PHAR 5515THE UNIVERSITY OF SYDNEY Faculty of Pharmacy Name: ........................ Practical Manual 2016 Microbiology in Pharmacy Contents LABORATORY RULES ........................................................................................... 2 ASSESSMENT .......................................................................................................... 3 PENALTIES .............................................................................................................. 4 PLAGIARISM ........................................................................................................... 4 PRACTICAL 1 ............................................................................................................. 5 TOPIC 1: ASEPTIC PRODUCTION .............................................................................. 5 Exercise 1.1: Aseptic transfer ............................................................................... 7 PRACTICAL 2 ........................................................................................................... 15 TOPIC 2: FILTRATION, DISINFECTION AND PRESERVATION .................................. 15 Exercise 2.1: Testing efficiency of filtration with the micro-organism Serratia marcescens (bacterial challenge test) .................................................................. 17 Exercise 2.2: Evaluation of preservatives ........................................................... 20 Exercise 2.3: Determination of MICs of antimicrobial agents ............................ 22 Exercise 2.4: Formulation compatibility and preservatives ................................ 24 PRACTICAL REPORT ............................................................................................ 27 TOPIC 1: ASEPTIC TECHNIQUE (COMPLETED BY PRACTICAL 1) ......... 32 TOPIC 2: DISINFECTION AND PRESERVATION (COMPLETE BY PRACTICAL 2) ......................................................................................................... 33 TOPIC 3: STERILISATION (COMPLETE BY VIDEO DEMONSTRATION) 35 APPENDIX 1 .............................................................................................................. 43 LABORATORY RULES As potentially practising Pharmacists, it is critical that you handle pharmaceutics carefully to avoid contamination. In the following laboratory classes you will be instructed with the correct aseptic technique to use to handle such sterile products. You will also learn how to handle microbes safely. COMPLIANCE WITH ALL LABORATORY RULES CONTRIBUTES TO YOUR ATTENDANCE AND PARTICIPATION MARK The following precautions MUST be observed for the safety of everyone working in the laboratory – any student NOT COMPLYING will be ASKED TO LEAVE. Protective Clothing and Dress 1. A laboratory coat MUST be worn at all times in the laboratory. It should be put on and fastened fully on entry to the laboratory and removed on leaving to reduce the risk of contaminating your clothing. 2. Safety glasses MUST be worn at all times in the laboratory. 3. Substantial shoes with closed in toes MUST be worn. Thongs and sandals are unsatisfactory protection and should not be worn in the laboratory. 4. Hair, if long, MUST be tied back. Safety Requirements 1. DO NOT EAT, DRINK OR SMOKE in the laboratory and never place pencils, pens, labels or other materials in the mouth. 2. MOUTH PIPETTING OF LIQUIDS IS BANNED. A rubber teat or filler must be used at all times. 3. STORE BAGS, COATS, UMBRELLAS ETC. AWAY FROM THE WORK AREA. 4. CULTURES ARE NEVER TO BE TAKEN from the laboratory. 5. Inoculated media must be PROPERLY LABELLED (i.e. with name, date and the nature of the specimen) and put in the appropriate box for incubation. 6. DO NOT SIT on the benches. 7. Turn gas burners down or off when not in use during the laboratory period to keep the laboratory as cool as possible. 8. Any personal accident must be reported to a demonstrator immediately. 9. Any spillage of culture material must be reported to a demonstrator immediately so that appropriate action may be taken. -2- 10. At the end of each practical session: • Discard all used tubes, pipettes, Petri dishes etc. in the designated containers. • Clear the bench top of all equipment except stain bottle rack, pipette canister and disinfectant bottle. • Wipe bench top with disinfectant. • Wash hands with the skin disinfectant supplied before leaving the laboratory at any stage. Infectious materials will be handled. 11. Ensure that you know where the nearest emergency exit is. ASSESSMENT a. Attendance and Participation b. Practical Report c. Video Demonstration Online Quiz a. • • • • • (2%) (5%) (3%) Attendance and Participation (2%) Classes commence promptly at 5 minutes after the hour. Attendance at all practical sessions is compulsory. An attendance list will be circulated at each session. You must sign the attendance list. Students are required to comply with the Laboratory Rules. Attitude to work, effort and understanding will also be assessed. Pre-lab tasks must be completed prior to coming to class. These tasks will be checked by your demonstrator during each practical session. b. • Practical Report (5%) The practical report must be uploaded on the Blackboard site by 4:00pm, 7 days after completion of the last practical class. A signed assignment sheet should be attached to the report. The 7 day due date is absolutely strict and the report will not be accepted and marked once the due date is passed. The report should be carried out in group of two students. c. • Video Demonstration Workshop – Week 3 (3%) The workshops contain a short film demonstrating different aspects, requirements and techniques to prepare sterilised pharmaceutical products. After the film, the students should log-on on the Blackboard and complete an assessment (a series of multiple choice questions) related to the film. Students have five days (by 4:00PM on the first Monday or Tuesday following the Wednesday or Thursday workshop, respectively) to complete the assessment (MCQs). The students are required to answer these questions without consulting other students. The allowed time and instruction to complete the • • -3- copying or reproducing them without acknowledging the source. specific and detailed collaboration with other students on assignments not specifically designated as “Group Assignments”. and is dishonest and unacceptable. Please note. explaining the ways in which students are permitted to work together. you must apply for Special Consideration. and range from not receiving any marks for the specific piece of work to expulsion from the University in extremely serious cases. • It is your responsibility to obtain all information and results for the class and to fill in your Practical Manual.assessment will be appeared on screen prior of beginning of the assessment. PLAGIARISM Plagiarism occurs when you present someone else’s words or ideas as your own by presenting. without plagiarising each other’s work. If you miss an assessable activity because of certified illness or misadventure. The University has very clear and strict guidelines about responding to plagiarism and the penalties for this are quite severe. -4- . The policy defines “Legitimate Cooperation”. or “Joint Work” is also considered plagiarism. Thus. IF YOU MISS A PRACTICAL CLASS • Inform your demonstrator of the reason for your absence and. it is very important that you avoid plagiarism. submit a Special Consideration form to the Faculty of Pharmacy Office. Plagiarism is a kind of stealing. Your demonstrator may be able to organise an alternative time for you to learn new practical skills. PENALTIES Absence from any assessable activity will result in a mark of zero being recorded for the task. PRACTICAL 1 TOPIC 1: Aseptic Production Learning Outcomes After completing these exercises you will: Understand the principles for aseptic process in the manufacturing of pharmaceutical products Become familiar with working under hood. aseptic techniques and condition Appreciate that good aseptic techniques can minimise the risk of contamination -5- . the definition of an aseptic technique is: the act of handling materials in a controlled environment in which the air supply. ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… -6- . List ten basic rules of the aseptic technique in the production of clean pharmaceutical products.…………………….. and personnel are regulated to control microbial and particulate contamination in pharmaceutical products within acceptable levels. equipment.Practical 1 Pre-work NAME.………………….……………….…………………. In pharmaceutics. materials. 