PHAR5515 Manual 2016

PHAR 5515

THE UNIVERSITY OF SYDNEY Faculty of Pharmacy

Microbiology in Pharmacy

Practical Manual 2016

Name: ........................

Contents

LABORATORY RULES ........................................................................................... 2ASSESSMENT .......................................................................................................... 3PENALTIES .............................................................................................................. 4PLAGIARISM ........................................................................................................... 4

PRACTICAL 1 ............................................................................................................. 5TOPIC 1: ASEPTIC PRODUCTION .............................................................................. 5

Exercise 1.1: Aseptic transfer ............................................................................... 7PRACTICAL 2 ........................................................................................................... 15

TOPIC 2: FILTRATION, DISINFECTION AND PRESERVATION .................................. 15Exercise 2.1: Testing efficiency of filtration with the micro-organism Serratia marcescens (bacterial challenge test) .................................................................. 17Exercise 2.2: Evaluation of preservatives ........................................................... 20Exercise 2.3: Determination of MICs of antimicrobial agents ............................ 22Exercise 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

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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.

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.

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.

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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 (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

a. Attendance and Participation (2%) b. Practical Report (5%) c. Video Demonstration Online Quiz (3%)

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assessment will be appeared on screen prior of beginning of the assessment.

PENALTIES Absence from any assessable activity will result in a mark of zero being recorded for the task. If you miss an assessable activity because of certified illness or misadventure, you must apply for Special Consideration. IF YOU MISS A PRACTICAL CLASS • Inform your demonstrator of the reason for your absence and, submit a Special

Consideration form to the Faculty of Pharmacy Office. Your demonstrator may be able to organise an alternative time for you to learn new practical skills.

• It is your responsibility to obtain all information and results for the class and to fill in your Practical Manual.

PLAGIARISM Plagiarism occurs when you present someone else’s words or ideas as your own by presenting, copying or reproducing them without acknowledging the source. Plagiarism is a kind of stealing, and is dishonest and unacceptable. The University has very clear and strict guidelines about responding to plagiarism and the penalties for this are quite severe, and range from not receiving any marks for the specific piece of work to expulsion from the University in extremely serious cases. Thus, it is very important that you avoid plagiarism. Please note, specific and detailed collaboration with other students on assignments not specifically designated as “Group Assignments”, or “Joint Work” is also considered plagiarism. The policy defines “Legitimate Cooperation”, explaining the ways in which students are permitted to work together, without plagiarising each other’s work.

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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

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Practical 1 Pre-work NAME.………………….…………………….SID..………………….………………. 1. In pharmaceutics, the definition of an aseptic technique is: the act of handling

materials in a controlled environment in which the air supply, materials, equipment, and personnel are regulated to control microbial and particulate contamination in pharmaceutical products within acceptable levels. List ten basic rules of the aseptic technique in the production of clean pharmaceutical products.

……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ……………………………………………………………………………………… ………………………………………………………………………………………

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Exercise 1.1: Aseptic transfer

Introduction In this practical you will be introduced to aseptic preparation of pharmaceutical products. In aseptic preparation, 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. This exercise demonstrates the aseptic process for transfer of liquids, dilution of liquids, weighing and dissolving of solids, and capping of containers. Protocol 1 does not follow the basic rules of aseptic preparation, therefore, 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. Aim

• To understand the principles of aseptic techniques. 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

1 pack of 3 rubber closures

Boil in purified water- rinse well and pack in autoclave bag

Moist heat 121°C for 15 min

Lactose 500 mg Double sealed in envelopes Dry heat 160°C for 2 hours

25 mL measuring cylinder, beaker and stirring rod

Double sealed in autoclave bags Dry heat 160°C for 2 hours

1 × 25 mL sterile water Moist heat 121°C for 15 min

1 × 25 mL Nutrient Broth Moist heat 121°C for 15 min

Method - Protocol 1 1. Gather all the materials and equipment required for this exercise in front of the

hood.

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2. Attach a small piece of autoclave tape to each multidose container and label them 1, 2 and 3.

3. Open the wrapping of beaker, measure, stirring rod, and lactose outside the hood.

Discard all wrapping material into the waste bin. Transfer all equipment and materials into the hood.

4. Open the sterile nutrient broth, pour the entire contents into a beaker and then

transfer 10 mL to the measure. 5. Remove the aluminium foil from multidose container no. 1 and transfer 10 mL of

nutrient broth from the measure. 6. Open the sterile water and pour 15 mL into the measure (in the same measure),

then transfer it to the remaining nutrient broth in the beaker. Swirl to mix the diluted broth.

7. Measure 10 mL of the diluted broth (in the same measure) and transfer it to the

multidose container no. 2 (using the same technique as for the container no. 1). 8. Unfold one end of the package containing lactose and transfer the contents to the

beaker (now containing 20 mL diluted broth). 9. Use stirring rod to dissolve the lactose in the broth. 10. Measure 10 mL of this lactose/diluted broth (in the same measure) and transfer to

the multidose container no. 3 (using the same technique as for the container no. 1). 11. Remove all the material, except the multidose containers. 12. Insert the closures in the neck of the multidose containers. 13. Before removing the containers from the hood, make certain that the closures are

pressed firmly into the necks, and then take them to the crimping unit. GENTLY crimp a metal closure onto the neck of each multidose container.

14. Bind the 3 bottles together with a tape then label with your name and protocol

number. Leave them on your bench: They will be collected and incubated for 7 days at 32oC then returned to you next week.

15. Next week, record the results in the following table.

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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



3 × 20 mL multi-dose containers (dry) Seal with foil Dry heat 160°C for 2 hours

1 pack of 3 rubber closures



Lactose 500 mg Double sealed in envelopes Dry heat 160°C for 2 hours

25 mL measuring cylinder, beaker and stirring rod

Double sealed in autoclave bags Dry heat 160°C for 2 hours

1 × 25 mL sterile water Moist heat 121°C for 15 min


Method - 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. 2. Clean your aseptic hood in accordance with SOP 2 in Appendix 1.

