24 Nov 2020

Do’s and Don’ts of tissue culture

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

The tissue culture is an efficient technique in the research and commercialization of secondary metabolites, horticulture crops, and plants that are difficult to grow by conventional methods. Some people have started utilizing and working with this technique on a smaller scale as well because of several advantages it provides.

This article will discuss the do’s and don’t of tissue culture which beginners often miss while experimenting and that leads to a great loss of money and effort. You can keep it handy to remind yourself what to do and what should be avoided and carefully performed during experimentation, especially if you are not regular in using this technique. Some of the things mentioned here are mandatory and some are just common sense. So let’s see what they are!

Do’s (Never miss these points and carefully perform your experiment)

1. Always wear personal protective equipment (PPE): To save your cultures from getting contaminated and yourself from harmful chemicals always wear a mask, gloves, disposable caps to cover hair, and a clean lab coat. While working with liquid nitrogen, wear thermally isolated gloves, eye protection, and a splash-proof apron.
2. Try to keep two PPE kits for the washing and media preparation area and the tissue culture area of the lab. You can make it easier by keeping two different colored coats in both labs.
3. Keeping the culture area, media preparation area, and transfer area clutter-free helps to reduce mistakes while performing a particular task. It clears your mind and you will be able to focus on the task at hand.
4. The most common mistake done while working in the lab is preparing multiple reagents or solutions simultaneously and not labeling them. This creates confusion and may lead to the disposal of solutions. So, always label the solutions or reagents with content and dates of preparation as soon as you prepare. This also helps to avoid the use of a solution after its expiration date.
5. Work with one kind of tissue or perform one experiment at a time. Don’t try to multitask but be arranged as much as possible. This will also reduce the chances of cross-contamination by mislabeling and the spread of microbes from numerous opened media bottles due to the generation of aerosols.
6. Always use 70% isopropanol or ethanol to clean the work area or surface. You can use a clean paper towel to wipe the area. This should be done before you initiate the experiment or in between operations.
7. If you are working with multiple cell lines then it’s advisable to maintain separate bottles of media for each cell line in culture/cultivation.
8. Regularly examine your cultures and commercially purchased media for any signs of gross bacterial and fungal contamination. This also prevents the spreading of microbial contamination (if any) invaded your cultures.
9. After you buy media or reagents always check its quality control information and ideally examine its performance before involving it in your experiments. Checking the information will tell you what conditions are required to store your chemicals to maintain their performance.
10. Keep minimum cardboard packaging in all the cell culture areas.
11. Always ensure at regular intervals that all the machines installed in the media preparation area, transfer area, and culture area are working properly.
12. Mycoplasmas are endophytic bacteria that live inside cells. So, always test explants/starting material whenever you are going to work with any for the presence of this bacteria.

Don'ts (You can protect your cultures by just avoiding these simple things)

1. The continuous use of antibiotics should be strictly avoided as this can lead to the generation of resistant strains in the culture and may mask the underlying contamination. The use of antibiotics is also avoided in plant tissue culture because it slows down the growth of cultures.
2. Avoid the accumulation of any kind of waste in microbiological safety cabinets or incubators.
3. Do not make the culture area crowded at one time. It is advisable to have only one person at a time while working especially in the culture or transfer area.
4. Don’t bring cells from unauthenticated sources in the main culture area without completing the quality control checks. You can also use a cell supplier that authenticates cells.
5. Don’t keep cell lines continually in cultures without returning frozen stock.
6. Avoid cultures to run out of media. So, always subculture them after 70% of the media is utilized or after a fixed interval of days suitable for a particular culture.
7. Do not allow the media to cross the expiration date. The shelf life is only 4-6 weeks at +4°C after adding glutamine and serum.
8. Don’t let the water baths get dirty. So, clean them regularly.
9. Don’t allow the essential equipment used in the labs to become out of calibration. This will reduce analytical mistakes.

References

1. https://www.sigmaaldrich.com/technical-documents/protocols/biology/basic-techniques.html
2. https://www.labmanager.com/laboratory-technology/the-dos-and-don-ts-of-cell-culture-19116
3. https://ocw.mit.edu/courses/biological-engineering...