Sterilization by Saturated Steam | Experiment
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Many microorganisms are non-pathogenic and can live in harmony with humans as they do not cause disease. However pathogenic microorganisms can be deadly and therefore need to be eliminated from certain environments. These environments can be hospitals; individuals are already unwell and their immune systems are compromised making them susceptible to infection, water treatment, food and pharmaceutical production; supply available to communities making everyone susceptible, and laboratories; contamination of microorganisms can cause conflicting results.
In order to eliminate microorganisms, sterilization of equipment, hospital supplies and production sites are necessary. Sterilization process may involve different methods using heat sterilization, radiation sterilization, filtration, and chemical sterilization. Radiation involves sterilising using gamma waves or ultraviolet light. Chemical sterilization involves using toxic chemicals such as ethylene oxide to sterilise equipment. Filtration sterilises by filtering out microorganism residues from gases and liquids that are sensitive to heat, making them unsuitable for heat sterilization (Goering et al., 2007). Heat sterilization is classified under dry heat and moist heat. Dry heat involves using heat to sterilize by causing denaturation of proteins and oxidative stress onto the cell (Goering et al., 2007).. Moist heat involves using heat and liquid to destroy microorganisms. The most common sterilization method is the use of moist heat in steam sterilization.
Steam is considered an easy and effective sterilant, as it is economical, fast working and is harmless to users. Steam is non toxic and economical as it is simply pressurised water in gas phase. Steam sterilization is a fast working process as steam production does not consume a lot of time and high pressure allows exposure to the entire compartment quickly.
Steam sterilization is an effective process as it can destroy living microorganisms and at high temperatures it can prevent regermination by destroying endospores as well. Steam sterilization acts by denaturing proteins within cells thereby killing the microorganism. Water vapour releases large amount of heat during condensation, this heat allows penetration of endospores to occur thereby killing endospores.
The steam steriliser works using gravity and is therefore often called a 'gravity sterilizer'. The steam sterilizer can have steam be generated from external source or can be produced from a water reservoir internally. Initially the water from a water reservoir or steam from external source enter the steriliser and is heated using a heating element. The steam being produced rises to the top of the chamber leaving cooler air at the bottom. There are drains at the bottom of the autoclave so the cool air can exit the compartment. As the steam fills the steriliser the thermostatic steam trap located at the bottom of the compartment closes. This allows the pressure of the system to build up causing high pressured steam. The timer begins at this point measuring the time set for sterilisation. To maintain the temperature and pressure at set point the heating element turns on and off. After the set time has finished the steam can be removed either to the water reservoir to cool and allow water to condense and be collector before venting to the room, or can be vented straight into the room or a designated safe zone (Dondelinger, 2008).
Problems may occur in steam sterilization where it may not work. This can be due to a variety of technical problems such as leaks in the steam line. To monitor the function of steam sterilisers a Sterikon® plus Bioindicator vial is added to every batch. Sterikon® plus Bioindicator is made of essential nutrients needed for bacterial growth including sugar, Bacillus stearothermophilus spores and a pH indicator. In a working steriliser these pores should be destroyed in steam at "temperature of 121°C and pressure of 1 bar" (VWR, 2002). When all the pores have been killed the vial should stay a pink/red colour. However if the sterilization did not work, in the next 24 hours the B. stearothermophilus spores within the incubated vial will get the opportunity to regerminate. The growth of B. stearothermophilus is facilitated by sugar fermentation producing acid. This acid causes the pH indicator to change colour to yellow and due to the microbe growth the vial will become turbid. (VWR, 2002). This provides an understanding if the steam steriliser is working to safe conditions and helps keep everything sterile.
