The overall goal of this procedure is to sort mycelial pellets formed by filamentous streptomyces according to size, using a large particle COPA flow cytometer. This is accomplished first by the growth and subsequent sampling of the desired bacterial strain. In the second step, the sample strain is measured by coppa.
The data is then analyzed in silico and the mycelial pellets are sorted according to user-defined parameters. Ultimately, these analyses can be used to further characterize the pellet size heterogeneity. Though this method can be used for streptomyces, it can also be applied to other organisms such as filamentous fungi, demonstrating this procedure will be MaRous Petrus, a PhD student from our lab.
First, grow the cultures by adding 100 milliliters of yeast extract, malt extract medium and 0.2 milliliters of 2.5 molar magnesium chloride to one sterile 250 milliliter erlenmeyer flask, equipped with metal coiled springs for each bacterial sample. Then inoculate each flask with one times 10 to the eight spores of streptomyces to obtain a spore concentration of one times 10 to the six spores per milliliter and grow the bacteria at 30 degrees Celsius with shaking at 180 RPM. After two days, use a sterile five milliliter pipette to collect a five milliliter sample from each culture while gently shaking the flask to evenly distribute the cells.
Then dispense each sample into individual 15 milliliter conical tubes. To evaluate the samples by copus analysis, confirm that the copus plus pump computer and 488 nanometer argon laser are turned on, and then open the bio source software. Confirm that the sheath fluid bottle is full and that the waste bottle is empty, and then click start and run.
To switch the 488 nanometer argonne laser from standby to on after approximately 60 seconds, the laser will be ready. Click, done, and then check the pressure gauges. Click the checkbox next to pressure.
Okay, and then after the system primes the flow cell, confirm that the delay setting is at 11 and that the width is at seven. Set the thresholds at 50 for signal and 40 for time of flight minimum. Then remove the leftover water from the sample cup.
Add about 50 milliliters of PBS, and then add 0.1 milliliter of one of the strep demy samples into the cup. Make sure the cup is closed properly and then click acquire to start collecting data. Manage the flow speed to obtain between 30 and 50 events per second when at least 2, 500 events have been collected.
Click, stop, and then store to save the data for subsequent statistical analysis to sort bacterial pellets for each sample. First set sorting limits and enter details about the minimal and maximal time of flight values. Alternatively, select regions and then define gate region to create an area for selecting the pellets with the desired dimensions and properties.
Place a 50 milliliter tube to collect the pellets that meet the sorting parameters. Then set the number of pellets to be sorted and click sort manually to sort for the selected parameters. After the sort, remove the leftover sample and rinse the sample cup with water twice.
Before shutting down the copus, clean the sample cup with 70%ethanol. Now run the system twice. Once with chlorinated water and once with plain water, then empty the overflow container After cleaning, click stop to release the pressure from the system.
When the motor stops, close the program and click shut down without purging. Then switch off the computer, the copa, the laser, and the pump streptomyces form. Mycelial pellets and liquid cultures that have a wide range of sizes.
To analyze the pellet size distribution, a two day old liquid grown streptomyces cila color culture was subjected to large particle flow cytometry using a copus plus profiler equipped with a one millimeter nozzle. Here, a typical copus output scatterplot is shown. The x axis represents the time of flight.
The larger appellate is the longer it takes to pass the laser beam. The Y axis indicates the extinction, which represents the optical density of the object, each point in the corresponds to a single pellet passing through the laser beam. Plotting the data points into a histogram, indicated that the sizes from this representative sample were not normally distributed.
The distribution appeared to be skewed to the right log. Transforming the dataset also did not lead to a normal distribution to assess whether the size distribution could be explained by assuming a mixture of two normal distributions, the data was mathematically modeled. Modeling indeed indicated a population of small pellets consisting of 92%of all the pellets with an average size of 248 micrometers, and a population of large pellets comprising 8%of all the pellets with an average size of 319 micrometers.
Here a representative analysis of micro colonies sorted from the large and small pellet populations are shown. The average pellet sizes of the two populations were used to define the boundaries for sorting so as to limit the sorting of the pellets from the overlapping portion of the two size distributions pellets smaller than 248 micrometers were considered to be from the small pellet population, whereas pellets larger than 319 micrometers were considered to be from the large pellet population. Microscopic analysis of the sorted pellets confirmed their distinct sizes Following this procedure.
Other methods like next generation sequencing or proteomics can be performed to unravel the underlying mechanisms of this heterogeneity.
