Microbiology "unknown" project

Blessed. I am so blessed. I am blessed that I have a super supportive...although mostly absent husband who encouraged me to go back to school and pays for it. I am blessed that I have two children who are older and require less hands-on child-rearing every minute of the day. I am blessed that Caleb has Autism because every bit of progress he makes is cause for great celebration. I am blessed that Jansen has a sweet and helpful spirit and is developing a wonderful, independent, and responsible nature. I am blessed to have parents who are always willing to help watch the children for me after school and/or on weekends while I study and do school work. I am blessed to have a part-time job that gives me just enough of what I need. 

I am feeling so relieved this evening as I am looking at the end of this semester square in the face. In lab semester my group has struggled. I struggled so much that several days, I sat at my table and cried. The material is really difficult and we were frequently left with little supervision. It took me quite a while to get used to this style and just accept it. I have yet to embrace it, but having gone through it, it has made me even more self sufficient. 

All semester, we have been told about this big "unknown" project. We have been working toward it the whole time. Each week, we "learned" new biochemical tests, different bacteria, and so on, and had no idea how it was all going to come together. I learned that the "unknowns" in life make me very anxious. For this particular unknown, I spent three days creating a detailed chart for a 14 Gram Positive bacteria and 16 Gram Negative bacteria. I spent two more days creating a flow chart full of tests to isolate each one, still not knowing how this is all going to play out. But having a plan on paper made me more comfortable. 

Last Thursday, we were told to choose a numbered test tube. I chose #48 because that is my favorite number. Each tube had both an unknown gram positive and unknown gram negative bacteria in it. We had 9 days to complete all tests and come up with results. 

I noted the color in the tube, it's cloudiness, and began testing and taking pictures. 

I gram stained once and got beautiful results. Purple gram positive rods and pink gram negative rods. We could have had rods or cocci, gram positive or gram negative or any combination of them. 

Next, I had to isolate both bacteria. So before I left, I did 4 streak plates on 4 agars (TSA, BHI, NA, and PEA) and put them in the incubator.

On Friday morning, I came in to analyze the growth. The best growth came out on BHI and I had two separate colonies of bacteria, visually quite different. After comparing that growth to the one on PEA, I knew that the large, flat, irregular colonies were my gram positives, and the small, orange, pin point colonies were my gram positives. 

I took a sample from each colony and did another gram stain on each...and although it took me four times, I finally got a pure sample of each. I transferred a bit from each plate to put on a slant for incubation over the weekend.

I also took my gram negative and did a streak plate on MacConkey's agar to incubate over the weekend. Those results would put me a bit ahead of the game on Monday.

Monday morning, I took out my gram negative and noted that it grew, but didn't turn red showing fermentation, so I noted that it was a negative result. 

So then I took a sample from my slant and did and Oxidase test, which turned blue in 4 seconds, yielding a positive result. 

Thankful, I moved on to prepare the next test that would give me differentiation between the two gram negative bacteria that met all the above requirements. It was a 48 hour test, so I wanted to get that one going quickly. I did the Methyl Red-Voges Proskaur test and got it in the incubator, hoping that in two days, I would have an answer. 

Knowing that I needed a confirmation test, and notating that the two gram negatives had different colored colonies, I thought that I had Aeromonas hydrophilus. So I went ahead and did a catalase test, hoping it would turn out positive, indicating a positive result and confirming AH. Bubbles appeared under oil in the microscope and I felt so thankful. 

So then I turned to my gram positive. I had a pure sample so I started with an Acid fast test, which turned purple indicating a negative result. 

That narrowed down my options. Then, instead of doing wet mount test for motility (which would give me a result the same day but is super, super difficult), I set up a SIM motility test, which takes 24-48 hours. That went into the incubator and I moved on. The nest test was a nitrate reduction test, so I set that one up, and got it in the incubator to check along with the motility test the next day. 

I did a catalase test for that one to record the results and got bubbling immediately, so it was positive. 

Tuesday morning, I came in to check my SIM motility and it was positive for growth and movement. 

I checked the nitrate reduction, and added the reagents, praying for a change to the color red, which meant I was on the right track. Red, it was!!

I was so close to the answer. When I checked the chart, I was down to Bacillus cereus, and needed a confirmation test. So I did an endospore test. That I was able to view immediately, within 20 minutes, and it was positive for endospores, confirming BC. 

Wednesday morning, I was able to take my 48 hour MR-VP test out and add the reagents. I waited an hour for results, and it turned out red for positive. I compared that to the chart and confirmed it to be AH. 

Now I had taken all my pictures and left the school itching to get home to put it all together and be finished with it. 

I created a neater version of my testing chart for each, my flow chart for each, and then added a Word document with the photos of my tests as I worked toward my result. Tomorrow, I will be heading to school in the morning to turn it all in and it only took 5 days. Oh, how I hope it is correct. 

This class has taken over my life. All that is left is a 10 minute presentation next week and a final after Thanksgiving. We took our 4th test, and turned in a lab quiz, and a lab report that we are waiting to be graded. 

Almost done!!!!!!!!