So much media

Hello everybody. It has been a long week but it's almost over. Just a small piece of information; it seems like we will be enjoying a cloudy weekend, at least according to the weather.com. This week I finally got my Yeast Mannitol Agar (YMA). I was so happy because like I previously said, it was ordered before spring break. Now I have been able to make my eight different combinations of media and I'll be ready to start testing next week. Fun little detail; the lab just got the YMA sample that Matt ordered before spring break. I'm still not sure what happened with the YMA from the first lab equipment company, why it took so long, but now I have extra YMA so I can make as much media as I need.

Now that I have finished pouring all of my plates this week I can work on a little side project this Friday. I'll be working with Josh and we'll both figure out how to prepare a bacteria for -80 degrees Celsius storage. I thought it would be just as easy as to put the bacteria into the freezer but this can damage the bacteria if not done properly. Luckily I was able to find quite a few protocols that are quite easy to follow. I did have to be careful about which ones I could refer to since some require the bacteria to be snap-frozen before putting it in the freezer. That kind of protocol asks for liquid nitrogen, dried ice or dry ice/ethanol slurry. Since the lab doesn't have access to any of those I'll be using another method. For future references here is a Protocol that I found at Ops Diagnostics, a lab equipment company.

First one needs to prepare a solution of 30% glycerol, 30ml glycerol plus 70ml water. Then transfer the solution to a screw cap glass bottle and autoclave at 121 degrees Celsius for 15 minuts.

Obtain 500 microliters of sterile 30% glycerol into two sterile microfuge tubes that contain 4 mm glass beads.

Add 500 microliters of the bacteria culture to the tube and mix with glycerol using a vortex mixer.

Label and place it in the freezer.

I hope everyone has a good weekend. I'll let you all know if this protocol works or if I decided to do another one. "Live long and Prosper."

Long Overdue

Two weeks after spring break and I can still feel the lingering effects of not coming to school for a whole week. Now I must focus on my new project. I was able to submit my new abstract to the Estrella Mountain conference in April. Since it is bad etiquette to submit the same abstract to different conferences I had to tweak my previous abstract a bit. I will also redesign my poster since I won't be able to keep the same methods and conclusion. I am excited about going to this upcoming conference specially since the last one was so fun.

There will be one major difference with my project, I will be testing different recipes that will increase the efficacy of Agrobacteria and Rhizobium growth. Matt came up with a few ingredients that would make a good media for Agrobacteria. Inspired by Macconkey media, Matt gave me a list of materials that I will combine to make better media; YMA (yeast manitol agar), crystal violet, bile salts and penicillin. I will make 4 litters of media and test each plate that comes out of those four litters to see which combination is more beneficial to my project. Needless to say I will be pouring a lot of media. This time around I will be using gloves and possibly a mouth guard when handling penicillin, seeing as how I am quite allergic to it.

Sadly I cannot start my new project yet because the YMA that was ordered, about two weeks ago, has not arrived at the lab. Matt had to reorder the media from a different company because the first one "couldn't find it" even though their website said it was being processed. I'm not even mad because I have been using my time to help around the lab. From making media and pouring it into plates, to washing dishes (which I find surprisingly enjoyable; it's easy, simple and kind of relaxing, specially when I get to sing and hum while I do it). I've also tried to help anyone that needed help with their projects even if it was just labeling things.

Funny story about the time I washed equipment at the lab. As I was scrubbing some trays, that previously held hearts, a blast of water pushed the faucet straight out of the sink. Luckily I had and apron on so I was not completely soaked; I hurried to screw the faucet back on. It turns out that every time the faucet is moved right or counterclockwise, it unscrews itself a little. The faucet was at its loosest point when I was there; I'm glad it was me and not someone else because that caused me to laugh for quite a bit.

This week was all about the poster I'm presenting to ASU. The topic is the development of a protocol for the isolation of agrobacterium and rhizobium from the environment. I was able put the finishing touches to my poster just in time for Matt and Josh to send it to print. What made making the poster easier was the help of Melina and Gilberto, since I probably wouldn't have been able to get the correct dimensions for the poster. I wanted to make it look fancy and intricate; Josh, however, advised me to keep it simple and keep it simple I did. This made my job a lot easier because the poster has only the information it needs, a couple images from my project and the design is simple and to the point.