3. Gather all the materials and equipment required for this exercise in front of the

hood.

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4. Attach a small piece of autoclave tape to each multidose container (vial) and label

them 1, 2 and 3. 5. Transfer the materials and equipment into the clean hood as follows: 6. 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, open it and place the lid on the other side of the work area.

7. Swab a pair of forceps with 70% ethanol and rest them on the sterile Petri dish. 8. In turn, lightly spray the bottom of each multidose container (outside the container)

with 70% ethanol, and as you transfer it into the hood loosen the aluminium foil cap so that it can be removed with forceps. Arrange the 3 multidose containers at the rear of the hood.

9. Open the outside wrapping of your equipment (beaker, measure and stirring rod)

and then transfer them (still single wrapped) under the hood. Do the same for the lactose.

10. Swab the McCartney bottles of sterile nutrient broth and sterile water with 70%

alcohol and place them under the hood. 11. Roll up your sleeves then following SOP 1 in Appendix 1 clean your hands and

forearms with chlorhexidine scrub. 12. 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). 13. Open the sterile nutrient broth, pour the entire contents into the beaker and then

transfer 10 mL to the measure. 14. Remove the aluminium foil with forceps from multidose container no. 1 and

transfer the 10 mL of nutrient broth from the measure. Immediately replace the foil.

15. Open the sterile water and pour 15 mL into the measure (in the same measure),

then transfer it to the remaining nutrient broth in the beaker. Swirl to mix the diluted broth.

16. Measure 10 mL of the diluted broth (in the same measure) and transfer to the

multidose container no. 2 (using the same technique as for the container no. 1). 17. Unfold one end of the package containing lactose and transfer the contents to the

beaker (now containing 20 mL diluted broth).

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18. Tear open the wrapper covering the sterile stirring rod, and use it to stir and completely dissolve the lactose in the broth.

19. Measure 10 mL of this lactose/diluted broth (in the same measure) and transfer to

the multidose container no. 3 (using the same technique as for the container no. 1). 20. Remove all the material, except the multidose containers, from the hood and

discard wrapping material and Petri dish into the waste bin. 21. Again, take a sterile Petri dish from the pack beside the hood and place, and open

it inside the hood as before. Re-swab the forceps with 70% alcohol and place them on one of the open halves of the Petri dish. Tear open the outer wrapping of the packet of rubber closures, transfer it under the hood.

22. 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. Using the forceps pick up a rubber closure and simultaneously, as you lift the foil

from the vial with your other hand, insert the closure in the neck of the multidose container. Repeat for all three containers.

24. Before removing the containers from the hood, make certain that the closures are

pressed firmly into the necks, and then take them to the crimping unit. GENTLY crimp a metal closure onto the neck of each multidose container.

25. Bind the 3 bottles together with a tape then label with your name and protocol number. Leave them on your bench: They will be collected and incubated for 7 days at 32oC then returned to you next week.





n




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Protocol 3



1 × 20 mL multi-dose containers (empty) Seal with foil Dry heat 160°C for 2 hour

2 rubber closures for multi-dose containers



Lactose 500 mg in multi-dose container Seal with foil Dry heat 160°C for 2 hours

1 disposable 20 ml syringe In disposable bag Radiation 25 G

1 × 20 mL vial Water for Injections

Filtered with 0.8µm membrane filter Moist heat 121°C for 15 min


Method - Protocol 3 Plan your work carefully before commencing. All manipulations are to be carried out under the plastic hoods to simulate using aseptic hoods. 1. Clean your aseptic hood in accordance with SOP 2 in Appendix 1. 2. Gather all the materials and equipment required for this exercise in front of the

hood. 3. Attach a small piece of autoclave tape to each multidose container and label them

1. (empty Multidose vial), 2. (Water For Injection) and 3. (lactose vial). 4. Expose the needle entry point under the metal crimp sealing the 20mL vial of

water for injections with your forceps. Do not take the crimp off the bottle. 5. Transfer the materials and equipment into the clean hood as follows: 6. 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, open it and place the lid on the other side of the work area.

7. Swab a pair of forceps with 70% ethanol and rest them on the sterile Petri dish. 8. In turn, lightly spray the bottom of each multidose container (outside the

container) with 70% ethanol, and as you transfer it into the hood loosen the foil

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cap so that it can be removed with forceps. Arrange the 3 multidose containers at the rear of the hood.

9. Swab the McCartney bottle of sterile nutrient broth, the multidose vial of water for

injections, the syringe and needle pack with 70% ethanol and place them under the hood.

10. Roll up your sleeves then following SOP 1 in Appendix 1 clean your hands and

forearms with chlorhexidine scrub. 11. Tear open the wrapping, remove the syringe and stand it vertically on the flat end

of the plunger (discard the packing to the bin). 12. Open the needle pack and while the needle is still in the cover attach it to the

sterile syringe. 13. Adjust the plunger until the syringe contains 10 mL of air then remove the needle

cover (discard) DO NOT RE-SHEATH NEEDLE. Pierce the rubber closure of the multidose container (no. 2) and inject 10 mL air into the container, 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. The multidose vial (no. 2) now has 10 mL of water for injection. Stand the syringe on its base again.

14. Open the sterile nutrient broth container and remove 20 mL of broth with the

syringe. 15. Remove the foil with forceps from multidose container no. 1 and transfer 10 mL

of nutrient broth from the syringe. Immediately replace the foil. 16. Transfer the remaining 10 mL of nutrient broth in the syringe into the multidose

container (no. 2) containing 10 mL water for injections. Remove the syringe and shake the container to dilute the nutrient broth. Withdraw 10 mL of diluted broth using syringe by injecting 10 mL air into the container.

