Evaluation and In Process Monitoring of

Sterilization Procedures

Prepared By :
Ms. Shubhangi Vitthal Shekade
DY Patil Institute of Pharmaceutical Sciences and Research, Pimpri
 Physical
1. Moist Heat :
Indicators
 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
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
 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 andits 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
 Chemical
Indicators
 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
 Browne’s Tubes Method of Sterilization Temperature Colour of Indicator
(°C)
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 180 Blue spot

oven

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
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
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
 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
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
 Biological indicators for monitoring sterilization
processes
Sterilization Process Species D-value
Autoclave at 121°C Bacillus 1.5 min
stearothermophilu
s
0.8 min
Clostridium
sporogenes
Dry heat at 160 °C Bacillus subtilis 5-10 min
var. niger
Ethylene oxide at 600 mg/lit. Bacillus subtilis 2.5 min
(Temperature- 54 °C & 60 % relative var. niger
humidity)

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