Week 10 – Unknown #3
Unknown #3: Identification of 2 organisms. (120 pts. total)
Due date: _____________________________
Name: _______________________________
Section: ______________________________
Unknown Number:_____________________
Background
Your third unknown is a mixture of two organisms. Each organism must be separated and characterized.
You must provide the correct genus and species for each. If your identification is incorrect, you can still receive
partial credit if a full account of all the tests performed is given.
Follow the instructions given for day one and day two and beyond. After that use the flowcharts for
identification (They are a different PDF). No shot gunning is allowed; this means you have to do one test at a
time once you are on the flowcharts. The following two notes are very important:
1. During the week, you should examine tests every 24 hours in order to get accurate results. This is
especially important for the oxidase, catalase, motility, sugar and amino acid utilization, LIA, and TSI
tests. Gram stains and the oxidase and catalase tests should be done using a fresh culture.
2. Gram staining controls (i.e. Staphylococcus sp. and E. coli) will be available during the first two weeks.
We suggest that you use them to make sure your staining technique was done correctly.
A. Day One
1. Pick an unknown and record your number on both the signup sheet and your lab book. Label the unknown
tube with your initials and section number.
2. Examine the visual appearance of the broth. Mix and Gram stain the broth with examination under the
microscope at 1000X.
3. Streak for isolation on two non-selective medium plates and record the medium type used. One plate will be
placed in your drawer (22°C) and one will be placed in the 37°C incubator.
4. Store your unknown broth at 4°C as a backup precaution until you are done with your unknown.
B. Day Two and Beyond
1. Record what temperature the organisms grow best at.
2. You should have two distinct colony morphologies since there are two unknown organisms in your broth.
If you do not, please discuss your situation with your instructor.
3. Transfer one colony of each type to two TSA slants (streak only the surface). You should end up with four
slant tubes (2 for each colony type). Make sure you use the same plate for the inoculations of both
organisms to make sure they are different.
4. Incubate all the slants at the organisms' best temperature. After you get good growth store them all at 4°C.
One slant will be used as your working slant and one will be your backup.
5. You must smell and do a catalase, oxidase, and Gram stain from the 24-hour-old working slants.
6. Everyone must then run the following test on each organism:
a. Fluid thioglycolate for their oxygen requirement.
b. Phenol red or purple glucose fermentation tube for fermentation and gas formation.
c. Motility test
7. Make sure you have filled out the first two tables in the results. Check your results with Tim to see if you
are on the right track. Using this data follow the flow charts to finish identifying your organisms.
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Week 10 – Unknown #3
The Results
Broth Results (18 pts.)
Organism #1
Organism #2
Gram Reaction
Cell Observations
Broth Appearance
Isolation Streak Results (16 pts.)
37°C type of medium:___________
Organism #1
Organism #2
22°C type of medium: ___________
Organism #1
Organism #2
Colony
Morphology:
Include color, size,
texture, margins,
elevation, and any
other unique
characteristics
Required Results (16 pts.)
Organism #1
Gram reaction
Cell Morphology
Oxidase
Catalase
Motility
Oxygen Requirement
Glucose Fermentation
Gas from Glucose
Odor
Optimal growth temp.
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Organism #2
Week 10 – Unknown #3
Test Results Used to Identify Organism #1 (35 pts.)
The Test
The Results
Organism #1 is:
Test Results Used to Identify Organism #2 (35 pts.)
The Test
The Results
Organism #2 is:
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