17. Transfer 10 mL of this diluted broth to the multidose vial containing the 500mg lactose (container no. 3).

18. Remove all the material, except the multidose containers, from the hood and

discard wrapping material and Petri dish into the waste bin. 19. Again, take a sterile Petri dish from the pack beside the hood and place, and open

it inside the hood as before. Re-swab the forceps with 70% alcohol and place them on one of the open halves of the Petri dish. Tear open the outer wrapping of the packet of rubber closures, transfer it under the hood.

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20. 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.

21. Using the forceps pick up a rubber closure and simultaneously, as you lift the foil

from the vial with your other hand, insert the closure in the neck of the multidose container. Repeat for all three multidose containers.

22. Before removing the containers from under the hood make certain that the

closures are pressed firmly into the necks, and then take them to the crimping unit. GENTLY crimp a metal closure onto the neck of each multidose container.

23. Once the closure is firmly inserted and crimped to the multidose vial containing

the lactose, this bottle can be shaken until the lactose has dissolved.

24. Bind the 3 bottles together with a tape then label with your name and protocol number. Leave them on your bench: They will be collected and incubated for 7 days at 32oC then returned to you next week.





n




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PRACTICAL 2

FOLLOW UP WORK FROM PRACTICAL 1 - Exercise 1.1

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

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Practical 2 Pre-work NAME.………………….…………………….SID..………………….………………. 1. Define: Antiseptic:……………………………………………………………………………… …………………………………………………………………………………………. Disinfectant:…………………………………………………………………………… …………………………………………………………………………………………. Preservative:………………………………………………………………………….... …………………………………………………………………………………………. Sterilisation:…………………………………………………………………………… ………………………………………………………………………………………… 2. What is the main anti-microbial agent of the following disinfectants / antiseptics? Milton:………………………………………………………………………………….. Dettol:…………………………………………………………………………………... Savlon:………………………………………………………………………………….. Betadine:………………………………………………………………………………...

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Exercise 2.1: Testing efficiency of filtration with the micro-organism

Serratia marcescens (bacterial challenge test)

Introduction Filtration is unique among other sterilisation techniques (e.g., steam and dry heat sterilisation) in that it removes, rather than destroys, micro-organisms. It is also capable of preventing the passage of both viable and non-viable particles. The ideal filter medium should offer efficient removal of particles above stated size, acceptably high flow rate, resistance to clogging, flexibility and mechanical strength, low potential to release fibres or chemicals. In this exercise you will perform the bacterial challenge test for efficacy of filtration with the Serratia marcescens. Aims

• To test the ability of a filter to produce a sterile filtrate from a culture of Serratia marcescens.

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. • Needle. • Suspension of Serratia marcescens (~107 organisms/mL). • 2 x nutrient broth in glass tubes • 1 x peptone glycerol agar plate.

Methods All manipulations are to be carried out in the biohazard laminar flow cabinet. 1. Only two students at a time can work at the Biohazard cabinet.

2. Wash your hands, according to SOP1 Appendix 1.

3. Label the McCartney bottles and plate with your initials and bench number and

indicate if they represent the filtered or unfiltered test. Transfer them into the biohazard hood and loosen the caps so they can be easily removed.

4. Remove a 5 mL sterile disposable syringe from its package, attach a sterile needle then stand it on the base of its plunger.

5. Remove the lid from a bottle containing a suspension of Serratia marcescens (about 107 CFUs)/mL. With the syringe remove 5 mL of the Serratia suspension from the bottle, remove the needle and discard it into the sharps container, then stand the syringe on its base. Re-cap the bottle of Serratia.

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6. Transfer 2 mL of the Serratia suspension (without filtration) to one of the tubes of

broth.

7. Attach the 5 mL syringe (containing approximately 3 mL of Serratia suspension) to your assigned filter. Please see SOP 3 in Appendix 1 for more details about filtration technique.

8. Transfer, through the filter, 2 mL of the suspension to the other tubes of broth and 2 drops onto a peptone glycerol agar plate.

9. Put the lid of the agar plate back and spread the S. marcescens suspension by shaking gently the agar plate.

10. Detach the syringe from 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.

11. Take the agar plate and broth tubes to your bench. Bind the tubes together and make certain they are labelled with your name, type of the filter that you used (filter in Swinnex or commercially disposable filter), and your bench number.

12. The tubes and agar plates will be collected and incubated at 25°C for 2 days and returned to you next week.

Results Individual Results Examine the tubes and plate for the growth of Serratia marcescens. The nutrient broth is cloudy if you have contamination. When incubated at 25°C Serratia marcescens produces a red pigment. The tubes or plates contaminated with Serratia have a light pinkish colour. 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. Class results will be displayed on the notice board 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. Membrane filters have a limited capacity as they are essentially surface filters for particles above or around their nominal pore size.

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Bacterial Challenge Test

Type of filter used:----------------- ----------------------------------------

Visible growth of microorganisms

Filtered (plate)

Filtered (tube)

Unfiltered (tube)

Class Results Two 0.22 µm filters are used in this exercise.

Bacterial Challenge test Challenge test on 0.22 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

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Exercise 2.2: Evaluation of preservatives

Introduction Pharmaceutical products are frequently intended for multiple use. 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.g. 28 days after opening). Oral mixtures and topical products are not required to be sterile, however it is important that they do not become heavily contaminated during their claimed shelf-life which is frequently much longer that 28 days. To reduce contamination level of pharmaceutical products during storage, the products are formulated with one or more preservatives. In this exercise a creamy lotion for topical application is formulated with different preservatives. The lotions are challenged with a defined inoculum of suitable micro-organisms and then stored at a prescribed temperature to evaluate the microbial growth. 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, we will use Saccharomyces cerevisiae and Staphylococcus epidermidis. Aims

• To evaluate the efficacy of three preservatives in preserving a lotion based on the APF17 formula for Cetomacrogol lotion.

