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While microorganisms are found on nearly every surface imaginable, certain industries such as the medical, and the food production industry depend on being able to minimize the adverse effects associated with some of these microbes. This is most easily accomplished by the annihilation of these organisms, before they pose a problem. This is what is known as sterilization, or more correctly "the elimination or destruction of all forms of microbial life" (Rutala and Weber, 2002). Sterilization may be performed by means of dry or moist heat (steam), chemicals, or radiation (Dutta, 2008). However in the case of microorganism 'death' is a phenomenon that is hard to categorize (Phillips and Warshowsky, 1958). This is because some of these methods meant to be bactericidal are merely bacteriostatic (Phillips and Warshowsky, 1958). In addition there are many drawbacks associated with a number of these methods, such as toxicity hazards, physical damage to equipment and expense in carrying out procedure (Bruch, 1961). As a result heat in the form of dry or moist heat (steam) is made use of for sterilization purposes (Dutta, 2008). Heat at 60oC is sufficient to kill most vegetative microorganisms such as bacteria, fungi and viruses. However bacterial endospores are known as thermoduric, as they are able to survive long term exposure to extreme temperatures, beyond the boiling point of water. These thermoduric bacteria most commonly "belong to the genera Bacillus, Clostridium, Mycobacterium, Micrococcus, Streptococcus and Lactobacillus" (Edema and Akingbade 2007). A significant concern with such thermoduric spores is that they are the causal organisms of diseases such as anthrax, caused by Bacillus anthracis (Lemieux et al., 2006). Furthermore these thermoducric spores also lead to significant negative economic consequences (Rajput et al., 2009). For instance by the spoilage of food products such as milk due to their heat resistant nature, enabling them to survive the process of pasteurisation (Edema and Akingbade, 2007). Pasturisation involves heating milk to 63° C for 30 min (Edema and Akingbade, 2007), and hence this method, successfully destroys vegetative microorganisms but not the thermoduric spores. In addition to these concerns, there is also high risk of of contamination due to inadequate sterilization in practical aspects such as in the medical field, especially in a surgical context. For instance the transmission of mycobacterium infections in various surgeries, as discussed by Wulc and Edmonson, 2006. Due to these reasons there needs to accurate and effective steam sterilization, this is done by means of machine known as an autoclave. The autoclave uses steam under pressure to sterilize materials, and it destroys microbes by "by denaturing proteins important to the organisms survival" for instance enzymes and proteins (Wulc and Edmonson, 2006). Steam is used within the autoclave as it is an ideal sterilant. This is because compared to other sterilizing media discussed previously steam is non toxic in nature, the process of sterilization by means of steam is rapid and effective. In addition it is cheap and readily available, and comparatively easy to use. Furthermore the steam sterilization cycle is in 2 phases, the penetration phase and the holding phase. The temperature that is maintained in the holding phase is built up in the penetration phase. These properties of steam make it the sterilant of choice to be used within the autoclave, and it is this functioning of moist air in correlation with the autoclave that will be explored in this investigation.
Appreciate the mechanisms and principles by which steam sterilization takes place within an autoclave
Understand the significance of moist heat (steam) in the process of sterilization, when compared to dry heat
Examine the methods of evaluating successful steam sterilization, within a practical industry application.
The industry standard for sterilization by means of moist heat, 121oC (101 kpa) for 15 mins is successful in its bactericidal nature, and does not simply function in a bacteriostatic manner. Furthermore industry methods of measuring effective sterilization by means of chemical integrators and biological indicators
Materials and Methods
As per practical manual, pp 35 - 38 (BMS 2052, Class Notes, 2010)
Observation of Thermalog strips
Two 'Thermalog' sterilization indicator strips were placed within 2 Schott bottles, one containing 2ml of water and loosely capped, while the other empty and tightly capped. These 2 bottles along with the 'Thermalog' strips were placed within the sterlizer and maintained at 121oC for 15 minutes. Immediately on completion of the sterilizing cycle the colour change on the 'Thermalog' strips was examined and noted down.
Table 1: 'Thermalog' sterilization indicator strips sterilised at 121oC for 15 minutes within Schott bottles in either dry or moist heat (saturated steam).
MOIST HEAT (SATURATED STEAM)
YES+ : Colour of the 'safe' or 'unsafe' zones on the 'Thermalog' strips changed colour.
On completion of the sterilisation cycle the 'Thermalog' strip exposed to moist heat (saturated steam) changed from transparent to blue in both the 'unsafe' and 'safe zones'. In comparison the 'Thermalog' strip exposed to dry heat only changed colour within the 'unsafe' zone.
