PHAR 5515

THE 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.

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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
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

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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 elses 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 others 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

160C for 2 hours

Moist heat

121C for 15 min

Dry heat

160C for 2 hours

Dry heat

160C for 2 hours

Moist heat

121C for 15 min

Moist heat

121C for 15 min

1 pack of 3 rubber
closures
Lactose 500 mg
25 mL measuring
cylinder, 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 - 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)

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

160C for 2 hours

Moist heat

121C for 15 min

Dry heat

160C for 2 hours

Dry heat

160C for 2 hours

Moist heat

121C for 15 min

Moist heat

121C for 15 min

1 pack of 3 rubber
closures
Lactose 500 mg
25 mL measuring
cylinder, 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 - 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.
26. Next week, record the results in the following table.
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)

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Class Results
(contaminated
broths as % of n)

Protocol 3
Materials

Special Pre-treatment
or Packaging

Sterilisation
Method

Conditions

1 20 mL multi-dose
containers (empty)

Seal with foil

Dry heat

160C for 2 hour

2 rubber closures for

multi-dose containers

Boil in purified waterrinse well and pack in

autoclave bag

Moist heat

121C for 15 min

Seal with foil

Dry heat

160C for 2 hours

In disposable bag

Radiation

25 G

Filtered with 0.8m

membrane filter

Moist heat

121C for 15 min

Moist heat

121C 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 - 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.
25. Next week, record the results in the following table.
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)

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Class Results
(contaminated
broths as % of n)

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 25C 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 25C 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 m filters are used in this exercise.

Bacterial Challenge test

Challenge test on 0.22 m 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

- 19 -

Number of
tubes &
plates

Filtration
Failure
Rate

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 microorganisms 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
Chlorocresol
Phenoxyethanol

0.1%
0.1%
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 20C to 25C 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:

Antimicrobial agent
Chlorhexidine gluconate 0.1%
Chlorocresol 0.1%
Phenoxyethanol 1%

Plate count of CFUs:

Microorganism

Code

S. epidermidis
S. cerevisiae
S. epidermidis
S. cerevisiae
S. epidermidis
S. cerevisiae

CGB
CGF
CSB
CSF
PEB
PEF

- 21 -

Average
Plate Count

Log Reduction
in CFUs

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 microorganisms 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 2phenoxyethanol 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.

- 22 -

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 32C 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 +
2-Phenoxyethanol

+ EDTA
- EDTA

- 23 -

Against Gram -

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 50C) 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 #3 with aqueous cream.

Fill hole #2 with cetomacrogol 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.

- 24 -

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
Diameter of hole
inhibition zone
dh (mm)
dz (mm)

# 1 Methylcellulose gel
# 2 Cetomacrogol Cream
#3 Aqueous cream
#4 Oily cream

- 25 -

dz2 - dh2

GROUP ASSIGNMENT COVER SHEET

Unit of Study Code: ________________ Unit of Study Name: _______________________
(Mandatory Field Please complete)
Course Coordinator: ______________________Tutors 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

- 26 -

Date

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
Aseptic Transfer Protocol 1
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)

Aseptic Transfer Protocol 2

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)
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)

- 27 -

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)
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....

- 28 -

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 m 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)

....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....

- 29 -

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+
2-Phenoxyethanol

4.

Against Gram-

+ EDTA
- 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 -

...........................................................................................................................................
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)

...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................

- 31 -

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

- 32 -

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 manufacturers 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.

- 33 -

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 121C (250F) 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.

- 34 -

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 121C (121F). 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 121C. Adequate time must be permitted
to attain the 121C 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 (-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.

- 35 -

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

- 36 -

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 246C (450 to 475F); 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

- 37 -

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

- 38 -

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,

- 39 -

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 intermediatelevel 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 intermediatelevel 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 m 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.

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