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Flashcards in Sterilisation Deck (118)
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1

What are the two general sterilisation processes to produce a sterile medicinal/medical product?

- produced under CLEAN conditions, but then terminally sterilised in the final container

- produced under conditions completely FREE of MOs via aseptic processing (this method is more prone to error and more unreliable)

2

Where do contaminants of medical/medicinal products come from?

- raw materials (synthetic/natural - intrinsic MOs)
- water (a requirement for ALL MOs)
- manufacturing environment (air, workers, equipment)

3

What types of organisms are found in the various contaminating environments?

- RESIDENT organisms - live in these environments
soil - gram positive, endospore forming, fungi
water - gram negative, yeast, mould
animals/humans - gram negative & positive, obligate anaerobes
plants - yeast, mould

- TRANSIENT organisms
these are organisms that travel via water and air

4

Define "sterile"

an absolute term, which means that a product is free of all MOs

a product is either sterile or is not

5

Define "sterilisation"

the killing or removal of ALL viable MOs

6

What are the different sterilisation processes?

KILLING
- Heat (moist heat using steam/ dry heat using an autoclave)
- Chemical (Ethylene Oxide)
- Radiation

REMOVAL
- Filtration

7

Who regulates the sterilisation standards?

- EN
- FDA

the standards vary slightly between the two, but if an American company wanted to sell their product in Europe, it would have to adhere to the EN standards

8

What are the sterilisation standards used for?

- to control the number of MOs in a manufacturing environment
- to VALIDATE sterilising agents and processes
- to MONITOR sterilisation processes

9

What is inactivation kinetics?

measuring inactivation kinetics allows you to assess whether a process is delivering a sterile product

10

How do you produce a kill curve?

either
- heat the culture at a certain temperature
- add EtO at a certain conc to the culture
- expose the culture to a certain dose of radiation

then take samples of the culture at regular intervals, dilute in order to culture and count colonies
- sample 0 is the sample before exposing to sterilisation
plot as the no. of survivors (no. of viable colonies) v time

11

What type of curve is a kill curve?

ASYMPTOTE curve
- as long as you take samples at regular time intervals, the same proportion of cells will be killed as the last sample
- the line will never reach 0, because the same proportion of cells are killed, which means that there is an infinite probability of survival

12

How do you produce a straight line relationship from the kill curve?

- create a semi-logarithmic plot
log the number of survivors v time

13

What is the semi-logarithmic plot of the kill curve used for?

to take measurements:
GRADIENT =
- thermal death rate of the organism (how quickly the organism dies at that temperature)

OR how quickly the organism dies at a specific concentration of EtO or dose of radiation

you can then repeat at different temperatures/concentrations/doses OR organisms - inactivation kinetics are organism specific, vary between organisms depending on their intrinsic resistance

14

How many colony forming units do you need to have when taking a sample to produce the kill curve?

between 30 and 300 colonies
- 30 is not statistically significant
- 300 would be too difficult to count

15

What order of kinetics are inactivation kinetics?

FIRST ORDER
- reaction proceeds at a rate that depends on only one reactant (proportional to the heat/conc/dose)

16

What is the D Value?

is the time taken, at a fixed temperature, dose, concentration to reduce the MO population by 90%, or ONE FULL LOG CYCLE

17

How do you calculate the D Value?

on the semi-logarithmic plot of the kill curve:
- choose a value on the log survivor's axis
- choose another value that is one full log cycle below the first value
- extrapolate the time information of the two values
- the difference of the two values gives the time

it doesn't matter WHICH value you choose, as long as you go one full log cycle below, will always give you the same D value

18

What is a thermal resistance curve?

a log of D Values v Temperature

looks at the effect of temperature on the viability of the microorganism population

19

How do you produce a thermal resistance curve?

- calculate at least three different D Values (three different temperatures)
- plot the LOG of the D Values v Temperature it was generated

produces a straight-line relationship graph

20

What is the Z Value?

the change in temperature needed to reduce the D Value by 90%, or ONE FULL LOG CYCLE

i.e. how much you have to change the temperature by to reduce the time taken to kill 90% of the MOs

- this is a measure of thermal resistance
- this is an indicator of efficiency
- can be used to compare organisms (by their D and Z values)

21

How do you calculate the Z Value?

on the thermal resistance curve:
- choose a value on the Log of D Values axis
- choose a value that is one full log cycle below the first value on the same axis
- extrapolate the temperature information of the two values
- difference of the two temperature values gives you the change in temperature = Z Value

22

What are the reference organisms when comparing Z Values?

how does the organism you're using compare to these, in the endospore form, most resistant (not vegetative):

Moist Heat Sterilisation:
- Bacillus stearothermophilus, Z Value = 10 degrees

Dry Heat Sterilisation:
- Bacillus subtilus, Z Value = 20 degrees

23

When is a product deemed sterile?

manufacturers have to reach the Sterility Assurance Level (SAL) of 10-6

this means that for every million products produced, only one will be contaminated

most manufacturers aim beyond this

24

How do you ensure the SAL 10-6?

calculate the time needed for a product to be processed (at a certain temperature, concentration or dose) to reach the 10-6 log of survivors (or 10 6 probability of survivors)

25

How do you calculate the time needed for processing to reach the SAL?

- choose a value from the log of survivors axis, positive log value
- work out how many log cycle reductions are needed to reach 10-6
- if you know one log cycle reduction = the D Value, then multiple the number of log cycle reductions with the D Value

e.g. to go from 10 2 to 10 -6, 8 log cycle reductions, 8 x D value = the time needed for processing

26

What are D Values affected by?

- bacterial species - different organisms have different intrinsic resistance
- vegetative/spore form of species - vegetative form of the species will be more sensitive to the sterilisation
- production method - how the product was produced
- nutrient environment - that the organism is exposed to
- treatment dose - of the sterilising agent

27

What is 'bioburden'?

a population of viable MOs on or in a product/package

28

Why is estimating the bioburden important?

estimate the starting population associated with materials the makes up the product

important to know the initial numbers of MOs in order to specify sterilisation parameters and inactivation kinetics

29

What are the 8 steps of bioburden estimation?

1. Sample Selection
2. Collection of Items for Test
3. Transfer to Test Laboratory
4. Treatment (if required)
5. Transfer to Culture Medium
6. Incubation
7. Enumeration and Characterisation
8. Interpretation of Data

30

What does transfer to test laboratory involve when estimating bioburden?

there may not be a microbiology lab on the site of the production of the product, have to consider
- cold temperatures - may get an UNDERESTIMATION of the bioburden as cells start dying
- warm temperatures - may get an OVERESTIMATION of the bioburden as cells will multiply
- time taken to transport, which would give a false representation of the bioburden