2. Operating instructions.

Sterilization is an essential process for the operation of a hospital, in which all surgical instruments, implants and many other devices must be used absolutely sterile. Drying and freezing kill many species of bacteria, but others simply remain in a vegetative state. Dry or moist heat eliminates all bacteria by properly combining factors such as the temperature to which they are subjected and the exposure time. It can be sterilized by dry heat in ovens at more than 160 °C for half an hour, or by moist heat in autoclaves at 120 °C for 20 minutes and at pressures above atmospheric. Boiling at 100 °C does not kill all pathogenic germs (including not only bacteria but also viruses and yeasts). Another common means of sterilization, used for objects not resistant to heat, are chemical means: carbolic acid, initiator of the era of antisepsis (see Phenol), hydrocyanic acid (see Hydrogen Cyanide), ethylene oxide, chlorhexidine, mercurial derivatives, iodine derivatives (especially povidone-iodine) and many other substances. Ethyl alcohol does not produce complete sterilization. Another means of current sterilization is ionizing radiation (beta, gamma).

Sterilization involves the destruction of all organisms, including spores, and disinfection involves the destruction of vegetative microorganisms that can cause disease, but disinfection does not necessarily kill spores.

The most commonly used methods in microbiological laboratories are:

• Red heat (flamed).

• Dry heat (hot air).

• Pressurized steam (damp heat)

• Flowing steam (tyndallization)

red heat: Instruments such as inoculating loops and wires and dry rods are sterilized by heating them in a bunsen burner flame until they turn red.

Dry heat: It is applied in an electrically heated oven that is controlled by thermostats and that are provided with a large circulating fan that ensures uniformity of temperature in all parts of the content. Modern equipment can bring the temperature to the required level, this device safeguards and protects personnel from accidental burns. Material that can be sterilized by this method includes petri dishes, flasks, glass pipettes, and metal objects. Air is not a good conductor of heat, so stoves must be loaded loosely, leaving plenty of space to allow hot air to circulate. When calculating run times for hot air sterilization equipment, three periods should be considered.

The temperature rise period, which is the time required for the entire load to reach sterilization temperature; it may take about an hour.

The maintenance periods at the different sterilization temperatures recommended by the Medical Research Council, which are 160ºC for 45 minutes, 170ºC for 18 minutes, 180ºC for 7½ minutes and 190ºC for 1½ minutes.

The cooling period, which is carried out gradually to prevent the breakage of the glass material as a result of a too rapid drop in temperature; this period takes two hours.

Pressurized steam: It is done by autoclaving. Bacteria are more easily killed by moist heat than by dry heat. The steam kills bacteria by denaturing proteins. An agreed safety condition for sterilization is to use steam at 121ºC for 15 to 20 minutes. Air has an important influence on the effectiveness of sterilization, because its presence modifies the pressure/temperature relationship, in addition, the existence of air pockets will prevent the penetration of steam, all air must be eliminated first, all the air that surrounds and penetrates in the load before steam sterilization can begin.


A good autoclave sterilization depends on the removal of all air from the chamber and the load, of the materials to be sterilized must be placed loosely. Clean items can be placed in wire baskets, but contaminated material should be in a solid-bottomed container no higher than 8 cm. Large air spaces should be left around each container and none should be closed.

There are two types of autoclave:

• The pressure cooker type.

• The displacement by gravity.

For practical reasons we will only deal with the pressure cooker type since it is the one we have in the laboratory.

The pressure cooker type is the most common, it is a device for boiling water under pressure. It has a vertical metal chamber with a strong metal lid that is tightened and sealed with a rubber ring. A spigot for air and steam outlet, a pressure indicator and a safety valve are arranged on the lid. The water at the bottom of the autoclave is heated by external gas burners, an electric immersion heater or a steam coil.

Operating instructions.

There must be enough water inside the chamber. The autoclave is loaded and the lid is tightened keeping the discharge spout open. The safety valve is then adjusted to the required temperature and connected to the heat source. When the water boils, the steam will flow through the discharge spout, dragging with it the hot air in the chamber. Air and steam are allowed to escape freely until all air has been removed. When this phase has been reached, the air−steam discharge tap is closed. Steam pressure rises in the chamber until the desired pressure, usually 1.054 kg/cm2, is reached and steam flows through the safety valve. When the load has reached the required temperature, the pressure is maintained for 15 to 20 minutes. At the end of the sterilization period, the heater is turned off and the autoclave is allowed to cool down. The discharge spout is opened very slowly once the gauge has reached zero, the material is allowed to cool down to a temperature where it can be held in the hands.

The autoclave should never be allowed to cool for a long time, since if it is not opened, a vacuum is formed, which can break the sterile material.

Bennet has developed an indicator tape, which is placed on the materials, and depending on the color of the tape, it is known if it was a good or bad sterilization. In addition, when autoclaves are unloaded, operators must wear full-face protective visors of the type that cover the skin of the chin and throat. They should also wear thermal protection gloves.

Tyndallization: You will use a Koch vaporizer, which is a metal box at the bottom of which water is boiled using a gas burner or steam coil. This method is used to sterilize culture media that may be altered by exposure to higher temperatures. These media are vaporized for 30-45 minutes a day for three consecutive days. The first time, vegetative bacteria are killed; any surviving spores will germinate in the nutrient medium overnight, giving rise to vegetative forms that will be killed during the second and third sprays.


A wide variety of chemicals can be used for this, all of which are commonly called biocides. The effects of time, temperature, pH, and the physical and chemical nature of the item to be disinfected and the organic matter present are often not fully considered. The types of disinfectants are many, and their effectiveness depends on the sensitivity of the microorganisms, the most sensitive being vegetative bacteria, fungi and lipid-containing viruses. Mycobacteria and viruses that do not contain lipids are less sensitive, and spores are generally resistant. When choosing disinfectants, some considerations must be taken into account regarding their toxicity and the harmful effects they can have on the skin, eyes and respiratory tract. The most commonly used disinfectants in laboratory work are phenols and hypochlorites. Aldehydes have limited application and alcohol and mixtures of alcohols are less popular, although they deserve more attention.


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