PRIMARY REQUIREMENTS TO TISSUE CULTURE
The primary requirement of tissue culture is to maintain the aseptic environment. It very much determines the success percentage of the process and impacts the production of the end product. Several sterilization techniques are designed to kill microorganisms invading your cultures. These microbes not only harm your cultures, but your hard work, time, and money you put into culturing the plants.
Once the microbes enter your lab, they spread rapidly to other cultures and kill them. Many scientists use antibiotics and plant preservative mixtures to avoid these tiny organisms from spoiling their beautiful cultures. But, apart from using chemicals, some other sterilization techniques are also must-know processes that are required to completely create an aseptic environment for your cultures.
In this article, you will learn about three different most commonly used sterilizing techniques, their principles, and what purpose they serve. So, let’s begin!
It’s a physical method of killing microorganisms including bacteria, viruses, and spores. This device uses steam under regulated pressure to achieve sterilization of the materials put inside the vessel. But how does it work?
Figure: Image of an Autoclave.
Principle of Autoclaving
It works on the principle of moist heat sterilization. The boiling temperature of the water is 100 °C under normal atmospheric pressure. But, when the atmospheric pressure is increased, the boiling temperature of the water also increases, thus helping achieve a higher temperature for sterilization. So, if the steam pressure inside the vessel is adjusted to 15 lb/inch2, the temperature rises to 121.6°C. Then, the autoclave maintains the saturated steam at a designated temperature and pressure.
The high pressure inside the vessel also facilitates the rapid penetration of heat into deeper parts of the material. The moisture in steam causes a change in the protein structure of microorganisms that leads to loss of function and activity of microbes.
Use of Autoclave
It can be used to sterilize your glass apparatus, solid or liquid media, distilled water, normal saline, discarded cultures, and contaminated media.
If you don’t have a bigger autoclave you can buy a reasonable size pressure cooker to sterilize your materials. You can refer to the plant cell technology Youtube video for more information on the pressure cookers and which one you should use.
2.Hot Air Oven
It uses dry heat to sterilize suitable laboratory materials. It is also known as forced air circulating ovens. It was developed by Pasteur. It can kill bacteria and spores by dehydrating them with dry heat.
Figure: Image of a hot air oven.
Principle of Hot Air Oven
It works on the principle of conduction. The temperature heats the surface of materials and then moves at their cores layer by layer or coat by coat. Slowly, a uniform suitable amount of heat is absorbed by the materials that completely sterilize them.
The device uses a thermostat to control temperature. The thin double-walled insulation on the device keeps the energy or heat conserved inside the device. The suitable temperature for sterile materials using a thermostat is 170 °C for 30 minutes, 160 °C for 60 minutes, and 150 °C for 150 minutes.
Uses of Hot Air Oven
You can sterilize Glassware (like Petri dishes, flasks, pipettes, and test tubes), Powder (like starch, zinc oxide, and sulfadiazine), Materials that contain oils, Metal equipment (like scalpels, scissors, and blades) by using hot air oven.
NOTE: Do not sterilize materials containing water or materials that can melt at higher temperatures. Some examples of materials that can not be sterilized using a hot air oven include surgical dressings, rubber items, or plastic material.
3. Filter Sterilization
Some substances like plant growth regulators or vitamins are heat-labile and can not be sterilized using any kind of heat sterilization method. In these cases, filter sterilization is the best solution! In this method, microbes are not killed but removed or filtered out from the materials.
Principle of Filter Sterilization
It works on the principle of trapping microorganisms. Filters are used in this technique depending on the size of microbes that need to be removed. A filter with a pore size smaller than the particle size is used to capture them. The factors that influence the efficacy of the filtration include:
- Pore size
- The electric charge of the filter
- Nature of the fluid being filtered
Uses of Filter Sterilization
It is used to sterilize growth substances that are thermolabile such as Zeatine, Gibberellic acid (GA3), Abscisic acid (ABA), urea, and certain vitamins. The sterilization of these chemicals is done by using filters of pore size 0.22 –0.45 µ. The technique is also used to sterilize the air in industrial processes. Filtration of air is achieved by using HEPA filters. These filters are designed to remove microorganisms larger than 0.3 μm from isolation rooms, operating rooms, and biological safety cabinets (laminar hoods).
So, these are the three most commonly used sterilization techniques used in tissue culture labs worldwide to sterilize the materials. In our upcoming articles, you will learn in-depth about all these techniques. And, if there is any topic that you want to learn more about, then you can write your suggestions at email@example.com.