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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 6.1: Aseptic Transfer

Aseptic Transfer – Protocol 1

Class Results
Results (contaminated n
broths as % of n)
Vial 1 (nutrient broth only) 46% 40

Vial 2 (diluted nutrient broth) 52% 40

Vial 3 (5% lactose in diluted nutrient broth) 82% 40

Aseptic Transfer – Protocol 2

Class Results
Results (contaminated n
broths as % of n)
Vial 1 (nutrient broth only) 25% 40

Vial 2 (diluted nutrient broth) 31% 40

Vial 3 (5% lactose in diluted nutrient broth) 48% 40

Aseptic Transfer – Protocol 3

Class Results
Results (contaminated n
broths as % of n)
Vial 1 (nutrient broth only) 10% 40

Vial 2 (diluted nutrient broth) 15% 40

Vial 3 (5% lactose in diluted nutrient broth) 7.5% 40

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)

The differences in results between protocols 1, 2 & 3 indicated a decrease in

bacterial growth in protocol descending order. The procedure undertaken to
complete protocol 1 lacks the fundamentals of aseptic preparation. Whereas
procedures undertaken by protocol 2 were better designed to prevent certain
contaminants, and protocol 3 even better designed so there will be a very low risk
of contamination. The main differences in procedures between the 3 protocols
were the ways in which the equipment were unwrapped, the place and way in
which the nutrient broth was opened, mixed and transferred between measures.
These differences either increased or decreased contamination.
Protocol 1 involved the procedures giving a high chance of contamination; this was
because firstly, the equipment was unwrapped and opened outside of the hood,
this allowed for falling particles in the air to contaminate the already sterilised
equipment. Also, the sterile nutrient broth was opened outside of the hood, and
was opened by unsterilized or “uncleaned hands” which would contaminate the
neck of the tube and hence the broth when being poured out.
Protocol 2 decreased contaminations slightly as the procedure was improved by
being carried out INSIDE the plastic hood along with having the broth bottles
being sprayed with 70% ethanol before being placed in the hood. This denatures
any microorganism carrying risk of contamination. Hands and arms were
sanitised with chlorhexidine scrub, and then the sterile equipment in wrapping
was opened under the plastic hood and again hands were sanitised before dealing
with the rubber closures of the multi dose containers. These procedures prevented
any contamination from skin as well as minimising contact with air borne
microorganisms – an improvement from previous protocol procedure. Also, to
minimise further risk of contamination forceps swabbed with 70% ethanol was
used to remove the foil entirely off the neck of the broth bottles, instead of using
hands. Step 22 also ensured minimal contamination at the final point of
procedure.
Significant improvements were made in aseptic transfer procedures undertaken in
protocol 3 compared to that of protocol 2. These included the use of a syringe and
needle in transferring nutrient broth to sterile vials, instead of openly transferring
nutrient broth to and from measures.
A possible reason as to why the trend in contaminated changed in protocol 3, was
because the lactose 500mg was already pre-made and pre-sterilised before its use
in this exercise. Whereas for protocols 1 and 2, the lactose was dissolved and
mixed and in contact with air which increased its risk to contamination.
 Exercise 7.1: 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 Number of tubes Number of Filtration
showing Growth of showing Growth of tubes & Failure
microorganisms microorganisms plates Rate
Cellulose acetate 4 3 22 32%
membrane filter
in Swinnex
Commercially 6 2 56 14%
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. (15 marks)

One of two filters was used in carrying out this exercise; the cellulose acetate
membrane filter in Swinnex or the commercially disposable filter. Although the
experiment yielded more microorganism growth in plates post-use of cellulose
acetate membrane filter, the filtration failure rate was higher than that of the
commercially disposable filter. This is most likely due to the way each filter was
assembled before use. Usually, the cellulose acetate membrane filters need assembly
to another part of a filter i.e. the Swinnex adaptor. This assembly procedure risks
contamination and can damage filter integrity, although it was pre sterilised in an
autoclave bag, due to the contact it has with air when removed from bag as well as
contact with skin when assembled. Also, further contamination can be associated
when the cellulose acetate membrane filter/Swinnex adaptor is attached to the
syringe from air borne microorganisms as well as micro organisms that weren’t
removed when hands/arms were cleaned AND microorganisms already present on the
neck of the syringe.
On the other hand, the commercially disposable filter is pre-packaged and pre-
sterilised before use. When it was connected to the syringe, greater care was taken;
the head of the filter was contained in its sealed and the syringe was connected. This
limited the risk of any contamination as the head of the filter was in minimal contact
with air borne micro organisms as well as skin contact.
Filtration as a use for sterilisation, has to be carried out very carefully and possible
ONLY when needed, following the most basic rules of aseptic preparations as the
exterior of the filters (not so much the interior) can be easily contaminated.
Especially for pharmaceutics preparations as the products are used on/in human
bodies which has a broad range of susceptibility to certain micro-organisms.
Following the results of this exercise, cellulose acetate membrane filters/Swinnex
adaptors are less reliable for filtration than the commercially disposable filters.
Exercise 7.2: 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 rather than Staphylococcus epidermidis? (10 marks)