1.SID. This exercise demonstrates the aseptic process for transfer of liquids. In aseptic preparation. dilution of liquids. sterile pharmaceutical products are prepared by assembly of sterile ingredients under aseptic conditions to prevent the introduction of any particulate matters or micro-organisms into the products. beaker and stirring rod 1 × 25 mL sterile water 1 × 25 mL Nutrient Broth Boil in purified waterrinse well and pack in autoclave bag Double sealed in envelopes Double sealed in autoclave bags Method . there will be a high risk of contamination. Protocol 2 follows some basic rules but it is purposely poorly designed so the products may still be contaminated. Protocol 3 is more carefully designed so there will be a very low risk of contamination. Students will work in pairs but each student should undertake each of the three protocols for the exercise. therefore. Protocol 1 Materials Special Pre-treatment or Packaging Sterilisation Method Conditions 3 × 20 mL multi-dose containers (dry) Seal with foil Dry heat 160°C for 2 hours Moist heat 121°C for 15 min Dry heat 160°C for 2 hours Dry heat 160°C for 2 hours Moist heat 121°C for 15 min Moist heat 121°C for 15 min 1 pack of 3 rubber closures Lactose 500 mg 25 mL measuring cylinder. and capping of containers.1: Aseptic transfer Introduction In this practical you will be introduced to aseptic preparation of pharmaceutical products. Protocol 1 does not follow the basic rules of aseptic preparation. -7- .Exercise 1.Protocol 1 1. weighing and dissolving of solids. Aim • To understand the principles of aseptic techniques. Gather all the materials and equipment required for this exercise in front of the hood. 13. Insert the closures in the neck of the multidose containers. Measure 10 mL of this lactose/diluted broth (in the same measure) and transfer to the multidose container no. Next week. GENTLY crimp a metal closure onto the neck of each multidose container. Open the sterile water and pour 15 mL into the measure (in the same measure). measure. Measure 10 mL of the diluted broth (in the same measure) and transfer it to the multidose container no. stirring rod. 1). Leave them on your bench: They will be collected and incubated for 7 days at 32oC then returned to you next week. Remove all the material. and then take them to the crimping unit. 14. Unfold one end of the package containing lactose and transfer the contents to the beaker (now containing 20 mL diluted broth). and lactose outside the hood. Swirl to mix the diluted broth. Before removing the containers from the hood. Remove the aluminium foil from multidose container no. Discard all wrapping material into the waste bin. 3 (using the same technique as for the container no.2. make certain that the closures are pressed firmly into the necks. Open the wrapping of beaker. Use stirring rod to dissolve the lactose in the broth. 1). 1 and transfer 10 mL of nutrient broth from the measure. 2 (using the same technique as for the container no. Bind the 3 bottles together with a tape then label with your name and protocol number. 3. Open the sterile nutrient broth. Transfer all equipment and materials into the hood. pour the entire contents into a beaker and then transfer 10 mL to the measure. except the multidose containers. 5. Attach a small piece of autoclave tape to each multidose container and label them 1. 4. 2 and 3. record the results in the following table. 12. -8- . 6. then transfer it to the remaining nutrient broth in the beaker. 8. 11. 15. 9. 7. 10. 3.Aseptic Transfer – Protocol 1 Results Individual Indicate growth (+) or no growth (-) Class Results (contaminated broths as % of n) n Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) Protocol 2 Materials Special Pre-treatment or Packaging Sterilisation Method Conditions 3 × 20 mL multi-dose containers (dry) Seal with foil Dry heat 160°C for 2 hours Moist heat 121°C for 15 min Dry heat 160°C for 2 hours Dry heat 160°C for 2 hours Moist heat 121°C for 15 min Moist heat 121°C for 15 min 1 pack of 3 rubber closures Lactose 500 mg 25 mL measuring cylinder.Protocol 2 Plan your work carefully before commencing. All manipulations are to be carried out under the plastic hoods to simulate using aseptic hoods. 1. Ensure all the materials left from previous exercise are removed from the hood. beaker and stirring rod 1 × 25 mL sterile water 1 × 25 mL Nutrient Broth Boil in purified waterrinse well and pack in autoclave bag Double sealed in envelopes Double sealed in autoclave bags Method . -9- . Gather all the materials and equipment required for this exercise in front of the hood. 2. Clean your aseptic hood in accordance with SOP 2 in Appendix 1. Tear open (at the base) the single wrapping protecting the measure and beaker and remove them from the packing (discard the packing to the front of the hood). measure and stirring rod) and then transfer them (still single wrapped) under the hood. 2 (using the same technique as for the container no. 7. Open the outside wrapping of your equipment (beaker. 13. 10. Transfer the materials and equipment into the clean hood as follows: 6.10 - . 2 and 3. and as you transfer it into the hood loosen the aluminium foil cap so that it can be removed with forceps. Unfold one end of the package containing lactose and transfer the contents to the beaker (now containing 20 mL diluted broth). Roll up your sleeves then following SOP 1 in Appendix 1 clean your hands and forearms with chlorhexidine scrub. In turn. 14. . 1 and transfer the 10 mL of nutrient broth from the measure. Swirl to mix the diluted broth. Arrange the 3 multidose containers at the rear of the hood. Open the sterile water and pour 15 mL into the measure (in the same measure). Open the sterile nutrient broth. 9. 15. 5. 12.4. Remove the aluminium foil with forceps from multidose container no. 11. Take a sterile Petri dish from the pack beside the hood and place it inside the hood to one side and toward the back of the work area. pour the entire contents into the beaker and then transfer 10 mL to the measure. Immediately replace the foil. lightly spray the bottom of each multidose container (outside the container) with 70% ethanol. Swab the McCartney bottles of sterile nutrient broth and sterile water with 70% alcohol and place them under the hood. 16. 8. Do the same for the lactose. Measure 10 mL of the diluted broth (in the same measure) and transfer to the multidose container no. Attach a small piece of autoclave tape to each multidose container (vial) and label them 1. Swab a pair of forceps with 70% ethanol and rest them on the sterile Petri dish. then transfer it to the remaining nutrient broth in the beaker. 17. open it and place the lid on the other side of the work area. 1). 25. record the results in the following table. Using the forceps pick up a rubber closure and simultaneously. Repeat for all three containers. Bind the 3 bottles together with a tape then label with your name and protocol number.11 - Class Results (contaminated broths as % of n) n . Re-swab the forceps with 70% alcohol and place them on one of the open halves of the Petri dish. Tear open the wrapper covering the sterile stirring rod. 3 (using the same technique as for the container no. Next week. GENTLY crimp a metal closure onto the neck of each multidose container. Remove all the material. as you lift the foil from the vial with your other hand. Leave them on your bench: They will be collected and incubated for 7 days at 32oC then returned to you next week. from the hood and discard wrapping material and Petri dish into the waste bin. 20. Aseptic Transfer – Protocol 2 Results Individual Indicate growth (+) or no growth (-) Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) . Tear open the outer wrapping of the packet of rubber closures. 22. 21. 26. Before removing the containers from the hood. make certain that the closures are pressed firmly into the necks. take a sterile Petri dish from the pack beside the hood and place. and then take them to the crimping unit. Again. Tear open one end of the packet (inner wrap) of rubber closures with the forceps and empty the closures into the second open half of the sterile Petri dish. 23. insert the closure in the neck of the multidose container. 24. except the multidose containers.18. 19. Measure 10 mL of this lactose/diluted broth (in the same measure) and transfer to the multidose container no. 1). transfer it under the hood. and open it inside the hood as before. and use it to stir and completely dissolve the lactose in the broth. 2. lightly spray the bottom of each multidose container (outside the container) with 70% ethanol. 3. 5. All manipulations are to be carried out under the plastic hoods to simulate using aseptic hoods. 2. Do not take the crimp off the bottle. Transfer the materials and equipment into the clean hood as follows: 6. Gather all the materials and equipment required for this exercise in front of the hood.Protocol 3 Plan your work carefully before commencing. 1. In turn. open it and place the lid on the other side of the work area. 4.12 - . (lactose vial). Expose the needle entry point under the metal crimp sealing the 20mL vial of water for injections with your forceps. Take a sterile Petri dish from the pack beside the hood and place it inside the hood to one side and toward the back of the work area. and as you transfer it into the hood loosen the foil . (empty Multidose vial).8µm membrane filter Moist heat 121°C for 15 min Moist heat 121°C for 15 min Lactose 500 mg in multi-dose container 1 disposable 20 ml syringe 1 × 20 mL vial Water for Injections 1 × 25 mL Nutrient Broth Method . 8. Swab a pair of forceps with 70% ethanol and rest them on the sterile Petri dish. (Water For Injection) and 3. Clean your aseptic hood in accordance with SOP 2 in Appendix 1. 7. Attach a small piece of autoclave tape to each multidose container and label them 1.Protocol 3 Materials Special Pre-treatment or Packaging Sterilisation Method Conditions 1 × 20 mL multi-dose containers (empty) Seal with foil Dry heat 160°C for 2 hour 2 rubber closures for multi-dose containers Boil in purified waterrinse well and pack in autoclave bag Moist heat 121°C for 15 min Seal with foil Dry heat 160°C for 2 hours In disposable bag Radiation 25 G Filtered with 0. 2) now has 10 mL of water for injection. 15. Withdraw 10 mL of diluted broth using syringe by injecting 10 mL air into the container. Roll up your sleeves then following SOP 1 in Appendix 1 clean your hands and forearms with chlorhexidine scrub. Swab the McCartney bottle of sterile nutrient broth. 9. 