Sterilisation Methods

Question 1

Sterilisation by HEAT

Answer 1

• heat produces thermal energy that is lethal to m.o, damages cytoplasmic membrane, cellular enzymes, DNA, RNA
• Moist heat (Protein Coagulation)
• Dry heat (Oxidation)
• Factors (HOT): sufficient quantity of heat i.e. temp, Organisms i.e. need to know population and their heat resistance, Time needs to be sufficient heat and holding time to destroy them

Question 2

Thermal death kinetics

Answer 2

At constant temp, same percentage of m.o will be destroyed per unit of time at the rate proportional to the number of surviving organisms

Log10Nt=Log10No-kTt

LogNT VS t

KT = thermal death rate constant at temp T

T in minutes

Question 3

Decimal Reduction Time (D)

Answer 3

Heating time required from 10 fold reduction (1 log cycle) in number of surviving organisms
D=1/Kt

Question 4

How do we know whether organisms are heat sensitive or heat resistant?

Answer 4

Z value

• defines heat sensitivity (small z) or resistance (large z) of m.o
• defined by increase in temp required to bring a 10 fold decrease in decimal reduction time

Question 5

Fo value

Answer 5

• defines the lethality in terms of equivalent heating time in minutes at 121 deg using reference organism with a z value of 10 deg
• takes into account the equivalent lethal effect go lower temp compared with 121 deg
  Fo = T x 10^( T-121 / z )

Question 6

Sterility Assurance Level SAL

Answer 6

• Defines the degree of sterility in the treated batch of product

Question 7

MOIST HEAT STERILISATION

Answer 7

• saturated steam at high temp holds load of latent heat which can transfer without drop in temperature when it condenses onto a cooler surface
• protein coagulation and denaturation of m.o
  Autoclaving
• moist heat sterilisation under high steam pressure in an autoclave oven
• steps involve remove air, heat to temp under high steam pressure, hold temp for required time, cool and depressurise
  Used for sterilising: dressings, equipment, aq injections only

Question 8

Monitoring steam sterilisation

Answer 8

Instrumental indicators: thermometers, pressure gauges, thermocouples
Chemical indicators: Browne’s tube red to green, Bowie-Dick tape (light-dark), Steam-Clox strip (purple-green)
Biological Indicator: Spore strips, B. stearothermophilus

Question 9

Biological Indicators

Answer 9

• strain stable
• non- pathogenic
• Resistant to method used
• reproducible recovery
• not less than 10^5 viable spores per unit

Question 10

Advantages of Moist Heat

Answer 10

Lower temp
Shorter exposure time
Aqueous thermostable preparations ie aq inj
Sealed in heat stable plastic containers

Question 11

Disadvantages of Moist Heat

Answer 11

Heat labile prep
corrosive
containers must be penetrable by steam
difficult to monitor
damp load
bursting of rigid containers

Question 12

DRY HEAT STERILISATION

Answer 12

• Heat transfer by convection or radiation
• Oxidative destruction of bacterial cell wall
• higher pressure and longer holding time (no rubber, plastic packages)
• suitable for metal instruments, glass ware, powders, oils
• also used for depyrogenation of glassware

Question 13

Hot air oven

Answer 13

Electric heater to heat the oven
Fan Circulation to distribute heat evenly, max variation in chamber <10 temp
Thermostat to maintain temp
Heat lining to prevent heat transfer from inside
Efficiency Indicator: B. Subtilis var niger

Question 14

Advantages of Dry Heat

Answer 14

Non-aq systems
Non-corrosive
Penetration
Dry process
Oily thermostable products i.e. oily inn

Question 15

Disadvantages of dry heat

Answer 15

Slow heating
Longer holding times
Higher temp
Not for aq preparations

Question 16

moist heat vs Dry heat

Answer 16

Condensation, protein coagulation MOA, 121 for 15 mins under high steam pressure 15psi, B. Stearothermophilus, D121 = 2 mins

Convection and radiation, oxidative destruction, 160-170 for 1-2 hours, B subtilise var niger, D160 = 5-10 mins

Question 17

Radiation

Answer 17

• electromagnetic radiation: energy in motion in the form of non-particulate electromagnetic rays
• Energy photons can induce excitation of atoms in living cells depending on level of energy and penetrating power
• chemical reactions in nucleic acid, cell mutation and death
• UV, GAMMA

Question 18

UV Radiation

Answer 18

Electromagnetic radiation of lower energy and poor penetrating power

• non-ionising, can cause excitation of atoms to higher energy state
• cause chemical reactions in nucleic acid, alter DNA, mutate, cell death

Uses:
Air - lower m.o counts in clean rooms by UV lamps fitted ducted air supply systems
Water - UV water steriliser equipment in water supply
Surfaces - Direct irradiation of sterile cabinet surfaces (LFC)`