Method You will perform part of a group experiment. Cetomacrogol lotion APF17 usually contains chlorhexidine gluconate as the preservative however it is possible to substitute other preservatives if required. Three lotions are to be investigated, preserved with either Chlorhexidine gluconate solution 0.1% Chlorocresol 0.1% Phenoxyethanol 1 % 10 g portions of lotions each will have been inoculated with either 106-107 CFU (Colony Forming Unit per millilitre) of S. cerevisiae or S. epidermidis and incubated at 20°C to 25°C for 7 days. Each student will perform a count of viable organisms for the one 10g portion of contaminated lotion supplied to them at their bench. 1. Unscrew the lid off a bottle of lecithin-Tween broth (LTB) until it can be removed

with one hand.

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2. Gently agitate the sample of lotion to ensure that it is homogeneous then unscrew the lid until it can be removed with one hand.

3. With a sterile disposable syringe remove 1 mL of the lotion and transfer it to the

bottle of 20 mL lecithin-Tween broth (LTB). Mix thoroughly on a vortex mixer. Discard the syringe into the contaminated waste container immediately, and recap the lotion sample and leave it on your bench.

4. Using a sterile Pasture pipette transfer 4 drops of the lotion/lecithin-Tween (LTB)

broth to a lecithin-Tween agar plate. Insert a wire spreader into the hot bead sterilizer for ten seconds, allow to cool, and then spread the lotion/broth liquid evenly with the spreader. Discard the pipette into the contaminated waste container and flame and insert the spreader into the hot bead after use.

5. Label the plate with your name, and the code letters that were marked on the

sample of lotion you were provided with. 6. Next week, inspect for growth and fill out the following table. Your demonstrator

will explain to you how to calculate the Colony Forming Unit (CFU) of your agar plate.

Lotion code: Plate count of CFUs:

Antimicrobial agent Microorganism Code Average Plate Count

Log Reduction in CFUs

Chlorhexidine gluconate 0.1%

S. epidermidis CGB S. cerevisiae CGF

Chlorocresol 0.1%

S. epidermidis CSB S. cerevisiae CSF

Phenoxyethanol 1%

S. epidermidis PEB S. cerevisiae PEF

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Exercise 2.3: 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 micro-organisms do not grow. There are two basic difficulties in interpreting this type of test however, firstly, it gives no indication as to whether the antimicrobial agent is bactericidal or bacteriostatic and secondly, it is performed in a complex nutrient in which a number of antimicrobials are inactivated. Aims

• To determine the Minimum Inhibitory Concentration (MIC) of 2-phenoxyethanol against a gram negative (Serratia marcescens) and a gram positive (Staphylococcus epidermidis) bacteria.

• 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.

Methods You will perform part of a group experiment. You will be supplied with one tube containing an antimicrobial system in Tryptone Soy broth. 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. Phenoxyethanol Systems If your assigned antimicrobial system is phenoxyethanol, one broth will be labelled PTS (20 mL) and four labelled MTS (10 mL). If your assigned antimicrobial system is phenoxyethanol + EDTA, one broth will be labelled PTSE (20 mL) and four labelled MTSE (10 mL). 1. Label the McCartney bottle containing 20 mL broth P1 and the bottles containing

10 mL broth P2, P3, P4, and P5 then unscrew the lids so that they can be removed with one hand.

2. 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. Re-seal the bottle and mix.

3. Withdraw 10 mL of antimicrobial/broth from the bottle 2 using the same syringe

and transfer to the bottle 3. Replace the needle cover again. Re-seal the bottle and mix.

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4. Withdraw 10 mL of antimicrobial/broth from the bottle 3 and transfer to the bottle 4. Replace the needle cover again. Re-seal the bottle and mix.

5. Withdraw 10 mL of antimicrobial/broth from the bottle 4 and transfer to the bottle 5. Replace the needle cover again. Re-seal the bottle and mix.

6. Assemble the 5 McCartney bottles at the “contaminated” work area (at the end of

the bench) and again loosen the caps. 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). Re-seal the bottles and mix.

7. 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.

8. The bottles will be incubated at 32°C until next week. Next week record the results in the following table. 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:------------------------------ (What is the minimum concentration that prevents microbial growth) Class Results Against Gram + Against Gram - 2-Phenoxyethanol + EDTA

- EDTA

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Exercise 2.4: Formulation compatibility and preservatives

Aims

• To observe whether the “incompatibility” of chlorhexidine with aqueous cream affects the antimicrobial activity of chlorhexidine.

• To observe the effect of three other cream bases on the activity of chlorhexidine using the agar diffusion technique.

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. To each, 0.5% chlorhexidine gluconate has been added and they have been packed into 10 mL syringes (placed on your bench). 1. Take a previously melted and cooled (to about 50°C) tube of nutrient agar. To

keep the bacteria alive, do not inoculate bacteria if the agar is still hot (ask your demonstrator if you are not sure).

2. Inoculate with 2 drops of a broth culture of S. epidermidis. 3. Roll the tube gently to mix so as to avoid any air bubbles. 4. Pour aseptically (without contaminating the agar or dish) into a sterile Petri dish

and allow to set. 5. Once the agar gel is set, drill four holes in the plate using a sterile borer (ask your

demonstrator to show you). 6. Discard the agar plugs and the borer into its container. 7. Label the holes 1 to 4 on the base of the plate and with your name and bench

numbers. 8. Fill hole #1 with the gel as a control Fill hole #2 with cetomacrogol cream Fill hole #3 with aqueous cream. Fill hole #4 with oily cream To fill the holes, 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. Do not overfill the holes. The cream should touch the wall of the hole. 2 1

4 3

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9. Leave the plate on the bench. It will be collected, incubated at 32 °C for 24 hours and returned to you next week. Next week record your data in following table.

