MPharm3 LAQ2
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Bioburden represents the number or concentration of microorganisms present on an unsterilised
surface. The aim is to have a low pre-sterilisation bioburden as this will reduce exposure of the product
to high sterilisation temperature. A low bioburden indicates a low endotoxin concentration, which
increases the probability of the product passing the bacterial endotoxin test. Furthermore, it means
shorter autoclaving cycles which saves more energy.
First, the aqueous sample should be homogenised to remove or neutralise any preservatives or
antimicrobial components. The sample would then undergo membrane filtration to determine the
pre-sterilisation bioburden. It involves less preparation and is a very efficient method for fluid samples.
In this method, the aqueous sample is filtered through a sterile membrane. This membrane is then
placed carefully on a nutrient agar plate and incubated at 37 degrees for 48 hours. The colony forming
unite are counted and this value is the pre sterilisation bioburden. D121°C is the rate at which bacteria
are killed at 121 degrees. It is determined using the Inactivation Factor equation ‘IF = No/N = 10t/D’.
Inactivation Factor is equal to initial bioburden divided by bioburden after sterilisation cycle, and this
is equal 10 to the power of time divided by D-value.
A sterility test assesses whether a sterilised injection vial is free from microorganisms. A sterilised
injection vial is placed into a suitable liquid culture and incubated for 14 days. A sterility test
recommended by the British Pharmacopoeia is membrane filtration. If there is no microbial growth
after 48hrs of incubating the membrane filter, then the product has passed the test. It is important
that the tests are performed under aseptic conditions to reduce risks of false positives.
Endotoxin safety of this product is tested using the Limulus Amoebocyte Lysate (LAL) test. A
component of endotoxin acts on the pro-clotting enzyme found in LAL and it becomes and active
clotting enzyme. This acts on a clotting protein and produces a gel clot. Standardised Limulus lysate is
made into a standard solution and is added to the vials. It is then incubated and tested for any gel clot
formation. The absence of gel clots means there are no endotoxins present.
It is important to always use aseptic techniques when preparing and administering the injection to
patients. All equipment must be sterile before use. The medication cannot be administered to multiple
patients with the same syringe. Each patient must have their own. Another unsafe practice is injecting
a single-use dose for multiple agents. Hands must be clean, or gloves must be worn. The patient’s
injection site should be disinfected. After administration, the syringe must be disposed of
appropriately in a sharps bin. Lack of these precautions can lead to blood-borne viruses e.g., HIV.
Damaged post-sterilised injection can result in microbial growth and can be harmful to the patient and
their foetus. The contaminated drug can cross the placenta and result in impaired foetus development
issues or other birth defects.