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IPM > Sterilisation > Flashcards

Sterilisation Flashcards

1
Q

What are the 5 recognised methods for sterilisation of pharmaceuticals?

A
  1. Steam/moist heat (autoclave)
  2. Dry heat
  3. Ionising radiation
  4. Gas sterilisation
  5. Filtration
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2
Q

Outline autoclave sterilisation

A
  • Exposure to dry saturated steam under pressure in an autoclave
  • 121 degrees for 15 minutes for thermostable aqueous preparations
  • 100% water vapour

Consists of:

  • Chamber + door
  • Steam let in via baffles
  • Thermocouples
  • Temperature of drain valve should = autoclave temperature (otherwise there is a fault and procedure needs to be stopped)
  • Some have a double walled jacket
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3
Q

How does autoclaving sterilise?

A
  • At high temperature, it kills microorganisms rapidly. Can transfer a large amount of heat quickly
  • Kills microbes via hydrolysis of proteins and nucleic acids (denaturation)
  • Convection
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4
Q

Why is there a large amount of heat obtained from steam?

A
  • Intrinsic heat as high pressures produce temperatures of 121 degrees
  • Latent heat- contribution from condensation
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5
Q

Why is there an issue if air enters the autoclave process?

A
  • Equilibrium between temperature and pressure is related to partial pressure of the steam
  • If there is a mixture of steam and air present - temperature and pressure is lowered (compared to pure steam in same conditions)
  • Related to Dalton’s law- Total pressure of a mixture of gases= sum of individual partial pressures
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6
Q

Outline the process of autoclaving sterilising bottled aqueous fluids

A
  • Does not rely on direct contact between steam and the product
  • Steam condenses on the surface of the bottle which is followed by heat transfer across the container walls
  • Pressure within the bottle increases
  1. Heating up
  2. Sterilisation (holding time) temperature within the product will stabilise and then be held at a sterilising temperature (121 degrees for 15 minutes)
  3. Cooling stage- can be accelerated by spraying with STERILE water

Jacket preheating and pulling a vacuum is NOT needed

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7
Q

Outline the process of autoclaving porous loads

A
  • Most efficient sterilisation process
  • Wrapped goods and porous materials
  1. Air removal via evacuation and steam injections and repeat
  2. Sterilisation holding time is low but temperature is high (134 degrees for 3 mins)
  3. Drying, steam is drawn off by pulling a partial vacuum
  4. Allows air admission via bacterial proof filter

Autoclave design must have:

  • Jacket to preheat the chamber to lower condensation (will avoid too much condensation within the porous load, e.g. dressing)
  • Bacteria proof filter over air entry
  • Must be able to draw a vacuum
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8
Q

In terms of autoclaving, what is heating affected by?

A
  • Size of container
  • Size of load
  • Shape of container
  • Porosity and entrapment of air
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9
Q

What are advantages and disadvantages of autoclaving?

A

Advantages:

  • Terminal process for aqueous solutions or suspensions
  • Wide safety margin
  • Kills bacteria and viruses
  • Use for products given by IV route

Disadvantages:

  • Only useful for thermostable solutions and suspensions
  • Cannot be applied to bottled products with oily bases as water is essential for the process
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10
Q

Outline the hot air oven (Dry heat Sterilisation) process and the criteria for it to occur

A
  • There is no steam
  • 170 degrees for 1 hour
  • Convection fan to give forced air circulation
  • Radiation from walls
  • Silvered shiney layer to reflect heat within chamber
  • Load should be same type and size, evenly spaces to reduce interference with air flow (heat transfer shouldn’t be obscured by neighbouring load)
  • Containers should be sealed
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11
Q

How does dry heat kill microorganisms?

What kind of products is it used for?

A
  • Causes cell death by oxidative processes although
  • Slower and less efficient proces
  • Used for oils, powders, apparatus, glassware
  • Not for samples vulnerable to high temp (Biologicals and liquids)
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12
Q

What are some important points about hot air ovens?

A
  • Maximum heat within the apparatus is at the walls, so contact with the walls must be avoided
  • Avoid packing of many small items in large scaled tins
  • Screw caps of containers should be unscrewed 1/2 a turn to prevent distortion of closure or bursting of containers
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13
Q

What are the advantages and disadvantages of hot air ovens?

