Decontamination Effectiveness of Esco Celsafe CO2 Incubator Sterilization Feature using High Heat Temperature By Bekti Tri Sumaryati

Transcription

1 Decontamination Effectiveness of Esco Celsafe CO2 Incubator Sterilization Feature using High Heat Temperature By Bekti Tri Sumaryati 1. Summary Esco Celsafe CO 2 Incubator sterilization feature, which is 200 C, was observed. The observation was done in its chamber and glass door using many microorganisms, included Geobacillus stearothermophilus spore. The 6 logs reduction of bacterial vegetative cell and spore, and 4 logs of fungi spore were observed. 2. Introduction CO 2 incubator is equipment for embryo cell incubation, one of the mostly used medical device currently. Its cleanliness becomes a concern because the presence of contaminant could bother the cell growth. Contaminant removal can be done by disinfection and sterilization. Disinfection and sterilization are essential for ensuring that medical and surgical instruments do not transmit infectious pathogens to patients (1). Disinfection is the elimination of all pathogenic microorganisms, but not included bacterial spore. Sterilization is the elimination of all forms of microorganism life. Physical and chemical sterilization methods are used in the last decade, with their advantages and disadvantages studies. Dry heat is one of the most common used physical sterilization methods in the health care facility. Non toxic, does not harm the environment, easy to install, low operating cost, can penetrates materials, and noncorrosive to metal are the advantages of dry heat sterilization method. Oxidation of cell constituents is the lethal process of this sterilization (1; 2). Spore treated with high temperature could not grow because of its DNA damage (3). The common temperature and time for this sterilization are 170 C (340 F) for 60 minutes, 160 C (320 F) for 120 minutes, 150 C (300 F) for 150 minutes (1). In the dry heat sterilization cycle, time for the item to reach the sterilization temperature, the holding time of sterilization temperature, and the cooling down of item need to be consider (4). Esco Celsafe CO 2 incubator adopts the hot air oven system (4). It s should keep the hot air at the sterilization temperature and defined time in order to sterilize its chamber and trays. The effectiveness of this feature was observed by biological controls which were vegetative cell and bacterial spore. Bacterial vegetative cell and spore tested was 6 logs, while it was 4 logs of fungi spore. 3. Material and Method 3.1 Material Materials used in this experiment were: 1. Esco Celsafe 170L CO2 Incubator (unit tested) 2. Biosafety Cabinet Class II (Esco Labculture) 3. Incubator (Esco Celculture) 4. Vortex model 231 (Fisher Scientific) 5. Pipette volume 10 μl and 100 μl (Biohit Proline) 6. Pipette volume 1000 μl (Nichipet Ex) 7. Pipette tips

2 8. Centrifugator (Beckman Coulter) 9. Freeze dried bacterial vegetative cells (sub cultured from culture purchased from Presque Isle Culture): Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 19433), Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 6538), and Pseudomonas aeruginosa (ATCC 27853); bacterial spore in spore suspension: Bacillus atrophaeus (ATCC 9372) and Geobacillus stearothermophilus (ATCC 12980); fungi spore in spore suspension: Aspergillus niger (ATCC 6275). 10. Tryptone Soya Broth medium (Oxoid), for bacteria and fungi inoculums 11. Tryptone Soya Agar medium (Acumedia), as bacterial agar plate 12. Peptone Dextrose Agar (Oxoid), as fungi agar plate 13. Enviroswab (3M) 14. Sterile 0.85 % NaCl solution (produced in house from Fisher Scientific crystal) mm stainless steel disc (produced in house and sterilized) 3.2 Method Bacterial vegetative cell preparation 1. Rehydrate microorganisms using TSB medium 2. Inoculate rehydrated microorganism into 5 ml TSB medium and incubate in 37 o C for 24 hours. 3. Inoculate 1 ml of these inoculums into 10 ml TSB medium and incubate in 37 o C for 24 hours. 4. Enumerate bacteria using surface plate method on TSA medium. 5. Centrifuge inoculums if their numbers don t reach 10 8 CFU/ml. Bacteria and fungi spore suspension Dilute spore suspension to reach 10 8 CFU/ml and 10 6 CFU/ml of bacteria and fungi spore suspension respectively. Decontamination effectiveness test (Moy and Speight (2011) with modification) 1. Set up the entire tray (figure 1). 2. Mark 4x8 cm area will be tested on the glass door. Make 7 areas. It will be use for G. stearothermophilus only. 3. Place 3 empty discs from sterilization container into TSB medium and incubate in the incubator to serve as negative controls. 4. Place in 4 discs for every microorganism on each tray (a total of 32 discs per tray). 5. On the second tray, add extra 4 discs for each microorganism to be used for enumeration prior to decontamination cycle and as positive controls. 6. Place 100 µl of G. stearothermophilus s spore suspension on the marked area of glass door. 7. Place 10 μl of bacterial vegetative cells or spore suspension per disc. 8. Wait until the suspension dries (approximately 0.5 h in 37 o C). 9. For every microorganism, take 3 discs from the extra discs placed on second tray (Step 3) as positive control. Inoculate these discs into TSB medium and incubate at 37 o C for hours and hours for bacteria and fungi respectively. 10. Take 1 disc for each microorganism from the extra discs placed on second tray and put into 10 ml 0.85 % NaCl solution to obtain the initial bacterial count before decontamination. 11. Swap 3 area of glass door using Enviroswab on each area. Add 20 ml of TSB into each Enviroswab as a positive control and incubate at C for 3-7 days. 12. Swap 1 area of glass door using Enviroswab and add 20 ml of sterile 0.85 % NaCl solution to obtain the initial number of G. stearothermophilus before decontamination. 13. Run the decontamination cycle by press the STERILIZE button.