17. remove the syringe and stand it vertically on the flat end of the plunger (discard the packing to the bin). take a sterile Petri dish from the pack beside the hood and place. the multidose vial of water for injections. 16.13 - . 19. Remove the syringe and shake the container to dilute the nutrient broth. 3). 1 and transfer 10 mL of nutrient broth from the syringe. 10. Arrange the 3 multidose containers at the rear of the hood. Tear open the outer wrapping of the packet of rubber closures. 11. then withdraw 10 mL of water for injection (the air pressure pushes the water into the syringe) and then discard it into the second half of the sterile Petri dish. except the multidose containers. Remove the foil with forceps from multidose container no. 18. from the hood and discard wrapping material and Petri dish into the waste bin. and open it inside the hood as before. Pierce the rubber closure of the multidose container (no. Re-swab the forceps with 70% alcohol and place them on one of the open halves of the Petri dish. Immediately replace the foil. Remove all the material. Adjust the plunger until the syringe contains 10 mL of air then remove the needle cover (discard) DO NOT RE-SHEATH NEEDLE. Tear open the wrapping. Again. 13. Transfer 10 mL of this diluted broth to the multidose vial containing the 500mg lactose (container no. 2) containing 10 mL water for injections. . 2) and inject 10 mL air into the container. 12. The multidose vial (no. Stand the syringe on its base again.cap so that it can be removed with forceps. Open the needle pack and while the needle is still in the cover attach it to the sterile syringe. 14. Open the sterile nutrient broth container and remove 20 mL of broth with the syringe. transfer it under the hood. Transfer the remaining 10 mL of nutrient broth in the syringe into the multidose container (no. the syringe and needle pack with 70% ethanol and place them under the hood. Bind the 3 bottles together with a tape then label with your name and protocol number. Next week. 24. 22. this bottle can be shaken until the lactose has dissolved. Using the forceps pick up a rubber closure and simultaneously. 23. as you lift the foil from the vial with your other hand. Once the closure is firmly inserted and crimped to the multidose vial containing the lactose. GENTLY crimp a metal closure onto the neck of each multidose container. Aseptic Transfer – Protocol 3 Results Individual Indicate growth (+) or no growth (-) Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) . and then take them to the crimping unit. record the results in the following table.20. 21. insert the closure in the neck of the multidose container. Repeat for all three multidose containers.14 - Class Results (contaminated broths as % of n) n . Leave them on your bench: They will be collected and incubated for 7 days at 32oC then returned to you next week. 25. Before removing the containers from under the hood make certain that the closures are pressed firmly into the necks. Tear open one end of the packet (inner wrap) of rubber closures with the forceps and empty the closures into the second open half of the sterile Petri dish. 1 TOPIC 2: Filtration. Disinfection and Preservation Learning Objectives After completing these exercises you will be able to: To become familiar with filtration sterilisation Understand the application of commonly used disinfectants Appreciate the usefulness and limitations of current tests for efficacy of disinfectants and preservative Demonstrate the in vitro activity of preservatives in pharmaceutical preparations Understand that any preservative added to a formulation must be compatible with the other ingredients .15 - .Exercise 1.PRACTICAL 2 FOLLOW UP WORK FROM PRACTICAL 1 . Practical 2 Pre-work NAME. What is the main anti-microbial agent of the following disinfectants / antiseptics? Milton:………………………………………………………………………………….. Savlon:………………………………………………………………………………….. Disinfectant:…………………………………………………………………………… …………………………………………………………………………………………..………………. .. ………………………………………………………………………………………….………………….SID. Sterilisation:…………………………………………………………………………… ………………………………………………………………………………………… 2. Define: Antiseptic:……………………………………………………………………………… ………………………………………………………………………………………….. 1.…………………..... Betadine:………………………………………………………………………………. Dettol:………………………………………………………………………………….…………………….16 - .. Preservative:…………………………………………………………………………. 4. Materials • • • • • • Assigned filter: Either cellulose acetate membrane filter in Swinnex (placed in an autoclave bag) or commercially disposable membrane filter (made of hydrophilic polyethersulfone membrane) 5 mL sterile disposable syringe. 1. low potential to release fibres or chemicals. Suspension of Serratia marcescens (~107 organisms/mL). then stand the syringe on its base. . attach a sterile needle then stand it on the base of its plunger. micro-organisms. steam and dry heat sterilisation) in that it removes. remove the needle and discard it into the sharps container. flexibility and mechanical strength. Wash your hands.Exercise 2.g.. In this exercise you will perform the bacterial challenge test for efficacy of filtration with the Serratia marcescens.17 - . Re-cap the bottle of Serratia. resistance to clogging.1: Testing efficiency of filtration with the micro-organism Serratia marcescens (bacterial challenge test) Introduction Filtration is unique among other sterilisation techniques (e. 2 x nutrient broth in glass tubes 1 x peptone glycerol agar plate. Remove a 5 mL sterile disposable syringe from its package. 3. 5. Transfer them into the biohazard hood and loosen the caps so they can be easily removed. Only two students at a time can work at the Biohazard cabinet. rather than destroys. Needle. acceptably high flow rate. With the syringe remove 5 mL of the Serratia suspension from the bottle. Remove the lid from a bottle containing a suspension of Serratia marcescens (about 107 CFUs)/mL. Methods All manipulations are to be carried out in the biohazard laminar flow cabinet. It is also capable of preventing the passage of both viable and non-viable particles. 2. The ideal filter medium should offer efficient removal of particles above stated size. Label the McCartney bottles and plate with your initials and bench number and indicate if they represent the filtered or unfiltered test. according to SOP1 Appendix 1. Aims • To test the ability of a filter to produce a sterile filtrate from a culture of Serratia marcescens. marcescens suspension by shaking gently the agar plate. Membrane filters have a limited capacity as they are essentially surface filters for particles above or around their nominal pore size. 2 mL of the suspension to the other tubes of broth and 2 drops onto a peptone glycerol agar plate. and your bench number. Take the agar plate and broth tubes to your bench. . type of the filter that you used (filter in Swinnex or commercially disposable filter). The tubes and agar plates will be collected and incubated at 25°C for 2 days and returned to you next week. Even though only 2 mL of the suspension of Serratia was filtered it is probable that some of you noticed an increase in the pressure required to push the liquid through the filter. place it in the labelled waste container and put the filter back in the beaker then place the beaker in the tray next to the biohazard hood. 12. Results Individual Results Examine the tubes and plate for the growth of Serratia marcescens. 8. The tubes or plates contaminated with Serratia have a light pinkish colour. through the filter. Transfer 2 mL of the Serratia suspension (without filtration) to one of the tubes of broth. 11. Class results will be displayed on the notice board next week. Detach the syringe from the filter. Attach the 5 mL syringe (containing approximately 3 mL of Serratia suspension) to your assigned filter. 10. Bind the tubes together and make certain they are labelled with your name. An estimate of the effectiveness of this sterilisation technique and the types of filters used can be gauged from the number of tubes filtered not showing growth. Please see SOP 3 in Appendix 1 for more details about filtration technique. 9. When incubated at 25°C Serratia marcescens produces a red pigment. Transfer. Put the lid of the agar plate back and spread the S.6. The nutrient broth is cloudy if you have contamination.18 - . 7. Bacterial Challenge Test Type of filter used:----------------- Visible growth of microorganisms ---------------------------------------Filtered (plate) Filtered (tube) Unfiltered (tube) Class Results Two 0.19 - Number of tubes & plates Filtration Failure Rate . Bacterial Challenge test Challenge test on 0.22 µm filters are used in this exercise.22 µm filters with Serratia marcescens Type of Filter Number of plates showing Growth of microorganisms Number of tubes showing Growth of microorganisms Cellulose acetate membrane filter in Swinnex Commercially disposable membrane filter . Exercise 2. epidermidis and incubated at 20°C to 25°C for 7 days.1% 1% 10 g portions of lotions each will have been inoculated with either 106-107 CFU (Colony Forming Unit per millilitre) of S.20 - . Products such as eye drops and ointments are originally dispensed as sterile units but must maintain zero or very low levels of contamination throughout the claimed shelf-life (e. Each student will perform a count of viable organisms for the one 10g portion of contaminated lotion supplied to them at their bench. Cetomacrogol lotion APF17 usually contains chlorhexidine gluconate as the preservative however it is possible to substitute other preservatives if required.g. To reduce contamination level of pharmaceutical products during storage. In this exercise a creamy lotion for topical application is formulated with different preservatives. Oral mixtures and topical products are not required to be sterile. 