The overall goal of these experiments is to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion and dye-release assay have utility for the functional discovery of new antimicrobials with lytic activity against specific targets such as a pathogenic bacterium of interest. The main advantages of these techniques are that they are simple to execute, provide flexibility for variation of experimental parameters and can be performed with laboratory equipment commonly found in most microbiology laboratories.

Prior to starting the microslide diffusion assay, the concentration of the antimicrobial enzyme to be evaluated should be determined using a BCA protein assay kit, according to the manufacturer's protocol. Perform a serial dilution of the antimicrobial enzyme, using PBS as reaction buffer. Dispense the protein into individual microfuge tubes and adjust volumes to 20 milliliters using PBS.

Next, prepare a 0.5%agarose solution by dissolving 0.25 grams of agarose in 50 milliliters of PBS. Heat this solution to a boil until the agarose is completely dissolved. Determine water lost during the melting process by weight and add back to the solution.

To inhibit contaminating bacterial growth during incubation of the assay, add 15 microliters of 10%sodium azide in water to the agarose solution, and adjust the temperature of the solution to 50 degree Celsius in a waterbath. If viable cells are used as the substrate, omit the sodium azide from the agarose solution. Thaw the heat-killed bacterial substrate on ice.

Resuspend the bacterial substrate in 12 milliliters of the agarose solution to approximately match the turbidity of a 2.0 McFarland equivalence turbidity standard. Compare the turbidities of the solutions by visualizing a black line on a white background through the solutions, adding bacterial substrate to the agarose until the approximate turbidity is achieved. Immediately but carefully, pipet three milliliters of the agarose substrate solution to each microslide, making sure the solution does not overflow the microslide.

After the slides have solidified, use a 4.8 millimeter diameter cork borer to punch three wells in the agarose substrate layer of each microslide. Add 20 microliters of each adjusted protein serial dilution to the respective wells. Use bovine serum albumin in a negative control well.

As a positive control, use known bacteriolytic enzymes appropriate for the substrate such as lysozyme. Incubate the slides in a humidity chamber at 37 degrees Celsius, or the optimal activity temperature of the enzyme. The length of incubation depends upon the concentration and specific activity of the antimicrobial enzyme.

A typical reaction time is approximately 16 hours. When the incubation is complete, observe the slides under indirect light, and capture images of the developing assay. Use a digital single-lens reflex camera with a 60 millimeter lens at a focal distance of approximately 30 centimeters.

In the dye-release assay to demonstrated in the next segment, the substrate is covalently linked to Remazol Brilliant Blue R dye or RBB. Begin this protocol by making a 200 milliMolar RBB solution. Dissolve 1.25 grams of RBB in a freshly prepared sodium hydroxide solution.

Next, resuspend heat-killed bacterial cells at a concentration of 0.5 grams wet weight in 30 milliliters of RBB solution. For purified peptidoglycan, resuspend the peptidoglycan at a concentration of 0.3 grams wet weight in 30 milliliters of RBB solution. Since subsequent steps are the same, regardless of the substrate used, the labeling will be demonstrated for only the bacterial cells.

Incubate the reaction mixture on a rotating platform for six hours at 37 degrees Celsius with gentle mixing. After six hours, transfer the reaction mixture to a four degree Celsius incubator and incubate for an additional 12 hours with gentle mixing. When the incubation is complete, harvest the dyed substrate by centrifugation at 3000 x g for 30 minutes.

Decant the dye solution from the substrate pellet. Resuspend the pellet thoroughly in Type I water followed by centrifugation. Water the dye-labeled cells three to five times in this manner to remove noncovalently-linked soluble dye from the substrates.

For final washes, transfer the pellet portion that will be used in the dye-release assay to 1.5 milliliter microfuge tubes. The supernatant of the last wash should be clear while the substrate remains blue. Store the dyed substrate suspended in a minimal amount of water at 20 degrees Celsius until ready for use.

To prepare for the dye-release assay, allow the frozen dyed substrate to return to room temperature, and wash twice with the assay buffer that was empirically determined for the enzyme. PBS is used in this case. Calculate the volume of the substrate suspension that is needed by multiplying the 200 microliter reaction volume by the number of dye-release assays to be performed.

To prepare the substrate suspension, first add the dyed substrate to the appropriate volume of PBS. Make a one to one dilution, and take an initial measurement of the optical density at 595 nanometers, using PBS as a blank. Add incremental amounts of the dyed substrate to the suspension until an optical density of 2.0 at 595 nanometers is achieved.

In this demonstration, the dye-release assay will be used to determine the optimal incubation conditions for a protein antimicrobial. First, prepare a stock protein antimicrobial suspension by adjusting the concentration to one milligram per milliliter using PBS as described in the protocol text. Then dilute the stock protein antimicrobial suspension to a concentration of 100 nanograms per microliter.

This concentration gives a final reaction mass of one microgram of protein per volume added to the assay reaction. Perform the reaction assays in 0.5 milliliter microfuge tubes for the thermal range to be assessed. For each thermal condition, add 10 microliters of the stock protein to 200 microliters of prepared substrate suspension.

Incubate in a thermal cycler for eight hours with mixing by inversion once per hour. Following the incubation, arrest the reactions by adding 25 microliters of ethanol to each microfuge tube. To remove undigested insoluble substrate, centrifuge at 3000 x g for two minutes.

After that, while being careful not to disrupt the pellet of undigested substrate, transfer 150 microliters of the reaction supernatant for each reaction mixture to a 96-well flat-bottom microplate. To quantify the enzymatic activity, use a microplate spectrophotometer to measure the absorbance of the supernatant at 595 nanometers. Increased absorbance by the soluble dye released into the supernatant from the labeled substrate is a quantitative measurement of enzymatic activity.

The microslide diffusion assay was used to qualitatively evaluate the activity of an unknown protein antimicrobial against heat-killed Salmonella enterica whole cell substrate. The antimicrobial was added in increasing amounts from wells A through F, and PBS was used for negative controls. After six hours, the size of the clear zone of hydrolysis that formed around each well was observed.

High enzymatic activity qualitatively relates to a larger zone while low enzymatic activity qualitatively relates to a smaller zone. The quantitative dye-release assay was used to determine the optimal reaction temperature for a protein antimicrobial. As seen from the amounts of blue color in the reaction supernatants, and the activity units derived from the absorbance measurements at 595 nanometers, 35 degrees Celsius is the optimal temperature.

The dye-release assay was also used to determine the activity range of an unknown protein antimicrobial against Bacillus subtilis heat-killed substrate. A comparison to the activity range of alpha-chymotrypsin revealed that the unknown antimicrobial enzyme has almost twice the affinity for the substrate. In addition, while the activity of alpha-chymotrypsin begins to plateau around 0.3 micrograms, the activity of the unknown enzyme continued to rise across all enzyme amounts.

Once mastered, the microslide diffusion assay can be completed within 30 minutes. The dye-release assay can completed within one hour. Incubation times for each assay will depend upon enzyme activity and their concentrations.

Expanding upon these procedures, the dye-release method can be modified to determine the chemical influences affecting the enzymatic activity of the antimicrobial enzyme by manipulating aspects of the reaction such as buffer pH and salinity. After watching this video, you should have a good understanding of how to rapidly screen and characterize enzymes for antimicrobial activity using the microslide diffusion and dye-release assays.

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