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MCB2000C Microbiology
Task
Worksheet for the identification of a bacterial unknown- Part A
Divided into different lab lessons, the students will follow the worksheet’s guidelines to identify an unknown bacterial organism. Students will prepare a dichotomous key to identify an assigned unknown organism based on its morphological, physiological, and cultural characteristics (on Worksheet B).
Following the identification of the unknown by interpretation of tests, the students will confirm their findings utilizing genetic information from the bacterium (on Worksheet C).
A. Flow Chart
Create a flow chart that isolates each bacterial species based on their morphological, physiological, and cultural characteristics. This flow chart will serve as a dichotomous key that will guide you in the identification of your unknown. The flow chart’s goal is to isolate all 13 species of bacteria based on the expected experimental results that have been provided.
To create this dichotomous key, each student will only use the information provided to complete the flow chart: List of possible bacterial unknowns, List of tests performed, and the Test results for the bacterial species.
Before proceeding with the exercise, review how to construct a dichotomous key, starting with “Part 2:
Creating your key” from the WikiHow link provided. Then explore how to utilize the shapes and text boxes on PowerPoint to create your digital version of your flowchart by following the links provided.
- WikiHow on creating dichotomous keys: https://www.wikihow.com/Make-a-DichotomousKey
- Microsoft’s Office PowerPoint training to Create a flow chart: https://support.microsoft.com/en-us/office/video-create-a-flow-chart-af4e3f4c-3854-486a88ff-eb35692663dc
- Slidesgo tutorial on How to create a flowchart in PowerPoint, follow “Creating a Flowchart from Scratch” set of instructions: https://slidesgo.com/slidesgo-school/powerpointtutorials/how-to-create-a-flowchart-in-powerpoint
Begin the exercise by exploring the list of possible bacterial unknowns and the table containing the list of tests performed with the possible results for each test. Then, utilize the provided descriptions for the bacterial organisms to complete your flow chart.
List of possible bacterial unknowns
Bacillus cereus
Bacillus megaterium
Bacillus subtilis
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Klebsiella pneumoniae
Klebsiella oxytoca
Mycobacterium smegmatis
Proteus mirabilis
Pseudomonas aeruginosa
Staphylococcus aureus
Staphylococcus epidermidis
List of tests performed to describe the bacteria
|
Test name |
Possible results |
Media used |
|
Aerotolerance test |
Facultative anaerobe, Anaerobe or Aerobe |
Fluid Thyoglycollate Broth (FTB) |
|
Catalase test |
Catalase enzyme present- Positive or Negative |
N/A |
|
Citrate test |
Citrate permease present- Positive or Negative |
Simmons citrate agar |
|
Fermentation of carbohydrate- mannitol |
Ferments mannitol with acid production or does not ferment mannitol Ferments with gas production or without acid production |
Mannitol Salt Agar or Phenol Red- Mannitol |
|
Gram stain test |
Gram positive or Gram negative |
N/A |
|
Indole production test |
Indole present- Positive or Negative |
Sulfur, Indole, Motility agar (SIM) |
|
Methyl Red test |
Mixed acid fermentation- Positive or Negative |
MR/VP |
|
Motility test |
Motile or non-motile |
Motility media with TTC |
|
Oxidase test |
Cytochrome C oxidase enzyme present- Positive or Negative |
N/A |
|
Sulfur reduction test |
Hydrogen sulfide production- Positive or Negative |
Sulfur, Indole, Motility agar (SIM) |
|
Vogues-Proskauer test |
2,3-butanediol fermentation- Positive or Negative |
MR/VP |
Test results for bacterial species
All test results according to Bergey’s Manual of Determinative Bacteriology 9th ed.
Bacillus cereus- Gram (+). Motile. Facultative anaerobe. Catalase (+). VP (+). Ferments mannitol with acid production. Citrate (+). Indole (-).
Bacillus megaterium- Gram (+). Motile. Aerobe. Catalase (+). Citrate (-).
Bacillus subtilis- Gram (+). Motile. Aerobe. Catalase (+). VP (+). Ferments mannitol with acid production. Citrate (+). Indole (-).
Enterobacter aerogenes- Gram (-). Motile. Facultative anaerobe. Indole (-). MR (-). VP (+). Sulfur reduction (-). Non-pigmented. Oxidase (-). Catalase (+).
Enterococcus faecalis- Gram (+). Non-motile. Facultative anaerobe. Catalase (-). Ferments mannitol with acid production.
Escherichia coli- Gram (-). Motile. Facultative anaerobe. Oxidase (-). Catalase (+). MR (+), VP (-). citrate (-). Sulfur reduction (-). Indole (+).
Klebsiella oxytoca- Gram (-). Non-motile. Facultative anaerobe. Oxidase (-). Catalase (+). Indole (+). MR (-). VP (+). Citrate (+). Sulfur reduction (-).
Klebsiella pneumoniae- Gram (-). Non-motile. Facultative anaerobe. Oxidase (-). Catalase (+). Indole (-). Citrate (+). Sulfur reduction (-).
Mycobacterium smegmatis- Gram (+). Non-motile. Aerobe. Catalase (+). Citrate (+). Ferments mannitol with acid production.
Proteus mirabilis- Gram (-). Facultative anaerobe. Oxidase (-). Indole (-). MR (+). Sulfur reduction (+). Motile. Citrate (-). Does not ferment mannitol. Catalase (+).
Pseudomonas aeruginosa- Gram (-). Motile. Aerobe. Catalase (+). Oxidase (+).
Staphylococcus aureus- Gram (+). Non-motile. Facultative anaerobe. Oxidase (-). Ferments mannitol with acid production. Catalase (+).
Staphylococcus epidermidis- Gram (+). Non-motile. Facultative anaerobe. Oxidase (-). Does not Ferment mannitol. Catalase (+).
Important note: Test results might be not provided for a given species, do not be concerned about that fact. Each student has been provided with all the information needed to construct this flow chart, and there are multiple ways to construct it. If a result of a test is not provided, you cannot assume it is a negative result. For example, S. pyogenes has no information associated with the Indole test, but you cannot assume the organism is Indole Negative.
Save your flow chart from PowerPoint as a PDF (.pdf) document. Upload the PDF of your completed flow chart to the appropriate Assignment submission on Blackboard.
Before completing Part B your instructor will assign you a number that represents the unknown microbe you have been assigned to identify, but you should be exploring the information about the tests.
MCB2000C Microbiology
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