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2.1: Aseptic Transfer Aseptic Transfer – Protocol 1 Results Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) Class Results (contaminated broths as % of n) 46% 52% 82% n 40 40 40 Aseptic Transfer – Protocol 2 Results Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) Class Results (contaminated broths as % of n) 25% 31% 48% n 40 40 40 Aseptic Transfer – Protocol 3 Results Vial 1 (nutrient broth only) Vial 2 (diluted nutrient broth) Vial 3 (5% lactose in diluted nutrient broth) Class Results (contaminated broths as % of n) 10% 15% 7. Exercise 6.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.5% n 40 40 40 1. Did you observe any difference in bacterial growth between protocol 1. and 3? .

Comment on your class results. Compare these three protocols. These included the use of a syringe and needle in transferring nutrient broth to sterile vials. and list the major factors that can cause contamination in the aseptic transfer protocol 1. instead of using hands. These differences either increased or decreased contamination. mixed and transferred between measures. Whereas for protocols 1 and 2. 2. 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. (15 marks) The differences in results between protocols 1. A possible reason as to why the trend in contaminated changed in protocol 3. Protocol 1 involved the procedures giving a high chance of contamination. The main differences in procedures between the 3 protocols were the ways in which the equipment were unwrapped. and was opened by unsterilized or “uncleaned hands” which would contaminate the neck of the tube and hence the broth when being poured out. 2 & 3 indicated a decrease in bacterial growth in protocol descending order. and protocol 3 even better designed so there will be a very low risk of contamination. 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. the equipment was unwrapped and opened outside of the hood. . the lactose was dissolved and mixed and in contact with air which increased its risk to contamination. the sterile nutrient broth was opened outside of the hood. 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. this allowed for falling particles in the air to contaminate the already sterilised equipment. Whereas procedures undertaken by protocol 2 were better designed to prevent certain contaminants. These procedures prevented any contamination from skin as well as minimising contact with air borne microorganisms – an improvement from previous protocol procedure. This denatures any microorganism carrying risk of contamination. The procedure undertaken to complete protocol 1 lacks the fundamentals of aseptic preparation. this was because firstly. Also. Hands and arms were sanitised with chlorhexidine scrub. the place and way in which the nutrient broth was opened. instead of openly transferring nutrient broth to and from measures. and 3. 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. Also. was because the lactose 500mg was already pre-made and pre-sterilised before its use in this exercise.

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. Filtration as a use for sterilisation. the Swinnex adaptor. the head of the filter was contained in its sealed and the syringe was connected.1: Testing Efficiency of Filtration with the micro-organism Serratia marcescens (Bacterial Challenge Test) Class Results Bacterial Challenge test Challenge test on 0. the cellulose acetate membrane filter in Swinnex or the commercially disposable filter. Especially for pharmaceutics preparations as the products are used on/in human bodies which has a broad range of susceptibility to certain micro-organisms. the cellulose acetate membrane filters need assembly to another part of a filter i. . following the most basic rules of aseptic preparations as the exterior of the filters (not so much the interior) can be easily contaminated. Although the experiment yielded more microorganism growth in plates post-use of cellulose acetate membrane filter. greater care was taken. 6 2 56 14% Comment on class result with respect to differences in filter types. 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. On the other hand.e. List the factors that may cause contamination during filtration. differences in filter assemblies. the filtration failure rate was higher than that of the commercially disposable filter. Following the results of this exercise. due to the contact it has with air when removed from bag as well as contact with skin when assembled.22 µm filters with Serratia marcescens Type of Filter Number of plates showing Growth of microorganisms 4 Number of tubes showing Growth of microorganisms 3 Number of tubes & plates 22 Filtration Failure Rate 32% Cellulose acetate membrane filter in Swinnex Commercially disposable membrane filter 2. the commercially disposable filter is pre-packaged and presterilised before use. Usually. Also. and overall on the confidence you would have in using this type of sterilisation process in preparation of pharmaceutical products. (15 marks) One of two filters was used in carrying out this exercise.Exercise 7. although it was pre sterilised in an autoclave bag. When it was connected to the syringe. This is most likely due to the way each filter was assembled before use. This assembly procedure risks contamination and can damage filter integrity. cellulose acetate membrane filters/Swinnex adaptors are less reliable for filtration than the commercially disposable filters. has to be carried out very carefully and possible ONLY when needed.

