Hi,
I'm working on phage therapy. In bacterial growth reduction assay, the O.D decreases during first 8 hours and then start to increase which maybe due to cell debris or emerging phage resistant strains. How to figure out the reason for increased turbidity?
In an article i came across the attached figure 2(B). Can anyone please elaborate how they have calculated the ratio of phage resistant strain?
The simple method from publication:
1. Strains were cultivated with the phage with a different multiplicity of infection (MOI).
2. The strains were incubated at 37 °C with moderate shaking for 6 hours.
3. To count the number of phage resistant bacterial cells, the phage was mixed with the upper semisolid medium with a final concentration of 10^8 PFU/ml, which could inhibit the growth of the sensitive strain, and poured onto the plate.
4. The strain cultures were gradient diluted and spread onto the upper-medium (prepared semisolid medium from point 3).
5. After overnight cultivation at 37 °C, the concentration of the phage-resistant strain was recorded and calculated.
6. The gradient diluted cultures were also spread onto an LB agar plate to determine the total bacteria concentrations.
obtained results were probably calculated with the use of the simple equation (to generate results in %):
Cp/Co x 100
Cp=number of colonies on the plate with phages
Co=number of colonies in LB plate
Fulltext:
Article Characterization of a newly isolated phage infecting pathoge...
Bartłomiej Grygorcewicz Can you please tell how do the phage resistant strains emerge in control? Control is supposed to be bacterial strain soley.
No phage is added to control so, the control should only contain sensitive bacterial strain.
here You have some publications about bacteriophage-insensitive mutants formation
Article Assessment of antibiotic resistance in bacteriophage-insensi...
Article Bacteriophage-Insensitive Mutants of Antimicrobial-Resistant...
Article Efficient method for generation of bacteriophage insensitive...
https://doi.org/ 10.1128/AEM.01733-17
Article Bacteriophage-insensitive mutants for high quality Crescenza...
You need to realize that mutations occur randomly, so in any given culture of a bacterium (at high enough cell density) you will have representatives of virtually all mutants (that are not selected against) - even in the absence of any positive selection. The frequency will be low but they will all exist. One thing I always remind my students "a tiny fraction of a really large population is still a large population"
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