Laboratory Sterilization and pH Measurement Equipment
Autoclave Sterilization
The laboratory apparatus designed to use steam under regulated pressure to achieve sterilization is called an autoclave.
Principle of Autoclave Operation
Water boils at 100°C under normal atmospheric pressure. If the atmospheric pressure is raised, the boiling temperature for water will also rise. If the steam pressure inside a closed vessel is increased to 15 psi, the temperature can reach 121.6°C. An autoclave is a device that can maintain saturated steam at a designated temperature and pressure at all times.
Working Mechanism
An autoclave is a pressure chamber used to sterilize equipment by subjecting it to high pressure (15 psi) saturated steam at 121°C for approximately 15-20 minutes (or more), depending on the size of the load and the contents. Steam penetrates objects in the autoclave; condensation creates negative pressure and draws in additional steam. Moist heat kills microorganisms via the coagulation of proteins. It is used to remove microorganisms (including viruses, bacteria, and fungi) and spores using high-pressure and high-temperature steam sterilization.
Common Uses
- Sterilization of nutrient media (solid or liquid)
- Glass materials and instruments
- Micropipette tips and distilled water
- Normal saline and discarded cultures
- Contaminated media, aprons, rubber tubes, and gloves
Safety Instructions
- Do not attempt to open the door while the autoclave is operating.
- Do not overload the autoclave.
- No other materials should be autoclaved together with waste in the same load.
- 121°C is the standard temperature, generally achieved when chamber pressure is 15-16 psi.
- Wear autoclave gloves when handling hot items.
- DO NOT autoclave liquids containing bleach, formalin, or glutaraldehyde.
Hot Air Oven
The hot air oven provides a temperature higher than that of the atmosphere. It is used for the rapid evaporation of materials, rapid drying, and the sterilization of materials that can be processed by dry heat.
Principle of Dry Heat Sterilization
Sterilizing by dry heat is accomplished by conduction. The heat is absorbed by the outside surface of the item, then passes toward the center of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization. Sterilization is carried out by dry heat at high temperatures, where bacterial cells and spores die due to dehydration.
Working Process
Inside the chamber, the air flows in a forced circulation manner, allowing for appropriate heat distribution. As the air inside the chamber becomes hot, it becomes lighter and moves in an upward direction. As it reaches the top, the fan inside the chamber pushes it back to the bottom. This creates a circular motion inside the cabinet and ensures a consistent flow of air, eventually achieving the optimum temperature.
Laminar Air Flow
Principle and Working
A laminar airflow cabinet is a highly suitable, convenient, and reliable instrument for aseptic work. It utilizes a number of small blower motors to move air through HEPA (High-Efficiency Particulate Air) filters. These filters remove particles larger than 0.3 µm. The ultra-clean air, which is free from fungal and bacterial contaminants, flows at a velocity of about 27±3 m/minute through the working area. All contaminants are blown away by the ultra-clean air, maintaining an aseptic environment over the working area.
pH Meter
A pH meter is an electronic instrument used to measure the hydrogen ion concentration (pH) of a solution. It is an essential instrument in plant tissue culture for adjusting the pH of nutrient media before sterilization, as pH greatly influences nutrient availability, enzyme activity, and the growth of plant tissues.
Principle of pH Measurement
pH is a measure of the acidity or alkalinity of a solution and is defined as the negative logarithm of hydrogen ion concentration (-log [H+]). A pH meter works on the principle of electrochemical measurement of hydrogen ion activity. It consists of a glass electrode sensitive to H+ ions and a reference electrode. When the glass electrode is immersed in a solution, a potential difference develops across the glass membrane due to the difference in hydrogen ion concentration between the internal solution and the test solution. This potential difference is measured and converted into pH units by the meter.
Working and Application
The pH meter measures the electrical potential developed between the glass electrode and the reference electrode when immersed in a solution. The signal produced is directly proportional to the hydrogen ion concentration. The meter amplifies this signal and displays the pH value digitally. In plant tissue culture, the pH of the nutrient medium is usually adjusted between 5.6-5.8 before autoclaving.