Sept 29

Our task for today's lab was to prepare a capsule stain. In order to prepare a capsule stain we had to prepare a negative stain.
(* all proper sterilization procedures were practiced throughout this lab*)

Materials used for the Negative stain:

1. 2 clean microscope slides that will be used for the staining process.
2. 2 clean microscope slides that will be used to spread the Nigrosin.
3. Nigrosin stain.
4. Staining rack over the sink.
5. Disinfectant solution.


The steps that were taken for both the environmental sample slide and for the unknown sample:

1. We placed a small drop of Nigrosin near one end of the slide.

2. With a loop, we transferred a small amount of bacteria from an agar plate into the Nigrosin drop and mixed the bacteria and Nigrosin together.

3. We touched (and slid) the short edge of another clean slide to the slide with the contaminated Nigrosin at a 45 degree angle.

4. After the contaminated Nigrosin drop was spread along the edge of the spreader slide, we quickly pulled the slide to spread out the drop.

5. We allowed the smear to air dry completely.

6. We repeat this process for the second slide

PICTURES OF NEGATIVE STAIN

Materials used for the capsule stain:

1. negative stained slides
2. safranin or crystal violet
3. distilled water


This stain is used to detect whether or not either of our environmental bacteria or unknown bacteria have capsules.

1. We took our prepared negative stains and, after allowing them to completely dry, stained our environmental stain with safranin and our unknown with crystal violet.

2. After allowing the stain to set in for about a minute each, we gently washed off the excess stain with distilled water.

3. Blot the water from the slide with bibulous paper.

4. We examined the smears under the microscope using the oil immersion lens.

5.We washed the slides in disinfectant solution

Results: Our unknown sample = cocci shaped, no capsule
              Environmental sample= Rod-shaped, no capsule

Sept 22

 Today we made 2 concave slides so that we would be able to look at a drop of both our environmental and unknown bacteria that had been growing in broth since September 20th.

Broth

Concave slide: environmental sample

Also, a video of the environmental sample....LOOK AT THEM GO!!!(Rod Shaped)

Concave slide: Unknown sample (Circular Shaped)

To make a concave slide one must add a drop of Vaseline to the four corners of a cover slip. Then, using a sterile inoculating loop, take a sample of contaminated broth and place a drop of the broth in the center of the covers slip. Then with the concave slide - making sure you are indeed using the concave side- place the concave side on top of the cover slip making sure the drop of broth is in the center of the concave section of the slide. The Vaseline sticks to the slide so that the drop of broth stays intact.

Below is a picture of the mobility test for our unknown bacteria. As you can see the bacteria only grew within the area where the inoculating needle punctured the semisolid agar.

We also looked at the stained slides we made last class

Stained Environmental sample (Rod shaped)

Stained unknown sample (Circular Shaped)

Sept 27

Today we prepared a Gram stain of both our environmental sample and of our unknown sample.

The materials we used to prepare a Gram stain included:

1. Two slides: one with a fixed smear of our unknown sample, and one with a fixed smear of our environmental sample.
2. Safranin stain
3. Crystal Violet stain
4. 95% Ethanol solution.
5. Gram's Iodine
6. Distilled water


(NOTE: Look to our previous postings for demonstrations as to how to fix bacteria to a slide.)

1.After fixing the bacteria to the slide, we covered the smear with crystal violet and let it sit for about 20 seconds.

2. We rinsed the slide with water to remove excess crystal violet, and covered the smear with Gram's iodine for 1 minute. We then rinsed the slide with distilled water to remove any excess iodine solution.

3. Then, while holding the slide at a 45 degree angle, we de-colorized the slide with an 95% ethanol solution, applying the solution drop by drop until colored solution stopped running down the slide. After decolorizing the slide, we immediately rinsed the slide with distilled water to remove the decolorizing agent.

4. We covered the smear with Safranin for about 1 minute, and rinsed the slide off with distilled water to remove any excess Safranin, and blotted the water off the slide using bibulous paper.

5. Lastly, we examined the stained smear under the microscope using the oil immersion lens.

We concluded that our unknown sample was a Gram negative because the stain stayed Red.

Pic: GRAM STAIN

 Safranin agent on slide

Sept 20

We were given four tasks to complete.
         1. Inoculate the broth with your environmental and unknown samples
         2. Prepare a streak plate for the unknown
         3. Repeat simple stain for both the environmental and unknown samples using the methylene blue stain.
         4. Motility test
*When doing each test all of the necessary sterilization processes were observed.*

1.
To inoculate the broth given to us we flamed the loop, took a sample of our unknown bacteria, and after      running the mouth of the tube over the flame about three times we stirred the bacteria in the broth. We flamed the opening of the tubes as well as the loop before doing the same for our environmental sample.