Another method to monitor steam sterilization is the use of Thermalog strips. Thermalog strips are made of two different outer layers, one side is made of foil and the other made of paper, this paper side allows steam to enter. Within these outer layers there is a chemical enclosed with a paper indicator. This chemical liquefies when steam and heat reaches it allowing it to flow along the paper indicator. The length this chemical moves is dependent on the time of exposure to steam, the temperature of steam and the volume of steam (3M, 2010). On the paper side there are two boxes labelled unsafe and safe. If the steam sterilisation occurs properly the chemical will move into the safe window of the strip. However if it does not there must have not been enough steam produced, not high enough temperature or not enough time within steriliser.
This experimental report addresses the necessities needed for complete steam sterilization and producing safe equipment. In order to understand the requirements needed for steam sterilization, the experiment is conducted using different methods and conditions for B. stearothermophilus spore strips. The experiment is important as steam sterilization has important applications in preventing spread of disease within the community by sterilising medical equipment and giving reliable results by sterilising laboratory equipment.
Moist heat may be more effective than dry heat in sterilization process as moist heat plays a substantial role in sterilising spores. Steam sterilization is the most used method of sterilization yet its affectivity may be dependent on specific operation conditions. Steam sterilization needs to be monitored as problems may arise with its function, determine these methods of monitoring steam sterilization process.
Materials and Methods:
BMS2052 - Microbes in Health and Diseases Practical Class Notes (2010), Department of Microbiology, Monash University. Pages 35 -37.
Thermalog strips were placed in Schott bottles, one with water and loose cap and the other tightly capped with no water added. After 15 minute sterilization at 121°C the Thermalog strips read either safe or unsafe in relation to microbial presence.
Two bioindicators, initially pink, were separated one underwent steam sterilization and the other had no sterilization. After incubation for 3 days at 56°C the bioindicators colours were recorded.
All four screw-capped bottles had one strip of B. stearothermophilus spores inside. These four bottles underwent different conditions, e.g. underwent steam sterilization or had liquids added. All these bottles underwent incubation for 3 days at 56°C.
Steam sterilization experiment shows the affectivity of steam sterilization, the operation conditions and monitoring the process using Thermalog strips and Sterikon plus Bioindicator vials.
In order to determine the requirements needed for steam sterilization Thermalog strips are used to measure affectivity of steam sterilization. In the experiment the Schott bottle with water that was loosely capped had a reading on Thermalog as safe. This is due to steam having direct contact to Thermalog strip as water inside the Schott bottle vaporises when inside steriliser and the loose cap on the bottle allows steam to enter during sterilization. However the other Schott bottle that has no water and is tightly capped has a reading on Thermalog strip as unsafe. The Thermalog strip remains in the unsafe window as it has not had enough contact with steam as the cap was tight thereby not allowing steam from the steriliser into the bottle and there was no water within the bottle so steam could not be produced within the bottle either. Thereby this shows for complete sterilization to occur there needs to be direct contact between equipment being sterilised and steam, a high enough temperature and enough time in the steriliser, all these properties are monitored by Thermalog strips. Thermalog strips are affective at monitoring temperatures and time exposure to steam yet it does not prove that say heat resistance pores will be destroyed at the specific conditions. Therefore Thermalog strips should be used but in combination with other monitoring items.
