An invitation: Case-by-Case Cell Ghosts preparation
Recently a new protocol for bacterial ghosts (BGs) preparation was introduced. The protocol was given the name “Sponge-Like Protocol” (Amara et al., 2013b). And in some cases it has been given the name “The critical chemical concentration method (CCM)” (Amara et al., 2014; Vinod et al., 2014). It is based on introducing pore(s) in the bacterial cell wall aiming to evacuate it, without damaging their 3D structure (Figure 1 and 2). The evacuation process using this protocol turns the viable microbial cells (or the used cells) to dead ghosts. The cells stay with correct 3D structure and mostly, only one pore is introduced for each. The steps of the protocol guarantee correct evacuation and remove the cells cytoplasmic contents completely. Upon that the viable cells are turned to dead ones but still maintain correct cell walls with correct surface antigens able to induce successful immune response. For that, they can be used as vaccines against themselves. Additionally, non-self-antigen(s) can be expressed on a suitable strain (before turning it to ghost). The microbial Ghosts (MGs) can be used as a drug delivery system and in many other applications. The main idea is to determine the critical concentrations of both of the minimum survive and the minimum killing concentration for the used chemical compounds. Such compounds were selected based on their effect on the cell’s wall; the cell’s DNA/RNA and their ability to introduce pore(s). For that, the serial dilution method was used to determine both of the Minimum Inhibition Concentration (MIC [+1]) and Minimum Growth Concentration (MGC [-1]) of the used chemical compounds (Amara et al., 2013b). Plackett-Burman design was used to optimize the protocol and to map the effective points during the BGs preparation. The best experimental conditions, which give the best results, were used to prepare the BGs. After that and for a simplification, the best two experiments' conditions obtained from the Plackett-Burman design (fourteen experiments) were used only. In some cases such as the virus, LaSota a single chemical compound was used (H2O2). In general, one could change the protocol main components (e.g. the used chemical compounds; the physical conditions (e.g. the shaking rats); the biological conditions (e.g. the microbial ageing time) based on the used microbes and the surrounding conditions. However, both of the MIC and the MGC must be calculated accurately. One should observe any factor could effect on the chemical compounds/cells interactions such as the temperature, time of the exposure; order of the used chemical compounds etc. Any step might be critical and in some cases, the rearrangement of the steps will improve the results particularly in cases such as when the microbe is able to resist one of the used compound but still sensitive to the others. In such case, the most effective compound should be used first to kill the microbe. Also, the resistance to certain compound(s) might give false MIC and MGC. However, in such cases one can use the E. coli (sensitive microbe) MIC or MGC with a particular compound and one can increase the concentration if there is a need for that. Using light microscope could help in precisely determine the best concentration for preparing the ghosts (after the determining of both the MIC and the MGC). For more details about the protocol, the bacterial strains (which were used) and the other Ghosts forms and their applications refer to Amara (2015b) and Amara (2016b) (Amara, 2015b; Amara, 2016b)
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