New Project

Hello again,

Well I wanted to start my blog with good news, that this week I started a new project beside my bioluminescent bacteria project, which I’m going to explain what it is about.

But before talking about my new project, let me tell you what I did related to bioluminescent bacteria project. This week I did the transformation bacteria experiment again but this time only for Agrobacteria, and Josh told us to change the few steps so maybe the new way gives us some result because so far we didn’t get any result from the old experiment. So what I did different this time was after I placed the mixtures of transformation solution and plasmid DNA (in +pGLO tube only) on ice, then rapidly transferred them to a hot water bath (95 ºC) and this time instead of 50 seconds I kept them there for 10 minutes, and then placed them back on ice again . After two minutes, that I incubated them on ice. I did the same steps as before and added 250µl of LB Nutrient Broth. After 10 minutes, I started adding the solution from tubes to agar plates and spread it across the plate with a sterile loop. After 24 hours in the 37 ºC incubator, I checked the Agrobacteria plates with UV light but unfortunately they didn’t glow!

Agrobacteria plates

And now what’s my second project?

Matt and Josh gave me a new project “Allelopathy test” which means I’m going to spending a little bit in the Phoenix College garden to do the Allelopathy test. But before Matt giving me any further information about this project and how to do the experiment, I needed to know what is “Allelopathy”? So after what he explained to me and do some research I find out that, “Allelopathy” refers to the beneficial or harmful effects of one plant on another plant, both crop and weed species, by the release of chemicals from plant parts by leaching, root exudation, volatilization, residue decomposition and other processes in both natural and agricultural systems (ufl.edu). Which means that different plant parts include leaves, flowers, root, and also soil can have allelopathic activity and that can affect many aspects of plant ecology.

According to Matt the reason that I’m doing this project is that it seems there’s a toxin in PC garden that inhibit seed germination and seedling growth. So what I suppose to do it’s to do a leaf extraction from 4 different trees that we have in PC garden ( Sycamore, Sun flower, Orange, and Oak), then prepare 100 petri dishes with radish, lettuce, chard and carrot seeds, 5 plates of each with 25seeds on them. After that I’ll water the seeds with extract of each tree, and then I have to wait for the germination. So far, I cut all my filter paper and labeled all the petri dishes with seeds on them. Hopefully next week I’ll start the leaf extraction.

100 petridishes with radish, lettuce, chard and carrot seeds

Second week of school, first week of internship

I’m glad to be back and I’m really happy that I started my internship since this week, which is the second week of spring semester (hopefully my last semester), so that gives us more time to learn from Josh and Matt and do more cool experiments. I’m so excited about our project this semester; I’ll keep my fingers crossed to get the results from bioluminescent bacteria because so far we couldn’t make it glow!

So I started my first week with making Tryptic Soy Broth (TSB). Tryptic Soy Broth (TSB) is a nutritious medium that supports the growth of a wide variety of microorganisms, especially common aerobic and facultatively anaerobic bacteria (bd.com). The steps were pretty much the same as TSA that I did before - weighting 30.0 g of the powder and add it into the 2L flask, containing 1L distilled water. Then you heat it up and warm slightly to completely dissolve the powder. 

Tryptic Soy Broth 

Heat up the solution 

Before autoclaving it, I made the solution to three racks of test tubes with autoclave tape on top of them. Finally after 46 minutes autoclaving, the TSB’s test tubes were ready. I left them for a couple minutes at room temperature and then put them in incubator.  

TSB test tubes ready for autoclave

TSB test tubes after autoclave

But besides working on bioluminescent bacteria and TSB in the first week, my sister and I checked the cadavers and sprayed the cadaver with moistening solution. We also tried to do the blood test ourselves to find out our blood type. According to the results, I am blood type A+.

I also did transformation bacteria for E.Coli and Agrobacteria two times during last week. Normally bacteria exchange genetic material with other bacteria and they try to change to the one that can survive better. Transformation in bacterial cells occurs when the cell joins naked DNA into its genetic material. I placed the mixtures of transformation solution and plasmid DNA (in +pGLO tube only) on ice, then rapidly transferred them to a hot water bath (42ºC ) for about fifty seconds, and then placed them back on ice again – this procedure is called heat shock and “increases the permeability of the cell membrane to DNA”. After two minutes, I incubated them on ice. I added 250µl of LB Nutrient Broth. After 10 minutes, I started adding the solution from tubes to agar plates and spread it across the plate with a sterile loop. After 24 hours in the 37 ºC incubator, I checked the plates with UV light and the +pGLO for E.Coli was glowing.

Gram Staining

In the past three weeks of my internship, I’ve learned more about biology by doing different experiments and doing some researches. Last week, which was the last week of fall semester, I was taught how to do Gram-staining. It was easy and super interesting for me. With this method (Gram Stain) you can categorizes bacteria based on the physical and chemical structure of their cell wall, also it distinguishes between Gram positive and Gram negative groups.

After making a slide of your sample, in my case was the bacteria that I growth before, Gram staining involves four processes: first add the crystal violet to the sample on the slide and leave it for 30 seconds, and then rinse it with distilled water. Then add the Gram iodine for 1 minute, and rinse the slide with water again. Next we need to use a Gram decolorize for almost 5 seconds and rinse it with water after 5 seconds. It decolorizes the sample if it’s a Gram negative and it will remove the crystal violet. For the last part, add the Safranin to the slide and after 30 seconds rinse the slide with distilled water for almost 5 seconds. If it’s a Gram positive bacteria, it keeps the crystal violet and it looks purple under microscope.

The slide that I got from Gram staining it shows on the picture below.

So when I viewed my slide under a microscope I got this picture, which it shows that it’s Gram positive and bacilli.

Tryptic Soy Agar (TSA)

I needed to make a media for my bioluminescent bacteria to grow. The best media for growing it, is Tryptic Soy Agar (TSA).

Tryptic Soy Agar (TSA) is a general purpose medium that contains enough nutrients for a wide variety of microorganisms to grow.

First, I weighed two 20 grams of TSA with scalar. Then, I measured two 500 mL of distilled water with the graduated cylinder and poured 500 mL of distilled water from the graduated cylinder into the Erlenmeyer flask. I added magnetic stir bar to the flasks and placed flasks on the hot stir plates. At the same time, I pour 20 grams TSA into each flask and turned on the stir plate selection for mixing Agar and heat selection for boiling solution. I continued heating and string for dissolving Agar. It took approximately 15 minutes to all Agar was dissolved.

I covered each flask with a piece of foil and cut off two strips of autoclave tape and place the tape around flasks neck. The tape should hold the foil and flask together.

I wore the gloves and placed my flasks into the metal autoclave plate. Then, I put the flasks in the chamber and close the autoclave door. For autoclaving the TSA, I chose the media and then I hit the start. It took around 45-50 minutes to autoclave TSA. When autoclave finished, I took out my flasks and placed them into a water bath at 60 ºC for cooling down about 30 minutes. After that, I set up the biological safety cabinet to pure the solution into petri plates. At the end I placed my plates into incubator to use them later.