28 days after opening). we will use Saccharomyces cerevisiae and Staphylococcus epidermidis.1% 0. The micro-organisms used for the challenge will vary according to the dose and type of the preservative but for our test on a creamy lotion.2: Evaluation of preservatives Introduction Pharmaceutical products are frequently intended for multiple use. Method You will perform part of a group experiment. cerevisiae or S. preserved with either Chlorhexidine gluconate solution Chlorocresol Phenoxyethanol 0. the products are formulated with one or more preservatives. The lotions are challenged with a defined inoculum of suitable microorganisms and then stored at a prescribed temperature to evaluate the microbial growth. 1. Aims • To evaluate the efficacy of three preservatives in preserving a lotion based on the APF17 formula for Cetomacrogol lotion. Unscrew the lid off a bottle of lecithin-Tween broth (LTB) until it can be removed with one hand. Three lotions are to be investigated. . however it is important that they do not become heavily contaminated during their claimed shelf-life which is frequently much longer that 28 days. cerevisiae S. cerevisiae CGB CGF CSB CSF PEB PEF . epidermidis S. Discard the pipette into the contaminated waste container and flame and insert the spreader into the hot bead after use. epidermidis S. With a sterile disposable syringe remove 1 mL of the lotion and transfer it to the bottle of 20 mL lecithin-Tween broth (LTB). and then spread the lotion/broth liquid evenly with the spreader. and the code letters that were marked on the sample of lotion you were provided with. Mix thoroughly on a vortex mixer. Next week. Lotion code: Antimicrobial agent Chlorhexidine gluconate 0. cerevisiae S.2. and recap the lotion sample and leave it on your bench. Insert a wire spreader into the hot bead sterilizer for ten seconds. Label the plate with your name. 6. 4. Using a sterile Pasture pipette transfer 4 drops of the lotion/lecithin-Tween (LTB) broth to a lecithin-Tween agar plate. Your demonstrator will explain to you how to calculate the Colony Forming Unit (CFU) of your agar plate. 5.21 - Average Plate Count Log Reduction in CFUs .1% Phenoxyethanol 1% Plate count of CFUs: Microorganism Code S. Gently agitate the sample of lotion to ensure that it is homogeneous then unscrew the lid until it can be removed with one hand. allow to cool. epidermidis S. Discard the syringe into the contaminated waste container immediately. 3. inspect for growth and fill out the following table.1% Chlorocresol 0. Label the McCartney bottle containing 20 mL broth P1 and the bottles containing 10 mL broth P2. Aims • • To determine the Minimum Inhibitory Concentration (MIC) of 2phenoxyethanol against a gram negative (Serratia marcescens) and a gram positive (Staphylococcus epidermidis) bacteria. and P5 then unscrew the lids so that they can be removed with one hand.3: Determination of MICs of antimicrobial agents Introduction A Minimum Inhibitory Concentration (MIC) determination is a simple and rapid method for determining a concentration of an antimicrobial agent at which microorganisms do not grow.22 - . You will be supplied with one tube containing an antimicrobial system in Tryptone Soy broth. If your assigned antimicrobial system is phenoxyethanol + EDTA. Phenoxyethanol Systems If your assigned antimicrobial system is phenoxyethanol. it is performed in a complex nutrient in which a number of antimicrobials are inactivated. Withdraw 10 mL of antimicrobial/broth from the bottle 2 using the same syringe and transfer to the bottle 3. Attach a sterile needle to a 10 mL sterile plastic syringe and withdraw 10 mL of antimicrobial broth from the bottle 1 using the syringe and transfer to the bottle 2.Exercise 2. . one broth will be labelled PTSE (20 mL) and four labelled MTSE (10 mL). it gives no indication as to whether the antimicrobial agent is bactericidal or bacteriostatic and secondly. Re-seal the bottle and mix. one broth will be labelled PTS (20 mL) and four labelled MTS (10 mL). Re-seal the bottle and mix. 1. P3. 3. Replace the needle cover again. To investigate whether or not there is an enhancement of the bactericidal activity of these antimicrobial agents when a chelating agent (such as EDTA) with no intrinsic antimicrobial activity is added to the formulation. There are two basic difficulties in interpreting this type of test however. 2. P4. firstly. Methods You will perform part of a group experiment. From this you will prepare serial dilution of the antimicrobial agent in Tryptone Soy (TS) broth or Tryptone Soy broth with added EDTA then inoculate all five tubes with your assigned micro-organism. Withdraw 10 mL of antimicrobial/broth from the bottle 3 and transfer to the bottle 4. Bind the 5 McCartney bottles together with a rubber band and attached a label showing your name and the micro-organisms used (either Serratia or Staphylococcus) added to the test tubes. Transfer 2 drops of your inoculum (either Serratia marcescens (gram-) or Staphylococcus epidermidis (gram+) bacteria using a sterile Pasture pipette (discard immediately the pipette into the contaminated waste unit). Replace the needle cover again. The bottles will be incubated at 32°C until next week. Assemble the 5 McCartney bottles at the “contaminated” work area (at the end of the bench) and again loosen the caps. Individual Results Your assigned phenoxyethanol (PTS or PTSE):------------------------------------Your assigned micro-organisms:-------------------------------------------------------Filling the following Table: No of bottle Concentration of preservative g/L Growth (+ / -) 1 2 3 4 5 The MIC you obtained:-----------------------------. Re-seal the bottle and mix. 7.(What is the minimum concentration that prevents microbial growth) Class Results Against Gram + 2-Phenoxyethanol + EDTA . 5. Replace the needle cover again. Re-seal the bottle and mix.4.EDTA . 8. 6. Re-seal the bottles and mix. Withdraw 10 mL of antimicrobial/broth from the bottle 4 and transfer to the bottle 5. Next week record the results in the following table.23 - Against Gram - . 4. 8. Take a previously melted and cooled (to about 50°C) tube of nutrient agar. Fill hole #2 with cetomacrogol cream Fill hole #4 with oily cream To fill the holes.Exercise 2. Fill hole #1 with the gel as a control Fill hole #3 with aqueous cream. 1 4 2 .5% chlorhexidine gluconate has been added and they have been packed into 10 mL syringes (placed on your bench). Roll the tube gently to mix so as to avoid any air bubbles. do not inoculate bacteria if the agar is still hot (ask your demonstrator if you are not sure). To keep the bacteria alive. Pour aseptically (without contaminating the agar or dish) into a sterile Petri dish and allow to set. 1. 7. Inoculate with 2 drops of a broth culture of S. To observe the effect of three other cream bases on the activity of chlorhexidine using the agar diffusion technique. 2. drill four holes in the plate using a sterile borer (ask your demonstrator to show you). Label the holes 1 to 4 on the base of the plate and with your name and bench numbers. Method Three creams that are very similar (but less viscous) to those of the APF17 formulas and a gel thickened with Hypromellose 4000 (control) have been prepared. initially expel a small amount of the product onto a piece of paper towel (to start flow) then place the tip of the syringe in the centre of the hole against the bottom of the Petri dish and slowly expel the cream/gel whilst raising the syringe.4: Formulation compatibility and preservatives Aims • • To observe whether the “incompatibility” of chlorhexidine with aqueous cream affects the antimicrobial activity of chlorhexidine. 6. 0. Once the agar gel is set. The cream should touch the wall of the hole. 3.24 - 3 . Do not overfill the holes. Discard the agar plugs and the borer into its container. epidermidis. 5. To each. Measure the diameters of the holes containing the formulations and the inhibition zones using a ruler and record in the table below. It will be collected. Leave the plate on the bench.25 - dz2 .9. incubated at 32 °C for 24 hours and returned to you next week.dh2 . Formulation Diameter of Diameter of hole inhibition zone dh (mm) dz (mm) # 1 Methylcellulose gel # 2 Cetomacrogol Cream #3 Aqueous cream #4 Oily cream . Next week record your data in following table. This work is substantially my own. the work of the others in my group only. To the extent that any part of this work is not my own or the work of others in my group. and. I have indicated that it is not my own by acknowledging the source of that part or those parts of the work.GROUP ASSIGNMENT COVER SHEET Unit of Study Code: ________________ Unit of Study Name: _______________________ (Mandatory Field – Please complete) Course Coordinator: ______________________Tutor’s Name: _______________________ Assignment: ______________________________ Date Submitted: ____________________ Declaration Each student must confirm that your assignment meets the academic honesty requirements by signing below: • • • • • • I have read and understood the University of Sydney Student Plagiarism: Coursework Policy and Procedure. Student Name SID Signature . I understand that failure to comply with the Student Plagiarism: Coursework Policy and Procedure can lead to the University commencing proceedings against me for potential student misconduct under Chapter 8 of the University of Sydney By-Law 1999 (as amended). I have not engaged someone else to complete this assignment. I have retained a duplicate hard copy of this assignment. to the best of my knowledge.26 - Date . I have not re-used previously submitted material in this assignment. 