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Y3 Med sterilisation sem 1 > Inactivation kinetics > Flashcards

Inactivation kinetics Flashcards

1
Q

What is a kill curve?

A

Experimentally take samples at regular intervals, diluting the culture so that you get colony forming units that you can count on an agar plate.
Rapidly decreasing curve.
It is called an Asymptote curve: the same proportion of cells is lost each portion of time (curve will never touch the axis)

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2
Q

What is the D-value?

A

The time taken at a fixed temperature (dose of radiation) to reduce the population by 90% (1-log). Gives a measure by which you can compare different temperatures and different organisms.

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3
Q

What is the thermal resistance curve?

A

• The temperature change required to produce a 90% reduction (1-log cycle) in D-value

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4
Q

What is a Z-value?

A

• A measure of thermal resistance
• Indicator of efficiency
• E.g. reference (indicator) organisms:
o Bacillus stearothermophilus, Z-value 10oC (moist heat sterilization) – endospore forming
o Bacillus subtilus, Z-value 20oC (dry heat sterilization)
o (STANDARDS)

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5
Q

When is a product deemed sterile?

A

Sterility Assurance Level (SAL) represents sterility

o S.A.L. = 10^-6 (minimum) – a millionth of a bacterial cell present

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6
Q

What is 1 log cycle equal to?

A

D value

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7
Q

What are D values influenced by?

A 
o	Bacterial species
o	Vegetative vs spore form
o	Production method
o	Nutrient environment 
	Suspension media, carrier materials, culture media 
o	Treatment dose
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8
Q

What is the definition of ‘bioburden estimation’?

A

‘a population of viavle microorganisms on or in a product and/or package

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9
Q

What is the importance of bioburden estimation

A

Initial population numbers required in order to specify sterilisation parameters and inactivation kinetics

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10
Q

Describe the process of bioburden estimation

A 
Sample selection (done statistically)
Collection of items for test
Transfer to test lab (variability at this stage - too hot/cold environments can damage)
Treatment (if required):
 - lots of variability 
 - treatment to remove cells 
 - difficult to handle 
Transfer to culture medium 
Incubation 
Enumeration and characterisation 
Interpretation of data
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11
Q

What techniques are there involving bioburden estimation?

A

Direct - contact with culture medium (IDEAL)
Indirect - contact with eluent (e.g. buffered saline)
- physical treatment (e.g. vortex will shaje at controlled level
(ultrasound or shaking with glass beads can also be used)
- transfer to culture medium

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12
Q

What is considered in the selection of a removal technique?

A
  • Make sure technique is able to remove microbial contamination
  • Doesn’t effect viability (e.g. antibacterial properties)
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13
Q

What is considered in the selection of culture medium conditions?

A

• Types of microorganisms likely to be encountered dependent on:
o Nature of product
o Method of manufacture
o Potential sources of microbial contamination (e.g. operator, packaging etc)
• Culture conditions
o No universal growth medium
o Conditions assessed during validation of a technique

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14
Q

What is process operation?

A
  1. Cycle development
  2. Cycle validation – proof that process works
  3. Cycle monitoring
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15
Q

What is process validation?

A

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
• Physical qualification – taking a physical measurement (better option)
• Microbiological – used as a backup to support physical – microorganisms used that have a defined and high resistance to sterilisation process
o More prone to error and variability

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16
Q

What are Biological Indicators (BIs)

A

‘an inoculated carrier contained within its primary pack ready for use and providing a defined resistance to the specified sterilisation process’

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17
Q

When are BIs used?

A
  • Used to asses directly the microbial lethality of a sterilisation process
  • They are standardised preparations containing selected microorganisms having known stable high resistance to sterilising agents
  • Used for validation (steam, dry heat, radiation and EtO) and monitoring (EtO) of sterilisation processes
  • In use, proportion of test organisms surviving the process are measured and related to the expected lethality of the process
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18
Q

What are BIs characterised by?

A
  • Strain of test organism
  • Reference to culture collection
  • Manufacturers name etc
  • Number (10^6) CFUs per test piece
  • D-value
  • Z-value
  • Recommended storage conditions
  • Expiry date
  • Disposal instructions
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19
Q

What are the factors governing choice of BI?

A
  • Stability
  • Resistance (high in comparison to product bioburden)
  • Non-pathogenic
  • Recoverability
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20
Q

What are some recommended tests BI’s?