3 14. After the decontamination cycle is fully completed, take 3 discs for every microorganism on each tray and inoculate into TSB medium and incubate in 37 o C for hours for bacteria and hours for fungi. 15. Swap 3 area of glass door using Enviroswab on each area. Add 20 ml of TSB into each Enviroswab and incubate at C for 3-7 days. 4. Result and Discussion Figure 1. Esco Celsafe CO 2 Incubator The STERILIZATION feature of Esco Celsafe CO 2 Incubator adopts the hot air oven system. This system is one of dry heat sterilization method, which its effectiveness depends on the prevalence of hot air in the chamber (4). Because of the objective of this feature is to sterilize its chamber, therefore it is important to make sure that the chamber temperature is same as the set up temperature. The set up temperature is 200 C during the sterilization process. The other challenge is the glass door. It should reach 200 C during the sterilization process as well. Figure 2 presents the temperature of chamber and glass door during the sterilization.

4 log reduction temperature ( C ) time (h) Chamber Glass door Figure 2. Graph of chamber and glass door temperature during sterilization Those figure showed that during the sterilization process, both chamber and glass door temperature run in row way. The ramp up time, where in the temperature increase from 37 C to 200 C, was 2 hours. The soaking time, where in the temperature 200 C, was hold for 1 hour. Then, the ramp down time or cooling time was 5 hours. The glass door temperature was slightly lower than chamber temperature because of it s the last part which received the thermal. The heater heated up the chamber by spread out the thermal. While heated up the chamber, the thermal lead to the glass door at the last. The challenge of sterilization feature was to reduce microorganisms as much as 6 logs. Microorganisms tested were vegetative cell of bacteria, and spore of bacteria and fungi. Vegetative cell and spore of bacteria numbers tested were 6 logs, while spore of fungi was 4 logs. Figure 3 presents the log reduction of microorganisms tested in Celsafe chamber Figure 3. Graph of microorganisms log reduction in the Celsafe chamber

5 Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 19433), Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 6538), and Pseudomonas aeruginosa (ATCC 27853) vegetative cell log reduction were more than 6 logs. Spore of Bacillus atrophaeus (ATCC 9372) and Geobacillus stearothermophilus (ATCC 12980) log reduction were more than 6 logs too. While fungi spore of Aspergillus niger (ATCC 6275) was more than 4 logs. Four logs of fungi spore was the highest number we got from the preparation. The 6 logs reduction of microorganisms cell indicating that this sterilization feature may oxidize the cell constituent and even damage DNA spore resulting no cell growth. In order to confirm this feature effectiveness toward microorganisms on the glass door, we observed the log reduction on it as well. We used Geobacillus stearothermophilus (ATCC 12980) spore as a challenge microorganisms because of its high heat resistance property. We put 6.3 logs and observed that there was no growth of this microorganism. This result indicating that the effectiveness of this feature was remaining even on the glass door. 5. Conclusion The 6 logs reduction of bacteria vegetative cell and spore, and 4 logs reduction of fungi spore was an indication of the succeed of Esco Celsafe CO 2 Incubator STERILIZATION feature. REFERENCE 1. Rutala,W.A, D.J. Weber, and Healthcare Infection Control Practices Advisory Committee (HICPAC).2008.CDC: Guideline for Disinfection and Sterilization in Healthcare Facilities 2. World Health Organization. The International Pharmacopoeia (4 th edition)-section 5.8 [last accessed 6 May 2015]. Available at 3. Setlow,P Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. Journal of Applied Microbiology. 101: Rao,S Sterilization and Desinfection [last accesses 5 June 2015]. Available at