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PR3151 Disinfectants and Sterilization > IC9 Concepts for Sterility > Flashcards

IC9 Concepts for Sterility Flashcards

1
Q

Compare terminal sterilization and aseptic processing

A

Terminal sterilization - lethal treatment on microorganism (can involve heat, radiation, chemicals) in order to destroy all life on material

Aseptic processing - continued exclusion/removal of harmful microorganisms to reduce contamination and bioburden

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2
Q

What are the 2 types of culture media used in sterility testing, and how are samples incubated?

A
  1. Fluid thioglycolate medium (FLM) - anaerobic, and some aerobic bacteria
  2. Soybean casein digest medium (SCDM) - fungi, aerobic bacteria

Not common: Sabouraud Dextrose Agar (SDA) for yeast

Samples are incubated for at least 14 days at 32.5dc and 22.5dc (two sep temperatures)

FYI (?):
- temp of incubators should be monitored, there should be records of calibration of the temp monitoring devices
- test containers should be inspected at intervals during the incubation period, observations recorded
- if pdt produces a suspension, flocculation or deposit in the media, suitable portions of the contents of the containers should be transferred to fresh media under clean room conditions, after 14 days, and reincubated for a further 7 days

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3
Q

How are outcomes assessed in sterility testing?

A

Turbidity in culture media
- Including morphology study

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4
Q

What factors determine the number of sterile, packaged units to be sampled for sterility?

A
  1. Number of units in a batch (select a representative sample size)
  2. Volume of liquid per container
  3. Method of sterilization
  4. Manufacturing requirements of the regulatory
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5
Q

What are the two types of sterility testing for products?

*These sterility testing involves removal/neutralization of any antimicrobial properties before incubation

A
  1. Membrane Filtration
  2. Direct inoculation
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6
Q

What membrane filter is used for membrane filtration sterility testing and why?

A

0.45um membrane filter is used => culture medium => incubation

Do not use 0.22um bacteria retentive filter, as we are only interested in filtering out the antimicrobial or preservative properties that may cause turbidity or inhibit growth

0.22um is used for aseptic filtration (sterility method) instead to remove microorganism

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7
Q

Which method (no longer used) was used to estimate the number of viable bacteria in the sample after membrane filtration sterility test?

A

Most probable number (MPN)
- estimate viable no. of bacteria in a sample by inoculating broth in 10 fold dilutions, based on principle of extinction dilution (dilute till microbes cant be found, go backwards to assess initial microbial load)

Why not used anymore?
- Use softwares to count CFU and assess morphology

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8
Q

Describe the steps involved in direct inoculation sterility testing.

A
  1. Aseptically remove sample
  2. Neutralize sample (if it has antimicrobial properties)
  3. Inoculate in culture medium
  4. Incubate
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9
Q

What are 2 disadvantages of direct inoculation sterility testing?

A
  1. Low sensitivity because of small sample/volume used (form whole pdt)
  2. Cloudy samples (e.g., suspension) may affect reading of microbial growth (turbidity)
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10
Q

How can sterility testing be conducted for medical devices?

Also explain how it should be carried out if medical devices have hollow spaces and pipes.

A

Medical device can be aseptically transferred into test media (placed in direct contact) for incubation

If device has many spaces and pipes, can consider rinsing with rinsing solution that will be incubated with the growth medium, or rinsing with growth medium directly
OR
can cut up long pipes to place in growth medium *ensures all surfaces of medical device comes into contact with the test media

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11
Q

What are some limits of sterility testing?

A

Sample size
Failure rate
Cost
Reliability
Time required - not so much a concern as production rate usually much higher

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12
Q

Sterilization method 3: Moist Heat (Steam) Sterilization
Explain the principle of this method.

  • what are the parameters
  • what is the ideal steam
  • how does it work (MOA)
  • role of water
A

Direct steam contact at the required temp and pressure for the specified time
(Generally 121dc for 15min)

  • Parameters: steam, temp, pressure, time (STPT)
  • Ideal steam: dry saturated steam and entrained water

How it works?
- Moist heat destroys microorganisms by the irreversible coagulation and denaturation of enzymes and structural proteins

Role of water: transmit heat better than air

Disadvantages:
- not used for heat-sensitive and moisture-sensitive instruments
- may leave instruments wet, cause them to rust

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13
Q

Sterilization method 3: Moist Heat (Steam) Sterilization
What are the 3 types of steam sterilizers (autoclaves)

A
  1. Gravity displacement autoclave
    - Let steam in, change temp and humidity in system, re-circulation of cold air sinking and hot air rising
  2. High speed prevacuum sterilizer
    - Inlet of steam, vacuum needed to be filled => movement of steam
  3. Steam flush pressure pulsing process
    - Steam flushes in at very high pressure, in pulses
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14
Q

Sterilization method 3: Moist Heat (Steam) Sterilization
Explain the F concept in determining temp and time for steam sterilization method.