Question 19

Disadvantages of UV radiation

Answer 19

Hazard to operators as they are also irradiated
- use reflectors to protect operator and direct and intensify UV exposure to defined areas

Poor penetration
Shielding and reflection

Question 20

GAMMA IRRADIATION

Answer 20

• High energy, powerful and penetrating electromagnetic radiation
• shorter wavelength than X-rays
• Emitted by decaying unstable nucleus (cobalt 60 - unstable radioactive form of cobalt 59)
• ionisation of vital cell components and cell death –> ionisation of DNA, ionisation of cell water and produce free radicals to react with DNA
  Biological Indicator: B. Pumilus

used in: drugs in dry state i.e. freeze dried antibiotics, vaccines, oil, creams

Factors:
Amount of energy absorbed
Oxygen
Temperature
Organic substances (protective)

Question 21

Advantages of Radiation

Answer 21

• materials not wetted or heated
• operated at ambient temps (not extreme heat)
• packed and sealed container in packaging
• continuous and automated

Question 22

Disadvantages

Answer 22

• Hazardous
• chemical changes in drug
• container deterioration
• small volume, low density
• expensive, needs a plant

Question 23

Chemical Sterilisation

Answer 23

Ethylene Oxide
Formaldehyde
Glutaldehyde

MOA

• alkylating agents
• Addition of hydrocarbons to reactive groups of protein molecules
• acts on cell during DNA replication
• Biocidal, mutagenic, carcinogenic

Question 24

Ethylene Oxide

Answer 24

Flammable
Highly explosive
used with inert diluent gases as non-flammable mixture
highly diffusable through packaging
freely soluble in water
dissolve in rubber, plastic

Uses: dressings, containers, equipment, implants e.g. breast implants

Question 25

Factors affecting ethylene oxide sterilisation

Answer 25

Concentration Temperature Moisture - dissolves gas to form conc solution for optimal effect at 33% RH

Question 26

Monitoring of ethylene oxide

Answer 26

Thermocouples Humidity sensors Chemical indicators - strip changes colours on exposure Biological indicators - attest vials, spore strips, indicator organisms B. subtilis var niger

Question 27

Limitations of ethylene oxide

Answer 27

Toxicity - carcinogenic Difficult to monitor Removal of residues Time consuming

Question 28

Sterilisation by filtration

Answer 28

- liquid and gas substances are removed by filters - no killing and no growth inhibited, only physical removal ``` Use: Cleanroom air supply, LFC heat labile liquids heat labile ointments QC tests using membrane filtration method purification of water ```

Question 29

Desired properties of filters

Answer 29

``` Inert Non-pyrogenic Efficiently removes particles Good flow rate Resistant to clogging Sterilisable Reusable Do not retain drug Do not release filter components ```

Question 30

Depth Filters

Answer 30

Compacted pads of fibres with pores and channels or variable diameters and directions Eg HEPA filters Mechanism: Sieving, adsorption, retention Under vacuum or positive pressure to assist flow Arrest depends on size, charge, velocity of particles High porosity Good pore size range Good dirt handling capacity

Question 31

Depth Filters Limitations

Answer 31

- Need uniform pressure, if too high pressure, filter compress and decrease flow rate - Retain liquid in filter = loss of solvent, can not suck out liquid in filter otherwise filter will crack - loss of formulation ingredients - slow speed of sterilisation - may not be suitable for sterilisation or reuse - filter fibres may shed into filtrate

Question 32

Membrane Filters

Answer 32

``` Polymeric membranes (one layer) Hydrophilic, hydrophobic, solvent resistant, gridded High porosity Fast flow and speed Low fluid retention no shedding Minimum solute adsorption narrow pore size range Poor dirt handling capacity rapidly clogging ``` Uses: screening/ sieving, retention, all particles greater than pore size retained

Question 33

Monitoring Filters

Answer 33

Bubble point pressure test | Bacterial retention test (microbial challenge test)

Question 34

Bubble point pressure

Answer 34

- the pressure of a test gas (N2) to push liquid out of a wet membrane filter - gas bubbles appear in liquid at that pressure - gas pressure is a measure of pore diameter - smaller pore size requires higher pressure (to overcome surface tension) Pass filter integrity test if bubble pressure is >/= to recommended pressure i.e. actual pore size is = or < specified test

Question 35

Microbial Challenge/ retention test

Answer 35

Test efficiency of sterilising filter (0.22um) using a challenge organism Br. diminuta 0.3um size in high conc slow filtration then the entire filtration is cultured in broth medium, or filtered again and culture the filter Filter passes if no growth after 72 hours at 30 deg