A 
o Filtration --> Brevundimonas diminuta
o Moist heat --> Bacillus stearothermophilus
o Dry heat --> Bacillus subtilus
o Irradiation --> Bacillus pumilus
o EtO --> Bacillus subtilus
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21
Q

What is a traditional sterilisation method for removal?

A

Filtration

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22
Q

What is a traditional destructive sterilisation method?

A

Heat (moist and dry)
Ethylene Oxide
Radiation

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23
Q

What is filtration sterilisation?

A

“Passage of a fluid (liquid or gas) across a filter, removing any contaminating solutes”

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24
Q

How big do the particles have to be in order to be filtered?

A

smaller than pore diameter

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25
Q

What are the types of filter?

A 
Depth:
  o	Non-fixed pore size (variable)
o	Inertial impaction
o	High retentative capacity 
o	Robust 
o	Cheap 
o	No sterility 
Screen (absolute filters):
  o	Uniform pore size (0.8µm. 0.45µm…)
o	Direct interception
o	Easily blocked 
o	Fragile 
o	Expensive 
o	Sterility (0.22µm)
26
Q

How do you get filter validation?

A

Bubble point pressure test.
• Challenge filter with Brevundimonas diminuta (0.4µm)
• Minimum requirement 107 / cm2
• Working capacity 109 - 1010 / cm2

27
Q

What is moist heat sterilisation?

A

Death by protein coagulation and hydrolysis. Used for aqueous products/devices/dressings

28
Q

What is dry heat sterilisation?

A

Death by oxidative processes. Used for dry powders/oil preparations/glassware and instruments

29
Q

What technology is used during dry heat sterilisation?

A

Dry heat ovens

Sterilising tunnels

30
Q

What are the mechanisms of heat transfer for dry heat sterilisation?

A

Conduction
Radiation
Convection

31
Q

What is the dry heat cycle?

A
  1. Drying
  2. Heating
  3. Exposure
  4. Cooling
32
Q

What technology is used in moist heat sterilisation?

A

Autoclave

  • self-boiler
  • mains steam
33
Q

What is the mechanism of heat transfer for moist heat sterilisation?

A

Latent heat of vaporisation

34
Q

What are the critical aspects of moist heat sterilisation?

A

Air removal
Saturated steam
Steam under pressure

35
Q

What are the critical lethal parameters for Moist heat sterilisation?

A
  1. Steam
    a. Dry saturated NOT wet or superheated
  2. Temperature
    a. Maintained within +/-5 kelvin of limit
  3. Time of contact
    a. Sufficient to give > 10-6 SAL
  4. Bioburden level
    a. Nature, number and location of M/Os
36
Q

Describe how an Autoclave operates?

A
  1. Air removal
    a. Downward displacement: evacuation
  2. Heating
  3. Sterilisation/holding period
  4. Cooling
  5. Drying
37
Q

What are the types of autoclave? (3)

A
  1. Fluid cycle
    a. Most common
    b. 2 hours
  2. Porous load cycle
    a. Porous materials such as fabrics and dressings
    b. 30 mins
  3. Air ballasted cycle
    a. Hermetically sealed products
    b. Very complex
38
Q

How is moist heat sterilisation validated and monitored?

A
  1. M.T.R – Master Temperature Record
    a. Test load
    b. Thermocouples
  2. T.R.C – Temperature Record Chart
    a. Drain probe Temperature
39
Q

How is the MTR determined?

A
  • Validation is specific to one process
  • Drain is coolest part of autoclave
  • Minimum of 12 thermocouples used in chamber
40
Q

What is the Fo concept specific to?

A

moist heat sterilisation

41
Q

What do compendial cycles require?

A

SAL of min 10^-6

42
Q

What does the typical compendial cycle consist of?

A

15 mins at 121 degrees celcius

43
Q

What is the Fo value?

A

The lethality expressed in terms of the equivalent time in minutes at a temperature of 121oC (standard) delivered by the process to the product in its final container with reference to microorganisms possessing a z-value of 10.
Alternative to compendial cycles, allows lethalities to be compared

44
Q

What is the minimum Fo value?

A

8 (equivalent to 8 mins at 121 degrees)

45
Q

What is the relationship between F and D values (equation) for biological data (cell count)?

A

F = D (logN0 - Log N)
o N0 is the initial number of microorganisms present (bioburden)
o N is the number of microorganisms surviving the process (actual or expected

46
Q

What F calculation is used for thermal data?

