The overall goal of this step-by-step protocol is to quantify the abundance of cortex fragments or reducing sugars during bacterial spore germination. This method will detail the steps required to detect the presence of cortex fragments or reducing sugars during spore germination. The main advantage of this technique is most laboratories have the reagents and equipment necessary to successfully complete the protocol.
Begin this procedure by placing a tube of Clostridium difficile spores in a heating block and incubating it 65 degrees Celsius for 30 minutes. After the incubation, place the tube on ice. The germination solution is specific to the species of bacterial spore being studied.
For assaying C.difficile spore germination, we use a solution of 10 millimolar Tris at pH 7.5 with 150 millimolar of sodium chloride, 100 millimolar glycine, and 10 millimolar taurocholic acid in deionized water. Prepare one milliliter of germination solution per sample plus an additional one milliliter. Before beginning the assay, transfer one milliliter of the germination solution to a microcentrifuge tube and set it aside.
This will serve as a blank for cortex fragment detection. Next, to the germination solution, add spores to an OD600 of about 3.0 and briefly vortex. This amount of spores works well for moderating cortex fragment release during C.difficile spore germination.
Immediately after adding the spores, remove a 1.2 milliliter sample to a new microcentrifuge tube and put it into the centrifuge. Then centrifuge at room temperature for one minute at 14, 000 times G.This is the zero time point. After the spin, transfer one milliliter of the supernatant to a fresh two-milliliter screw-cap tube and freeze it at negative 80 degrees Celsius.
Repeat the process of taking aliquots, spinning down, and freezing at regular intervals until the sample has been collected for each time point. Because of time required for centrifugation and sampling, time points of less than two minutes are not possible during this procedure, so these samples are taken once every two minutes for 14 minutes. While preparing the cortex samples, should dipicolinic acid need to be monitored, remove a separate 100 microliter sample and dispense in a microcentrifuge tube on ice.
Also prepare samples for generating a standard curve with the following amounts of N-acetylglucosamine. Zero, 12.5, 25, 50, 100, 250, 500, and 5, 000 nanomoles in germination solution. After a sample has been collected for each time point, remove the samples from the freezer and lyophilize them along with the blank and the standard curve samples.
Then store all of the samples at negative 80 degrees Celsius until use. On the day of the assay, resuspend the lyophilized samples, including the controls and standard curve samples, in 120 microliters of hydrochloric acid with phenol and beta-mercaptoethanol. Next, transfer all of the resuspended samples to fresh two-milliliter screw cap tubes.
Then incubate them in a recirculating water bath at 95 degrees Celsius for four hours. After the incubation, place the samples on ice until they are cool. Then add 120 microliters of three molar sodium hydroxide to neutralize them.
Next, add 80 microliters of a saturated sodium bicarbonate solution and 80 microliters of a 5%acetic anhydride solution to each sample and vortex it. Then incubate the samples at room temperature for 10 minutes. Following the incubation, transfer the samples back to the 95 degree Celsius water bath for exactly three minutes.
Note that this step is time sensitive and longer times can affect downstream signal development. Remove the samples from the water bath and cool them on ice. Add 400 microliters of 6.54%potassium tetraborate tetrahydrate to each tube and vortex.
Incubate the samples in a recirculating water bath at 95 degrees Celsius for exactly seven minutes. This step is also time sensitive. Remove the samples and place them on ice for five minutes.
While the samples are on ice, prepare the color reagent. Dissolve 0.320 grams of DMAB in 1.9 milliliters of glacial acetic acid. Note that this reagent is time, temperature, and light sensitive and should not be made in advance.
After the DMAB completely dissolves, add 100 microliters of 10 normal hydrochloric acid and vortex the sample. Then add five milliliters of glacial acetic acid and vortex again. For the colorimetric reaction, transfer 100 microliters of each cooled cortex or control sample to a new 1.5 milliliter microcentrifuge tube.
Add 700 microliters of the freshly prepared color solution to each sample. Immediately transfer the samples to a 37 degree Celsius water bath and incubate the samples for exactly 20 minutes. After the incubation, transfer 200 microliters of each sample to a clear 96-well plate.
The samples should be yellow in color to begin with but will shift to purple if reducing sugar, which indicates the presence of cortex, is present. Here, many of the samples do not show a visible purple color. However, the 250, 500, and 5, 000 nanomole samples appear visually different from the negative control zero nanomole.
Using a plate reader, determine the absorbance at 585 nanometers. When measured in an OD of 585 nanometers, the signal generated from the standard samples best fits a linear regression. This table shows typical results using N-acetylglucosamine standards.
These data were then used to generate the standard curve shown here. A germination assay was performed under germination promoting conditions with germination buffer supplemented with 100 millimolar glycine and 10 millimolar taurocholic acid. In non-germinating conditions, 100 millimolar glycine only.
In the absence of taurocholic acid, C.Difficile spores do not germinate, and there is little change in the presence of cortex fragments in the solution. However, in the presence of both taurocholic acid and glycine, C.difficile spores germinate and release cortex fragments into the solution. This is absorbed as an increase in the OD585 over time of the reacted samples.
The quantity of cortex fragments released during this assay was calculated using a standard curve. As can be seen here in this assay, most of the cortex was hydrolyzed or released by 15 minutes post-germinant exposure. This suggests that most of the spores have germinated in this time frame.
After watching this video, you should have a good understanding of how to detect cortex fragments or reducing sugars in a germination solution during bacterial spore germination. While attempting this procedure, it is important to pay close attention to reaction times so that the assay will develop correctly.