Measure the diameters of the holes containing the formulations and the inhibition zones using a ruler and record in the table below.

Formulation Diameter of hole dh (mm)

Diameter of inhibition zone dz (mm)

dz2 - dh2

# 1 Methylcellulose gel

# 2 Cetomacrogol Cream

#3 Aqueous cream

#4 Oily cream

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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.

• 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).

• This work is substantially my own, and, to the best of my knowledge, 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, I have indicated that it is not my own by acknowledging the source of that part or those parts of the work.

• I have not re-used previously submitted material in this assignment. • I have not engaged someone else to complete this assignment. • I have retained a duplicate hard copy of this assignment.

Student Name SID Signature Date

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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 1.1: Aseptic Transfer


Results Class Results (contaminated broths as % of n)

n






n






n




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1. Did you observe any difference in bacterial growth between protocol 1, 2, and 3? Comment on your class results. Compare these three protocols, and list the major factors that can cause contamination in the aseptic transfer protocol 1, 2, and 3. (15 marks)

………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ……………………………………………………………………………………….....

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Exercise 2.1: Testing Efficiency of Filtration with the micro-organism Serratia marcescens (Bacterial Challenge Test) Class Results

Bacterial Challenge test Challenge test on 0.22 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. Comment on class result with respect to differences in filter types, differences in filter assemblies, and overall on the confidence you would have in using this type of sterilisation process in preparation of pharmaceutical products. List the factors that may cause contamination during filtration. (12 marks)

……………………………………………………………………………………….... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ……………………………………………………………………………………….....

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Exercise 2.2: Evaluation of Preservative 3. Although chlorocresol (a phenolic component) is a strong anti-microbial agent,

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. (6 marks)

………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ………………………………………………………………………………………..... ……………………………………………………………………………………….....

………………………………………………………………………………………..... ………………………………………………………………………………………..... Exercise 2.3: Determination of MICs of Antimicrobial Agents Class Results Against Gram+ Against Gram- 2-Phenoxyethanol + EDTA

- EDTA 4. Are the MICs dependent on the micro-organisms chosen for the test? Gram+ or

Gram–: which one is more resistant? Why? (6 marks) ........................................................................................................................................... ........................................................................................................................................... ........................................................................................................................................... ...........................................................................................................................................

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........................................................................................................................................... 5. Comment on the effect of adding EDTA to the antimicrobial agent and suggest a

reason for that effect. (6 marks) ........................................................................................................................................... ........................................................................................................................................... ........................................................................................................................................... ........................................................................................................................................... ........................................................................................................................................... ........................................................................................................................................... Exercise 2.4: Formulation Compatibility and Preservatives 6. What is the nature of the “incompatibility” of chlorhexidine with aqueous cream

and does this “incompatibility” affect its antimicrobial activity? (5 marks) ........................................................................................................................................... ........................................................................................................................................... ........................................................................................................................................... .......................................................................................................................................... ..........................................................................................................................................

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TOPIC 1: Aseptic Technique (Completed by Practical 1)

• Work on a disinfected surface, preferably in a laminar flow cabinet;

• Use sterile equipment held in appropriate wrapping/containers until ready for use;

• Disinfect hands and forearms before working, do not touch sterile equipment with hands or with contaminated objects or surfaces;

• Work as rapidly as good technique allows;

• Do not speak, cough or sneeze while working;

• Eliminate unnecessary air currents;

• Loosen caps before opening bottles;

• Place the cap on the bench top with the inside up;

• Use one hand to hold both the cap and inoculating loop or pipette;

• To pour plates, quickly pour molten agar into the base of the sterile plate, replace the lid, swirl the plate and allow to set;

To open sterile pipettes, break the paper seal around the top and not the tip of the pipette and pull the pipette straight out. Discard paper into waste bin

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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. Failure to clean up after a procedure, to properly clean used glassware, 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. General housekeeping of the facility can reduce the chances of accidental exposures, however a more thorough cleaning of equipment and work surfaces is needed to eliminate the potential for accidental contamination. The cleaning of laboratory equipment, work surfaces and work areas by decontamination, 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. Selecting the most effective decontamination procedure or disinfectant will be dependent upon the physical limitations of the material being cleaned, how thorough the cleaning needs to be and the other potential contaminates that may be present. Physical sterilisation processes; i.e., heat and gas sterilisation, are used on laboratory equipment and labware that are capable of withstanding the exposure to the process. 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. By the general nature of the product, gas sterilants and chemical disinfectants are toxic and are to be handled according to the manufacturer’s directions. Appropriate personnel protective equipment is to be worn when these materials are in use. Definitions Antiseptics: Chemical disinfectants that are designed to be used on living tissue surfaces. Antiseptics are less toxic than disinfectants used on inanimate objects. Due to the lower toxicity, antiseptics can be less active in the destruction of normal and any pathogenic flora present. Autoclave: An apparatus used for sterilising using superheated steam under high pressure. To sterilize using superheated steam under high pressure. (See Steam Sterilisation) Chemical Sterilant: A germicide that can destroy all forms of microbial life when adequate exposure conditions are realized. Chemical sterilants are often used as high level disinfectants when shorter contact times are utilized. Decontamination: The killing of organisms or removal of contamination after use, with no quantitative implication, generally referring to procedures for making items safe before disposal. Disinfectant: A germicide that inactivates virtually all recognized pathogenic microorganisms but not necessarily all microbial forms. May not be effective against bacterial spores.

