Capsule Staining :]

Today, we practiced staining even more and stained our samples to see if they had capsules or not. First, we had to prepare a slide with a little bit of a different technique than usual. After placing a drop of nigrosin on a clean slide, I added a little of my unknown sample bacteria to the drop and mixed it carefully, employing the asceptic technique. Then came the tricky part, after mixing the bacteria, I used a second clean slide to smear the drop of nigrosin across the slide, and let it air dry. 

After it dried, I flooded the slide with Gram's safranin, and gently rinsed it off, which took off a lot more dye than I think it was supposed to. I then examined the slide under the oil immersion lens and determined the small cocci did not have a capsule. 

Gram Stains!

Today, we prepared Gram Stains in class, which is a slightly more sophisticated technique than preparing a simple stain.  First, using the asceptic technique, I made a slide by smearing my unknown sample with a drop of water, letting it air dry, then heat fixed it. I covered the stain with crystal violet for 20 seconds, rinsed the slide, covered it with Gram's iodine for a minute, and rinsed that off. I then decolorized the stain with EtOH, and rinsed it off. After covering the slide with safranin for one minute and rinsing it, I finally got to check out the sample under a microscope! I used the oil immersion lens, but had a little trouble getting it into focus. Luckily, Dr. Pathakamuri found the stained bacteria, and we identified it as Gram-negative bacilli. :]

Motile Bacteria

 Today, We   observed the  semisolid agar motility test tube, finding out our unknown bacteria was motile, and our environmental bacteria was not. Following this, we performed the hanging drop test for motility using a special concave lens and a lens cover to observe the bacteria swimming! Then we classified our bacteria. Our unknown bacteria, L 25, is red, rod shaped (bacillis) and motile. In the agar slant tube, the unknown was filiform. In the broth tube, it was turbid, having sediment, a ring around the top, and flocculent simultaneously. Our environmental sample, was circular, cream/tan in color, entire  and raised.  In the broth tube, it had sediment, turbid, and was tan in color. We also studied the reaction of bacteria to antibiotics, finding out Dr. Pathakamuri did not have strep throat because the bacteria did not completely lyse the red blood cells, even though it was killed by bacitracin.  

Inoculating Broth Tubes and Streak Plates

Today, we inoculated broth tubes with both environmental and our unknown samples using the asceptic technique. We also created a streak plate of our unknown bacteria using the slant tube and asceptic technique. We performed a motility test for both unknown and environmental samples using the inoculating needle and a special 0.4% agar semisolid solution. Next we prepared slides and stained both unknown and environmental bacteria with methylene blue dye. Monica also swabbed our wonderful professor's throat to test for strep throat and the virus's reaction to different antibiotics, more specifically bacitracin and penicillin. We also learned about bacteriophages and prepared a streak plate with a bacteria and T4 bacteriophage to  observe on Thursday.

Simple Staining Inspection

Last week, we looked at our simple stains of environmental bacteria under the microscope. My safranin red staid showed best the little clusters of four bacteria, also known as tetrads. Attached is the picture! We also transferred our unknown bacterial slants from the incubator to the fridge. The movie about the flu virus and how it mutates and moves from species to species was an incredible inside look at the virus and precautions I can take everyday to avoid transfer of it. I also thought how scientists predict the next flu strain to create a vaccine for was really interesting.

Preparing and Staining a Bacterial Smear

Today we took our pure cultures from last week from the incubator, and chose a colony to make a smear plate from. Using the asceptic technique, I chose a colony and used my inoculating loop to create a thin film on the glass sides. After air-drying the slides, I fixed the bacteria to the plate by swiping it through the flame 3 times. Then I stained one slide using crystal violet stain for 25 seconds, and the other using safranin for a minute. I rinsed the excess dye and blotted the plate dry to observe next week. Then each lab group got a sample of bacteria to make a slant tube from. Once again, using the asceptic technique, we sterilized our inoculating loops and smeared the sample in the new slant tubes, and put them in the incubator for next week. Enjoy the pics!

Isolating a Pure Culture by creating a Streak Plate

Today, I got my bacteria out of the incubator, and found that it had grown. I saw mostly little yellow, shiny colonies of bacteria and one larger, green, moldy looking colony. After observing them under a microscope, I chose to isolate bacteria from one of the yellow colonies. I sterilized my inoculating loop, obtained a colony from the agar plate, and streaked a fresh agar plate in quadrants using the inoculating loop, sterilizing the loop between quadrants. After labeling the new agar plate, I placed it in the incubator at about 25 degrees centigrade and placed the original agar plate in the refrigerator. Next week, I will hopefully be able to more closely examine an isolated pure culture of my bacteria.

Isolation of Bacteria

Today, we isolated bacteria from different objects in the environment to prepare a smear plate! I chose to isolate bacteria from an item utilized daily, my student ID. First, I took a sterile swab, dipped it in nutrient agar broth, swabbed all over the front of my student ID, then swabbed the bacteria onto the hardened agar broth in the petri dish. I labeled the dish, and put it in the incubator at 25 degrees centigrade. Enjoy the pictures!