27 - n .1: Aseptic Transfer Aseptic Transfer – Protocol 1 Class Results (contaminated broths as % of n) Results n Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) Aseptic Transfer – Protocol 2 Class Results (contaminated broths as % of n) Results n Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) Aseptic Transfer – Protocol 3 Class Results (contaminated broths as % of n) Results Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) .PRACTICAL REPORT Please note that the quality and clarity of writing will be taken into account in the determination of your mark for each question. Exercise 1. . Compare these three protocols.... and list the major factors that can cause contamination in the aseptic transfer protocol 1............ ………………………………………………………………………………………... ………………………………………………………………………………………. ………………………………………………………………………………………. ……………………………………………………………………………………….. ………………………………………………………………………………………......... ………………………………………………………………………………………. and 3...1....... ……………………………………………………………………………………….... Did you observe any difference in bacterial growth between protocol 1. ………………………………………………………………………………………..... .......... ………………………………………………………………………………………... ………………………………………………………………………………………. 2.... ………………………………………………………………………………………. ………………………………………………………………………………………... ………………………………………………………………………………………. ………………………………………………………………………………………. ……………………………………………………………………………………….. ………………………………………………………………………………………...... ………………………………………………………………………………………. ………………………………………………………………………………………....28 - ........ ………………………………………………………………………………………. 2.. (15 marks) ………………………………………………………………………………………... and 3? Comment on your class results...... ……………………………………………………………………………………….... .29 - ....... ………………………………………………………………………………………. ……………………………………………………………………………………….. ………………………………………………………………………………………...Exercise 2..1: Testing Efficiency of Filtration with the micro-organism Serratia marcescens (Bacterial Challenge Test) Class Results Bacterial Challenge test Challenge test on 0..... ……………………………………………………………………………………….. ... ………………………………………………………………………………………. ……………………………………………………………………………………….. ………………………………………………………………………………………......... ………………………………………………………………………………………........ ………………………………………………………………………………………... ………………………………………………………………………………………. ……………………………………………………………………………………….... List the factors that may cause contamination during filtration. differences in filter assemblies....22 µm filters with Serratia marcescens Type of Filter Number of plates showing Growth of microorganisms Number of tubes showing Growth of microorganisms Number of tubes & plates Filtration Failure Rate Cellulose acetate membrane filter in Swinnex Commercially disposable membrane filter 2. (12 marks) ………………………………………………………………………………………. and overall on the confidence you would have in using this type of sterilisation process in preparation of pharmaceutical products......... Comment on class result with respect to differences in filter types. ……………………………………………………………………………………….. .................................... ……………………………………………………………………………………….................. Against Gram- + EDTA ........................ ………………………………………………………………………………………........ .......................... ……………………………………………………………………………………….................................... ...................................... ……………………………………………………………………………………….................Exercise 2....................... Exercise 2... ........................................................................................3: Determination of MICs of Antimicrobial Agents Class Results Against Gram+ 2-Phenoxyethanol 4........................ ………………………………………………………………………………………............................................ ……………………………………………………………………………………….. Although chlorocresol (a phenolic component) is a strong anti-microbial agent..................2: Evaluation of Preservative 3..............................................EDTA Are the MICs dependent on the micro-organisms chosen for the test? Gram+ or Gram–: which one is more resistant? Why? (6 marks) .................. ………………………………………………………………………………………................................... ............................... ………………………………………………………………………………………...................30 - ...................... (6 marks) ……………………………………………………………………………………….................. why in this exercise it did demonstrate the lowest antimicrobial activity? Why did you observe lower CFU reduction when the lotion was inoculated with Saccharomyces cerevisiae rater than Staphylococcus epidermidis.................. ………………………………………………………………………………………... ......................................................................................4: Formulation Compatibility and Preservatives 6.............................................................................................................................................................................................................................................................................................................31 - .................................................................................... (6 marks) ........................................................................................................................................................................................................ ......................................................................................................................................................... ................................ 5................................................................ ............................. What is the nature of the “incompatibility” of chlorhexidine with aqueous cream and does this “incompatibility” affect its antimicrobial activity? (5 marks) ................... ........................................................................... . ........................... .................................................................................. ............................................................................................................................................................................................................................................................ Exercise 2........................... Comment on the effect of adding EDTA to the antimicrobial agent and suggest a reason for that effect............................................................ ..................................................... ............................................................................................................................... • Use one hand to hold both the cap and inoculating loop or pipette. do not touch sterile equipment with hands or with contaminated objects or surfaces. Discard paper into waste bin .TOPIC 1: Aseptic Technique (Completed by Practical 1) • Work on a disinfected surface. cough or sneeze while working. • Place the cap on the bench top with the inside up. preferably in a laminar flow cabinet. • Do not speak.32 - . • Eliminate unnecessary air currents. • Use sterile equipment held in appropriate wrapping/containers until ready for use. swirl the plate and allow to set. • Work as rapidly as good technique allows. • Loosen caps before opening bottles. To open sterile pipettes. • Disinfect hands and forearms before working. replace the lid. • To pour plates. quickly pour molten agar into the base of the sterile plate. break the paper seal around the top and not the tip of the pipette and pull the pipette straight out. with no quantitative implication. Autoclave: An apparatus used for sterilising using superheated steam under high pressure.33 - . Decontamination: The killing of organisms or removal of contamination after use. . are used on laboratory equipment and labware that are capable of withstanding the exposure to the process. Definitions Antiseptics: Chemical disinfectants that are designed to be used on living tissue surfaces. i. however a more thorough cleaning of equipment and work surfaces is needed to eliminate the potential for accidental contamination.. how thorough the cleaning needs to be and the other potential contaminates that may be present. Antiseptics are less toxic than disinfectants used on inanimate objects. By the general nature of the product. antiseptics can be less active in the destruction of normal and any pathogenic flora present. generally referring to procedures for making items safe before disposal. Selecting the most effective decontamination procedure or disinfectant will be dependent upon the physical limitations of the material being cleaned. To sterilize using superheated steam under high pressure. heat and gas sterilisation. gas sterilants and chemical disinfectants are toxic and are to be handled according to the manufacturer’s directions. (See Steam Sterilisation) Chemical Sterilant: A germicide that can destroy all forms of microbial life when adequate exposure conditions are realized. Due to the lower toxicity. Appropriate personnel protective equipment is to be worn when these materials are in use. to clean up after spills or even to have routine cleaning of the laboratory floors by custodial personnel on a scheduled basis can all contribute to the incidental inoculation of research material and of the unsuspecting laboratory worker. Physical sterilisation processes. disinfection and sterilisation methods is essential to eliminate or inhibit the ability of a contaminate microorganism from being spread throughout the work area or inoculating research material. Failure to