A

Advantages:

  • Can be used for substances which can be harmed by moisture
  • Suitable for assembled apparatus (glass syringes)
  • Less damaging to glass and metal equipment compared to moist heat

Disadvantages:

  • Drastic conditions are not tolerated by packaging material (plastics, rubber)
  • Not suitable for surgical dressings
  • Does not penetrate as well as moist air
  • Non-terminal as you have to repackage materials
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14
Q

Outline Gas sterilisation

A
  • Ethylene oxide (volatile- boiling point is 10.8 degrees)
  • 40 degrees
  • 2 hours (lengthy process time)
  • Gas tight chamber with vacuum pump
  1. Air is removed by drawing a vacuum
  2. Water vapour is admitted to the required RH (minimum 30% is optimum antimicrobial activity)
  3. Sterilisation at a pre set pressure and and a set time
  4. Gas is removed by drawing a vacuum. Air is admitted via bacterial filter
  5. Total air replacement allows air entry via a filter to atm pressure then chamber may be opened up
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15
Q

What are the problems associated with ethylene oxide?

A
  • Toxic gas so sterilisation must take place within enclosed chamber fitted with vacuum pump
  • Forms an explosive mixture with air (overcome this by mixing it with inert gases such as carbon dioxide)
  • 12% ethylene oxide with 88% dychlorodifluoromethane - under these conditions it is not explosive but does not interrupt the killing action
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16
Q

What can gas sterilisation be used for?

A
  • used for single use medical devices such as catheter, pacemaker wires
  • Not for products which may absorb ethylene oxide such as porous products (toxicity may stay in it however it penetrates well)
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17
Q

What are the advantages and disadvantages of gas sterilisation?

A

Advantages:

  • Few materials damaged as minimal heat
  • Good penetration into porous loads and plastics
  • Effective against all organisms (bacterias, moulds, viruses)
  • Effective at low humidity

Disadvantages:

  • Expensive
  • Need to control RH closely
  • Toxic gas is being used and risk of explosion if mixed with air
  • Resides of ethylene oxide must be allowed to disperse and be fully removed as it is dangerous if it stays in the product
  • What do you do with the toxic gas after processing?
  • Slower process (especially with plastics and nylon wraps which have to be left open and sealed aspetically)
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18
Q

What is the MOA of ethylene oxide?

A
  • Alkylation -CH2 addition
  • Reacts with amino groups, OH
  • Destroys function of essential proteins and nucleic acids preventing reproduction of contamination
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19
Q

Name some other gases that can be used in sterilisation

A
  • Hydrogen peroxide- cheap but reacts with many things
  • Formaledehyde (CH2O)
  • Pereacetic acid
  • Ozone (hazardous)
  • Chloride dioxide
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20
Q

What are the ideal properties of a sterilant gas?

A
  • Effective at low humidity and low temperature
  • Non-toxic (safe to operator)
  • Volatile, high penetration
  • High activity against a broad range
  • Ease of disposal
  • cheap
  • Compatibility with wide range of products and materials
  • Non-explosive in air
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21
Q

Outline ionising radiation as a method of sterilisation

A

Exposing product in final container to:
- Gamma radiation e.g. from Cobalt-60 OR electron beam e.g. linear acceleration with high energy electrons

  • Needs minimum absorbed dose at 25KGy (kilograys)
  • Terminal sterilisation

Aspects of the process:

  • Irradiator containing the source
  • Sample area (in a box)
  • Conveyor belt passing samples through
  • Measured time of exposure
  • Dosimeter to measure exposure dose
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22
Q

What is the MOA of ionising radiation?

A
  • Targets site in microorganisms in DNA
  • Ionising radiation induces structural damage in microbial DNA
  • Ionising radiation causes the ejection of an electron that produces highly reactive radicals and positively charged ions
  • Primary event

Interaction with gamma and water:
- Short lived hydroxyl radical that disrupts ability of cfu to reproduce as it interacts with DNA

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23
Q

Outline ionising radiation as a method of sterilisation

A

Exposing product in final container to:
- Gamma radiation e.g. from Cobalt-60 OR electron beam e.g. linear acceleration with high energy electrons

  • Needs minimum absorbed dose at 25KGy (kilograys)
  • Terminal sterilisation

Aspects of the process:

  • Irradiator containing the source
  • Sample area (in a box)
  • Conveyor belt passing samples through
  • Measured time of exposure
  • Dosimeter to measure exposure dose
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24
Q

What is the energy of ionising radiation expressed in?

