. The green thumb isn’t in my DNA structure, but I still have six pots of regular soil and another six of vermiculite. Also, the Sonora Wheat is still being used but the soy beans were change to Anasazi Beans for germination. Figure 1 below shows the arrangement of seeds planted.

Figure 1. I should start to see some sprouting next week by Monday at least if not then I'm a horrible planter. Furthermore, these seeds grow in places with less water like Arizona or other states along the Southwestern States.

I haven’t sampled this week for my fomite project but I have some potential ideas of where to sample at. The gym has some exercise equipment that can be sampled, and maybe the smartphones students use around campus can be sampled. I can prepare and pour TSA in the contact plates with no problems now.

Furthermore, I took my sample from the residential property across the street from the DB building. Figure 2 and 3 below show how I took my sample with a couple of carpenter tools although the incremental borer would make it easier. Once the sample arrived in the lab, a simple protocol was used to isolate the potential specimen for Agrobacterium. On the other hand, the results were not what I expected because of lack of ascetic techniques and storing areas.

Figure 2. Approaching the area where the sample was taken is shown above. The inside volume is like a pyramid that is scraped out with tools like a flat head screwdriver, mini sludge hammer, box cutter, and some tweezers.

Figure 3.  This side view shows the measured depth of the sample obtained on Wednesday October 16, 2013.

Side Project

The easiest way to perform a natural molecular biological transformation with plants is using bacteria. The bacteria being used is Agrobacterium tumefaciens (A. tumefaciens) that has a plasmid that can be easily transferred to plant cells. Thus, this Gram-negative agent is used as a tool for engineering desired genes into plants. The major cause of plant disease that this bacterium causes is crown gall. Figure 1 and 2 below show some of the pictures I took around off campus sites.

Figure 1. This tree whole trunk is covered with crown gall. The tree is located just across the street from the DB Building at Phoenix College.

Figure 2. The second site of this crown gall is located just a few more feet away near Thomas Rd.

I started preparing my subjects below for germination and the seeds used are soybeans and wheat. Figure 3 and 4 show the pots and location of each per pot.

Figure 3. The seeds used are shown below the pots and the pots are numbered 1-6 just above the rim.

Figure 4. The pots were placed in a 12 hour light incubator for growth around 1 o'clock on October 08, 2013. Note that the typo above in the picture is "seeds" for the "plants"

I hope to identify these bacteria with positive growth of the tumor once introduced to the plant with some type of identification marker like pGLO added to the bacteria. I plan on thinking about how to introduce these bacteria to the growing plants. For example, should I inject the disease causing tumor in the stem, on the leaf, or on the roots? Should I mix with soil or dip plant in solution of A. tumefaciens? Moreover, I look forward to a positive Agrobacterium tumefaciens.

Exciting, But Exhausting This Week

I sampled this week the school text books at the bookstore and library of the campus. The used rental books at the bookstore of Phoenix College were sampled on the cover. The picture below shows the titles of each book sampled below.

 

Figure 1. The numbers on the left side of the picture correspond with the plate sample. Some books were sampled twice on the covers.

Used Rental Books at Bookstore
Plate #	# CFU	Plate #	# CFU	Plate #	# CFU	Plate #	# CFU
1	21	6	10	11	TNTC	16	35
2	4	7	10	12	32	17	TNTC
3	19	8	30	13	6	18	20
4	2	9	15	14	41	19	28
5	11	10	51	15	43	20	41

Figure 2. The books shown above are the reference books used throughout the school year if the student doesn't have enough money for the books in the campus bookstore or elsewhere. They can be checked out for 2 hours maximum if there isn't anyone else waiting for the book.

Reserved Books at Campus Library
Plate #	# CFU	Plate #	# CFU	Plate #	# CFU	Plate #	# CFU
1	5	6	15	11	10	16	9
2	5	7	8	12	1	17	86
3	15	8	42	13	3	18	26
4	2	9	27	14	0	19	15
5	4	10	7	15	18	20	20

The reserved books behind the counter of the campus library were, also, sampled. The picture above show the books used in this experiment.

Although I haven’t had much time in the lab this week, I was pondering if it is possible to insert pGLO into plants for an illumination. Basically, a plant that glows as it grows. For example, there are enzymes that will break open an E.coli cell and combine the pGLO DNA into the ring DNA of the cell for reproduction of glowing bacteria colonies. The process of transferring exogenous DNA information and up taking it into another cell membrane is called Transformation in molecular biology. The video above shows how to conduct animal DNA transformation. The second video below shows how the start of this project might begin with the first steps. Maybe?

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