Sterilization Techniques in Microbiology Laboratory

Sterilization is destruction of all microorganisms and their spores from the surface of equipment and from certain environments. Sterilization is essential in microbiology laboratory to carry out testing in the microorganism’s free environment because unwanted or untargeted microorganisms can be contaminated with testing. It may give wrong results which leads to make wrong decisions. As an example, if the culture media where there is no gram positive is contaminated with gram positive bacteria, it give positive  results which require further testing for confirm. After usage, culture media must be sterilized before disposal into the environment because culture media contain thousands of microorganisms. The situation become serious, when the culture media contains pathogens which causes extremely health risks. Sterilization techniques are important for the health protection of working people in the microbiology laboratory. Because when they handle pathogenic microorganisms, there is a high probability to infect the people who have low immunity.

                              Several sterilization techniques are available in microbiology laboratory such as heat sterilization, radiation and filtration. Combinations of these approached are used to achieve better sterilization rather than one approaches.

                              Heat sterilization is physical sterilization techniques. The period of heating or temperature of heat can be increased to achieve better sterilization. Moist heat or dry heat can be used.

                              Under moist heat, two methods are available such as boiling and autoclave. In boiling technique, equipment are just boiled at 100◦C. Boiling is only capable of killing microorganisms. It fails to kill their spores. So it is only used when other methods are not available.

                              Autoclave uses steam to kill microorganism. It is able to kill both microorganisms and their spores. It produces steam at elevated temperature and elevated pressure. Steam penetrates the equipment’s surfaces and sterilizes them.  It is operated either at 115◦C for 60 minutes or at 121◦C for 40 minutes at 15 ibs/sq. It is used to sterilize glassware and culture media.

                              Moist heat method has some advantages as well as disadvantages.it does not use toxic material or chemicals. It is expensive. It is highly effective because steam penetrates rapidly the equipment. It cannot be used for sterilization of thermo unstable substances. After sterilization, autoclave left some moisture on the surface of the equipment. It causes the corrosion.

                              In dry heat, three methods are available such as flaming, hot air oven and alcohol dip. Equipments are flaming directly at bunson flame until red hot. Metallic equipment such as loop and needles are sterilized by flaming directory at buson flame.

                              Hot air oven use hot air to sterilize. The heat is evenly distributed throughout the oven by using conventional current. Sterilization is applied in the hot air oven at 115◦C for 60 minutes for one hour period. For better sterilization, either temperature can be increased to 160◦C or the period of the heat can be increased to 2-3 hours. Hot air oven is used to sterilize glass equipment such as petri dishes, flasks and pipettes.

                              After dipping in alcohol, equipment is directly flamed directly in bunson flame. It kills fungi, bacteria and viruses via dissolving their cell membranes.  Glass equipment such as glass spreader and glass rod are sterilized by using dip alcohol. Alcohol is also used to swab the lab bench top.

                              Use of dry heat is also nontoxic. It has low operation cost and easy to install. It is non-corrosive. It is time consuming because it slowly penetrates the equipment than steam does. It cannot be also used for sterilization of thermo unstable substances.

                              Two types of radiations are available for sterilization such as ionizing and non-ionizing radiation. Ionizing radiation uses the short wave length and high intensity radiation. It kills the microbe via reaction with their nuclei. ɤ rays and x rays are used for ionizing radiation.

                              Non-ionizing radiation involve the use of long wave length and low intensity radiation. UV rays are used for non-ionizing radiation. It is impossible to penetrate equipment. Therefore it is only used for sterilize surfaces such as lab bench top.

                              Radiation sterilization is reliable. It does not leave any chemical residues. It can be used to sterilize thermo unstable substances. It is expensive techniques. It requires highly specialized equipment. It is harmful to workers when work with radiation for long time and exposure to radiation in high dose. It is not only killing the microorganisms but it also destroys the properties of media.

                              Filtration sterilization allows the microorganisms to separate based on their size. Membrane filtration, Hamming’s filtration and syringe filtration are used for filtration sterilization. Membrane filtration use membrane to spate microorganisms. The microorganisms that are larger than the pore size are trapped on the surface of the membrane. The microorganisms that are too small than pore size can pass through the membrane. Therefore with the decreasing size of the microorganisms, the pore size is also reduced.  Usually membrane with pore size 0.04µm or below 0.04µm is used. Vacuum is used to increase efficiency of the filtration. Hamming’s filtration is used to filter low volumes. Syringe filtration is used to filter even least volumes. Here pressure is applied to achieve better filtration. Vacuum is not used in both Hamming’s filtration and syringe filtration.

                              Filtration sterilization is used to sterilize thermo unstable substances such as culture media, enzymes, vitamins and antibodies. It filters large volumes more rapidly. It helps to separate microorganisms based on their size. It is inexpensive. The smallest microorganisms are able to pass through the membrane. Membrane absorbs significant volume of filtrate. It is impossible to separate two microorganism species with same size.