Common asked Exam Q Flashcards
ii. Draw a well-labelled flow diagram of the entire procedure. (Spread plate)
Label agar plates -> date ,group, course title and solution
Prepare samples -> creating a series of ten fold dilutions
Transfer the sample to the plate using 1ml pipette
Spread inoculum evenly over surface of agar using sterile spreader
Leave to stand in an upright position until sample is fully absorbed
Incubate at 35-37 degrees for 24-48 hours
Observe and record results
List appropriate control tests to be included to validate this analysis (Spread plate)
Specificity Testing: To confirm that the method can accurately distinguish the target microorganism from other microorganisms
Negative control: To check for contamination in the media. This is done by plating the media without inoculation
Positive control: To ensure the media is capable of growth. This is done by plating a known bacteria that is capable of growth on the media alongside the sample
• Equipment Sterility Checks: To verify that the equipment used in the tests is sterile.
(a) Explain the following microbiology terminology in relation to the microbial flora on the human body?
i. Resident Flora (2 marks)
Resident flora are microbes that are always present on the human body.
(a) Explain the following microbiology terminology in relation to the microbial flora on the human body?
ii. Transient Flora (2 marks)
Transient flora are microbes that live in or on the body for a period of time but then move on or die off.
(a) Explain the following microbiology terminology in relation to the microbial flora on the human body? iii. Mutualistic Flora and use one example to explain your answer. (3 marks)
Mutualistic Flora are when both organisms benefit from the each other’s Prescence an example being E.coli and humans
(a) Explain the following microbiology terminology in relation to the microbial flora on the human body?
iv. Opportunistic Flora or pathogens and use one example to explain your answer.
Opportunistic Flora is when a microbe does not cause disease under normal circumstances but can become pathogenic if conditions change. An example being E.coli, E.coli is normally found in the digestive tract and causes no problems but when it enters the urinary tract it can become pathogenic
(b).Describe the characteristics on the pathogenicity of one specific opportunistic
pathogen which are considered objectionable by the regulators and with
which you are familiar. 4x times
E.coli is a gram negative bacteria found in human intestines .
Pathogenic e.coli strains possess adhesions that allow them to attach to host cells. This ability helps E.coli to colonize and invade the host tissue making infection more likely.
These adhesions increase the risk of infection including the elderly and immunocompromised people.
E.coli has also developed antibiotic resistance in many strains which has limited treatment options for infections. This has resulted in a global health threat .
Regulators such as the food and drug administration have implemented guidelines to control the spread of E.coli including: food safety reglations, water quality control and healthcare protocols
(a).Discuss the phenomenon of stressed / injured cells and their significance,
in microbiological analysis. (10 Marks) 5 times
Stressed/injured cells are when microorganisms are subjected to environmental stresses and become structurally damaged/injured.
They are viable, however they are not as culturable as normal unstressed microorganisms are.
They are unable to replicate in selective environments that normal cells are able to tolerate.
Selected agents in media can mask their presence by inhibition or toxicity, causing death.
There are many reasons as to why the cells become injured, some examples including: Processing and handling, Heating/thermal treatment ,chilling, cold shock, dehydration and starvation of nutrients.
In a favourable environment the microorganisms can be resuscitated and become functionally normal .
The types of injury include leakage of cellular components into the medium, decreased enzyme activity, extended lag phase and degradation of RNA
(b).Indicate how stressed cells may be resuscitated as part of microbiological
analytical procedures. Support your answer with two appropriate examples of
such procedures. (5 Marks) 5 times
Analytical methods used in the lab should detect both normal and injured cell.
There is no universal resuscitation medium although the use of complex media is recommended.
A liquid repair could be used in the resuscitation of stressed cells. This is where holding the product is homogenated in saline for two hours at 25 degrees before proceeding with the analysis.
Another method is solid medium repair by the double agar technique.
This is where the sample and dilutions are plated into a non selective solid medium like TSA.
The cells are then allowed to resuscitate in this medium at room temperature, the plates are them overlayed with the selective medium
Describe the scientific principles of the Colilert Test. (5 Marks)
The scientific principles of the Colilert Test are:
Nutrient indicators , selective growth environment creators where it inhibits the growth of non target bacteria. There are two types the presence and absence tests and the quantification test
Describe the Colilert Test Procedure.
A water sample must be collected in a sterile container, a packet of colilert reagent is added directly into the sample.
The container is capped and shaken gently until completely dissolved.
The water sample is then incubated for 35 degrees for 24 hours.
After 24 hours if there is a colour change and the sample is yellow, there are coliforms present.
If the sample is clear, there is no coliforms. The vessel is then checked for fluorescence by placing it under a UV light .
Quantification tray: 100ml of the water sample is gathered and stored in a sterile container.
A snap pack of the colilert medium is opened and added to the water sample. The medium has to be fully dissolved before progressing.
The sample is then placed into a quanti tray and is then sealed using a quanti tray sealer.
The pack is then incubated at 35 degrees for 24 hours. After incubation the tray is placed into the sealer.
The sealer distributes the DST reagent to each well.
The results are then read clear indicates negative yellow indicates positive
(b) Discuss ‘Raw Materials’ as potential sources of microbial contamination in
pharmaceutical manufacturing. Support your answer with specific examples.
(10 Marks)
Raw materials especially those of animal or plant origin are responsible for a high proportion of the microbes introduced into the production line during manufacture.
Animal products are particularly susceptible to contamination including faecal contamination and it can carry human pathogens like salmonella and E.coli.
Plant materials can be contaminated with soil microbes.
Tight specifications and quality control testing are needed to control raw material contamination.
For contamination of an animal origin, it can come from animal tissues, blood or serum which may be contaminated with bacteria that includes pathogens.
For plant origin, plant extracts can be contaminated with bacteria and mould spores.
There are other excepients like starch, sugars and thickeners that can be contaminated with various microorganisms depending on their origin.
Faecal coliforms like E.coli are gram negative rod shaped bacteria. Faecal coliforms ferment lactose at 37 degrees which produces acid and gas which is used to separate them from other bacteria.
Write an essay under the following headings on the factors to be considered when
designing a Microbiology QC Programme for the pharmaceutical /Biopharmaceutical
industry. [5 Marks each]
a) Typical Samples to be analysed Microbiologically
Typical samples that need to be analysed microbiologically include:
raw materials, in line samples, end products sterile and non sterile, water and personnel.
Raw materials:
To evaluate their safety and hygiene levels.
Until these specifications are met no clearance can be given.
In line samples: taken at intermediate stages of production. Regular assessment of contamination pressure at all stages of production is more important than end product testing.
End products sterile/non sterile: Non sterile, microbial test limits should be conducted and based on a risk assessment of the product. Sterile, sterility testing and endotoxin testing
Water: Process water ,cooling water, WFI water and sterile water for injecting
Personnel: Routine hygiene checks which include swab tests, finger dab tests
Write an essay under the following headings on the factors to be considered when
designing a Microbiology QC Programme for the pharmaceutical /Biopharmaceutical
industry. [5 Marks each]
b) Choice of tests to be carried out
A limited number of tests should be performed. This allows examinations of more samples and is statistically better, the risk assessment on the sample, its end use, route of administration. General counts like TVC, Indicator organisms and sterility testing
Write an essay under the following headings on the factors to be considered when
designing a Microbiology QC Programme for the pharmaceutical /Biopharmaceutical
industry. [5 Marks each]
c) The frequency of testing
Testing should be related to the products history, associated hazards, infection and its likelihood to cause spoilage. Testing may be daily, weekly, monthly or biannually depending on the product