The cetomacrogol lotion used in this exercise, is a derivative of the polyethylene

glycol group which comes from the ether family. The chlorocresol, anti microbial
agent used has a phenol group. On its own, chlorocresol is an effective anti-
microbial, but it varies in solubility between alcohols, water, ethers and other
components. When mixed with cetomacrogol, chlorocresol loses some of its affectivity
as its phenol is partially deactivated by the ether group in cetomacrogol (in which it
is fairly soluble)i
A lower CFU reduction indicates more microbial growth on the plate. In this exercise
the reason why s.cerevisiae had a lower CFU reduction than s.epidermis was because
s.cerevisiae is a species of yeast, belonging to the fungi kingdomii. Generally, yeast
grows under extreme conditions; between temperatures 10-37°Ciii as well as
anaerobic and aerobic growthiv and due to their fungi tendencies take on filamentous
growth, which enhances the rate at which they spread and grow.

Exercise 7.3: Determination of MICs of Antimicrobial Agents

Class Results
Against Gram+ Against Gram-
2-Phenoxyethanol + EDTA 0.13mg/L 1mg/L
- EDTA 0.7mg/L 1.5mg/L

4. Are the MICs dependent on the micro-organisms chosen for the test? Gram+ or
Gram–: which one is more resistant? Why? (6 marks)

The MICs are dependent on the micro organism chosen, as the average MIC for
either gram positive or gram negative bacteria varied. According to the results
obtained, the gram negative bacteria are more resistant as its MIC is greater than
that of the gram positive tested. This is because the outer cell wall of gram negative
bacteria is double layered compared to that of gram positive. The double layer
consists of a layer of lipopolysaccharides, and phospholipidsv. The presence or
absence of EDTA made no difference in relative MIC.

5. Comment on the effect of adding EDTA to the antimicrobial agent and suggest a
reason for that effect. (6 marks)

The addition of EDTA to phenoxylethanol, enhanced its anti-microbial activity

against both gram positive and gram negative bacteria. The reason behind this, is
that EDTA works as a chelating agent and generally affects the permeability of the
cell wall in gram negative bacteria.vi This is why the addition of EDTA was more
affective against s.cerevisia.
Exercise 7.4: 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? (6 marks)

Exercise 8.1: Zinc Sulphate Eye drops APF

7. Why did you first filter the solution through the membrane filter and discard it?
(6 marks)

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Exercise 8.2: Potassium Chloride Ampoules

8. Why is steam sterilisation used to sterilise the potassium chloride ampoules? (6

marks)

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iNelson Paint Company. (2011). PEG 3350 50 lb. bag (Carbowax)
0425E.Available:http://www.nelsonpaint.com/0425E.html.Last accessed 5/05/2011.
U.S. Pharmacopeia. (between 2008-2010). Reference Tables: Description and Solubility
C. Available: http://www.pharmacopeia.cn/v29240/usp29nf24s0_alpha-2-12.html. Last
accessed 05/05/2011.
ii BioWeb. (2007). Saccharomyces cerevisiae. Available:
http://bioweb.uwlax.edu/bio203/s2007/nelson_andr/. Last accessed 05/05/2011
iii Dr. Trudy Wassenaar. (-). Yeast and Temperature. Available:

http://www.newton.dep.anl.gov/askasci/bio99/bio99693.htm. Last accessed 06/05/2011.

iv Dakota Yeast. (-). Bakers Yeast Production Principles. Available:

http://www.dakotayeast.com/yeast_production.html. Last accessed 06/05/2011.

v Anthony Goodell. (Last updated 2008). Lab-On-a-Chip Applications Development (LOCAD)

Science. Available: http://locad.nasa.gov/science.html. Last accessed 06/05/2011.

Russell, Hugo & Ayecliffe (2004). Principles and Practice of Disinfection, Preservation
vi

and Sterilisation. 4th ed. Australia: Blackwell Publisher. 57-58.

http://www.sageproducts.com/education/pdf/20785.pdf
http://www.vaccinetruth.org/2-phenoxyethanol.htm