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Disinfection: The elimination or destruction of all pathogenic microorganisms. The term has been extensively misused and generally applies to the destruction of any pathogenic vegetative bacteria. High-level: The elimination or destruction of all microorganisms with the exception of high numbers of bacterial spores. Intermediate-level: The elimination or destruction of all vegetative bacteria including the Mycobacteria, most viruses, and most fungi but does not necessarily kill bacterial spores. Low-level: The elimination or destruction of pathogenic vegetative bacteria, some viruses, and some fungi but not Mycobacteria or bacterial spores. Germicide: An agent that destroys microorganisms, particularly pathogenic microorganisms. Sanitization: The process of reducing microbial contamination to an acceptable “safe” level. The process of cleaning objects without necessarily going through sterilisation. Steam Sterilisation: Autoclave, the process of sterilisation by the use of heated steam under pressure to kill vegetative microorganisms and directly exposed spores. Common temperature and pressure for being effective is 121°C (250°F) at 15 psi (pounds per square inch) over pressure for 15 minutes. Special cases may require a variation of the steam temperature and pressure used. Sterilisation: The complete elimination or destruction of all forms of life by a chemical or physical means. An absolute not a relative term.

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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, glassware, and contaminated materials. All pathogenic bacteria, both vegetative and spore forms, are destroyed within twelve minutes of exposure and direct contact to pure steam heat of 121°C (121°F). Most are destroyed within seconds of exposure. Pure steam at a pressure of 15 psi (pounds per square inch), one atmosphere over pressure, corresponds to the temperature of 121°C. 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. 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 duration of time needed to adequately heat sterilize material will be dependent upon the quantity and type of material being sterilized at one time, the larger the load the longer the time needed to achieve the needed temperatures deep within the load. The effectiveness of a routine steam sterilising cycle can be determined by using the appropriate biological indicator, ampoules or test strips containing Bacillus stearothermophilus spores or a spore enzyme (α-D-glucosidase) based rapid readout test. There are also several chemical indicators that can also provide reliable information. 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. The use of the spore enzyme test is increasing in popularity because of its ability to provide results within 3 hours of exposure. The use of temperature sensitive autoclave tape can be misleading since the tape is only capable of indicating that a general temperature was reached. It does not indicate how long the material was exposed to the high temperature. Autoclaved biological indicator samples should be examined for growth following an exposure to an actual autoclave cycle. 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. 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. The researcher must understand and handle potentially infectious materials accordingly to reduce the potential for exposure. The types of materials that may be steam sterilized in an autoclave can be varied in form; by shape and size, solid or liquid in composition or a combination of all, and the autoclave must be capable of accommodating for the type of load. The type of load to be autoclaved will determine the type of steam sterilising cycle to be used; a liquid load requires a slow depressurization to prevent the liquid from boiling over once the autoclave pressure is reduced.

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There are a number of different manufactures and different model designs of steam autoclaves. Before using any steam autoclave, the operation instructions for proper use and timing requirements must be reviewed. Operators of a steam autoclave must remember that a steam autoclave is operated under pressure and at elevated steam temperatures. Failure to review the operational directions can result in improper sterilising cycle being used, damage to the materials being exposed to the steam heat, damage to the autoclave and potentially serious or fatal injuries of the operator. Personal injuries can result from steam burns and from not allowing the autoclave to depressurize properly. If a steam autoclave is not working properly do not use the unit until it is repaired, contact the responsible person for the unit and inform them of the problem and label the unit “Out of Service”. Not all materials are capable of being exposed to steam sterilisation in an autoclave. 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, the desired level of disinfection and potential hazards associated with handling of the item and the disinfectant. Gas Sterilisation Ethylene oxide and formaldehyde gases are generally used for gas disinfection as fumigants under controlled conditions. Ethylene oxide and formaldehyde require special handling procedures to minimize potential personal exposure. Both materials are considered to be suspect carcinogens according to OSHA and an occupational carcinogens according to NIOSH. Ethylene Oxide Ethylene Oxide (ETO) is used primarily as a means of sterilising materials that are not designed to be exposed to steam sterilisation. The use of ethylene oxide on sensitive plastics, medical and biological preparations and other heat sensitive equipment has contributed to revolutionizing developments in the medical field. Early testing found that ethylene oxide was very effective as a killing agent of bacteria, spores, molds and viruses. 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, carboxyl, sulfhydryl, amino and phenolic groups. The alkylation of these compounds in organisms affects cellular function and structure which leads ultimately to inactivation of cellular function and ultimately death. As effective as ethylene oxide is as a gas sterilizer, it has some major drawbacks that are potentially hazardous that limit its use in a general laboratory environment. Ethylene oxide is a highly flammable and potentially explosive gas. The gas has an explosive concentration range of 3 to 100 percent, and it is listed as a suspect human mutagen and carcinogen. Because of the potential health risks and flammability potentials there are special handling and ventilation requirements that must be used when handling ethylene oxide. Due to the hazards associated with potential exposures OSHA has listed an exposure limit of 1 ppm for the duration of a work day. Ethylene

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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. 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. 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. The venting of the sterilizer following use is necessary, exposure to the residual material can be damaging to skin and may present a potential fire hazard. To test for proper operation of an ethylene oxide sterilizer the biological indicator Bacillus subtilis var. niger is used. The spores from B. 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, handling and disposal of this material and to be certain that others working in the laboratory receive appropriate training and warnings. Contact the Department of Environmental Health and Safety for assistance in assessing the potential for personal exposures and evaluation of laboratory handling procedures. 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. At the present time the only accepted method available for decontaminating a biological safety cabinet is to use formaldehyde gas. 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. Paraformaldehyde generates formaldehyde gas when it is depolymerised by heating to 232 to 246°C (450 to 475°F); the depolymerised material reacts with the moisture in the air to form formaldehyde gas. Using a balanced amount of ammonium bicarbonate neutralizes the formaldehyde gas within the biological safety cabinet. Only individuals that have specific training are permitted to decontaminate biological safety cabinets. 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. Clear all materials out of an area where formaldehyde may be used to minimize the chance of a possible reaction with incompatible chemicals. Formaldehyde can react violently or explosively when exposed to incompatibles; 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. Most notable however, formaldehyde may combine with hydrochloric acid or hydrogen chloride to form bis(chloromethyl) ether (BCME), a carcinogenic compound. OSHA, NIOSH and IARC recognize formaldehyde as a suspect carcinogen. OSHA has established an exposure limit of 0.75 ppm during a workday. The Department of

