Pharavee Jaiprasart

Bacterial Cell Culture

How to culture bacterial cell 1. Acquire bacterial strains: One might receive bacterial cells as lyophilized vial, agar plate, or stabbed culture. 2. Sub-culture the acquired bacteria: For lyophilized vial-reconstituted bacterial cells with normal saline. For agar plate-pick up one colony and subculture it in another media. For stabbed culture-put some media on the top of the stabbed media, then transfer the media in to another tube. This step has to be done in laminar air flow hood. 3. Develop the infection model. Infect animals with the cultured bacteria. There are several ways to infected animal. For example, injected to peritoneal cavity, to the lungs. How to study bacterial growth and virulence of bacterial strain 1. Collect organs from animal that was infected with the bacteria. 2. Weight the sample collected and put in normal saline solution. 3. Reduce size of the sample with mechanical force such as homogenizing the organ with beads then centrifuge. 4. Use the supernatant portion to make 1 to 10 serial dilutions of sample in normal saline in order to have the right concentration of bacteria that one would be able to count its colony on the agar plate. This step has to be done in laminar air flow hood. 5. Put the bacterial samples from each dilutions and undiluted (neat) on the TSA plates and spread all over the plate with spreader. This step has to be done in laminar air flow hood. 6. Incubate the plate upside down in incubator at 37C for 24 hours. The plate need to be upside down because if there are contaminations, we dont want the precipitated water to drop down onto the agar and let contaminated bacteria grow on the agar. 7. After 24 hours count the colonies in the agar plate. Precaution 1. Wear gloves at all time. 2. Keep 70% Ethanol with you. Spray anything you want to bring into the laminar air flow hood, including your gloved hands with 70% Ethanol. 3. Maintain sterile condition. Use sterile equipment and do everything in a laminar air flow hood.

Pharavee Jaiprasart

Mammalian Cell Culture

How to culture mammalian cell 1. Acquire mammalian strains: Mammalian strains will come in a frozen state. DMSO 510% is used as a freezing medium. 2. Thaw the frozen cells then transfer the growth medium (pre-warmed at 37C) appropriate for the cell line into the centrifuge tube containing the thawed cells. Compositions of growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. 3. Centrifuge the cell suspension. The centrifugation speed and duration varies depending on the cell type. 4. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Get rid of the supernatant. 5. Resuspend the cells in growth medium, and transfer them into the appropriate culture vessel and into 37C incubator with 5%CO2 condition. How to change media 1. Cultured cells are kept incubated in the 37C incubator with 5%CO2 condition. 2. Inspect the cultured cells for the change in color of the media (change in pH can be caused by cell death), the turbidity (turbidity of the media can be caused by bacterial contamination), and inspect cells under microscopes. 3. For cell lines that are floating in the media transfer media out from the culture vessel and centrifuge at the speed and time recommend for that particular cell line. Get rid of the supernatant (old media) and resuspend the cell in new media. Transfer cells back to the culture vessel. 4. For cell lines that are attached to surface use cell scraper to scrape cells from surface of the vessel. 5. For cell lines that are strongly attached to surface and resilient such as cancer cells use enzyme Trypsin to dissociate the cell from surface Precaution 1. Switch on UV light in the laminar air flow hood at least 30 minutes before using the hood. Switch off the UV light while using the hood. 2. Wear gloves at all time. 3. Keep 70% Ethanol with you. Spray anything you want to bring into the laminar air flow hood, including your gloved hands with 70% Ethanol. 4. Maintain sterile condition. Use sterile equipment and do everything in a laminar air flow hood.