Using the data provided, calculate the count per m

Using the data provided, calculate the count per mL for each of the enumeration methods.

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Conjunctivitis case coursework assignment

MPharm 1

PY162 Therapeutics




This coursework task contributes 15% towards your PY162 module mark, i.e. half of your overall PY162 coursework marks. Your assignment should be submitted as a single typed document via the Turnitin submission point which can be found within the ‘Assessment’ area of your PY162 site on StudentCentral. Please consult the guidance on eSubmission, which you have been provided with, to ensure that you submit your document successfully. The table below indicates the submission deadline and the date by which you may expect to receive feedback on your work. In accordance with University requirements, this is within 20 working days from the date of submission. Feedback will be provided to you electronically via Grade Centre.

Table 1 – Coursework submission and feedback deadlines

Dates of practical classes

Coursework submission

Feedback deadline







03.11.16 & 04.11.16

Time 23.59, date 27.11.16


10.11.16 & 11.11.16






The assignment is based on a scenario in which an inexperienced technician has made an estimation of the bioburden of a pharmaceutical product. Due to their inexperience, some of the tests carried out may not have been necessary. In order to help them understand their results, you are required to consider their experimental processes and data then answer a number of questions, some of which are based around interpretation of data similar to that you will have generated within the two microbiology lab classes. For some of the questions, it will be necessary to support or supplement your answer using appropriate literature. In order to gain full credit for this, you must ensure that you fully reference your

work using a standard referencing format (10 marks out of 110 will be allocated to this).

Where there are calculations to perform, please show all of your working out. The marks available are shown against each question. Please ensure that in your submission, each answer is clearly and appropriately numbered. Your mark out of 110 will be converted to and reported as a percentage.




An inexperienced technician has been tasked with assessing the bioburden of an oral medicine, in the form of an aqueous solution. Any preservative was inactivated prior to analysis. You have been asked to assist with the interpretation of the results. Please study the experimental information and corresponding data given below and answer the accompanying questions.


It is known that the sample is contaminated with a single organism, specified to be objectionable by the British Pharmacopoeia, at a level of at least 500 colony forming units (CFUs) per mL.

Dilution of the product

A number of dilutions of the product were prepared before enumeration was carried out. The dilution series were prepared as follows:

Using a Micropipette and sterile tip, 1 mL of the aqueous solution was placed into 9 mL of phosphate buffered saline (PBS) measured using a sterile 10 mL plastic pipette (dilution A)

1 mL of dilution A was added to 9 mL of PBS (B)

1 mL of dilution B was added to 9 mL of PBS (C)

1 mL of dilution C was added to 9 mL of PBS (D)

1 mL of dilution D was added to 9 mL of PBS (E)

1 mL of dilution E was added to 9 mL of PBS (F)

Dilutions prepared in this manner were then subjected to both membrane filtration and spread plate counts, as shown in the methods below.


Membrane filtration

xA sterile 0.45 Pm filter was placed in a sterile filter unit

x9 mL of dilution A was passed through the filter

xThe filter was removed with forceps and transferred to a Petri dish containing 20 mL of casein soya bean digest agar

xThe same procedure was then carried out with 9 mL of dilutions B to E, and finally 10ml of dilution F was filtered and plated. All operations were performed using the same filter unit

xAll plates were incubated 30-35qC for 3-5 days.

xThe resulting colony counts are shown in Table 2 below.

Table 2 – Colony counts resulting from membrane filtration





Undiluted aqueous solution

Too many to count




Too many to count




Too many to count




Too many to count




Too many to count











Spread plates

xUsing a Micropipette and sterile tip, 0.2 mL of dilution A was placed onto the surface of a casein soya bean digest agar plate and spread using a sterile spreader.

xThe plating procedure was repeated in the same manner for dilutions B-F.

xAll plates were incubated 30-35qC for 3-5 days.

xThe resulting colony counts are shown in Table 3 below.


Table 3 – Colony counts resulting from spread plates





Undiluted aqueous solution

Too many to count




Too many to count























Task 1

1a) Using the data provided, calculate the count per mL for each of the enumeration

methods. (10 marks)

1b) Explain why you have chosen the units you have used to express your calculated answer. (4 marks)

1c) Please comment on the experimental design by discussing the following;

i. Were both methods necessary? Explain your answer.

(5 marks)

ii.How do you think the experiment could be improved? Give your reasoning.

(6 marks)

iii.What are the potential sources of error that could have occurred? Explain the effect

they may have had on the resulting data.

(6 marks)



Gram stain

The colony morphology observed on the incubated plates obtained from the unknown contaminant in the enumeration tests were all the same in terms of colony colour, shape, elevation and margin.

The technician has performed a Gram stain (most often the first step in identifying an unknown contaminant) on a heat-fixed smear prepared from a representative colony of the contaminant. The following image was taken down a microscope using a x100 oil immersion objective.

Figure 1 – Appearance of the contaminant following Gram staining.



Task 2


What do the Gram stain results obtained tell you about the organism?

(2 marks)


Explain the basis of this test.

(10 marks)


Do the results of this test prove that this is a pure culture? Explain your answer.



(3 marks)

Selective/differential media

Following the methods outlined in the British Pharmacopoeia, the technician prepared samples in order to detect specified pathogens (Escherichia coliPseudomonas aeruginosa,

Salmonella Staphylococcus aureus). You may assume that all of the necessary controls were performed. Briefly, appropriate amounts of product were incubated in casein soya bean digest broth for 18-24 h at 30-35◦C. Where appropriate, samples were then transferred to liquid enrichment media. Subsequently, all samples were sub-cultured onto the following selective/differential media; Blood agar, Baird-Parker agar, Cetrimide agar, MacConkey agar, Mannitol Salt agar and Xylose Lysine Deoxycholate agar (XLD). Following incubation at 30-35◦C for 18-24 h, the plates were observed for growth. Results are shown in figures 2a-2f.














Figure 2a-f: The unknown contaminant streaked onto a) Blood agar showing colony growth (NOTE: the colony areas appearing yellow are due to reflected light and do not represent colony pigmentation), b) Baird-Parker agar showing no colony growth, c) Cetrimide agar showing no colony growth, d) MacConkey agar showing colony growth, e) Mannitol Salt agar showing no growth and f) XLD agar showing colony growth.

Task 3


What is the purpose of incubating the sample with an enrichment medium prior to


subculturing on solid selective/differential media?

(2 marks)


Based on the appearance of the plates, combined with the Gram stain results, what


do you suspect the unknown contaminant to be? Based on the properties of the


various media, explain in detail your reasoning and how you eliminated other


organisms as the contaminant.

(15 marks)


Providing brief detail, give an additional test that could be used to confirm the


presumptive identification.

(2 marks)


Was it appropriate that the technician employed all of the media used? Explain your



(5 marks)

Task 4

4)Making use of primary literature, discuss the general pharmaceutical and clinical

significance of the contaminant identified (600 words maximum).

(20 marks)



Task 5

5)Write a short discussion (no more than 300 words) of how understanding the material covered in the practical classes is of importance to pharmacy. (10 marks)

Total assignment marks available: 110.


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