The environmental sample was placed in the regular incubator were as the unknown sample was placed in the incubator at 37 degrees Celsius.

2.
We then prepared a streak  plate from the unknown sample that we were given last class.
To create a streak plate we used a sterile loop, sterilized the top of the tube using the flaming technique, took a sample of bacteria, and sterilized the top of the tube again. We then streaked the bacteria on the plate by using a back and forth motion. When streaking the plate we divide the plate into 4 imaginary quadrants.

This culture was then placed in the incubator at 37 degrees Celsius. 

 3.
We prepared a stain  for both the environmental and the unknown samples. The same procedure was done for both.

We first placed a drop of distilled water on our cleaned slide. Then after flaming the loop, we took a sample of bacteria and spread it in with the water until a thin mixture developed. Our drop of contaminated mixture was left to air dry.

After drying we passed the slide quickly through a flame three times with the smear side up causing the bacteria to be fixed to the slide.

The methylene blue stain was placed on top of the contaminated area. After allowing the bacteria to be stained with the methylene blue for about 1 minute, we rinsed the slide off with distilled water so that any excess stain would be removed. The slides were then placed in our drawer until the next class.

slide with a drop of distilled water and bacteria stirred into it.

Above are a picture of our environmental and unknown slides after being stained.  

4.
The motility test was done so that we might be able to determine if the bacterium was mobile or not.

We took a sample from our unknown slant with an inoculating needle and stabbed the now contaminated needle into the center of semisolid agar- about halfway deep. We did the same for our environmental slant. 

The environmental tube was placed in the incubator at 35 degrees and the unknown was placed in the incubator at 37 degrees C.

placing the sterile inoculating needle in the semisolid agar.



 

15 Sept

We looked at our stained slides under the microscope using the oil immersion lens. To use the oil immersion lens we placed a drop of oil on top of our slide. We slowly turned the fine focus until we created a cylinder with the oil. This allowed better light refraction which allowed for better overall image resolution.

 
Above is our slide with a drop of immersion oil on it.

                                               Our slide under oil immersion lens.
Below is a photo of one of our "K" bacteria slants after they have been in the incubator for two days. The growth appeared to be raised, cream, translucent with undulating margins.  

Dr. Pathakamuri helped us to make a new pure colony. He used sterile applicators to re-streak the Petri dish. We placed the new streaked plate in the incubator. The next day we placed the said Petri dish in the fridge to slow down the growth.

Second Lab

13Sep11 Lab Work




Today's experiment involve staining a slide with a bacteria sample taken from the pure culture. After sterilizing the loop, we transferred said bacteria from the Petri dish to a slide. (Which was prepped by placing a drop of distilled water on it.) We spread the bacteria (using the loop) around the area of the slide with the water-drop. After waiting for the water drop (now contaminated) to dry, we heated it over a Bunsen burner by stroking the slide over the burner several times so that the bacteria would be fixed to the slide. Lastly we stained the slide with crystal violet and allowed it to stain the fixed bacteria for 20-30 seconds. Lastly we rinsed off the slide with distilled water to remove any excess stain. The stained slide was placed in a drawer for later use.

Our second task was to isolate an unknown bacteria that was given to us by Dr. Joseph into two slants. We (using a loop) transferred the bacteria named "K" into two separate sterile slants, and put said slants into the incubator at 37 degrees. The original slant was given back to the professor. We transferred the bacterial by using the same technique that was used in the last lab class with the Petri dish... i.e. zig zag motion.



Don Caldwell
Melanie Seitz 

First Lab

PETRI DISH LAB EXPERIMENT (FIRST LAB) 

08SEP11

 

 Pictured above is our Petri dish for our sample taken from the bottom of a shoe (Rain Boot). The Petri dish shown illustrates separate bacteria colonies, after two days in the incubator. Below are 3 images taken from the same sample under a microscope at various levels of magnification. Notice the different colors of the separate colonies.

Color: Cream & yellow
Elevation: Raised, Crateriform, flat, convex, hilly
Form: Circular and irregular
Margin: Undulate
Appearance: mostly Opaque and very little translucent

Our objective was to isolate and create a pure culture of a single bacterial colony by using the Streak Plate Method. After we examined and documented what kinds of colonies we had in our Petri dishes, we sterilized an inoculating loop, picked a single colony from our Petri dish using the loop and transferred the colony onto another Petri dish that contained an agar medium. Using the loop we spread the colony onto the agar into four quadrants. We placed the new Petri dish into the incubator to be examined next week. The original Petri dish was placed in the refrigerator.  

Conclusion: Our lab experiment demonstrated the growth and quality of the various bacteria found from our sample. It is perhaps surprising to learn how quickly bacteria can grow, and all the places of which it can be found.

Don Caldwell
Melanie Seitz