Sterilisation Flashcards
Define clean conditions:
microbiologically clean (reduced number of microorganisms) – favoured by manufacturers.
TWO General Approaches to sterile products:
- Produce under ‘CLEAN’ conditions, then TERMINALLY sterilise in the final container.
2) Produce and assemble under conditions ‘FREE’ of microorganisms and other particulates.
Examples of microbial Contaminants within the Manufacturing Environment
- Raw materials (synthetic/semi synthetic tend to have low counts of microorganisms) (natural materials will have their own intrinsic populations of microorganisms depending on nature of product)
- Water – primary requirement for microbial growth
- Environment – air (vector for particles), personnel, equipment
Resident organisms:
Soil –> gram positive, endospore forming, fungi
Water –> gram negative, yeast and moulds
Animals and humans –> gram -ve, obligate anaerobes, gram positive
Plants –> yeasts and moulds rather than bacteria
Transient organisms:
Carried by air and water (two main vectors). Humans as operators can also act as vectors
What does sterile mean?
Free of viable (living) microorganisms (sterile is an absolute term, no such thing as “quite sterile”)
Sterilisation = killing or removal of ALL viable microorganisms
Name some methods of killing organisms:
Moist heat steam (autoclaving), dry heat sterilisation (absence of moisture e.g. oils), radiation (cobalt 60), chemical (ethylene oxide)
Sterilisation standards:
Used to: - Control no. of microorganisms in manufacturing environment
- Validate sterilising agent – based on experimental data
- Validate sterilisation process – e.g. length of time for autoclaving
- Monitor sterilisation process – e.g. what controls have been put in place
- Regulated by EN (European), FDA (USA)
Methods of removing organisms?
Removal – Filtration (porosity taken into consideration),
Antibiotic sterilisation?
Filter sterilisation (heat sensitive, chemical could contaminate)
Vial sterilisation?
Steam sterilisation
Stopper sterilisation?
EtO sterilisation
Sterilisation standards are used to:
- Control no. of microorganisms in manufacturing environment
- Validate sterilising agent – based on experimental data
- Validate sterilisation process – e.g. length of time for autoclaving
- Monitor sterilisation process – e.g. what controls have been put in place
- Regulated by EN (European), FDA (US
What is a kill curve?
Curve which plots number of survivors against time
How is a kill curve made?
- Part of a development phase which occurs in a lab
- Take samples at regular intervals and dilute culture so you get colony forming units that you can count on a agar plate.
- Treat sample to sterilise it before you dilute and identify colonies.
- Serial dilutions so when you plate out on to agar you want 30-300 colony forming units
Why do you specifically want 30-300 colony forming units’?
Anything under 30 is not statistically liable and anything above 300 is too hard to count
What type of curve represents a working kill curve?
Decrease in numbers as a function of time, whatever we are doing we are getting a decrease in number – this means it is working
This is an ASYMPTOTE CURVE – regular intervals you get the same proportion of cells killed as per the unit of time. The blue line will never reach zero as It will lose the same number of cells each function of time.
How to make kill curve a straight line?
If you want to make this a straight line you take a log of the survivors, this is a semi-athymic graph. You can then use the gradient to work out the rate of survival at a given temperature. We can then repeat this at different temperatures so you can build up a portfolio of survival at different temperatures (you can change what it is you are measuring).
= First order kinetics
Why use a kill curve?
This is good to see any patterns and trends when you change what is affecting the colonies.
If you are doing different organisms you will also be able to see which organism is more sensitive to the change.
What is the D value?
The time taken, at a fixed temperature, to reduce the population by 90% (1-log). This gives a measuere you can compare at different temperatures. It doesn’t matter which number you have on the y axis as long as it is a full log cycle- it’s a straight-line relationship.
Whats is a thermal resistance curve?
The temperature change required to produce a 90% reduction (1-log cycle) in D-value. Can only be used for temperature changes not any other sterlisation agent
What is the Z value?
- A measure of thermal resistance
- Indicator of efficiency
- Reference (indicator) organisms
- Bacillus stearothermophilus under steam sterilisation has a Z value of 10 degrees
- Bacillus subtilus under dry sterilisation has a z value of 20 degrees.
When is a product deemed sterile?
- Inactivation on log scale, therefore no 0 on the log scale. Therefore, need a way to determine there is no organisms due to 0 not existing
- Measure via sterility assurance level (SAL)
- SAL= 10-6
- Minimum is the SAL value, even better if it is less then this value.
Sterility assurance:
- Start of with 102 and end with 106 you have an 8D reduction, if you know how long your D value is then you can see 8x1=8 minute reduction cycle. This allows you to see how long you need to carry it out for in order to reach you minimum SAL level.
- D values are always expressed in minutes.
our processing is effected by the resistance of the organisms and also the population size of the contaminating organism. - Therefore, even if they have the same d value but a different populations they will have a greater different reduction time so may take longer to get to SAL minimum.