clean up after a procedure. Chemical disinfectants are used on those items or materials that are not designed to withstand heat or gas sterilisation or may involve contact with living tissue. Chemical sterilants are often used as high level disinfectants when shorter contact times are utilized. May not be effective against bacterial spores.e. work surfaces and work areas by decontamination. General housekeeping of the facility can reduce the chances of accidental exposures. The cleaning of laboratory equipment.TOPIC 2: Disinfection and Preservation (Complete by Practical 2) The Purpose of Disinfection and Sterilisation Good housekeeping in a research laboratory should be a principle of operation at all times. Disinfectant: A germicide that inactivates virtually all recognized pathogenic microorganisms but not necessarily all microbial forms. to properly clean used glassware. Common temperature and pressure for being effective is 121°C (250°F) at 15 psi (pounds per square inch) over pressure for 15 minutes. The term has been extensively misused and generally applies to the destruction of any pathogenic vegetative bacteria.Disinfection: The elimination or destruction of all pathogenic microorganisms. The process of cleaning objects without necessarily going through sterilisation. some viruses. Steam Sterilisation: Autoclave. particularly pathogenic microorganisms. Intermediate-level: The elimination or destruction of all vegetative bacteria including the Mycobacteria. High-level: The elimination or destruction of all microorganisms with the exception of high numbers of bacterial spores. most viruses. Sanitization: The process of reducing microbial contamination to an acceptable “safe” level. Sterilisation: The complete elimination or destruction of all forms of life by a chemical or physical means.34 - . the process of sterilisation by the use of heated steam under pressure to kill vegetative microorganisms and directly exposed spores. . and most fungi but does not necessarily kill bacterial spores. Germicide: An agent that destroys microorganisms. An absolute not a relative term. and some fungi but not Mycobacteria or bacterial spores. Special cases may require a variation of the steam temperature and pressure used. Low-level: The elimination or destruction of pathogenic vegetative bacteria. It does not indicate how long the material was exposed to the high temperature. Because of the necessity to allow for adequate exposure for all portions of the materials that are being autoclaved it is necessary to increase the minimum exposure time to 15 minutes. The use of temperature sensitive autoclave tape can be misleading since the tape is only capable of indicating that a general temperature was reached. corresponds to the temperature of 121°C. are destroyed within twelve minutes of exposure and direct contact to pure steam heat of 121°C (121°F). solid or liquid in composition or a combination of all. There are also several chemical indicators that can also provide reliable information. the larger the load the longer the time needed to achieve the needed temperatures deep within the load. Adequate time must be permitted to attain the 121°C for an exposure of at least 12 minutes for all portions of the articles that are being steam autoclaved. The presence of growth in a Bacillus stearothermophilus sample or the presence of a color or of a fluorescing color change in other indicators after being steam autoclaved indicates that the exposure cycle was not adequate and must be repeated. glassware. The researcher must understand and handle potentially infectious materials accordingly to reduce the potential for exposure. All pathogenic bacteria. . and contaminated materials. The type of load to be autoclaved will determine the type of steam sterilising cycle to be used. The effectiveness of a routine steam sterilising cycle can be determined by using the appropriate biological indicator.TOPIC 3: Sterilisation (Complete by Video Demonstration) Steam Sterilisation The use of steam under pressure is perhaps the most efficient means of sterilisation and is widely used in laboratory and medical facilities to sterilize equipment. both vegetative and spore forms. one atmosphere over pressure. Autoclaved biological indicator samples should be examined for growth following an exposure to an actual autoclave cycle.35 - . The use of the spore enzyme test is increasing in popularity because of its ability to provide results within 3 hours of exposure. In addition to the use of a biological indicator for determining the effectiveness of an autoclave cycle it is important that the researcher be aware of any special handling requirements that may be needed to effectively neutralize their cultured microbial agent or contaminated laboratory equipment. and the autoclave must be capable of accommodating for the type of load. Most are destroyed within seconds of exposure. The standard biological indicator that is used in monitoring the effectiveness of steam sterilisation are the Bacillus stearothermophilus spores because the spores are highly resistant to high temperatures. by shape and size. Pure steam at a pressure of 15 psi (pounds per square inch). The duration of time needed to adequately heat sterilize material will be dependent upon the quantity and type of material being sterilized at one time. ampoules or test strips containing Bacillus stearothermophilus spores or a spore enzyme (α-D-glucosidase) based rapid readout test. a liquid load requires a slow depressurization to prevent the liquid from boiling over once the autoclave pressure is reduced. The types of materials that may be steam sterilized in an autoclave can be varied in form. Personal injuries can result from steam burns and from not allowing the autoclave to depressurize properly. contact the responsible person for the unit and inform them of the problem and label the unit “Out of Service”. Before using any steam autoclave. amino and phenolic groups. Ethylene oxide and formaldehyde require special handling procedures to minimize potential personal exposure. Operators of a steam autoclave must remember that a steam autoclave is operated under pressure and at elevated steam temperatures. sulfhydryl. Early testing found that ethylene oxide was very effective as a killing agent of bacteria. Gas Sterilisation Ethylene oxide and formaldehyde gases are generally used for gas disinfection as fumigants under controlled conditions. The gas has an explosive concentration range of 3 to 100 percent. If a steam autoclave is not working properly do not use the unit until it is repaired. it has some major drawbacks that are potentially hazardous that limit its use in a general laboratory environment. spores. the desired level of disinfection and potential hazards associated with handling of the item and the disinfectant.There are a number of different manufactures and different model designs of steam autoclaves. Ethylene oxide is a highly flammable and potentially explosive gas. carboxyl. Not all materials are capable of being exposed to steam sterilisation in an autoclave. Because of the potential health risks and flammability potentials there are special handling and ventilation requirements that must be used when handling ethylene oxide. Ethylene Oxide Ethylene Oxide (ETO) is used primarily as a means of sterilising materials that are not designed to be exposed to steam sterilisation. Both materials are considered to be suspect carcinogens according to OSHA and an occupational carcinogens according to NIOSH. Studies that were conducted to identify the method of activation involved in the destruction of exposed microorganisms found that ethylene oxide caused the replacement of a labile hydrogen with an alkyl group on hydroxyl. The use of ethylene oxide on sensitive plastics. and it is listed as a suspect human mutagen and carcinogen. medical and biological preparations and other heat sensitive equipment has contributed to revolutionizing developments in the medical field. Failure to review the operational directions can result in improper sterilising cycle being used. the operation instructions for proper use and timing requirements must be reviewed. For those items that cannot be steam sterilized there are other alternatives in the form of gas sterilisation or chemical disinfectants that can be used given proper consideration to practicality. damage to the autoclave and potentially serious or fatal injuries of the operator. Due to the hazards associated with potential exposures OSHA has listed an exposure limit of 1 ppm for the duration of a work day. As effective as ethylene oxide is as a gas sterilizer. molds and viruses. damage to the materials being exposed to the steam heat. The alkylation of these compounds in organisms affects cellular function and structure which leads ultimately to inactivation of cellular function and ultimately death. Ethylene .36 - . Paraformaldehyde generates formaldehyde gas when it is depolymerised by heating to 232 to 246°C (450 to 475°F). Formaldehyde gas for decontamination of a biological safety cabinet is generated by heating flaked or powdered paraformaldehyde in the presence of an elevated humidity of nearly 65 percent. Most notable however. niger is used. The Department of . Before an ethylene oxide sterilizer is to be used the unit should be checked for integrity and the operator must be familiar with operational procedures. In areas where formaldehyde may be used for fumigation it is important to be aware of potential contacts with incompatible materials that could cause the formation of dangerous reaction products. The exposure