sterilization / autoclave

STERILIATION / AUTOCLAVE

Sterilization: is the process used to make the item free from any viable organism.

Different type of sterilization process:

·         Moist heat sterilization (steam sterilization):

Mechanism of action (M/A) – Irreversible denaturation and coagulation of enzymes and structural protein.

Items to be sterilized by this method – Thermostable aqueous solutions, glassware, plastic wares, garments and rubber articles.

·         Dry heat sterilization:

M/A – Oxidation of cell constituents

Uses – glass, metal objects thermostable powders and fatty substances.

·         Other heating sterilization methods:

Flaming = Expose the item to flame, Incineration = Burn the item to ash,

Tyndalization – In this method items are boiled for certain time (̴ 20min ) then cool and incubate at room temperature, This step is repeated for 3 to 4 times.

Glass bead sterilization – In this glass bead heat at 250 ͦ c and then item doused in it). 

·         Filtration:

M/A – Remove live organism by filtration mechanism

Uses – Thermolabile solution, Media and buffer filtration

·         Exposure to ionizing radiation:

M/A – Ionizing the molecules in organism, mutation in DNA, radiations create free hydrogen radicals, hydroxyl radicals and some peroxides, which cause different kinds of intracellular damage

Uses – Sterilization of filter, cell bag/buffer bag, thermolabile drugs

·   Gaseous sterilization (Ethylene oxide sterilization, formaldehyde, glutaraldehyde, propylene oxide.):

M/A – Alkylation reaction

Uses - surgical instruments and gloves, plastic syringes, disposable needles, tubing sets, and dialysis units.

Basic terminologies:

D value (Decimal reduction value): It is the time in minute required at specific temperature to reduce the microorganism by factor of 10 or one log reduction.

Z value (Thermal death time): It is the degree of temperature changes necessary to change the D value by factor of 10 or one log reduction.

To find out Z value, note down the D value with different temperature and plot the graph D value Vs Temperature

(log1000 = 3, log100 = 2, log10 = 1, log1 = 0)

In above case Z value is 20 ͦ c.

F₀ Value (Lethality factor): F₀ Value is the time in minute required to kill specified number of microorganism at 121.1 ͦ c with Z value of 10 ͦ c. (1 F₀ is equivalent to 1 minute at 121.1 ͦ c).

Formula for F₀ calculation: F₀ = Σ10^(T-121.1)/Z Δt

Σ = sum of all recorded data from start to end time of sterilization hold period.

T = Temperature at specific time

Z = 10 ͦ c

Δt = Time interval of data recording (if data record at every 4sec than Δt = 4/60)
Minimum F0 Value required for process is depends on the D value of the bioburden.
F0 = D121 × (log A - logB)
A = microbial count per container
B= maximum acceptable probability of survival

F Value: F Value is the time in minute required to kill the specified number of microorganism with a specified Z value at a specific temperature. 

SAL (Sterility assurance level): is the level of microbial inactivation. eg. SAL 10^-6 means probability of viable microorganism being present on a sterilized item is one in one million post sterilization.

Overkill approach: is the sterilization approach with worst-case condition.

Autoclave (steam sterilizer): Is the equipment use high pressure and temperature to sterilize the items.

Working Principle of Autoclave:

Based on type of load cycles are design with and without vacuum. For empty load vacuum cycle is design in which pre and post vacuum incorporate. Pre vacuum play role to remove air with subsequent steam pulse, follow by heat up period with steam to achieve desire temperature and pressure. Post vacuum is used to remove steam condensate. For liquid load non vacuum cycle is design, where air remove by gravity displacement by steam and desired temperature and pressure will achieve.

Methods to achieve air removal

1)      Downward displacement / gravity type

As steam is less dence than air hence steam covered the upper area and force the air downward and compress the air at the bottom and forcing it out through the drain.

2)      Steam pulsing

Air dilution by using a series of pulses in which chamber is alternately pressurized and depressurized to near atmospheric pressure.

3)      Vacuum pumps

Vacuum pump to suck air or air/steam mixture from the chamber

DRY HEAT AND MOIST HEAT

Steam having less than 3% humidity called dry steam/superheated steam (reduce energy transferability) while steam having more than 3% humidity called wet steam.

All kind of loads are divided in three types and autoclave cycle designed based on that:

1)      Hard goods (vacuum) – suitable for the items, which are easily, sterilize. Air removal from these items are not much difficult so single pre vacuum pulse is enough to remove air.

eg. Many types of glass wares and plastic wares, large diameter piping

2)      Wrapped goods (vacuum) – Utilize for the items, which are difficult to sterilize. Air removal from these items require more pre vacuum pulses.

eg. Gown, long lengths of tubing, tank/vessel with small inlet outlet ports, vent filter

3)      Liquids (Non-vacuum): Suitable for the items that contain liquid. Here vacuum is not useful as vacuum pull out the liquid from the items so in this case gravity displacement principle used where air is removed by pulsing the steam from the top of the chamber.

eg. Media cycle

Load configuration:

1)      Fixed load/Fixed position: In this situation all of the load items as well as the position of the items are fixed from run to run, we can’t change the position of items and number of items.

2)Fixed load/variable position: In this situation load items are fixed but the position of these items can be varied.

3) Variable load/variable position: In this situation, load items as well as the position of these items can be varied.    

Autoclave validation

General procedure for autoclave validation

All measuring units used in validation exercise are calibrated.

Carried out pre and post calibration of the external temperature sensor to be used for autoclave validation and the variation shall not be more than 0.5 ͦ c. Chemical indicator (sterilized tap) and biological indicator (Geobacillus stearothermophillus) are used to check the efficiency of sterilization cycle, Biological indicator fix with the temperature sensor to the innermost part of item.

Different cycle to be carried out for autoclave validation

1)      Vacuum leak test (VLT): VLT is use to check the integrity of chamber. VLT is performed daily before any sterilization cycle, During validation VLT is performed pre and post insertion of external temperature sensor as well as daily basis.

2)      Bowie dick test for steam penetration: Objective of this test is to ensure vacuum pulse applied the sterilization hold period are sufficient to remove the entrapped air so as to facilitate rapid and even steam penetration into all parts of the load and maintaining these condition for the specified holding time.

3)      Empty chamber heat distribution (3 cycles): Objective of this test is to check the capability of the sterilizer, whether it can achieve 121-124 ͦ c temperature at 1.1-1.3 kg/cm² steam pressure during hold time. The position where temperature could not achieve is called cold spot.

4)      Loaded chamber heat distribution and penetration (3 cycles):  Heat distribution is performed to determine the effect of any loading pattern on the temperature distribution within the chamber. Objective of heat penetration test is to ensure that the steam is sufficiently penetrating into the innermost most portion of the load subjected for sterilization to achieve 121-124 ͦ c temperature at 1.1-1.3 kg/cm² steam pressure during hold time.

How to design cycle time:

Suppose the objects having bioburden 75CFU, D value 2 min/log, at 121.1 ͦ c, required SAL = 10^-6

Then the bioburden of object= log 75 = 1.88

Log reduction required = 1.88log + 6log (as SAL = 10^-6) = 7.88log

Ideal cycle time at 121.1 ͦ c = 7.88log * 2 (Dvalue) =15.76 minutes