On the Way to be Pathologists……… Isolation and Culturing of Bacterial and Fungal Phytopathogens

By Taiaba Afrin

On Tuesday, June 6th the outpace team was supposed to go for a field trip to the UAB community gardens to collect infected leaf samples. But it was heavily raining, so our Dr. Karolina and Xiaoyu (our TA) brought some samples for us, it’s very nice of them. We did the lab into two different sections: Isolation and culture of (1) Bacterial and (2) fungal phytopathogens.

(1) Isolation and culture of bacterial phytopathogens: We had our bacterial infected leaf samples. We cut 4 smaller pieces of infected areas and washed them with 10% Clorox solution for 20 seconds to avoid any other contamination other than our desired bacteria and blot on kimwipe. We rinsed the leaf tissues with sterile water and put them into small tubes of sterile water with a grinding beads and grinded them with a homogenizer. We made serial dilution of 1:10, 1:100 and 1:1000 of our bacterial samples with distilled water. We plated our bacterial samples on three different YPD medium plates with glass beads, which was the fun part. Finally, we sealed the plates with paraffin strips and leave them to room temperature to grow the bacteria. After seven days of inoculation we’ve seen the difference for different diluted samples. From 1:1000 plates we have seen the single colonies.

Picture1

Figure: 1:10 Bacterial Inoculation plate after 7 days

 

Picture2Figure 2: 1:100 Bacterial Inoculation plate after 7 days

 

Picture3Figure 3: 1:1000 Bacterial Inoculation plate after 7 days

(2) Isolation and culture of Fungal phytopathogens: We had our fungal infected leaf samples. We cut 4 smaller square pieces of infected areas and washed them with 10% Clorox solution for 20 seconds to avoid any other contamination other than our desired fungi and blot on kimwipe to remove excessive Clorox. We plated those smaller pieces into V8 medium plates with sterile forceps to avoid any unwanted contamination. Finally, we sealed the plates with paraffin strips and let them grow for seven days at room temparature. After seven days of inoculation we’ve seen the fungal colonies with their spores on the plates.

Picture4

Figure 4: Fungal Inoculation plate after 7 days

This lab was very exciting and effective. I believe everyone was very careful to avoid contamination which an expert pathologist does. We are baby pathologist .

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