Observation of Sterikon plus Bioindicator vials
One of the Sterikon plus Bioindicator vials was placed in the sterilizer, and maintained at 121oC for 15 minutes. The second Sterikon plus Bioindicator vial was not sterilized. On completion of sterilization both vials were incubated at 56oC for 3 days. Following this the colour changes of each vial were observed.
Table 2: Sterikon plus Bioindicator vials incubated at 56oC for 3 days, with or without prior sterilization at 121oC for 15 minutes.
NO+ : Sterilization/incubation performed - : Sterilization/incubation not performed
It was seen that vial 1 remained pink (showed no colour change), while vial 2 which was not sterilized prior to incubation changed colour from pink to yellow.
Observation of spore impregnated strips in TSB
Strips of paper impregnated with Bascillus stearothermophilus were placed in bottles 1 to 4. Several drops of water were added to bottle 2, while paraffin oil was added to bottle 4 until the spore strip was completely immersed in oil. The caps of bottles 2,3 and 4 were screwed on tightly and sterilized at 121oC for 15 minutes, bottle 1 was not sterilized. On completion of sterilization 3ml of tryptone soy broth (TSB) media were added to bottles 1,2,3 and 5. The spore strip from bottle 4 was then aseptically transferred to bottle 5, and bottles 1,2,3 and 5 were incubated at 56oC for 3 days. Upon completion the turbidity of each of the bottles was examined and recorded.
Table 3: Bascillus stearothermophilus spore impregnated strips in tryptone soy broth
media (TSB), incubated at 56oC for 3 days with or without prior sterilization in
different conditions at 121oC for 15 minutes.
of 'Thermalog' strip
Few drops of water
NO+ : Sterilization performed - : Sterilization not performed
Upon observation it was seen that bottle 1 had the highest amount of turbidity of all the bottles. Both bottles 3 and 5 showed turbidity, but to a lesser degree when compared to bottle 1. Of these bottle 3 showed the lowest turbidity. In contrast bottle 2 showed no signs of turbidity.
Bioindicators such as Sterikon plus Bioindicator vials and Chemical indicators such as 'Thermalog' strips are used in autoclaves, in order to ensure satisfactory sterilization has taken place. This is because the gauges on the autoclave used to measure temperature and pressure are not accurate on their own, due to the possibility of pockets of air forming or leakages in the autoclave that alter temperature within the autoclave. (Lee et al.,1979).
The first part of the investigation focused on the sterilisation of the 'Thermalog' strips within Schott bottles, from this it can be seen that the 'Thermalog' strip exposed to moist air/saturated steam (Table 1) changed colour within both the 'unsafe' and 'safe' zones. The colour change within the 'safe' zone indicates that the correct criteria for sterilisation have been met, and the conditions of moist heat are adequate for sterilisation. In comparison the 'Thermalog' strip exposed to dry heat (Table 1) only changed colour within the 'unsafe' zone, it can be seen that the correct criteria for sterilization has not been met.
In the part of the investigation that focused on the usage of Sterikon plus Bioindicator vials, each vial consists of the medium known as the broth, a pH indicator, sugar and spores from Geobacillus stearothermophilus, which are non-pathogenic in nature (Merck ,12th edn). The heat resistance of the Geobacillus stearothermophilus spores is such that they are destroyed if exposed to compressed steam at temperatures of 121oC or higher and pressures of for approximately 15 minutes (Merck, 12th edn). The temperature within the autoclave used for the experiment is 121oC and the exposure time is 15 minutes, and hence any vial of Sterikon plus Bioindicator should be properly sterilized. If however the time or temperature the spores were subjected to was lower than this level, a small proportion of the spores will be able to survive and become active in favourable conditions (Merck , 12th edn). After being autoclaved the adequacy of sterilization is investigated by means of incubation (Merck, 12th edn). This is also done to ensure that these organisms have been killed and not simply been immobilised, that is, after sterilization it is observed if they multiply to form new offspring when placed in a favourable environment, presented during incubation (Phillips and Warshowsky, 1958).
Upon completion of incubation vial 1, which was sterilized prior to incubation (Table 2) remained red-violet (showed no colour change). This lack of colour change is indicative of no spore activity, meaning that successful sterilization has taken place and that all the Geobacillus stearothermophilus bacterial spores have been destroyed (Merck , 12th edn). Hence the media of vial 1 does not change colour on incubation.
In comparison vial 2 which was not sterilized prior to incubation (Table 2) changed colour from red-violet to yellow, which is indicative of bacterial spore growth. This is due to the fact that the spores were not autoclaved and hence not sterilised. Therefore these spores, when exposed to more favourable conditions for bacterial growth become active and produced the colour change in the medium of the vial. This colour change is brought about by an acid produced during the fermentation of the sugars by the bacteria. The acid is detected by the pH indicator, and the turbid yellow colour associated with bacterial activity is produced. (Merck , 12th edn).