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between temperatures 10-37°Ciii as well as anaerobic and aerobic growthiv and due to their fungi tendencies take on filamentous growth. chlorocresol is an effective antimicrobial. The double layer consists of a layer of lipopolysaccharides.7mg/L Against Gram1mg/L 1. and phospholipidsv. water.cerevisiae is a species of yeast.2: Evaluation of Preservative 3. belonging to the fungi kingdomii. The presence or absence of EDTA made no difference in relative MIC. The chlorocresol. yeast grows under extreme conditions. but it varies in solubility between alcohols. as the average MIC for either gram positive or gram negative bacteria varied. Comment on the effect of adding EDTA to the antimicrobial agent and suggest a reason for that effect.5mg/L 4. the gram negative bacteria are more resistant as its MIC is greater than that of the gram positive tested.vi This is why the addition of EDTA was more affective against s.cerevisiae had a lower CFU reduction than s. Exercise 7. This is because the outer cell wall of gram negative bacteria is double layered compared to that of gram positive.Exercise 7. is that EDTA works as a chelating agent and generally affects the permeability of the cell wall in gram negative bacteria. ethers and other components.cerevisia.EDTA 0. Although chlorocresol (a phenolic component) is a strong anti-microbial agent.3: Determination of MICs of Antimicrobial Agents Class Results 2-Phenoxyethanol Against Gram+ + EDTA 0. Generally. which enhances the rate at which they spread and grow. 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. When mixed with cetomacrogol. (6 marks) The addition of EDTA to phenoxylethanol. On its own. The reason behind this. is a derivative of the polyethylene glycol group which comes from the ether family. 5.13mg/L . enhanced its anti-microbial activity against both gram positive and gram negative bacteria. 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. 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.epidermis was because s. anti microbial agent used has a phenol group. In this exercise the reason why s. . According to the results obtained.

... .......................... Why is steam sterilisation used to sterilise the potassium chloride ampoules? (6 marks) ………………………………………………………………………………………............................... ……………………………………………………………………………………….......Exercise 7......................... Why did you first filter the solution through the membrane filter and discard it? (6 marks) ....................................................................................................... ............ ....................................................................................................................2: Potassium Chloride Ampoules 8..................................................... ………………………………………………………………………………………................... ………………………………………………………………………………………................................................... Exercise 8..............................4: Formulation Compatibility and Preservatives 6.................................................................................... .........................1: Zinc Sulphate Eye drops APF 7............................................................................................... ………………………………………………………………………………………................................. . What is the nature of the “incompatibility” of chlorhexidine with aqueous cream and does this “incompatibility” affect its antimicrobial activity? (6 marks) Exercise 8.............

bag (Carbowax) 0425E. Bakers Yeast Production Principles.pdf http://www.com/0425E.cn/v29240/usp29nf24s0_alpha-2-12. Pharmacopeia.com/education/pdf/20785. Last accessed 05/05/2011 iii Dr. (-).nasa.newton.uwlax.Available:http://www. ii Russell.dakotayeast. Australia: Blackwell Publisher.htm . vi http://www.gov/askasci/bio99/bio99693. PEG 3350 50 lb. Available: http://www.html.gov/science. Preservation and Sterilisation. Last accessed 05/05/2011.dep. (Last updated 2008). Last accessed 06/05/2011. Last accessed 06/05/2011. Available: http://www. 57-58. (2007).edu/bio203/s2007/nelson_andr/. i U. Available: http://www. Lab-On-a-Chip Applications Development (LOCAD) Science.Nelson Paint Company.html. Reference Tables: Description and Solubility C.htm. Available: http://bioweb.anl. Hugo & Ayecliffe (2004).html.nelsonpaint. 4th ed. (2011). Last accessed 06/05/2011.Last accessed 5/05/2011. Saccharomyces cerevisiae.html. Trudy Wassenaar. (between 2008-2010). v Anthony Goodell.S. BioWeb.pharmacopeia. iv Dakota Yeast.vaccinetruth. (-).com/yeast_production. Available: http://locad. Principles and Practice of Disinfection.org/2-phenoxyethanol. Yeast and Temperature.sageproducts.

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