Steam sterilization monitoring can also be done with Sterikon® plus Bioindicator vials. This experiment shows how the Bioindicator vials work and how effective they are at monitoring the process. Bioindicator vials have B. stearothermophilus spores in a nutrient broth with a pH indicator. Initially both these vials appear to be clear and pink in colour. The Bioindicator vial that is placed in the steriliser stays pink and clear whereas the vial that was not sterilised became cloudy and yellow. This means that the Bioindicator vial sterilised has no bacterial growth, as regermination has not occurred while the vial not steam sterilised did have regermination. Regermination of spores allows formation of bacteria. These bacteria facilitate their growth by fermenting sugar. This fermnattion process generally procuces acidic end products, family of Bacillus do mainly produce lactic acid as an end product. As these products are acidic the pH indicator will change colour in respose to the formation of these products. The pH indicator changes colour from pink to yellow. The bacterial growth will also cause the vial to look cloudy due to 'turbidity' within. The results showed the Bioindicator vials work consistent with what was expected showing that they are an asset in monitoring steam steriliser function as they show
Monitoring the needs to facilitate complete steam sterilisation occurs in the third part of the experiment. Bottle 1 is used as the control showing that the B. stearothermophilus spores have the ability to regerminate from the initial spore strip. If bottle 1 had not shown microbe growth the results obtained would not prove steam sterilization has occurred as the spores may not have had the potential to regerminate at all. Bottle 2 shows that steam sterilization can occur when water is added to the bottle. As the heat within the steam steriliser increases the water within the bottle will vaporise forming steam. This steam will have direct contact with the spores allowing the spores to be completely eradicated. Bottle 3 was tightly capped and had no liquid added to it making it impossible for steam to have direct contact with the spore strip. As the spores were still alive during incubation the spores regerminated and formed bacterial growths within bottle 3, viewed as cloudy. Bottle 3 as it had no contact with steam had only dry heat sterilization working within which is not effective in killing of spores and thereby is less effective than steam sterilization method in bottle 2. Bottle 4/5 was tightly capped and had paraffin oil added to it. It would be expected that this bottle would have bacterial growth as there is no steam in direct contact with the spore strips. The oil could even act as a barrier for any steam, entering through the tight cap, to get in contact with the spores. However the results obtained in the experiment showed that there was no bacterial growth in bottle 4/5. This is most likely due to experimental errors where the spore strip was not completely submerged in paraffin oil and the cap of bottle 4/5 was not tight enough. This would allow steam to enter the bottle and have direct contact with the spore strip as the oil was not covering the whole strip. This experiment showed that for effective steam sterilisation to occur the equipment and instruments must have direct exposure to steam.
Steam sterilization experiment has showed that for steam sterilization to occur direct contact with steam is needed; this can be from direct steam from steriliser or water within vaporising. Steam sterilization experiment could have included a few more alternative conditions such as a loosely capped bottle with no water and a loosely capped bottle with oil. This would have showed steam can enter a bottle and cause sterilization. Also a loosely capped bottle with oil would have been able to tell the effect of oil on direct steam sterilization.
Steam sterilization is a more effective and time efficient process than dry heat sterilization techniques. Steam sterilization can manage to kill heat resistance bacterial spores whereas most dry heat sterilization cannot. There is a dry heat sterilization method that is effective in killing bacteria regerminating from spores called Tyndallization. Tyndallization involves heating equipment and instruments for a certain time ranging from a few minutes to an hour depending on temperature of heating for three to four days. Initially this will kill all existing bacteria and other microorganisms. On the second day the spores would have regerminated allowing the second row of bacteria to also be killed. The third day will allows time for the late germinating spores to regerminate and heating allows them to be killed (Aminot and Kerouel, 1997). This procedure despite its affectivity this procedure still takes several days to complete therefore steam sterilization is the better option.
Sterilization is an important process in hospitals, water treatment facilities, food and pharmaceutical production and laboratories. In hospitals sterilization can prevent the spread of diseases caused by opportunistic pathogens such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia (Goering et al., 2007). Steam sterilization is therefore an ideal form of sterilization in hospitals to prevent spread of disease with the aid of Bioindicator vials to monitor function in every batch and occasional use of Thermalog strips.
Steam sterilization can only occur if the equipment being sterilised has direct contact with steam from steam provided in steriliser or from heat causing water within to vaporise into steam. Without steam contact the equipment is having only sterilization by heat which is an ineffective sterilization method on spores. Oils, fats and other hydrophobic substances should cause barriers for steam penetration making sterilisation less likely. It is important to monitor steam sterilisers as many mechanical interruptions could prevent complete sterilisation. Sterikon plus Bioindicator vials are an effective way to monitor steam sterilisers as they produce consistent results showing whether sterilisation has occurred or not. Thermalog strips can also be used to monitor if steam sterilising machines are reaching conditions that allow safe sterilisation to occur, for example the right amount of steam, temperature and pressure.
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