Thus it can be seen that sterilization of bacterial spores within an autoclave has a negative effect on bacterial growth, even if environmental conditions following sterilization become more favourable to growth.
From the part of the investigation which focused on the use of strips impregnated with Bascillus stearothermophilus spores, placed in tryptone soy broth media (TSB), it can be seen that bottle 1 was not sterilized prior to incubation (Table 3). This unsterilized (unexposed) spore strip was used within the investigation as it acts as a positive control, exhibiting the normal growth of the spores. This growth is important for showing that the correct medium was used for the growth of the spores, eliminating any potential problems of the spore and medium not being compatible with each other. Furthermore the presence of viable spores is necessary for the successful reaction that results in turbidity (Table 3).
This bottle 1 (Table 3) showed the highest turbidity, indicating highest bacterial spore activity. The favourable environment provided during the incubation period offers a very favourable environment to the unsterilized bacterial spores, which become active and makes the TSB media turbid due to to the fermentation of nutrients within the TSB media
Of the bottles sterilised bottles 3 and 5 showed turbidity, with bottle 5 showing greater turbidity that bottle 3. Bottle 5 contained the spore strip from bottle 4 which was filled with paraffin oil. The presence of the oil along with the lid being capped tightly meant that no moist heat (steam) could reach the spore strip, and so it was only exposed to dry heat. Furthermore the presence of paraffin oil whos boiling point is 220oC (and hence in liquid form while in the autoclave at 121oC) means that the spore strip is also somewhat protected by the dry heat due to the liquid nature of the oil. Together this meant that while it was placed in the autoclave and sterilized, the sterilization was not adequate as it did not kill all the spores. These surviving spores when placed in the favourable environment during the incubation period became active and made the media turbid.
Bottle 3 was tightly capped before being placed in the autoclave. This meant that the moist heat (steam) could not reach the spore strip, and so the spores were only exposed to dry heat, this again meant that once conditions became more suited to bacterial growth the spores became active and made the media turbid as in the case of bottle 5, although to a lesser degree than was in bottle 5 due to less number of spores surviving in bottle 3.
Bottle 2, showed no signs of turbidity in the TSB media, which is indicative of no bacterial spore activity. As was mentioned in the results section, before being placed in the autoclave several drops of water were added to bottle 2. These droplets of water having a boiling point of 100oC quickly boiled over and turned to steam within the autoclave, which was at 121oC. This meant that although the cap on this bottle was screwed on tightly, preventing moist heat (steam) from the autoclave reaching the spore strip, the water vapour within the bottle functioned as its source of steam sterilizing the spores in the spore strip. Furthermore the fact that the bottle was tightly capped meant that none of the steam could escape the bottle and were contained within it. As the spores were completely sterilized, even when the conditions became more favourable during the incubation period, there were no surviving spores to become active and make the media turbid, and hence the media remained clear, and it can be seen that this bottle has been successfully sterilized.
These findings lead to an important issue, that of the biocidal nature of moist and dry heat. It is when compared to dry heat that the significance of moist heat (steam) can truely be understood. The temperature and time parameters for successful steam sterilization are 121°C for 15 minutes (Wulc and Edmonson, 2006). In comparison the minimal parameters for successful dry heat sterilization is 160°C for 2 h (Wulc and Edmonson, 2006). Hence the use of steam enables faster sterilization rather than the use of dry heat. (3M Technical information sheet). Furthermore steam is a more efficient sterilizer when compared to dry heat as "the intrinsic heat resistence" of bacterial cells is much higher "in a completely dry state" (Dutta, 2008). Thus as a result of this rate of death of cells in a dry condition are lower than those in a moist condition (Dutta, 2008).
However an important feature associated with sterilization by means of moist heat, is that for steam to be a successful sterilant there needs to be physical contact between the material being sterilized and steam. This is because saturated steam needs to condense onto a surface, when it does this it releases its latent heat to the material, which results in destruction of any microbes present.
The industry standard for sterilization by moist heat set out in the hypothesis (121oC ,101 kpa for 15 mins) was successful in sterilizing microorganisms, as well as thermoduric bacterial endospores. This success or failiure of sterilization was readily observable by means of the indicators used in conjunction with moist or dry heat sterilization within the autoclave. The success observed with the autoclave in this small scale investigation is indicative of the usefulness of sterilization by means of moist heat in conjunction of autoclaves in large scale industrial processes. With a more effective research and development, the use of steam sterilization using higher temperatures and greater pressure could be explored, leading to greater practical applications.