Unknown Report

0 Microbiology Unknown Project: #69 Enterococcus Faecalis ARIANNA HERNANDEZ BIOL-2420 PALO ALTO COLLEGE DR.LANCE SANDBERG FALL 2022

Table of Contents Table of Contents .................................................................................................................... 1 Introduction ......................................................................................................................... 2 Aseptic Transfer .................................................................................................................. 3 Gram Stain ........................................................................................................................... 3 Quadrant Streak .................................................................................................................. 4 Phenol Red Fermentation .................................................................................................. 5 Methyl Red and Voges- Proskauer Test (MR-VP) ........................................................... 5 Catalase Test ....................................................................................................................... 6 Oxidase Test ........................................................................................................................ 7 Nitrate Reduction Test ....................................................................................................... 7 Citrate Utilization Test ........................................................................................................ 8 Urease Test .......................................................................................................................... 8 Sim Media ............................................................................................................................. 9 Indole Production ............................................................................................................... 9 Motility .................................................................................................................................. 9 Figure 1: Phenol Red Broths ........................................................................................... 11 Figure 2: Quadrant Streak Plate ..................................................................................... 12 Figure 3 Nitrate Reduction test ....................................................................................... 13 Discussion ......................................................................................................................... 14 Conclusion ......................................................................................................................... 14 Appendix A ............................................................................................................................ 15 Appendix B ............................................................................................................................ 16 Resources .............................................................................................................................. 17 1

Introduction Microbiology is the study of microscopic organisms such as, viruses, bacteria, algae, fungi, slime molds, and protozoa. These microscopic organisms live everywhere, 2

on surfaces, foods, even on your body. We just don’t see them physically on our bodies or surfaces, but you better believe they are there. On October 18 we got our unknown specimen. We preformed many different tests to identify specimen 69. We were given a chart with Biochemical test which had the results of 15 different cultures. Some tests were more accurate and more directed toward my organism, which worked out in my advantage. I came to the conclusion that my microorganism was Enterococcus Faecalis. In this report I describe all the test and techniques used to uncover this unknown. Aseptic Transfer To minimize potential contamination of other microbes getting into our test tubes I used an aseptic transfer technique. As soon as I got my unknown it was in a broth, I decided to aseptically transfer it to an agar slant. The reason why I did this was so I was able to refrigerate it and keep it for several weeks while I ran test to identify specimen 69. To do a proper aseptic transfer, the inoculating instrument is a loop, sterilized from the loop to one third of the handle making sure it gets orange-hot, using the Bunsen burner flame. Make sure the loop is pointing downwards at a 45degree angle ass you pass the top of the flame. Then you pick up the broth with the your unknown, mix the broth gently. Remove the cap with your hand that has the sterile loop. Make sure the loop is cooled for at least 5 seconds or you will kill all the microbes. Hold the tube at an angle, flame the tube’s cap and lips by passing it quickly through the flame two or three times. This is to kill all the microbes around the tube. I did this aseptic transfer for every tube I opened. 3

Gram Stain Using a differential stain like this one allowed us to see the differences between organisms or differences between parts of the same organisms. Using the grams stain helped me to determine the cell size, morphology, and arrangement . To conduct a successful gram stain, you have to have use a 3 heat-fixed slide. Then aseptically inoculating broth and putting on the slide, letting it dry. Once dry you cover the smear with crystal violet for 1 minute. Hold the slide at an angle gentle rinse the slide with distilled water. Cover the smear with gram iodine for 1 minute. Hold the slide at an angle, while gently rinsing with distilled water. Still holding the slide at an angle decolorize with gram decolorizer until the runoff is clear. After the runoff is clear immediately rinse the slide with distilled water. Counterstain the smear with safranin for 1 minute. Hold the slide at an angle and rinse with distilled water, lastly blot dry with bibulous paper. Microbiology: Laboratory Theory &Application lab manual was used to describe the gram stain technique. The result of this stain was a gram positive, Cocci spherical, 0.6-2.0 micrometers, they appear to look as short chains or in pairs. Quadrant Streak The quadrant streak plate method is to see an isolated colony on an agar plate. This is also helpful because you can take a portion of the isolated colony then it may be transferred to a sterile medium to start a pure culture. To have a perfectly isolated colony, obtain a sample of your unknown on a sterile loop. Leave the sterile agar plate on the table and lift the lid while still hoovering the lid over the plate, then starting at one edge of the plate lighting drag the loop back and forth across the agar surface. You have to be sure not to cut the agar. Once you get the whole side of the plate you 4