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.
D-Values
Influenced by:
o Bacterial species
o Vegetative vs spore form
o Production method
o Nutrient environment (suspension media, carrier materials, culture media)
o Treatment dose (temperature, radiation, dose)
Importance of bioburden estimation?
Initial population numbers required in order to specify sterilisation parameters and inactivation kinetics.
What is bioburden?
a population of viable microorganisms on or in a product and or package.
Describe the bioburden estimation process:
- Sample selection = choosing samples statistically
- Collection of items for test
- Transfer to test lab = variability depending where the lab is, may need to be transported long distances, so how do you prepare them for them for this?
- Treatment (if required) = to remove the cells. What techniques will you use to remove microorganisms from the product, some of which will have complex designs
- Transfer to culture medium
- Incubation
Bioburden estimation techniques:
Direct contact between product and culture medium (agar plates). For some products this is easier than others. But sometimes you may be unable to do this (indirect approach).
This means you may have to wash the product to wash off any microorganism as bacteria like to stick to surfaces – usually full of nutrients.
We need to check that the eluent we are using isn’t causing effect by osmosis. Sometimes very mild detergents are used to remove cells from surface of the product. But some mild detergents have very weak antibacterial activity so we will need to check that this won’t kill the cells – lots of background checks that need to be carried out.
Some are more difficult to remove, and this means you may have to use physical treatment – vortex, ultrasound (high frequency causes vibration on surface), shake with glass beads to knock off the bacteria – but if they are too big you will harm the organisms you are looking at.
Selection of a Removal Technique.
Considerations:
- Ability to remove microbial contamination
- Effect of removal method on microbial viability = don’t want to cause change to numbers and get a false representation
- Types and location of microorganisms
- Nature of product
- Culture conditions
Selection of Culture Conditions
Types of microorganisms likely to be encountered dependent upon:
- Nature of product = natural are more likely to have a bioburden population
- Method of manufacture = have we added any microorganisms to the product how we made it
- Potential sources of contamination (operator, packaging)
Selection of Culture conditions: process operation:
- Cycle development is the heat killing the microorganisms you want to remove in test tube studies then moving to lab based
- Cycle validation proving that your experiment works. Key to the entire process.
- Cycle monitoring when you have all the evidence monitor every time to make sure you get the deserved end result
What is process validation?
Process validation the establishment of documentary evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications. This applies to all sterilisation techniques.
Stages of process validation:
What is installation qualification?
when you build sterilisation unit is everything you want to use working properly
Stages of process validation:
What is performance qualification?
does it work and do the job we want it to do properly. You can assess this in two different ways. The better of the two is physical qualification it is not subject to change (taking a physical measurement i.e. temperature profile). Microbiological is used as a back up to support physical qualification, this is when we use microorganisms which have a high resistance to the sterilisation process – but they are more prone to error and variability.
Biological Indicators (BIs) definition:
‘An inoculated carrier contained within its primary pack ready for use and providing a defined resistance to the specified sterilisation process‘. Usually they are simple things like spores’ strips, as endospores are the most resistant to remove, so you stick a stick in and you will be able to kill the most resistant form.
BI Use?
‘To provide a means of assessing directly the microbial lethality of a sterilisation process‘
• Standardised preparations containing selected microorganisms having known stable high resistance to sterilising agents – can collect D vales for endospores
• Used for validation (steam, dry heat, radiation, EtO) and monitoring (EtO) of sterilisation process
• In use, proportion of test organisms surviving the process are measure and related to the expected lethality of the process
BI’s Characterised By:
- Strain of test organisms
- Reference to culture collection
- Manufacturers name, details of who produced
- Number (usually 106) CFU’s per test piece
- D-value
- Z-value
- Recommended storage conditions
- Expiry date – can last to 50-100 years
- Disposal instructions – how to get rid if you need to remove of them
Factors Governing choice of BI:
- Stability
- Resistance (high in comparison to natural bioburden)
- Non-pathogenic – think of the people manufacturing the product
- Recoverability – you want to be able to check 100% where killed and if some don’t die you want to be able to recover the surviving spores to dispose of them properly
Recommended Test BI’s?
Filtration - Brevundimonas diminuta Moist heat - Bacillus stearothermophilus Dry heat - bacillus subtilus Irradiation - bacillus pumilus EtO - bacillus subtilus
