BAP Media Procedure
Name: _____________________________________________________
Course Section: ______________________________________________
The following activity will teach students how to inoculate a blood agar plate. Post-inoculation, students will use their colony growth to differentiate hemolytic patterns.
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Materials
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1 blood agar plate
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3 stock plates
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Stock plates will contain 1 of the following species:
Escherichia coli, Bacillus subtilis,
or
Staphylococcus epidermidis
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Sharpies
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Parafilm
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Inoculation loops
or
sterile cotton swabs
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Incinerator
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Step 1:
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Get into groups of 2.
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Label the bottom of your blood agar plate with your initials, course section, and date.
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Using a sharpie, split the bottom of the agar plate into
3
sections and label each section with a different bacterial species.
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Step 2:
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If using a sterile cotton swab, move to step 3.
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Heat your inoculation loop in the incinerator for at least 10 seconds until the wire is red/orange.
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Allow the loop to cool, but do not wave it around the air.
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You can test if the loop is cool by touching it to an area of agar that does not have bacteria and listening for a "sizzle" burning sound.
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Step 3:
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Aseptically collect 1 colony from the stock plate. You do not need to swipe or gather a large amount of bacteria.
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Partially remove the lid from your labeled sample plate, enough that you can reach the agar with the loop/swab while still shielding the plate.
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Gently touch the loop (or swab) to the agar and move it back and forth in a zig-zag motion over one-third of the plate.
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Be careful not to press too hard as you can cut or damage the agar.
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Make sure you are adding bacteria to the correctly labeled areas.
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Step 4:
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Repeat Step 2 for the remaining bacterial species.
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Make sure you are sterilizing your loop between bacterial species.
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If using sterile cotton swabs, make sure to obtain new swabs for each species.
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Do not drag bacteria into the different labeled areas of agar. This is not a quad streak.
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When you have inoculated all 3 bacterial species on 1 agar plate, seal your plate with parafilm and place it upside down in the 37°C incubator for 24 hours.
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Results
In the table below, record your observations for colonies grown in blood media.
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Species
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Growth (+/-)
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Hemolytic Activity
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Interpretation
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Bacillus subtilis
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Staphylococcus epidermidis
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Escherichia coli
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General Questions
1. How can contamination be minimized when inoculating blood agar plates?
2. Define fastidious bacteria.
3. Define hemolysis and differentiate between the three types.
4. How can one distinguish between the three hemolytic patterns on blood agar plates?
5. How can blood agar be used to identify pathogenic bacteria in clinical samples?
6. If a patient has a throat swap cultured on blood agar, and the resulting bacterial growth induces beta-hemolysis, what diagnosis would you expect for the patient?