Bacteria Growth Inhibition
Comparing Antibacterial Potency
See the related protocol on Agar Overlay Technique
EQUIPMENT AND SUPPLIES:
Fresh overnight cultures of Escherichia coli B and Staphylococcus aureus
2 prewarmed nutrient agar plates (or tryptone soy agar) per student
melted top agar (Nutrient Broth + 0.75% agar)
sterile 13 x 100 mm capped test tubes
45°C hot block
micropipetters with sterile yellow tips
assorted putative anti-bacterials
sterile (thick) filter paper plugs, 1/4 inch in diameter
Compile a list of the putative anti-bacterial agents which you wish to test for their ability to inhibit bacterial growth. Up to seven may be used on a plate if they are carefully applied and are not allowed to spread across the plate. The list of possible agents which you could test is very long.
garlic, fresh, crushed
garlic, fresh, cut with razor blade, not crushed
Draw the plate in your book, life-sized and identical to original. List the agents in a table and describe how each is prepared and quantities applied to the plate. Leave an empty column to record the size of the zone of inhibition observed.
Mark the plate bottoms with seven well-spaced small (1/4 inch in diameter) circles:
one in center, six arranged hexagonally around it, at least 1/2 inch from the edge).
Label each circle with the agent to be tested.
Write your initials and the date in small letters at the upper edge of the plate bottom.
Prepare the inoculated agar overlay blanks: Add 0.1 mL of a fresh ON culture of E. coli B to 2 mL of 45°C melted top agar.
Use of an Eppendorf repipet simplifies the addition.
Repeat for Staphylococcus aureus blanks.
Diagram for making the agar overlay.
Mix and pour over the surface of a pre-warmed nutrient agar plate.
Shake gently side to side to ensure that the entire surface of the plate is covered. Do not take too long or the top agar will begin to solidify.
Let sit undisturbed for several minutes to gel. (See protocol: Agar Overlay Technique.)
Place a filter paper plug at each spot where a liquid agent will be tested. (Solids need no filter paper).
Apply the putative anti-bacterials: Add 10 to 15 uL of each liquid diluted according the directions for use to the filter paper plugs. (Deliver slowly so not to overshoot.) If they are viscous, dilute them 10x so they can be pipetted. If solid, deliver to the agar surface a crystal about the size of a head of a pin for known poisonous agents, 10x that much for probable non-toxics. Carefully note in your notebook how they were prepared and/or the quantities applied. Let sit to allow liquids to absorb
Press the disc into the top agar to ensure exposure to the agent.
Invert the plate, incubate at 37°C for 24 to 48 hours.
Measure the zones of inhibitions around each agent in millimeters. Draw the zones of inhibition onto your notebook illustration of the plate, with circles corresponding to the zone of inhibition. Record the size of the inhibition zone in your table.