A 
F0 = F1 + F2 + F3…. + FN
•	Cumulative 
•	Measure of total process lethality 
F0 = [log-1(T-121)/Z)] x dt
•	121oC is the reference temp (Bacillus stearothermophilus)
•	T is the temp of heating 
•	Z is 10oC
•	dt is the time of heating
47
Q

Whar is the Fh value?

A 
Fh = [Log-1(T-170)/z] x dt
•	170o  is the reference temperature (Bacillus subtilus)
•	T is the temp of heating 
•	Z is 20oC
•	dt is the time of heating
48
Q

What is Ethylene Oxide sterilisation?

A
  • Used for disposable items and 50% of all medical devices
  • Alkylation of sulphhydryl, amino, hydroxyl and carboxyl groups on Prt and NA
  • Lethality affected by concentration, temperature and RH (non-uniform)
  • NOT same degree of sterility assurance as other methods
  • Requires standard Prd load containing suitable BI
  • Toxic residues
  • Operator safety
  • Difficult to detect
  • EtO in air is highly explosive – dangerous for operators
49
Q

What are the critical parameters for Ethylene Oxide sterilisation?

A 
• Time 
     o 1-24 hours 
• Temperature 
     o 25 – 65OC
• Humidity 
     o 40-85%
• EtO concentration 
     o 250 – 1200mg/L EtO
     o EtO distribution & penetration issues
• B. subtilus used for BOTH Validation and Monitoring
50
Q

What is the EtO process flow?

A

Pre-conditioning –> sterilizer –> aeration

51
Q

What is the EtO sterilisation cycle?

A
  1. Evacuation
  2. Vacuum hold (leak test)
  3. Conditioning
  4. Sterilant injection
  5. Exposure
  6. Sterilant removal
  7. Flushing
52
Q

What is preferred of a product in a final container: terminal sterilisation or aseptic processing?

A

Terminal sterilisation

53
Q

What is the microbiological method of test of sterility?

A

• Performed on devices exposed to a fraction of the specified sterilisation process, ie part of process development
• Purpose: validation of a sterilisation process
*similar to bioburden estimation

54
Q

What are false positives in microbiological methods?

A

• Frequency of occurrence
• Perform simulated test of “sterile” samples
• Precautions to minimise level:
o use environmentally controlled area/room
o use aseptic techniques
o avoid introducing contamination
o decontaminate test surfaces
o sterilize test equipment and materials
o minimise manipulations
o monitor and control incubator environment
o minimise aerosol production
o train personnel

55
Q

What are false negatives in microbiological methods?

A
  • Inadequate culture conditions
  • Presence of microbiostatic/cidal substance
  • Interval between treatment and testing
56
Q

What is the probability of rejection a batch of sterility testing?

A

• Probability of rejecting a batch as a function of
o Frequency of contamination
o Number of times tested
• Probability of rejection = 1-(1-p)^n
o where p = proportion contaminated
n = number of items tested
• presumption is that the sample tested represents the batch

57
Q

How many re-tests are allowed followinf failure?

A

2
* o reject batch on 2nd test if same m/o found
o retest if 2nd fail due to a different m/o

58
Q

What are pyrogens?

A

endotoxins produced by the LPS from Gram-negative bacteria

59
Q

What is the LAL test?

A

Bacterial endotoxins test
• LAL (Limulus Amebocyte Lysate) test is used for detecting endotoxin
• LAL is an aqueous extract of blood cells (amoebocytes) from the blue-blooded horsehoe crab. Limulus Polyphemus
•Based on clotting reaction of horseshoe crab lysate by endotoxin
o Equal volumes of test solution and LAL reagent are mixed in glass tubes
o After incubation at 37oC for 1h, the tubes are observed for clot formation after inverting them
o Formation of a solid clot that withstands inversion of the tube constitutes a positive test

60
Q

What are the types of LAL test? (3)

A

Gel clot
Turbidometric (kinetic)
Colorometric (chromogenic)

61
Q

What is depyrogenation?

A

Better to prevent endotoxin accumulation than remove from product once present
• Rinsing or dilution is a good way to eliminating pyrogenic activity
• Pyrogens in vials or glass components may be destroyed by dry heat sterilization at high temperatures (250C for 45mins)
• Pyrogens removed from Water for Injections by distillation

Y3 Med sterilisation sem 1 (2 decks)

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