9: Gram Stain Unknown
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- Valeria Hochman Adler
- Reedley College
Learning Outcomes
By the end of this lab period you will be able to:
- Compile data on a single microorganism from multiple sources (visual analysis of colony morphology, cell morphology, and Gram stain results).
- Use a dichotomous key to identify an unknown organism based on multiple features or characteristics.
Introduction
At this point in the semester, you have learned how to isolate microorganisms on streak plates, produce subcultures aseptically, and perform several different kinds of useful differential stains. We are almost ready to embark on our capstone Bacterial ID project!
Today, we will complete a “mini” unknown project using gram staining and morphological features of the bacterial colonies as well as their cell shape and arrangement. We will also learn how to read a dichotomous key.
Dichotomous keys allow us to identify an organism by asking questions about specific features. Does the organism have wings? Does it have feathers? Typically, a dichotomous key is a series of questions that allow you to narrow down the possible options until a single species of organism has been identified.
We are going to use the following dichotomous key to differentiate between six different species of bacteria.
- Proteus mirabilis
- Escherichia coli
- Micrococcus luteus
- Pseudomonas aeruginosa
- Staphylococcus aureus
- Bacillus subtilis
Materials
- Inoculating Loop
- Bunsen Burner
- Slide Warmer
- Gram Stain Solutions (Crystal Violet, Gram’s Iodine, Decolorizer, Safranin)
- Slides
- Microscope
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Blinded (labeled with numbers but not names!) cultures of each of the following organisms both on NA slants and plates.
- Proteus mirabilis (TSA plate is used)
- Micrococcus luteus
- Bacillus subtilis or Bacillus megaterium
- Pseudomonas aeruginosa (Mueller Hinton plate is used)
- Escherichia coli
- Staphylococcus aureus
Experiment
- Your instructor will provide you with two unknowns on slants.
- Prepare smears and Gram-stain each one. Your lab partner will gram-stain the same two organisms independently so that you can compare your results.
- After looking at your stained bacteria under the 100x objective on your microscope, complete the chart below for your organisms.
- Look at the growth on the plates on the instructor’s desk to see more colony morphology and growth characteristics.
- You will fill out the rest of the chart in class as we discuss everyone’s results.
Data
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Unknown Number |
Gram Stain Results |
Cell Shape |
Color/Growth on Media |
Swarming Growth? |
Organism Identification |
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1 |
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2 |
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3 |
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4 |
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5 |
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6 |