Sterilization efficiency: Brief Introduction, Evaluation and monitoring of sterilization, Different Indicators used to show complete sterilization

1. Evaluation and In Process Monitoring of Sterilization
Procedures
Prepared By :
Ms. Pradnya Raju Thakar
MS Pharm
DY Patil institute of Pharmaceutical Education and Research, Pimpri

2. Physical Indicators
1. Moist Heat :
❑ A master process record (MPR) is prepared as part of the validation procedures for a particular autoclave and for
each specified product and load configuration
❑ Used as a reference for the process record obtained from a single thermocouple placed in a strategic part of each
load (Batch process record)
❑ MPR should be check at annual interval & whenever significant changes occur in the BPR when compared with
MPR
❑ Thermocouples can be used for the determination of the temperature inside the autoclave
❑ Sterilization is considered to be proper if a particular standard temperature is reached inside the autoclave
❑ Microprocessor controlled sterilization cycles are now a part of modern autoclaves
❑ Pressure is measured by gauges or through pressure transducers

3. 2. Dry Heat :
❑ A temperature record chart is made of each sterilization cycle and compared against a master temperature
record
❑ The temperature should be taken as the coolest part of the loaded sterilizer, further information on heat
distribution and penetration within sterilizer can be gained by the use of thermocouple place at selected site
in the chamber or injected into test packs or bottles
3. Radio Sterilization :
❑ In radiation sterilization a plastic or perspex dosimeter which gradually darkens in proportion to the
radiation it absorbs
❑ It gives an accurate measure of the radiation dose and is considered to be the best technique currently
available for the radiation sterilization process
4. Gaseous Methods :
❑ Elevated temperatures are monitored for each sterilization cycle by temperature probes and routine leak
tests are performed to ensure gas-tight seals

4. ❑ Gas concentration is measured independently of pressure rise, often by reference to the weight of gas used
❑ Pressure & humidity measurements are recorded
5. Filtration :
❑ Sterilizing filters are subjected to a bubble point pressure test
❑ This is a technique for determining the pore size of a filter, and may also be used to check the integrity of
certain types of filters.
❑ The principle of the test is that the filter is soaked in an appropriate fluid and its assembled unit is
subjected to an increasing air or nitrogen gas pressure difference
❑ The pressure difference recorded when the first bubble of gas breaks away from the filter is related to
maximum pore size
❑ When the gas pressure is further increased slowly there is general eruption of bubble over the entire surface
❑ The pressure difference here is related to the mean pore size
❑ Pressure difference below the expected value would signify a damage or faulty filter

5. ❑ Chemical monitoring of a sterilization process is based on the ability of heat, steam, sterilant gases &
ionizing radiation to alter the chemical/ physical characteristics of a variety of chemical substances
1. Browne’s Tubes :
❑ Chemical indicators for heat processes
❑ These are small sealed tubes containing a reaction mixture and an indicator
❑ Exposure to high temperature completes the reaction producing a change in the colour of the indicator
❑ These tubes are placed inside the autoclave and dry heat sterilizers along with the articles
❑ All four type tubes are red in color, which change from red through yellow brown to green, the latter colour
only developed after a specified time at the given temperature
❑ This helps in determining the proper sterilization of the articles
Chemical Indicators

6. Browne’s Tubes Method of Sterilization Temperature
(°C)
Colour of Indicator
Type I Moist Heat 126 Black spot
Type II High Vacuum moist heat 130 or more Yellow spot
Type III Dry Heat 160 Green spot
Type IV Dry heat infra red conveyer
oven
180 Blue spot
2. Witness Tubes :
❑ It consists of single crystalline substances of known melting point contained in glass tubes
❑ EX. Sulphur (115° C), Succinic anhydride (120°C), benzoic acid (121°C)
❑ A dye may be included to show more clearly that the crystals have melted
❑ Such a device only indicates that a certain temperature has been reached
❑ Exposure time can be calculated by putting the crystals in one end of an hour-glass tube
❑ The volume of the crystals and the diameter of constriction of the tube being adjusted so that the time for the transfer of
the melt is the same as that required for sterilization at the required temperature

7. 3. Heat Sensitive Tapes :
❑ It is used quantitatively in the Bowie-Dick test
❑ It determines that all air has been removed from the dressings and that subsequent steam penetration has
been even and rapid
❑ The tape is placed on suitably wrapped at the centre of a test pack
❑ All the bars on the tape should change colour to demonstrate full penetration of the steam
4. Royce Sachet :
❑ Chemical indicator for ethylene oxide sterilization
❑ It consists of a polythene sachet containing magnesium chloride , HCL and bromophenol blue indicator
❑ A given concentration-time exposure to ethylene oxide results in the formation of ethylene chlorohydrin and
a colour change from yellow to purple

8. 5. Chemical Dosimeters :
❑ Chemical indicator for radiation sterilization
❑ Chemical dosimeter acidified with cerric ammonium sulphate or cerric sulphate solution
❑ These responds to irradiation by dose change in the applied density
❑ It gives an accurate measure of the radiation dose absorbed and are considered best for controlling radiation
sterilization
❑ Qualitative indicators made of radiosensitive chemicals impregnated in plastic are also available
❑ The indicator changes from yellow to red during irradiation

9. Biological Indicators
❑ The biological indicators are the standardized bacterial spore preparations which are usually in the form of
suspension in water or culture medium
❑ or of spore dried on paper or plastic carriers, they are placed in sterilizer
❑ After the sterilization process the aqueous suspension /spores are on carriers are aseptically transferred to an
appropriate nutrient medium, which is then incubated and occasionally seen for the growth
❑ It measures the sterilization processes directly and is able to integrate all sterilization parameters
❑ The selected organism should possess high and reproducible resistance to the sterilizing agent
❑ Should be genetically stable, readily characterizable and non-pathogenic
❑ The viability of the organisms, the storage conditions before use and the incubation and culture conditions after
sterilization must be standardized for the results
❑ Organism should be resistant bacterial spores

10. Filtration sterilization :
❑ Filtration sterilization requires a different approach from biological monitoring, the test effectively
measure in the ability of a filter to produce a sterile filtrate from a culture of suitable organism S.
marcesence, a
❑ small gram negative rod shape bacterium.
❑ B. diminuta used as a biological indicator having a dimension 0.5 micrometres and 0.3 micrometre
respectively has been used for filters of 0.45 micrometre and 0.22 micrometre
❑ The extent of the passage of this organism through membrane filter is enhanced by increasing the
filtration pressure
❑ Thus successful sterile filtration depends markedly on the challenge condition
❑ Such tests are used as the part of filter manufacture characterization and quality assurance process, and
user’s initial validation procedure

11. Sterilization Process Species D-value
Autoclave at 121°C Bacillus
stearothermophilus
Clostridium
sporogenes
1.5 min
0.8 min
Dry heat at 160 °C Bacillus subtilis
var. niger
5-10 min
Ethylene oxide at 600 mg/lit.
(Temperature- 54 °C & 60 % relative
humidity)
Bacillus subtilis
var. niger
2.5 min
Ionizing Radiation Bacillus pumilus 8kGy (0.3 M rad)
Membrane filters (0.45 µm pore size) Serratia
marcescens
-
Membrane filters (0.22 µm pore size) Pseudomonas
diminuta
-
Biological indicators for monitoring sterilization processes