Busy Week

During last week while almost all of the other inters were working hard on their posters and getting prepare for their presentation for ASU conference, I had a busy week. I did lots of different things for my project, but now I feel that I didn’t do any useful thing in the last week.  On Monday, I decided to set up another PCR from my purified Agrobacteria’s DNA to extract it with Agarose Gel Electrophoresis technique because I was not able to get a good result. I used three different primers to prepare my PCR (Polymerase Chain Reaction). When I finished it, I found out that  PCR machine would be used by one of the Biology classes for their lab for a whole week and I would not able to use it. Then I incubated my PCR tubes in the refrigerator at temperature -4 degree C to do PCR in the next week. On Tuesday, I cultured my Agrobacteria in the three different kinds of bacterial growth mediums. I used TSA (Trypticase soy agar), YEB agrobacterium, and MacConkey’s (MAC).  Then, I place them in the incubator at temperature 37 degree C.Another interesting thing that I did on Tuesday was planting 10 soy beans for my experiment. Now, I feel that my experiment is getting more serious.

 On Wednesday, I checked my bacterial growth mediums and Agrobacteria grown in the TSA and YEB medium but it did not grow in the MAC. I placed back MAC in the incubator for 24 more hours. Then, I decided to do gram staining my Agrobacteria which grown in the TSA and YEB mediums.  The Gram stain is one of the common methods for staining procedure in microbiology. This method is very useful in identifying bacteria.

 

 At first, I made a smear of Agrobacteria then I started gram staining. 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.At the end, i viewed my slide under a microscopic and took a picture of it. Pictures of Agrobacteria in both mediums show that Agrobacteria is gram negative.