Please write a report on identification of your unknown that should include the following:
An introduction outlining the significance of metabolic testing for identification of bacterial isolates.
A complete, properly formatted dichotomous key for identification the 32 potential unknown cultures.
Narratives of the procedures used, and results obtained, from identification of your culture.
The purpose of each test, and how it is used in diagnostic bacteriology, should be
A brief discussions of the species that you identified, including consideration of it’s clinical and/or ecological significance, and a report on some aspect of current research.
Properly formatted References (NOT URLs).
Finding Microbiology Literature
You should already be using PubMed, http://www.ncbi.nlm.nih.gov/PubMed/, to search for biomedical literature.
For microbiology literature, the American Society for Microbiology publishes a range of highimpact
journals, for which Wilkes is site-licensed. You can search ASM journals at http://
Emerging Infectious Diseases, http://www.cdc.gov/ncidod/EID/index.htm, is a journal of the Center for Disease Control and Prevention. EID is a good source of current events in infectious disease.
For Materials and Methods: I obtained a stock mixture that contained one-gram positive and one-gram negative bacterial species. Then I created a dichotomous key was to differentiate the 32 species of bacteria provided in the instructor handout. The following tests were done:
Gram stain Test
• For an agar culture, place a very small drop of water on the slide. Touch a sterile inoculating loop to the culture (don’t try to pick up a clump!), then use this to smear the drop of water into a large patch.
Limiting the volume of liquid used is critical, because nothing else can be done until the smear has air-dried. After the smear has been air-dried, it can be fixed by passing the slide, smear up, through the flame of a bunsen burner a couple of times.
Gram Staining Procedure
1. Prepare a dried, fixed smear as described above. Label the slide with pencil to identify the culture used. Place the slide on a staining rack over a sink.
2. Cover the smear with crystal violet, and leave it covered for one minute.
3. Rinse off the crystal violet by holding the slide at an angle over the sink, then directing a stream of water above the smear. (This way, the water can’t dislodge the smear.)
4. Cover the smear (now stained) with Gram’s iodine, and leave it covered for one minute.
5. Rinse off the Gram’s iodine with ethanol/acetone. Again, hold the slide at an angle and direct the rinse above the smear. Rinse for about three seconds, or until the solvent dripping off the slide is clear. Then, immediately rinse off the ethanol/acetone with water. This is critical to avoid having the solvent in contact with the cells for too long.
6. Cover the smear with safranin, and leave it covered for one minute. Rinse the safranin with water as before.
The slide can be dried by blotting with bibulous paper.Then bacteria must be observed at the highest magnification available,which means using the 100X (white band) oil immersion objective.
My result was: I isolate the Gram negative
Streak bacteria to be tested onto a plate of tryptic soy agar. For this exercise, use
Acinetobacter baumannii and Aeromonas veronii. Both cultures can be placed on the same
plate. Incubate overnight at 37°C.
Using forceps, transfer a 1/4″ filter paper disk to the growth on the plate. Add a drop oxidase
reagent to the disk (the reagent must have been prepared the same day.) Oxidase activity is
indicated by development of a deep purple color on the periphery of the disk within 5 min.
My result was: oxidase pozitive
1.Inoculate duplicate tubes of O-F glucose medium (six tubes total) with Acinetobacter
baumannii, Aeromonas veronii, and Moraxella catarrhalis. Use your inoculating wire to
make a clean stab through the medium.
2.Cover one tube of each culture with ~1 cm Vaspar.
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