time for a sterilisation cycle is usually 4 to 6 hours in duration followed by a period of ventilation to allow for thorough dissipation of absorbed gas. Using a balanced amount of ammonium bicarbonate neutralizes the formaldehyde gas within the biological safety cabinet.oxide is a gas at room temperature and is not to be used in the open environment of the laboratory due to its volatility and health effects. Ethylene oxide sterilizers are specifically designed to either use a mixture of ethylene oxide and carbon dioxide (10:90) or to use 100 percent ethylene oxide. the depolymerised material reacts with the moisture in the air to form formaldehyde gas. Only individuals that have specific training are permitted to decontaminate biological safety cabinets.75 ppm during a workday. a carcinogenic compound. The spores from B. formaldehyde may combine with hydrochloric acid or hydrogen chloride to form bis(chloromethyl) ether (BCME). The venting of the sterilizer following use is necessary. At the present time the only accepted method available for decontaminating a biological safety cabinet is to use formaldehyde gas. Contact the Department of Environmental Health and Safety for assistance in assessing the potential for personal exposures and evaluation of laboratory handling procedures. Clear all materials out of an area where formaldehyde may be used to minimize the chance of a possible reaction with incompatible chemicals. subtilis were found to be highly resistant to the effects of exposure to ethylene oxide If ethylene oxide is being used in the laboratory it is the laboratory supervisor's responsibility to review all relevant safety information in the safe use. Formaldehyde can react violently or explosively when exposed to incompatibles.37 - . OSHA. To test for proper operation of an ethylene oxide sterilizer the biological indicator Bacillus subtilis var. OSHA has established an exposure limit of 0. NIOSH and IARC recognize formaldehyde as a suspect carcinogen. in the presence of strong oxidizers there is a chance of fire and explosion or when exposed to hydrogen peroxide there is a violent reaction. Formaldehyde Formaldehyde gas is most frequently used in the process of performing space fumigation of a room or of a piece of laboratory equipment that operated with a controlled environment. exposure to the residual material can be damaging to skin and may present a potential fire hazard. handling and disposal of this material and to be certain that others working in the laboratory receive appropriate training and warnings. grease. unlike many of the other disinfectants. These varying recommendations occur primarily because of chlorine's easy inactivation by organic material (serum.e. flammable. The recommended concentration of sodium hypochlorite for "clean surface" disinfection is 200 ppm.. etc. soaps) can reduce the effectiveness of the disinfectant. corrosive. All of the vegetative . a decision that requires a fairly detailed knowledge of the target organism. a basic knowledge of disinfectants. increases as the concentration increases.38 - . Always consult the product information. the material safety data sheet (MSDS). Environmental factors such as air bubbles.Environmental Health and Safety can evaluate work tasks and perform monitoring tests to determine the potential for an occupational exposure. toxic) • Stability of the disinfectant • pH.e.. chlorine has one of the most extensive ranges of organisms that are susceptibility to destruction under ideal circumstances. representing approximately a 1:250 dilution of household bleach. The CDC recommends a 1:10 dilution of household bleach as the disinfectant of choice for blood spills while many laboratory safety texts recommend the use of undiluted household bleach for biohazard spill containment. No one disinfectant is universally ideal and the decision as to the optimum disinfectant involves the consideration of factors such as: • Organism susceptibility • Material or surface to be disinfected • Organic load of the material being disinfected • Potential health risks to laboratory personnel • Hazardous properties of the disinfectant (i. Of all the disinfectants. therefore. on a disinfectant before using the material. dirt. For the chosen chemical disinfectant to be affective when used it must be able to make direct contact with the target organism. proteins. temperature and presence of other contaminants in media and water for dilution • Required contact time for effective disinfection • Requirements for disposal of the disinfectant • Cost Choosing a disinfectant is. Halogens Chlorine Chlorine is one of the least expensive and most effective disinfectants. and careful consideration of the above factors as they apply to the unique potential conditions in which your laboratory will employ the disinfectant. a dense concentration of microorganisms and the presence of other chemicals (i. Appropriate personnel protective equipment is required to be worn when materials are being mixed and used. Chemical Disinfectants Choosing a Chemical Disinfectant A variety of concerns must be addressed when choosing a disinfectant for use in a biohazard area. blood.) and the fact that chlorine's disinfectant activity. 39 - . acid-fast bacteria. must never be autoclaved. While these compounds offer specific advantages. First. Bacterial spores are also susceptible although longer exposure times are generally required. Chlorine solutions. household bleach remains one of the best disinfectants available. often resulting in damage to the substrate materials being disinfected. and both enveloped and non-enveloped viruses although longer contact times are generally required under similar conditions. The limited pH range in which chlorine is effective. Most of the iodine-based disinfectants utilized in laboratory and medical situations are combinations of elemental iodine or triiodide with a neutral polymer carrier molecule. Like chlorine disinfectants. Chlorine disinfectant solutions are also extremely sensitive to pH and the sensitivity has dramatic implications on the effectiveness of these solutions. Iodophors are excellent disinfectants and antiseptics and are extensively used for surgical scrub solutions. therefore. Lastly. iron. A number of alternative forms of chlorine exist to use in the form of household bleach (sodium hypochlorite).bacteria that have been tested are susceptible to chlorine destruction. the contact of chlorine solutions with formaldehyde produces the carcinogen bis-chloromethyl ether. Chloramine-T and other organic chlorine compounds also offer increased resistance to organic inactivation but at the cost of decreased activity. One of the main disadvantages of chlorine as a disinfectant is the ease with which it is inactivated by organic material. Both enveloped and non-enveloped viruses are susceptible to chlorine inactivation. Iodine Iodine-based disinfectants share the same properties as the chlorine-based disinfectants but are somewhat less reactive with substrates and microorganisms. the heating of chlorine solutions produces the carcinogen trihalomethane. Chlorine is also easily inactivated by a variety of metals including copper. the addition of acid to hypochlorite solutions will produce a rapid production of toxic chlorine gas. Chlorine solutions are most active under slightly acid conditions (pH 6 to pH 7). the activity level decreases rapidly under conditions where the pH goes from a pH 7 to pH 8. . Second. zinc. Materials to be disinfected should be first cleaned to remove the organic material or the concentration of the chlorine must be increased to compensate for the organic material inactivation. These compounds are collectively referred to as iodophors. including the acid-fast bacteria. the iodines are effective against vegetative bacteria. As the pH of chlorine solutions increases the disinfectant activity levels decreases. Chlorine dioxide compounds are high level disinfectants/steriliants that offer somewhat increased activity and resistance to organic inactivation in comparison to household bleach. slightly acid to slightly basic. etc.5. bacterial spores. and the use of chlorine as a disinfectant on these materials requires increased concentrations of chlorine. is also a limiting factor necessitating the use of nonionic detergents or precleaning followed by thorough rinsing. Important Information when Considering to Use Hypochlorite Solutions: Three situations exist where the uses of hypochlorite solutions pose a potential risk to personnel using the compound. nickel. ninety percent (60 . have explored the various chemical substitutions and their effect upon germicidal properties. Both are rapidly bacteriocidal against vegetative bacterial forms. and virucidal activity against lipophilic viruses (enveloped viruses). The disinfectant properties of both ethanol and isopropyl alcohol rapidly drop at concentrations below fifty percent (50%) and above ninety percent (90%). however. Neither inactivates bacterial spores and isopropyl alcohol fails to inactivate hydrophilic viruses. Unlike the elemental chlorine and iodine. Phenol should never be used for sterilisation purposes. Overall.40 - . Phenolic compounds may exhibit dramatic toxic effects. as disinfectants are o-phenylphenol. phenols have been extensively used. o-benzyl-p-chlorophenol. the only phenolic derivatives found in extensive use.hand-washing compounds. and virucidal. tuberculocidal. tuberculocidal. Alcohols.90%) by volume. The mode of action of phenolic compounds appears to be a generalized cytoplasmic poisoning at higher concentrations and an inactivation of