I am working with macrophages and want to optimize infection of macrophages with fast growing Pseudomonas and slow growing non-TB causing Mycobacterium abscessus (one bacteria at a time, not co-infection). Things to consider
1) MOI: People use different MOI. How do I know, which MOI to use
2) Infection duration: How long to infect cells with fast and slow growing bacteria?
3) Bacterial escape from phagosome: If I want to study whether the bacteria escape from phagosome, at time point should I study?
First of all, I want to know if you are using macrophages derived from cell line or isolate from the animal model?
1) For MOI, you can perform studies by infecting the macrophages with different MOI of Pseudomonas and Mycobacterium abscessus. Allow the bacteria for internalization into macrophages and you can check this by Gram-staining after 1 hour or 2 hour. This will give you an idea about the internalization time. And study macrophage cell death after 24 hour if the macrophages are isolated from animal model. This will give you an idea about the MOI, I mean the percentage of macrophage death at particular MOI, and this experiment will also give you an indication about the morphology of the infected macrophage (whether the cell is living, undergoing apoptotic death or necrotic death).
2) Gram staining after infection at different time points (eg., after 1 hour of infection and so on) will give you an idea about this. You can calculate the phagocytic index as well.
3) This differs from bacteria to bacteria, you can check it in the literature if it is available with the macrophage and bacteria you want to study. In case if is not available, you can perform TEM of the infected macrophages after 3-4 hour of infection. This will give an idea about phagosomal escape of the bacteria and additional information as well such mitochondria morphology, interaction between ER and mitochondria, autophagy etc.
For better understanding, you can look into this paper: Aeromonas hydrophila induced head kidney macrophage apoptosis in Clarias batrachus involves the activation of calpain and is caspase-3 mediated
Establishing a bacterial infection model involves several steps to ensure reliable and reproducible results. such as
Selecting the Bacterial Strain: Choose a bacterial strain relevant to your research question or the specific infection you want to model. Consider factors such as virulence, pathogenicity, and availability of mutant strains if genetic manipulation is required.and Culture Preparation: Grow the bacterial culture under appropriate conditions, including temperature, pH, and media composition. Ensure that the culture is in the logarithmic growth phase, as bacteria in this phase are metabolically active and most virulent.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology