Nurul Aisha Zainal Abidin
Microorganisms are tiny living cells that inhabit our environment. Most of these microorganisms are harmless, where they do not cause any diseases, hence they are known as non-pathogens (Benowit-West et al., 2009). However, there are some that can cause infections which are termed pathogenic. Certain environments make them necessary that the population of these microorganisms are controlled especially health care facilities, laboratories, food industries, pharmaceutical industries, and more (Hoffman et al., 2004).
Sterilisation is an important method to control the microbial population, where it is the process of removing or killing viable microorganisms from equipment or substances. Sterilisation process can be done via several means including heat sterilisation, filtration, chemical sterilisation and radiation sterilisation (Schlegel and Zaborosch, 1993). Among these methods, the most commonly used is heat sterilisation in moist (steam) and dry form as others each have their own disadvantages such as toxic residues, risk of radiation, high cost, and capability of causing physical damage to equipment (Rogers, 2005). Other than heat being used as physical agents for sterilisation, ionising radiation and filtration can also be used.
Moist heat (steam) sterilisation uses liquid, heat and pressure to form steam which will kill the microorganisms. This process is recognised for its speed of operation, effectiveness, low risk and cost because steam is only pressurised water in gas phase (Block, 1983). It is known that vegetative cells of most bacteria can be killed within 5-10 minutes at a temperature of 60oC however bacterial spores are thermoduric, where they can survive long exposure to high temperature (Bonewit-West et al., 2009). Thus, steam sterilisation is considered to be effective as it destroys viable microorganisms at 121oC for 15 minutes and prevents them from germinating into bacteria. The high temperature applied denatures the proteins within the bacterial endospores, destroying them (Jha and Ghosha, 2005).
Dry heat sterilisation also uses heat to denature the proteins of the bacterial cells. This process involves exposing heat stable solid equipment to a temperature of 160oC for 1-2 hours. However, moist heat is more effective in destroying microorganisms compared to dry heat because water vapour can penetrate into microorganisms more readily than dry air. This heat sterilisation method is done in a metal vessel known as autoclave (Jha and Ghosha, 2005).
Another method for sterilisation is tyndallisation which is usually for materials that cannot withstand high temperatures in the autoclave. They are exposed to 100oC heat for 30 minutes to inactivate vegetative cells but not the spores. These spores that survive will germinate to form bacterial cells during incubation at 37oC and then the material being sterilised is again subjected to steam at 100 oC for 30 minutes to kill these bacteria. This cycle is repeated for the next 3 days (Talaro and Talaro, 1993).
This experiment was done to determine whether there is a difference between efficacy of heat sterilisation (moist heat and dry) and the requirements necessary for this sterilisation process to be adequate.
The aims of this experiment were to understand how a steam sterilizer operates, the role and the importance of having to implement moist heat sterilization process, as well as to identify the basic requirements needed for successful steam sterilization.
Materials and Methods
As per practical manual from page 56-57
Two Thermalog strips were each placed in two Schott bottles; one bottle was tightly capped with no water and the other was loosely capped with water present. Five bottles were prepared and spore strips impregnated with B. Stearothermophilus were placed in bottles 1-4. Some water and paraffin oil was added in bottle 2 and 4 respectively. Schott bottles, bottles 2-4 and a Sterikon plus Bioindicator were sterilised in the autoclave. Thermalog strips were observed after the sterilisation process. 3ml of TSB was added into bottles 1, 2, 3, 5 and the spore strip from bottle 4 was transferred to bottle 5. All the bottles were then incubated. The tables below show the observation made from the experiment.
Table 1. The observation of the “Thermalog” strips in the two Schott bottles with different conditions
No water added
Presence of water
Table 2. The observation of Sterikon vials with one being sterilized and one without being sterilized
Sterikon Vial 1
Clear (Not turbid)
Sterikon Vial 2
Clear (Not turbid)
Clear (Not turbid)
Table 3. B. Stearothermophilus spore strips in TSB with different conditions after sterilization and incubation
3ml of TSB
Presence of water
3ml of TSB
Growth not observed
3ml of TSB
Spore strip mmersed in paraffin oil
3ml of TSB
In the first part of the experiment, steam sterilisation indicator, Thermalog strips are placed in two Schott bottles under different conditions. The tightly capped Schott bottle without any water only had a change of colour within the “unsafe” zone. This is due to the steam from the steriliser that was not able to enter through the tight cap and reach the Thermalog strip. With no water present in the bottle either, steam could not be produced, giving exposure to dry heat. Hence, complete sterilisation was not achieved as direct contact between the steam and the bottle is needed, alongside its temperature and time parameters (121oC and 15 minutes respectively). In comparison, the loosely capped Schott bottle with added water had a change of colour until the “safe” zone. Because the Thermalog strip was exposed to moist heat in the form of steam during the sterilisation process, complete sterilisation is achieved.
In the second part of the experiment, two Sterikon plus Bioindicator vials are used, which could determine the effectiveness of steam sterilisation. These vials have B. stearothermophilus spores along with a pH indicator in a nutrient-filled broth. Both vials were pink at the beginning of the experiment and incubated for several days. The Bioindicator vial that was put in the steriliser showed no colour change while the vial that was not sterilised turned yellow and only slightly turbid. The sterilised vial had no bacterial growth because the spores did not undergo germination to form bacteria due to successful sterilisation which have completely destroyed all bacterial spores. Therefore, the vial retained its pink colour after incubation. However, the colour change from pink to yellow in the other vial indicates that the spores had germinated into bacteria. This is because the vial was not sterilised, thus the spores were able to grow in a favourable condition, whereby they take up nutrients and produce acid which causes the colour change.
These findings show that they are vital for monitoring steam steriliser, ensuring that all spores are properly destroyed. If they are not exposed to its temperature and time parameters, some spores might still survive and germinate. In order to determine that sterilisation process is successful, incubation process is implemented to observe whether these spores could still form new bacteria or whether they really have been destroyed.
In the experiment which used strips impregnated with spores of B. stearothermophilus in tryptone soy broth (TSB), bottle 1 appeared to be the most turbid among other bottles, which suggests bacterial growth is sustained. Because bottle 1 was not autoclaved, it did not go through proper sterilisation process prior incubation, thus allowing the spores to germinate from the spore strip. Culturing this unexposed spore strip in bottle 1 therefore acts as a control, as it would not have demonstrated that steam sterilisation was actually successful if bacterial growth was not observed because they could have not been able to germinate at all.
Bottle 2, however, shows that steam sterilisation was done successfully as the TSB media does not show any turbidity, thus bacterial spore activity was not there. As mentioned, water was added to bottle 2 before it was tightly capped and put into the autoclave, which evaporated into steam (or moist heat) at a 121oC within the steriliser. The steam formed will then kill the spores directly.
Apart from that, bottle 3 was tightly capped and had no water added before it was placed in the autoclave. As a consequence, the moist heat could not possibly have direct contact with the spores to be able to kill them. This meant that the spores were only subjected to dry heat sterilization within the bottle, which clearly showed to be a less effective of a method compared to moist heat sterilisation. Because these spores survived the dry heat sterilisation process, they were able to germinate and form bacterial growth under the favourable conditions during incubation, making the TSB media turbid. If dry heat sterilisation was to be implemented to eradicate spores, a higher temperature would suffice. Furthermore, bottle 5 also showed turbidity to almost the same degree as bottle 3. The spore strip in bottle 5 was initially immersed in paraffin oil in bottle 4, before it was transferred into bottle 5. Other than the tightly capped bottle preventing the moist heat from entering, the oil somewhat acts as a protective barrier for the spores, not even allowing dry heat to have direct contact with the spore strip.
Based on these findings, it demonstrates to a certain extent to how the biocidal action of moist and dry heat is different and can be compared. Most importantly, the role and the significance of the requirements needed for each sterilisation method. For moist heat sterilisation, a holding time of 121oC for 15 minutes under a pressure of 101kPa is required. In contrast, dry heat sterilisation needs a holding time of 1-2 hours at a temperature of 160oC (Arora, 2003). Therefore, it can be said that moist heat (steam) can perform faster sterilisation, with higher penetrating power as compared to dry heat (Vasanthakumari, 2007). Furthermore, sterilisation using moist heat is more efficient as it uses a lower temperature for the denaturation of protein and the heat in water is also transferred to substances easily (Greenwood et al., 2007).
Hence, it is important to note that for steam to be an ideal sterilant, it must be able to have direct contact with the object (external and internal surface) or substance being sterilised. The reason for this is for its stored energy to be transferred to the object through condensation onto all the surfaces which releases its latent heat. As a result, microorganisms are destroyed. Without this direct steam contact, the sterilisation process would be inadequate (Slatter, 1985).
Even so, moist heat sterilisation still has a limitation, where it is not capable of destroying prions in the same way as bacteria and spores. Prions, which are stable self-replicating proteins, are highly infective in the central nervous system tissue and they are highly resistant to heat (Hanlon and Hodges, 2013). Therefore, in order to destroy these prions, dry heat sterilisation may be implemented with a temperature of 200oC.
Successful and complete steam sterilisation can only be achieved if the material being sterilised have physical contact with moist heat (steam) either from the steriliser or from the water inside the material being vaporised. Without the steam, sterilisation process will not be effective because the dry heat cannot destroy the heat-resistant spores. Furthermore, barriers like oils or fats would also prevent the steam from penetrating. Because there are many interruptions or factors that could influence the efficacy of sterilisation, it is necessary to monitor the process. Thermalog strips can be used to determine if the sterilisation process has met its criteria where the material has been exposed to conditions to be safely sterilised. Sterikon plus Bioindicator vials are also used to monitor whether steam sterilisation has occurred.
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