enzyme systems and cell wall integrity at lower concentrations. They lack sporicidal activity and are ineffective against noneveloped viruses. Reports of phenolic disinfectant induced skin depigmentation. Phenolic Compounds Ever since the adoption of carbolic acid by Lister as the first germicide. fungicidal. Peak disinfectant activity occurs at approximately sixty-seven percent (67%) concentration. fungicidal. Today. are destructive to rubber compounds and to most of the cement and glues used in instruments. especially optics. Phenol compounds rapidly penetrate porous compounds and tend to accumulate in the body fat of exposed animals. iodophors are extremely sensitive to concentration and are quite expensive. Their use also results a residual activity on surfaces. beginning with a study by Kronig and Paul in 1897.to intermediatelevel disinfectants appropriate for general use in noncritical or semicritical areas. in general. and disinfectants for small laboratory objects. Both ethanol and isopropyl alcohol are volatile and flammable compounds and must only be used with adequate ventilation. Numerous studies. One of the most critical factors in the use of alcohols as disinfectants is concentration. nerve . Phenols have a high tolerance to both organic load and hard water. phenolic derivatives are best classified as low. The recommended concentration for use is sixty . Both ethanol and isopropyl alcohol should be considered as intermediatelevel disinfectants. and p-tertamylphenol. Overall the phenolic derivatives are all characterized by a broad-spectrum of activity against grampositive and gram-negative bacteria. Alcohols Ethyl and Isopropyl Alcohol Ethanol and Isopropyl alcohol are both excellent disinfectants whose germicidal properties are generally underestimated. are commonly used as antibacterial agents in soaps and scrubs as well as preservatives in a number of products. etc. Chlorhexidine's primary advantage over other disinfectants and antiseptic agents involves both its rapid rate of bacteriocidal activity and its strong binding to skin and mucous membranes. low mammalian toxicity. Bacterial spores are not killed but germination is inhibited while in contact with chlorhexidine. Chlorhexidine compounds are generally active against gram-positive and gram-negative vegetative bacteria and lipophilic viruses. advantages. Quaternary Ammonium Compounds (QAC) Quaternary ammonium disinfectants first appeared in the late 1930's. and a strong affinity for binding to skin and mucous membranes. As an antiseptic. and disadvantages. and ethyl groups resulted in increased activity and the generation of the second generation of quaternary ammonium ..to intermediate. there has been the addition of numerous compounds. all of which are desirable characteristics for an antiseptic.g. Chlorhexidine's activity at relatively low concentrations involves a series of related cytologic and physiologic changes culminating in ion leakage from the cytoplasmic membrane and cytoplasmic precipitation. Many fungi are sensitive to chlorhexidine and acid-fast bacteria are generally inhibited but not killed (bacteriostatic).demyelination and skin contact dermatitis that requires personnel using phenolic disinfectant be provided with appropriate protective clothing and equipment. Chlorhexidine) Discovered during a search for potential anti-malarial drugs.41 - .4. Common disadvantages include a lack of sporicidal activity and a lack of activity against acid-fast bacteria (except for some of the latest generation of QACs).level disinfectant appropriate for noncritical and semicritical area disinfectant. and a relatively low level of mammalian toxicity. different adjunctive agents. Substitution of the aromatic ring hydrogen with chlorine. blends. Triclosan is now commonly used in antibacterial soaps and deodorants as well as being incorporated into plastics as a "permanent" (but questionable) antibacterial. Chlorhexidine is best classified as a low. Two halogenated phenolic derivatives. The first generation of quaternary ammonium compounds were the standard benzalkonium chloride compounds developed in the 1930's. Biguanides (e.. chlorhexidine proved to have a high level of antibacterial activity. the lack of direct tissue toxicity and the rapidity of action makes chlorhexidine an excellent bacteriocidal skin cleanser and wound cleaning agent. making the entire group of quaternary ammonium disinfectants a rather broad group with a variety of activities.hydroxydiphenol (Triclosan. The major advantages that are common to the group are an inherent surfactant activity. methyl. PCMX has become the most widely used antiseptic scrub in surgery and is used as a preservative in products ranging from printing inks to cosmetics to shoe polishes. Irgasan). parachlorometaxylenol (PCMX) and 2. Since the original introduction. allowing them to also serve as cleansing agents.4'trichloro-2. Inactivating substances vary dramatically between the generations of QACs with the later generations generally much less susceptible to inactivation by extraneous material such as organic load or hard water. ophthalmic solutions. now polymeric and polysubstituted quaternary ammonium compounds. it is critical to read the label directions on the bottle.42 - . The newest generation of QACs possess a wide spectrum of activity with minimal mammalian host damage and are used in pharmaceuticals. are in the seventh generation of development. . The third generation of quaternary ammonium compounds.compounds. were developed in 1955 and represented compounds with superior microbiological activity. the quaternary ammonium compounds. The antimicrobial activity of quaternary ammonium compounds appears to be by inactivation of critical enzyme systems. etc. Presently. or the dual QACs. As far as choosing a quaternary ammonium disinfectant. Organism susceptibilities differ dramatically between different generations of QACs and different formulations. and contact lens solutions. This procedure should take about 1 minute when using soap and about 2 minutes when using an antiseptic scrub as the contact time for the antiseptic needs to be adequate. Rub right palm with fingertips of left hand. After handling microorganisms. 7. repeat with each finger . Before leaving the laboratory. 3. Wash palm to palm interlacing fingers. 3. nails must be kept short and clean and the hands of staff and students must be washed with soap or an antiseptic scrub depending on the situation: 1. Wash right palm over back left hand. Repeat as necessary. reverse the action. Hand Washing 1. then using soap or antiseptic scrub. Wash palm to palm. 4. reverse the action. 10. 9. 2. 8. 6. reverse the action. Initially rinse hands under running water. Before making clean or sterile products.Appendix 1 SOP 1: Hand Washing Policy To minimise contamination in products. Dry your hands with paper towel or the hot air dryers BUT Do not use paper towel to dry your hands before making a parenteral product as fibres may be transferred to your product. . Rinse well after washing. 5. Hold right thumb with left hand and rotate. Keep hands palm downwards to allow water to run into sink.43 - . 2. 6. starting at the back and working towards the front. 2. Bench. 7.44 - . 4. The order shall be: a. Never go back to an area already cleaned. 5. Remove all extraneous materials from underneath the hood. Wipe the base again. Sides d. Use broad sweeping motions. 8. . Wipe the hood with 70% w/w ethanol in water. Front e.SOP 2: Cleaning Aseptic Hood Cleaning Aseptic Hood 1. Leave the ethanol aerosol to settle for 2 minutes. Top b. Spray the interior with 70% w/w ethanol in water. Back c. 3. remove the Swinnex filter holder. place it on the (dry) support grid of the Swinnex adaptor then place an O ring on the filter and screw the other section of the filter device in place.8 µm pore size assembled in Swinnex 25mm filter holders are used. Filtration of small volumes using a syringe 1. eye preparations and aqueous solutions required to be sterile are to be filtered using membrane filters. . To refill the syringe. 4. Attach the Swinnex filter holder (assembled with membrane filter) to the syringe and with the syringe at an angle of 45o (filter unit uppermost) carefully expel the air from the filter holder. 3.45 - . Use techniques that minimise particulate and biological contamination and filter the solution as soon as possible after preparation. Transfer the filtered solution directly to the final container (using a needle if necessary) measuring the volume by the travel of the plunger relative to the volume scale on the syringe. refill the syringe and eliminate any air then re-attach the filter holder. Filters of 0. This will minimise contamination of the product with extractable components of the filter and avoid loss of actives by adsorption to the membrane. This ensures the solutions have low particulate contamination and significantly reduces the bioburden prior to sterilisation. Filter a small volume of the solution and discard. 5. Tighten firmly. It is only supported when liquid flows from the syringe. Remove the filter from between the waxed paper spacers with a flat faced pair of tweezers. 6. Fill a syringe with the maximum syringe volume or a volume in excess of the product volume and then exclude any entrapped air. Preparation of solutions for filtration Prepare the solution to be filtered with appropriately cleaned glassware and equipment. Never draw liquid into the syringe through an assembled Swinnex filter holder. 2. The filter is fragile.SOP 3: Membrane Filtration of Small Volume Aqueous Solutions Policy All parenteral products.