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Environmental Health and Safety can evaluate work tasks and perform monitoring tests to determine the potential for an occupational exposure. Chemical Disinfectants Choosing a Chemical Disinfectant A variety of concerns must be addressed when choosing a disinfectant for use in a biohazard area. 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.e., flammable, corrosive, toxic) • Stability of the disinfectant • pH, 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, therefore, a decision that requires a fairly detailed knowledge of the target organism, a basic knowledge of disinfectants, and careful consideration of the above factors as they apply to the unique potential conditions in which your laboratory will employ the disinfectant. Always consult the product information, the material safety data sheet (MSDS), on a disinfectant before using the material. Appropriate personnel protective equipment is required to be worn when materials are being mixed and used. For the chosen chemical disinfectant to be affective when used it must be able to make direct contact with the target organism. Environmental factors such as air bubbles, grease, dirt, a dense concentration of microorganisms and the presence of other chemicals (i.e., soaps) can reduce the effectiveness of the disinfectant. Halogens Chlorine Chlorine is one of the least expensive and most effective disinfectants. The recommended concentration of sodium hypochlorite for "clean surface" disinfection is 200 ppm, 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. These varying recommendations occur primarily because of chlorine's easy inactivation by organic material (serum, blood, proteins, etc.) and the fact that chlorine's disinfectant activity, unlike many of the other disinfectants, increases as the concentration increases. Of all the disinfectants, chlorine has one of the most extensive ranges of organisms that are susceptibility to destruction under ideal circumstances. All of the vegetative

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bacteria that have been tested are susceptible to chlorine destruction, including the acid-fast bacteria. Bacterial spores are also susceptible although longer exposure times are generally required. Both enveloped and non-enveloped viruses are susceptible to chlorine inactivation. One of the main disadvantages of chlorine as a disinfectant is the ease with which it is inactivated by organic material. 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. Chlorine is also easily inactivated by a variety of metals including copper, zinc, nickel, iron, etc. and the use of chlorine as a disinfectant on these materials requires increased concentrations of chlorine, often resulting in damage to the substrate materials being disinfected. Chlorine disinfectant solutions are also extremely sensitive to pH and the sensitivity has dramatic implications on the effectiveness of these solutions. 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 8.5. As the pH of chlorine solutions increases the disinfectant activity levels decreases. The limited pH range in which chlorine is effective, slightly acid to slightly basic, is also a limiting factor necessitating the use of nonionic detergents or precleaning followed by thorough rinsing. A number of alternative forms of chlorine exist to use in the form of household bleach (sodium hypochlorite). Chlorine dioxide compounds are high level disinfectants/steriliants that offer somewhat increased activity and resistance to organic inactivation in comparison to household bleach. Chloramine-T and other organic chlorine compounds also offer increased resistance to organic inactivation but at the cost of decreased activity. While these compounds offer specific advantages, household bleach remains one of the best disinfectants available. 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. First, the addition of acid to hypochlorite solutions will produce a rapid production of toxic chlorine gas. Second, the contact of chlorine solutions with formaldehyde produces the carcinogen bis-chloromethyl ether. Lastly, the heating of chlorine solutions produces the carcinogen trihalomethane. Chlorine solutions, therefore, must never be autoclaved. Iodine Iodine-based disinfectants share the same properties as the chlorine-based disinfectants but are somewhat less reactive with substrates and microorganisms. Like chlorine disinfectants, the iodines are effective against vegetative bacteria, acid-fast bacteria, bacterial spores, and both enveloped and non-enveloped viruses although longer contact times are generally required under similar conditions. 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. These compounds are collectively referred to as iodophors. Iodophors are excellent disinfectants and antiseptics and are extensively used for surgical scrub solutions,

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hand-washing compounds, and disinfectants for small laboratory objects. Unlike the elemental chlorine and iodine, however, iodophors are extremely sensitive to concentration and are quite expensive. Alcohols Ethyl and Isopropyl Alcohol Ethanol and Isopropyl alcohol are both excellent disinfectants whose germicidal properties are generally underestimated. Both are rapidly bacteriocidal against vegetative bacterial forms, tuberculocidal, fungicidal, and virucidal. Neither inactivates bacterial spores and isopropyl alcohol fails to inactivate hydrophilic viruses. Both ethanol and isopropyl alcohol should be considered as intermediate-level disinfectants. One of the most critical factors in the use of alcohols as disinfectants is concentration. The disinfectant properties of both ethanol and isopropyl alcohol rapidly drop at concentrations below fifty percent (50%) and above ninety percent (90%). Peak disinfectant activity occurs at approximately sixty-seven percent (67%) concentration. The recommended concentration for use is sixty - ninety percent (60 - 90%) by volume. Both ethanol and isopropyl alcohol are volatile and flammable compounds and must only be used with adequate ventilation. Alcohols, in general, are destructive to rubber compounds and to most of the cement and glues used in instruments, especially optics. Phenolic Compounds Ever since the adoption of carbolic acid by Lister as the first germicide, phenols have been extensively used. Numerous studies, beginning with a study by Kronig and Paul in 1897, have explored the various chemical substitutions and their effect upon germicidal properties. Today, the only phenolic derivatives found in extensive use, as disinfectants are o-phenylphenol, o-benzyl-p-chlorophenol, and p-tertamylphenol. 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. Overall the phenolic derivatives are all characterized by a broad-spectrum of activity against grampositive and gram-negative bacteria, fungicidal, tuberculocidal, and virucidal activity against lipophilic viruses (enveloped viruses). Phenols have a high tolerance to both organic load and hard water. Their use also results a residual activity on surfaces. Overall, phenolic derivatives are best classified as low- to intermediate- level disinfectants appropriate for general use in noncritical or semicritical areas. They lack sporicidal activity and are ineffective against noneveloped viruses. Phenol should never be used for sterilisation purposes. Phenolic compounds may exhibit dramatic toxic effects. Phenol compounds rapidly penetrate porous compounds and tend to accumulate in the body fat of exposed animals. Reports of phenolic disinfectant induced skin depigmentation, nerve

