asepsis 3 Flashcards
what is bioburden?
•Most items come with a limited number of microbes already on them (bioburden)
what are the categories in viable counting between alive and dead?
actively metabolising cell
cell with reduced metabolic activity
some metabolic activity and plasma membrane intact, but RNA content is reduced
plasma membrane intact,but no detectable metabolic activity
extensive damage to plasma membrane
cellular DNA degraded
cell fragments
define sterile
sterile: An absolute term meaning free from all viable microorganisms–Not possible to have degrees of sterility
define sterilisation
The process by which sterility is achieved, usually through the application of a biocidal agent or physical microbial removal
define disinfection
The reduction of microorganisms on an inanimate surface
what is a survivor curve?
Microorganism viability lost in exponential fashion following exposure to a killing process (not just a sterilization process)
•Independent of the starting number of organisms
what do the 3 lines represent on a survivor curve?
•Continuous (A)•Initial reduced rate of kill during early stages (B)•Reduced rate of kill at low survivor levels (C)
what microorganisms are the most resistant to sterilisation?
prions spores gram negative bacterial small non-envoloped viruses fungi large non-envloped viruses gram positive bacteria lipid envloped viruses
what are the 5 processes with standard methods for sterilisation?
steam( autoclave) filtration dry heat ( oven) gas sterilisation ionising radiation
what happens in practical sterilisation?
procedures are designed and validated with “Overkill” in mind
•Start with a measurable amount and extrapolate
in most cases what is the initial bioburden?
lower than the required level of kill needed (i.e. a 6 log reduction)
what is the D value?
The decimal reduction time (D-value) originated from assessing the thermal resistance of microbes
•The D-value is the amount of time required to reduce the number of viable organisms by 10% (a 1 log reduction) at defined experimental conditions
•Only true in steam sterilization
how is the D value usually quoted?
•Usually quoted with reference to the temperature tested•i.eD121of 5 minutes
when do D values increase/decrease?
- D-value increases at lower temperatures
* D-Value decreases at higher temperatures
how can D values also be quoted?
- D-values can be quoted for other sterilisation methods as well
- In these cases it may not be based on exposure time
what is a z value?
change in temperature that produces a tenfold (1 log reduction) change in D value
For named microorganisms a Z value is a measure of its resistance to heat
what do physical and chemical indicators show?
they do not guarantee sterilization only show procedural error/ equipment malfunctions
what is a physical indicator of a preformance verification test?
- Measurement of physical parameters with mechanical or electronic recorders
- Includes things like temperature sensors, pressure sensors
- Will provide some form of log for quality documentation
how many different classes of chemical indicators are there for steam sterilisation?
there are 6 different classes
what do the different classes in steam sterilisation show?
class 1 only shows exposure to process- e.g. autoclave tape class 5 is designed to react with all essential variables during a cycle e.g. thermalog colour change is directly related to death curve of B
What is the only type of indicator that shows that a process has killed organisms?
biological process
this is uaully for high resistance organisms
what are the two different techniques used for sterility testing?
- Direct Inoculation
* Membrane filtration
what media is used for sterility testing?
Soya-bean casein digest medium (or as you know it TSB)
•Allows for the growth of anaerobic and aerobic organisms
when there is no growth over a 14 day period the product is sterile unless:
- Environmental monitoring of facility shows a fault
- There is a fault in the testing procedure
- There is growth in the negative control
what is direct inoculation?
- As it sounds: Add product directly to media and incubate
* Preferred methods for materials that cant be easily filtered
how does membrane filtration work?
- Lets large volumes of sample be filtered•Also can rinse membrane to remove inhibitory substances
- Helps to minimize human intervention
how do you inturpurate sterility testing results?
•When looking at the sterility testing of a product the number of microbes don’t matter!
–If anything grows = fail
–If stays clear= pass
what are pyrogens?
•Pyrogens are fever inducing substances;
–Endogenous pyrogens are usually cytokines which induce a shift in temperature regulation once they have exceeded a threshold
–Exogenous pyrogen are usually microbial in origin–For pharmaceuticals usually means endotoxins (LPS) from G-bacteria
where do the different types pyrogens live?
•Both live and dead bacteria have the same pyrogenic potential
what is depyrogenation?
•Depyrogenation is the process by which pyrogens can be removed or destroyed
how cab pyrogens be removed?
•Pyrogens can be removed by;–Ion exchange chromatography–Ultrafiltration–Distillation
how can pyrogens be destroyed?
•Pyrogens can be destroyed by;–Acid base hydrolysis–Oxidation–Heating–Sodium hydroxide
what are the 3 main tests used to detect endotoxins and pyrogens?
- Rabbit pyrogen test (introduced to USP in 1942)
- Limulus amebocyte assay (introduced to USP in 1980)
- Monocyte activation test (introduced to EP in 2010)
how does the rabbit pyrogen test work?
- Product initially injected into marginal ear vein of 3 rabbits (≤10ml/Kg)
- Number increases if product fails•Temperature monitored every 30 mins for 3h
what are the limitations of rabbit pyrogen test?
- Not very sensitive
- Not very sensitive to some bacterial endotoxins (e.gLegionella)
- Variation in rabbit species response when standard endotoxin solutions used
- Incompatible with certain types of drugs (e.g. radiopharmaceuticals)
what is LAL assays specific for?
- Specific for bacterial endotoxins
- 3 different types of LAL assay recognised;
- Gel clot technique
- Turbidimetric technique
- Chromogenic technique
what does LAL assay rely on?
•Relies on the clotting cascade of horseshoe crab blood
what is the problem with LAL assay?
factor C needs to be activated in order to generate the clot
other things can also be activated thus giving a false positive result
how does monocyte activation test work?
Uses monocytes from human blood to detect pyrogens–Either cell lines, whole blood or peripheral blood mononuclear cells–Can be used as an alternative to Rabbits
•Relies on the monocyte to generate pro-inflammatory cytokines which can be detected by ELISA
what are endotoxin limits? and how do you calculate it?
are based on the dose of the product given per hour
EL=K/M
K = minimum pyrogenic dose (i.e. the maximum IU of endotoxin which a patient can get without a toxic reaction)•M = maximum dose of the drug substance per person (or Kg) per hour
