Day 2: All About the Stain

We brought out our bacteria samples from the fridge and observed them for any new growth. Turns out, all of our environment samples did not really grow overnight so we decided to collect new samples at the end of the lab. However, the bacteria from the fingerprint samples grew enough overnight for us to see different kinds of colony shapes. Our plate had bacteria with puntiform and filamentous colonies.

Our fingerprint bacteria with colonies

After viewing our samples, we observed a few on the 3D screen and even got to wear some pretty fly looking glasses.

Lab is a good time

Following this, we got some practice using the microscopes with slides provided for us. We were able to get comfortable using the microscope and then reviewed microscope information like resolutions and magnifications. We learned about the use of oil increases the resolution because it captures all of the light to the lens.

We then made a working stock and a reserve stock of our unknown bacteria to be able to use for future experiments.  We also made a streak plate and spread plate using our unknown bacteria.  The streak plate was used to get a pure culture by spreading out the bacteria to grow separately. For the streak plate, using the aseptic technique, we touched the unknown bacteria and spread the bacteria on the top region of the plate.  After doing the aseptic technique, we streaked the bacteria from the first region to the second region.  Then, after the aseptic technique was done again, we streaked the bacteria from the second region to the third region.  Finally, the aseptic technique was done again and the bacteria was streaked from the third region to the fourth region.  In the center of the plate, a squiggle line was drawn.  The plate was placed in the incubator at 37°C overnight.
        The spread plate was next and we used a spreader with 70% ethanol and a micropipet using 200 mL of our bacteria sample.  We placed 200 mL of our sample onto the agar culture plate and after doing the aseptic technique with the spreader, we spread the bacteria to cover the entire plate.   The plate was placed in the incubator at 37°C overnight.

Heating our spreader to kill off bacteria

 Next, it was time for the staining to begin!
Our first stain was the gram stain, which would tell us if our unknown bacteria was gram positive or gram negative.  After doing the aseptic technique, we placed our slide on the slide holder over the sink and covered the bacteria with crystal violet dye.  After 30 seconds, we rinsed off the excess crystal violet with water.  We added iodine to the slide and allowed it to sit for 1 minute. After the minute was up, was rinsed the excess iodine off with water and then added a decolorizing reagent (95% ethanol) until the color on the slide stopped running. Finally, was covered the slide in safranin dye, and let it sit for 1 minute.  Then, we rinsed the slide with water and blotted the slide dry with bibulous paper.
Gram-positive bacteria do not decolorize in the decolorizing reagent, meaning the crystal violet color is retained.  However, gram-negative bacteria decolorize in the decolorizing reagent allowing them to accept safranin's reddish color. Because our bacteria was reddish, our unknown bacteria was gram-negative!

Dr. P explaining gram-positive cell wall

Our gram-negative bacteria (RED!)

 Our staining days weren't over today! Day 3 continues with many more stains...

Day 1: The Beginning

           After final exams were done, 8 students decided the semester wasn't long enough and decided to take on one more class.  We entered the laboratory and Dr. Pathakamuri gave a small introduction to the class. He told us how this class is important to nursing majors and the health science relations, in general.  He said that patients will have germs and illnesses and we, as professionals, need to be able to aid in the treatment of the illnesses quickly.  We need to be able to properly identify the bacteria to be able to treat it efficiently.  Also in our introduction, Dr. Pathakamuri gave a brief introduction about the laboratory safety.  Once we enter the lab, we need to put on our lab coats, wash our hands thoroughly, and clean our bench area with disinfectant to get rid of any bacteria.  When we left the lab, we need to clean our bench area, wash our hands, and hang up our lab coats. 
                                                                                                 

 
 To really see and understand how clean our hands were after they were washed, we did an experiment.  Lindsey and I received a Petri dish with a culture medium. We divided our dish into four sections using a China marker. We pressed our thumbs into the medium and quickly covered the dish to prevent any contamination.  Then we washed our hands, we pressed our thumbs again into the medium.  We covered the dish and put them in a 37°C incubator.  This allowed the bacteria to grow overnight. 

Our Petri Dish with our thumb prints before and after washing

          Next, we made our own media culture from Nutrient Agar or Nutrient Broth.  Our group was assigned Nutrient Agar.  We measured 4.6 grams of Nutrient Agar and added it to a beaker of 200 mL of distilled water and covered the beaker with foil to prevent water loss.  The mixture was placed in the autoclave for 20 minutes under 15 lbs of pressure at 121°C.  The autoclave uses moist heat for sterilization.  Once the mixture cooled to approximately 30°C, the mixture was poured into Petri dishes to cover the bottom. The dish was covered to prevent contamination and the liquid had turned to gel within minutes. The dishes were placed in a 24°C incubator. 
                                                   

Weighing our Nutrient Agar

Placing our mixture in the autoclave

Removing our mixture from the autoclave

Gel media cultures

         
                       We learned about Aseptic techniques to transport bacteria from one tube to another under sterile conditions.   We lit the Bunsen burner and were given two test tubes of liquid.  The inoculation loop was sterilized over the Bunsen burner, the test tube was slightly heated to remove bacteria, the inoculation loop was placed in the test tube to get a thin film covering the loop, and the tube was heated and closed.  The second test tube was heated slightly and the bacteria was placed in the second tube.  The second tube was heated and closed.  The inoculation loop was heated a final time for sterilization. 

Sterilizing the inoculation loop

         Our final experiment of the day was to realize how many colonies of bacteria surround you. For this experiment, we decided to swab the silverware water container in the cafeteria.  We figured this would have tons of bacteria since this is where all the dirty silverware is placed.  We swiped the swab in the dirty water and spread it across the Petri dish.  We put the dish in the 24°C incubator (room temperature). We left the bacteria grow over two nights.
         We examined the colony morphology of the bacteria.  The whole colony form had three types of bacteria: puntiform, circular, and rhizoid.  The colony pigmentation was white/colorless and the colony margin was entire. The elevation of the puntiform colony was raised. The elevation of the circular colony was raised. The elevation of the rhizoid colony was undulate. 

Silverware Bin Bacteria