To ensure biosafety, we want to reduce the amount of mothercells in
the last tank as much as possible. The lambda phage in the genome is quite
effective already. However, the
lysis characterization (see here) showed that it does not lyse all the cells. In fact, some cells are remaining in the sample that can potentially regrow.
Therefore, we also tried to remove the mother cells with a physical method.
Affinity chromatography is not an option we can take, as the minicells have
very similar membrane compositions as the mother cells. The main difference between them is the size. This is why we went for the filtering option. We
wanted to find a new way to filter mothercells from minicells, that is cheap
and with easily accessible materials. In literature, the effective use of sand
for filtration of bacteria has been stated (1).
Hence, we tried out the filtering with aquarium sand, glass beads that have the
size of the sand stated in the literature, and also a column stuffed with cotton.
A culture of MG1655 with an OD of 0.1 was passed through the different filter techniques.
Figure 1. Setup of filtering experiment with sand, glass and cotton (left). Optical density measurements after the filtering experiment (right). Data was normalized to the unfiltered sample.
The OD after filtering with the 0.8µM filter was removing all the
bacteria. The OD of the cell culture was 0. On the contrary, the OD of filtering with sand and glassbeads was even higher than the initial OD of the cell culture. This is probably due to microparticles of sand and glass that were flushed through
as well and that increased the turbidity of the sample.
The column stuffed with normal cotton was delivering surprisingly good results.
The OD of the cell culture was decreased to 12.4% of the initial OD.
To confirm these positive results of the cotton column, we did a second round of
measurements where we also performed CFU counts after filtering. We were wondering
if FFP2 masks could also be used as a filter, so we included them in the following
filtering experiment as well. However, the material of the masks is hydrophobic, which
made the filtering quite difficult. We had to separate the 3 layers of the masks and
take out the middle, most hydrophobic one. The other two layers had to be soaked in the
cell culture for a few hours, in order to be able to pass liquid through. Filtering a
MG1655 culture with an OD of 0.1 through one of the layers and both of the layers were
tested. Then, the OD of the bacterial culture was measured before and after filtering
through the different approaches. Additionally, diluted samples were plated on LB plates
for acquiring CFU counts. The data were normalized to the unfiltered cell culture and plotted.
Figure 2. Optical density measurements and CFU counts after the second filtering experiment. Data was normalized to the unfiltered sample.
The filtering through one layer of the FFP2 mask decreased the OD to 71.4% of the initial OD. The addition of a second layer decreased the OD a little further to 66.3%. On the other side, the CFU counts were indicating higher cell numbers than in the unfiltered sample. We have not used a sterile mask, bacteria being already on the surface of the masks were most likely responsible for those increased CFU counts. The masks should have been sterilized before and LB should have been passed through them and analyzed to account for contaminations.
Another important property the filter must-have is the permeability for minicells. For this, we tested only the layer of the mask and the column stuffed with cotton with a purified minicell sample. The initial OD was 2.25, the data was normalized again to the unfiltered sample and plotted.
Figure 3. Optical density measurements after filtering a purified Minicell sample. Data was normalized to the unfiltered sample.
Filtering through the mask only decreased the OD to 98.7% of the initial one. It lets us assume that almost all of the minicells were passing through. The sample which went through the cotton column had a decreased OD of 40.7%. It seemed like approximately half of the minicell population was retained in the filter.
To confirm the presence of minicells in the filtered samples, microscopy images were taken.
Filtering with cotton column
Filtering with 0.8µM filter
Figure 4. Microscopy data of unfiltered, cotton- and 0.8µM filtered samples in order to analyse the permeability for minicells.
The microscopy images firstly show that the minicell sample was not completely purified. There was still some bacteria present. After filtering with the cotton column the amount of normal-sized E. coli is lower and there are also minicells visible in the image. Surprisingly, also in the sample that was passed through the 0.8µM filter, a lot of minicells are visible, even if the OD measured was 0.
The microscopy was delivering important data, as it confirmed the presence of minicells in the samples after filtering. It has also shown that the decreased OD in the minicell-filtering experiment of the cotton column might not be due to a loss of minicells, but due to partial removal of the mothercell population. The experiment also demonstrated that the 0.8µM filter has optimal filtering properties with complete removal of mothercells and permeability for minicells. But also the self-made cotton stuffed column was providing good results. It is able to filter about 90% of bacteria while being permeable for at least 50% of minicells. For our proof of concept in our hardware, we could therefore use either of them. As the cotton column is cheaper and easier to implement, as no pressure is required to pass the liquid through, we decided to use this method in our minicell-machine design.