A

F value measures EQUIVALENT time (to achieve 121dc for 15min) - note the time here starts from when the pdt reaches this temp
Significance: heat transmission from autoclave to product is NOT instantaneous

E.g., 15min at T = Fmin at 121dc

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15
Q

Sterilization method 4: Dry Heat Sterilization
Explain the principle of dry heat sterilization.

(Dry heat sterilization can be used for products sensitive to moisture e..g., powders, petroleum pdts, sharp instruments)

A

Dry heat 180dc for 30min OR 170dc for 1h OR 160dc for 2h

Dry heat greater than 220dc used for sterilization and depyrogenization (destroy pyrogen) of glassware

Slower rate of heat penetration (slower conductivity of heat) compared to moist heat sterilization, therefore dry heat sterilization require HIGHER TEMP + LONGER DURATION

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16
Q

Sterilization method 4: Dry Heat Sterilization
What are the 2 types of dry-heat sterilizers?

A
  1. Static air type
  2. Forced air type/mechanical convection sterilizer
    - Ventilated oven, equipped with motor-driven blower than circulates heated air throughout the chamber at high velocity to permit more rapid rate of transfer of energy/heat from the air to the instruments/products
17
Q

Which sterilization method is the only one capable of destroying endotoxins and other pyrogens?

A

Dry heat sterilization

*Absence of endotoxins is important for the sterility for parenteral applciations

18
Q

What are some methods to detect pyrogens?

A

Detect pyrogens:

  • Rabbit pyrogen test
    => low sensitivity due to pyrogen tolerance or stress from rabbits
  • Monocyte activation test - can produce cytokines that can be detected with immunooassays such as ELISA

Detect endotoxins only:

  • Bacterial endotoxin test (LAL test) - coagulate
    => high sensitivity for endotoxin
  • Recombinant factor C (rFC) - genetically engineered protein normally found in LAL cascade, Factor C reacts with endotoxin and couples with a marker to produce a quantifiable, fluorescent end product
19
Q

Sterilization method 5: Chemical Sterilization
Explain the principle of chemical sterilization.
What are the 4 sterilants commonly used.

A

Heat-sensitive products can be sterilized by liquid sterilants in “cold” systems ~60dc

Ethylene oxide, Formaldehyde, Hydrogen Peroxide, Peracetic acid

20
Q

Sterilization method 5: Chemical Sterilization

Explain the use of Ethylene Oxide.

A

Ethylene Oxide (ETO)

MOA: alkylating agent, can attack proteins, nucleic acids, and other organic compounds, particularly reactive with SH and other enzyme-reactive groups
=> induce irreversible activation of the microorganism

How is it used?
- pure or mixed with CFC => affects ozone layer
- CO2 or HCFC (hydrochloroflurocarbons)
- combi with peracetic acid, hydrogen peroxide gas plasma, ozone

How is it removed?
- Toxic residue removed via proper aeration

Properties?
- Flammable, explosive gas
- Carcinogenic
- Can penetrate packaging materials, device lumen

21
Q

Sterilization method 5: Chemical Sterilization

What are the toxic effects of Ethylene Oxide?

A
  1. Possibly carcinogenic/mutagenic
  2. Can cause eye pain, sore throat, difficulty breathing, blurred vision, dizziness, nausea, headache, convulsions, blisters, vomiting, coughing
    - Low conc: affect lungs
    - High conc: penetrate skin, cause blisters
22
Q

Sterilization method 5: Chemical Sterilization

Explain the use of Peracetic Acid.

What agent might be needed with Peracetic acid?