This week I also decided whether I will keep my project with Agrobacterium going or just start another project, in this case it would be video game physiology.I decided to keep going with my initial research and try to come up with different ways to isolate agrobacterium. At the same time I decided to do more research on video game physiology because I'm going to be doing both projects at the same time. I already have a decent amount of volunteers to play video games when needed.

It's still early on my research about Video Game physiology to start constructing a protocol. Seeing as how I'm a big fan of video games, being a gamer myself, I'm really excited to start this project and learn the effects of video games on the brain. Lots of studies have been done on this subject so I shouldn't run short of resources. But like I said it is still too early to even begin thinking about specifics.

Next week I will focus my efforts into increasing the amount of data from my experiment with agrobacterium, and I will also come up with better media for better growth of isolated bacterias. At the same time I will begin my research on video games. These two project have plenty of null time in between procedural steps to allow work to be done to both of them at the same time.

So close and yet.....

 Finally a breakthrough in my project. A colony of bacteria that meets the morphology of agrobacterium on a Macconkey plate appeared in one of my plates. It has pink coloration, it is round, convex and appears to be engulfed in a mucous coating. It is also Gram negative like agrobacterium. This colony grew from the sample of soil I obtained from the school's garden, which I believe might have given Matt the idea of getting soil samples from different places in Phoenix College. From here on, I'll be collecting a sample of soil from all over PC, diluting 1g each soil in 1% peptone water and inoculating them into Mac plates. Since Macconkey media is Gram-negative selective I decided to use this kind of media for future diluted soil inoculations.

The first day I will bring the soil sample to the lab and after the dilution process I'll be streaking Macconkey plates. The next day I will repeat this process with new soil. The same day I will be testing any colonies that grew from the previous soil sample. I will administer tests such as maltose, lactose and glucose, among others. The third day I'll repeat the process of the previous day and I'll continue this pattern until I obtain enough data samples to increase the validity of my results.

Also, if anyone out there has seen or owns a plant with crown gall on it please let me know. It would be a great addition to my research. They may look like this:

week 1/28. Orginizing my thoughts

This week I did not spent as much time in the lab as I would've wanted to; it might have seemed like I was there a lot but I wasn't there for long periods of time. Even so, I was able to get a lot done in the short amount of time I had. I watered my seeds, which I don't forget to check anymore, I inoculated two plates of the following, YEB, TSA and Macconkey media with a 1% peptone solution mixed with soil from Phoenix College's garden, I prepared two tests of antibiotics for agrobacterium and rhizobium, I streaked a YEB plate specifically to get more isolated agrobacterium colonies and I found out agrobacterium grows really quick so I can't leave my YEB plate for too long in the incubator........... which reminds me that I didn't take it out of the incubator!!!  hopefully I won't have to redo it, although even if I do it will be fine; I like to inoculate plates.

     Since I don't think I have been very clear on what it is exactly that I'm doing and why I'm doing it here it goes. Agrobacterium has an interestic characteristic that separates it from other bacteria; it can transfer its genetic information into a plant making it an important tool in genetic engineering. I am simply trying to get to know this bacteria more closely. So far I've been able to verify that it is a Gram negative bacteria and that it is bacilli, rod shaped. I've also performed different tests like oxidase and catalase, the bacteria was positive to both. Tomorrow I should be able to see if agrobacterium is resistant to the antibiotics rifampicin and tetracyclin.

Furthermore, this project has allowed me to learned tons of things, from performing tests on bacteria to researching and making the best media for my bacteria. I am also growing 12 pea plants that I plan to test the pathogenicity of agrobacterium on. I am planning on doing this with two different kind of bacteria, agrobacterium tumefaciens and rhizobium leguminosarum. Rhizobium used to be called agrobacteria until it was reclassified which is why I keep calling rhizobium agrobacterium sometimes. If everything goes well, after infecting the plants with both bacteria (one per plant), I should be able to see tumors in the roots of the plant and in others above the soil.