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demyelination and skin contact dermatitis that requires personnel using phenolic disinfectant be provided with appropriate protective clothing and equipment. Two halogenated phenolic derivatives; parachlorometaxylenol (PCMX) and 2,4,4'-trichloro-2- hydroxydiphenol (Triclosan, Irgasan), are commonly used as antibacterial agents in soaps and scrubs as well as preservatives in a number of products. 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. Triclosan is now commonly used in antibacterial soaps and deodorants as well as being incorporated into plastics as a "permanent" (but questionable) antibacterial. Biguanides (e.g., Chlorhexidine) Discovered during a search for potential anti-malarial drugs, chlorhexidine proved to have a high level of antibacterial activity, low mammalian toxicity, and a strong affinity for binding to skin and mucous membranes, all of which are desirable characteristics for an antiseptic. Chlorhexidine compounds are generally active against gram-positive and gram-negative vegetative bacteria and lipophilic viruses. Many fungi are sensitive to chlorhexidine and acid-fast bacteria are generally inhibited but not killed (bacteriostatic). Bacterial spores are not killed but germination is inhibited while in contact with chlorhexidine. 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. 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. Chlorhexidine is best classified as a low- to intermediate- level disinfectant appropriate for noncritical and semicritical area disinfectant. As an antiseptic, the lack of direct tissue toxicity and the rapidity of action makes chlorhexidine an excellent bacteriocidal skin cleanser and wound cleaning agent. Quaternary Ammonium Compounds (QAC) Quaternary ammonium disinfectants first appeared in the late 1930's. Since the original introduction, there has been the addition of numerous compounds, blends, different adjunctive agents, etc., making the entire group of quaternary ammonium disinfectants a rather broad group with a variety of activities, advantages, and disadvantages. The major advantages that are common to the group are an inherent surfactant activity, allowing them to also serve as cleansing agents, and a relatively low level of mammalian toxicity. 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). The first generation of quaternary ammonium compounds were the standard benzalkonium chloride compounds developed in the 1930's. Substitution of the aromatic ring hydrogen with chlorine, methyl, and ethyl groups resulted in increased activity and the generation of the second generation of quaternary ammonium

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compounds. The third generation of quaternary ammonium compounds, or the dual QACs, were developed in 1955 and represented compounds with superior microbiological activity. Presently, the quaternary ammonium compounds, now polymeric and polysubstituted quaternary ammonium compounds, are in the seventh generation of development. The newest generation of QACs possess a wide spectrum of activity with minimal mammalian host damage and are used in pharmaceuticals, ophthalmic solutions, and contact lens solutions, etc. The antimicrobial activity of quaternary ammonium compounds appears to be by inactivation of critical enzyme systems. 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. As far as choosing a quaternary ammonium disinfectant, it is critical to read the label directions on the bottle. Organism susceptibilities differ dramatically between different generations of QACs and different formulations.

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Appendix 1 SOP 1: Hand Washing Policy To minimise contamination in products, 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. Before making clean or sterile products, 2. After handling microorganisms; 3. Before leaving the laboratory.

Hand Washing

1. Initially rinse hands under running water, then using soap or antiseptic scrub; 2. Wash palm to palm; 3. Wash right palm over back left hand; reverse the action; 4. Wash palm to palm interlacing fingers; 5. Hold right thumb with left hand and rotate; reverse the action; repeat with each

finger ; 6. Rub right palm with fingertips of left hand; reverse the action; 7. Rinse well after washing. Keep hands palm downwards to allow water to run into

sink; 8. 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.

9. Repeat as necessary. 10. 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.

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SOP 2: Cleaning Aseptic Hood Cleaning Aseptic Hood

1. Remove all extraneous materials from underneath the hood; 2. Wipe the hood with 70% w/w ethanol in water; 3. Use broad sweeping motions, starting at the back and working towards the

front, 4. The order shall be:

a. Top b. Back c. Sides d. Front e. Bench.

5. Never go back to an area already cleaned; 6. Spray the interior with 70% w/w ethanol in water; 7. Leave the ethanol aerosol to settle for 2 minutes; 8. Wipe the base again.

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SOP 3: Membrane Filtration of Small Volume Aqueous Solutions Policy

All parenteral products, eye preparations and aqueous solutions required to be sterile are to be filtered using membrane filters. This ensures the solutions have low particulate contamination and significantly reduces the bioburden prior to sterilisation. Filters of 0.8 µm pore size assembled in Swinnex 25mm filter holders are used.

Preparation of solutions for filtration Prepare the solution to be filtered with appropriately cleaned glassware and equipment. Use techniques that minimise particulate and biological contamination and filter the solution as soon as possible after preparation.

Filtration of small volumes using a syringe 1. Remove the filter from between the waxed paper spacers with a flat faced pair of

tweezers, 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. Tighten firmly.

2. Fill a syringe with the maximum syringe volume or a volume in excess of the product volume and then exclude any entrapped air.

3. 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.

4. Filter a small volume of the solution and discard. This will minimise contamination of the product with extractable components of the filter and avoid loss of actives by adsorption to the membrane.

5. 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.

6. To refill the syringe, remove the Swinnex filter holder, refill the syringe and eliminate any air then re-attach the filter holder.

The filter is fragile. It is only supported when liquid flows from the syringe. Never draw liquid into the syringe through an assembled Swinnex filter holder.

PHAR5515 Manual 2016

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Transcript of PHAR5515 Manual 2016