A

Electron volts = Energy gained by an electron moving through a potential difference of 1 volt

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25
Q

What is the absorbed dose of ionising radiation measured by?

A 
Unit gray (Gy)
Defined as 1 joule of energy absorbed per kilogram of material irradiated
26
Q

What are the advantages and disadvantages of ionising radiation?

A

Advantages:

  • Effective against a large number of bacteria, yeast, moulds, some viruses
  • Terminal
  • Efficient
  • Can be used on thermo-labile samples

Disadvantages:

  • Cost of installation, training, disposal
  • Not effective against some viruses and prions (bent proteins that cause disease)
  • Dangerous
  • Perceived s/e as people still think sample is radioactive afterwards
27
Q

Outline filtration sterilisation

A
  • Membrane with holes in it and you can sterilise fluids/gases
  • Non-terminal
  • Liquid is passed through a sterile bacteria retaining filter of suitable construction
  • Probability of organism in filtrate < 1 in 1 million
  • After filtration, the liquid has to be placed in pre-sterilised containers
28
Q

What size is pseudomonas?

A

0.22-1 microns

29
Q

What are the two types of filters?

A

Depth and screen

30
Q

Outline a screen (membrane) filter

A
  • Better type of filter
  • e.g.) cellulose acetate
  • Absolutely retentive

Integrity test must be applied:

  • Before use- check for possible defects in the filter
  • After use- reveals leaks which may develop during use
31
Q

Outline a screen filter

A
  • Better type of filter
  • e.g.) cellulose acetate
  • Absolutely retentive

Integrity test must be applied:

  • Before use- check for possible defects in the filter
  • After use- reveals leaks which may develop during use
32
Q

What are the two integrity tests for filters?

A
  1. Pressure retention test

2. Bubble pressure test

33
Q

Outline the pressure retention test

A
  • Method to test filters
  • Push fluid through it and there should be a back pressure (if there is no pressure, liquid is going through filter too easily and there is a fault, such as too big holes)
34
Q

Outline the bubble pressure test

A
  • Allows you to calculate the pore diameter (D)
  • Will change between liquids, but generally D is proportional to liquid (e.g. if it is viscous, it is harder to push through the pores and D is higher)
35
Q

Outline criteria for an ideal aspetic sterilising filter

A
  1. Unlimited flow rate
  2. Inert
  3. Thermal and strength stability (won’t tear/puncture and can withstand pressures and steam sterilisation)
  4. Low cost
  5. Longevitity - with high particle contamination use a pre-filter such as Seitz
  6. Target should be able to withold a certain number of organisms
36
Q

What are the factors affecting efficiency of filtration?

A
  1. Initial microbial count
  2. Duration of filtration - prolonged filtration (> 1 day) must be avoided in case a contaminant grows through the filter medium
  3. Types of organisms:
    - Cannot filter viruses
    - Cannot filter mycoplasms (a form of bacteria that has no cell wall, making it not rigid so can squeeze through pores)
37
Q

What are the advantages and disadvantages of filtration?

A

Advantages:

  • Used with thermolabile products (those unstable with heat)
  • Removes bacterial bodies, so no remnants of dead cells are floating about the product (so no endotoxins and no LPS from gram -ve)

Disadvantages:

  • Not for suspensions as active ingredient may be filtered out
  • Non-terminal process (contamination risk and further sterility testing is needed)
  • Sterility test required (7-14 day delay)
  • Viruses and mycoplasms not removed
38
Q

What is enumeration?

A
  • Measurement of number of microbial cells in a sample

- Total viable count/cfu

39
Q

What is the simplest method for enumeration?

A
  1. Dilute the sample with an appropriate media and then inoculate growth media with this sample.
  2. It is then incubated for a set length of time
  3. Number of CFUs is determined and the numbers are related back to the original sample
40
Q

What are the methods of enumeration for viable counts?

A
  • Pour plate (counting colonies in agar)
  • Surface spread- counting colonies on agar surface
  • Membrane filter methods- sample passed through a filter then the colonies are grown on the membrane on an agar surface
41
Q

What are the methods of enumeration for total counts?

A
  • Direct microscopic counting using a counting chamber
  • Turbidity methods - measures opacity in suspensions/cultures
  • Dry weight determinations
  • Nitrogen, protein or nucleic acid determinations
42
Q

What are the methods of enumeration for indirect viable counts (rapid methods)?