By the way, 9 of my pea plants died due to special circumstances......... I didn't water them. Now I have replanted new ones, so I will have 9 peas without names, so if anyone is interested in naming a seed send me the names at blackboard.

Week 01/22/2013

Its a brand new year and the sun is getting brighter, both literally and metaphorically. I'm really excited because this semester I'll have a lot more time to actually complete my project and probably a second one as well. Last semester I was only able to get as far as my protocol but now I'll be able to complete the experiment. Just really quick though for those that are new to the S-Stem, my name is Jose Luis and I'm a biology major, now interested in genetic research. I have finished six semesters at Phoenix college and I will be attending ASU West in the fall, if everything goes well, with an associates in science.

Last semester I took it upon my self to do research with agrobacterium, a really cool bacteria utilized in the genetic engineering of plants. Sadly last semester was a short one and wasn't able to get too far in the project. This time around I have already made some cultures into a few media that I had to learn how to make. Basically I am to isolate agrobacteria from the environment and then infecting a plant to observe the results. The cool thing about this bacteria is that it is actually beneficial to the host, when it develops in the roots it increases the nitrogen intake of the plant. This specific type of agrobacterium, that grows in the roots, is called rizobium and its actually sold in stores to boost plant growth.

To speed up the process last semester Matt and Josh ordered a pure sample of agrobacterium that I used to inoculate a plate of YEB, a media that is great for growing agrobacterium, The image to the right contains the YEB plate that I'll be using from now on to make more cultures of agrobacterium. Since Rizobium is different from agrobacterium I also streaked other plates with soil that contains rizobium. This will give me more to experiment with.

This year I planted some peas, butter bean, to infect with agrobacterium and rizobium. Below there is an image of the twelve pots containing my plants. I got so excited planting them that I had to name them. It's been a week since I planted them and already two of them germinated to a point were they are coming out of the soil. And fun fact, Sora and Elise were the peas that germinated. I will try to upload more pictures of my plants as they grow.

This week. Dec 10/12

This week is finally... almost over, along with finals. I am still surprise of how much I've learned in these past few weeks during my internship at the biology lab in Phoenix College. I've learned to do Gram Staining, the methodology behind making media and streaking a plate with a bacteria sample.
Gram Stain is simple and yet easy to mess up, specially when its the first time doing it. After three or four trials I started to get the hang of it. There are four major steps during staining; first crystal violet is applied for 30 seconds, then iodine is added for 60 seconds.  A decolorizer is then added to wash away the crystal violet, if it's a Gram negative bacteria, it will not wash it away if its a Gram positive bacteria. The tricky part with this step is that if the decolorizer is left for too long on, the crystal violet gets washed away from both Gram positive and Gram negative bacteria, but if not left for enough time a Gram negative bacteria might have purple or blue dyed bacteria as well. At the end Safranin is added for about 30 seconds. Between all steps the sample must be rinsed with water for about 5 seconds.
 Just yesterday I was able to perform a Gram Stain on a bacteria that showed antibiotic characteristics. The sample turned out to look like rods which indicates bacili and it was also Gram Positive. The image shows what I saw in the microscope.

I also made my own Macconkey agar, with the instructions of Kimberly, all I know about her is that she works at the lab but I am very thankful. Fun fact, macconkey agar kind of smells like dog food so during the process of making it I remember a German shepherd I had years ago. Hopefully I will be able to use these plates to cultivate some agrobacterium for my research project. Right now I have only written my protocol and cultivated 8 plates with what, after three tests (Gram stain, catalase test and oxidase test, I believe to be Rhizobium. Rhizobium is a plant growth promoting rhizobacteria, or PGPR, that a Gram Negative bacili. It is also positive for a Catalase test and Oxidase test.  I used 4 plates with TSA media and 4 plates with Macconkey media. I have placed those plates in an incubator and I'm just in the waiting processes to see if anything grows, fingers crossed.