Cell counter of Bacillus subtilis spores using traditional methods can be a laborious task, as these methods can be completely manual and their accuracies depending on the operator's experience. This protocol makes the enumeration of dormant and germinating spores possible. In addition, the technique also makes it possible to determine the percentage of coping of fluorescent proteins on its surface.
Given the setup step present in this protocol the vision demonstrates with facilities and the technical understanding and the detail of care in this execution. Begin by logging into the cytometer software. In the software workspace, select Cytometer, followed by Fluidic Startup.
Then choose Cleaning Modes. And finally, initiate SIT Flush. Take 50 microliters of autoclave spores and incubate them with ethidium bromide at a dilution factor of 0.05%volume by volume for 30 minutes protected from light.
Wash the spores three times with PBS by centrifuging at 17, 949 G for 10 minutes, and re-suspending in PBS. Next, add 10 microliters of beads following the manufacturer's recommendations for dilution and analyze the sample using a flow cytometer. Define the gating strategy based on the morpho-metric and fluorescence characteristics of the particles using the negative control as a reference.
Then mix the tube gently. Attach it to the flow cytometer probe and click Acquire. To set the laser power, go to the parameters tab in the cytometer window, adjust forward scatter to 375 and side scatter to 275.
Then select the threshold tab and set it to 500. Next, analyze the dye-free sample containing only spores to eliminate autofluorescence. In the parameter tab, adjust the filter detector three's voltages to 603 and filter detector five's voltages to 538 to differentiate between negative and positive populations using fluorescence in the controls.
Then click Compensation, and set filter detector 5 by 3 setup offset to one. To configure the device for acquisition, select Experiment, choose the experiment layout and set the acquisition to 30, 000 events. After adjusting the parameters, acquire data for the samples that are labeled and contain beads in the flow cytometer.
Centrifuge 50 microliters of spores at 17, 949 G for 10 minutes. Next, re-suspend the spores using 25 microliters of 1-ethyl-3-3-dimethylaminopropyl carbodiimide and incubate for 15 minutes. Afterward, add 25 microliters of N-hydroxysulfosuccinimide at a concentration of 50 millimolar to the spore suspension.
Incubate the spore suspension at room temperature for 30 minutes. Wash the spores three times with PBS by centrifuging as shown earlier. Add fluorescent protein to the samples and incubate overnight at 15 degrees Celsius.
Add 10 milligrams per milliliter of ethidium bromide diluted at 1 to 50, then leave the samples on ice for one hour, protected from light. After washing the spores and defining the gates as shown earlier, change the parameters for filter detector three on the x axis, and filter detector five on the Y axis of the dot plot. The counting beads method detected two times 10 to the third spores per microliter in autoclave spore samples.
A ethidium bromide staining of autoclave spore samples showed higher mean fluorescence intensity than the staining of non-autoclaved spores, indicating greater staining of genetic material. The autoclave spore surface, coupled with APC labeled, anti-human interleukin 10 antibody showed greater coupling efficiency compared to the non-autoclaved spores. The presence of spores permeable to ethidium bromide indicated the possible presence of germinated spores in the total population.
Based on the flow cytometry analyses, it was observed that the fluorescent antibody showed a higher percentage of coupling with the dormant spores in comparison to the germinated ones. Moreover, as the concentration of fluorescent antibodies increased, an increase in the percentage of coupled spores and mean fluorescence intensity was observed. It is important to perform a thorough homogenization of the current beads to ensure precise readings through flow cytometry and accurate enumeration of spores.
Standardizing the concentration of antigens attached to spore, Canadian studies analyze the use of spore as vaccine antibodies.