A
  • Epifluorescence - dyes that have a characteristic fluorescence when interacting with living cells
  • ATP methods- bioluminescence measures ATP production in living cells (firefly luciferase)
  • Impedance - measures change in resistance on growing cultures
  • Manometric methods - measures oxygen consumption/CO2 production by growing cultures
43
Q

How do calculate log % survivors?

A

log % S= log (Nt/No x 100)

Nt= survivors after time t
No = original number of organisms

in cells/mL

44
Q

What is the inactivation factor and how do you calculate it?

A

Degree of reduction in the viable population

IF= No/Nt

45
Q

Death of microbial contamination is a probability function. What is it based on and what can assure all viable organisms are absent?

A
  • It is based on the length of time of exposure to the lethal agent
  • Only an infinite exposure to the lethal agent can absence of all viable organisms be assured with certainty
46
Q

What does the BP state is an appropriate sterilisation assurance level?

A

1 viable microorganism in 1 million sterilised items of the final product

47
Q

What is the decimal reduction time and what is it used for?

A

Time to achieve 90% kill
Time for reduction in number by one log 10

Used to:

  • Characterize the resistance of spores under the influence of heat
  • To measure the efficacy of sterilisation process

Each spore has a characteristic D value

48
Q
  1. Under what temperature is Lethality given the symbol Fo?
  2. What does the BP recommend as an Fo value?
  3. What is the equation for Fo?
A
  1. 121 degrees
  2. Fo=8
  3. Fo = D x log(No/Nt)

Therefore, Fo= D X log(IF)

Fo means total thermal effect

49
Q
  1. Under what conditions if Lethality given the symbol Fo?
  2. What does the BP recommend as an Fo value?
  3. What is the equation for Fo?
A
  1. 121 degrees
  2. Fo=8
  3. Fo = D x log(No/Nt)
50
Q

Inactivation factor can be expressed in terms of D. How?

A

IF= 10^(t/D)

51
Q

What is validation?

A

The process of demonstrating that a sterilisation method/protocol will consistently produce the results that it intended to

52
Q

What is validation?

A

The process of

53
Q

What 3 ways do you carry out validation and in-process monitoring?

A
  1. Physical Indicators
  2. Chemical Indicators
  3. Biological Indicators
54
Q

Outline physical indicators for validation and in-process monitoring.

A

For autoclaving and hot air ovens:
- Temperature recording chart as part of the batch record

For ionising radiation:
- Plastic dosimeter measures radiation dose absorbed

For filtration:
- Measures pressure retention by the filter

Gas Sterilisation:
- Temperature, relative humidity

55
Q

Outline chemical indicators for validation and in-process monitoring

A

Autoclave and hot air oven:
- Browne’s tubes and tape- tube will go from red to green if heat sterilisation conditions are adequate

Gas sterilisation:

  • Chemical sachets that show whether the appropriate gas concentration has been present for the appropriate time
  • Shows whether a product has been processed or not
56
Q

What are biological indicators?

A
  • Placed directly into the container
  • Contain known number of microorganisms deposited on a carrier
  • Often in the form of metal foil/paper strips/discs
  • D value and z value known
  • Uses various Bacillus strains depending on the sterilisation method
57
Q

How do biologically monitor sterilisation?

A
  1. Put the organism in the batch being sterilised
  2. This introduces a risk and see if it is there at the end of sterilisation

Only in a few procedures is this essential

58
Q

What is the general process for sterility testing?

A
  1. Sterilise
  2. Take sample e.g. from injection
  3. Transfer to a suitable growth medium
  4. Observe to see if growth has occurred
59
Q

What is the membrane filtration technique for biological monitoring of validation?

What is the advantage of this?

A
  1. Filter sample through 0.45 microns
  2. Wash filter with suitable diluent such as peptone
  3. Transfer filter to suitable liquid medium (in filter holder)

Advantage:
You can filter the whole contents of the container and any contamination will end up on the filter

60
Q

What is the direct inoculation technique for biological monitoring of validation?

A

Inoculation into culture medium

Sample is diluted 10 times if it is a liquid and diluted 100 times if it is a solid

Disadvantage:
Can only take a small portion of the sample to be tested i.e if you had 1L you would need to dilute it to 10L which is a lot

61
Q

What is the direct inoculation technique for biological monitoring of validation?

A

Inoculation into culture medium

Sample is diluted 10 times if it is a liquid and diluted 100 times if it is a solid

IPM (10 decks)

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