A

Peracetic Acid
(effective at low temp + low conc., 100ppm)

MOA: denature proteins and enzymes, increase cell wall permeability by disrupting SH and SS bonds in enzymes and proteins

How is it used? *effective at LOW CONC, LOW TEMP
- mostly used as sterilant for medical device such as endoscopic tubes
- 35% in combi w anticorrosive agent (sulfonate)
- diluted to 0.2% with filtered water at temp of 50dc
- process time 12min

Properties?
- decompose to safe by-products, acetic acid + O2
- free from decomposition by peroxidase (unlike H2O2)
- activity may be affected by organic residue - but still retain some activity (from 100ppm to 5000ppm)
- need anticorrosive agent, sulfonate
- may be used in combi with other sterilants to improve spectrum of activity (+ virus)

23
Q

Sterilization method 5: Chemical Sterilization

What material is sensitive (compatibility issues) to Peracetic Acid?

A

Plastic, anodized aluminium

24
Q

Sterilization method 5: Chemical Sterilization

Explain how Hydrogen Peroxide Plasma is generated.

A

Hydrogen Peroxide Plasma is generated in an enclosed chamber under deep vacuum to pull liquid H2O2 to create a vaporized plasma. Radio frequency or microwave energy is used to excite the gas molecule and produce charged particles, which consist of many free radicals.

Vaporized H2O2 carried to sterilization chamber by a carrier gas

Process operates in range of 37-44dc, cycle time of 75min
(LOWER TEMP but LONGER TIME required)

25
Q

Sterilization method 5: Chemical Sterilization

Explain how H2O2 works.

What agent might be needed with it?

A

MOA: H2O2 is a strong oxidizing agent, produce hydroxyl free radicals that induce oxidation in the outer layer of microorganisms and attack essential cell components such as lipids, proteins, DNA
=> wide range of efficacy, against mycobacteria, MRSA, clostridium

Properties:
- non toxic (by products water vapor + O2)
- no aeration needed
- low penetration (may need penetration enhancer - e.g., dimethyl formamide, polyethylene glycol, lauryl carbomoyl)

26
Q

Sterilization method 5: Chemical Sterilization

Explain the use of diffusion enhancer for hydrogen peroxide plasma sterilization

A

Small vial/capsule of concentrated H2O2 that can be broken and released into the system to increase the amount of hydrogen peroxide
- can help reach cranny or nook of medical device

27
Q

Sterilization method 5: Chemical Sterilization

What are some concerns with regards to chemical sterilization?

A

Requires tight wrap to avoid leaks, possible contaminate adjacent areas

Compatability issues

28
Q

Sterilization method 6: Gamma Radiation

What might be used for ionizing radiation?

A

Ionizing radiation primarily by: cobalt 60, cesium 137, or electron accelerators

29
Q

Sterilization method 6: Gamma Radiation

Explain the principle of gamma radiation

What is it commonly used for?

A

MOA: disrupt nucleic acid

Low temperature sterilization (but require high radiation)
Suitable penetration and high dose rate (able to go through many layers of material)

Commonly used for sterilization of containers + packaging + medical products

30
Q

Sterilization method 6: Gamma Radiation

What formulations/materials may gamma radiation not be suitable for?

A

Not suitable for aq pdts with proteinaceous components as it can inactivate proteins

Not suitable for polyethylene, as it can cause oxidation, deamination, cracking

Not suitable for nitrile gloves - cause gloves to crack and become brittle

31
Q

Using Ozone for sterilization:

What is the principle of using Ozone?

A

Ozone consists of O2 loosely bonded with third oxygen atom
Third oxygen makes ozone a powerful oxidizing agent (radical O. is highly reactive), can destroy microorganisms including spores

However, it is highly unstable with very short half life of 22min at RT

*Ozone is formed by reacting oxygen with high electric charge

32
Q

Using Ozone for sterilization:

What is its advantage?

A
  1. Compatible with wide range of commonly used materials (stainless steel, titanium, anodized aluminium, ceramic, glass, silica, PVC, teflon, silicon, polypropylene, polythylene, acrylic)
  2. Can be used for water treatment, water pipes
  3. Instability: any residue that is not O2 can be eliminated simply by leaving pdt is sterile chamber (no need additional step to remove sterilant - same with gamma radiation) *react quickly
33
Q

Filling of products for terminal sterilization should be done in at least a Grade __ environment

Filling of aseptically prepared product should be done in a Grade __ environment

A

If going to be terminally sterilized: Grade C

If handling aseptic components: Grade A

PR3151 Disinfectants and Sterilization (10 decks)

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