PHARMACEUTICAL MICROBIOLOGY
(BP303T)
Unit-Ii
Part-3
Evaluation of the efficiency of sterilization methods.
Sterility indicators
Name: Mrs. Pooja Deepak Bhandare
Assistant Professor
G H RAISONI UNIVERSITY
SCHOOL OF PHARMACY

Content:
•Evaluation of the efficiency of sterilization
methods.
•Sterility indicators
12/8/2022
Sample Footer Text 2

Sterility criteria
• Bioburden is normally defined as the number of bacteria living on
a surface that has not been sterilized.
• The term is most often used in the context of bioburden testing, also
known as microbial limit testing, which is performed on
pharmaceutical products and medical products for quality control
purposes. •
• Time and temperature relationship for steam sterilization to ensure
that a large number of the most resistant pathogens would be killed.

Sensitivity of microorganisms
•Microorganisms shows resistance to heat, radiation and
chemicals. •
•The vegetative forms of bacteria and fungi are most
sensitive. •
•The thermophilic bacteria, smaller viruses and mould
spores are killed at temperature between 70 to 90°C,
while bacteria spores may be destroyed at 90 to 120°C
temperatures.

Death rate or Survivor curve
• It is determined by assessing the reduction in the number of
viable microorganisms resulting from contact with a given
destructive force.
• This can be represented graphically with a ‘survivor curve’
drawn from plot of the log of the fraction of survivor against
the exposure time or dose.

D- Value or Decimal reduction time
• Time in minutes at any
defined temperature to destroy
90% viable microorganisms is
called D- value

Z- value or Thermal reduction time
• The slope of the TDT curve is defined as “Z” which is equal to
the number of degrees on the temperature scale when the curve
traverse one log cycle.
• Z is the change in temperature necessary to cause a ten fold
change in D-value.
• The value of Z for C. botulinum is 10˚C
• Every 10˚C change in temperature there is a ten fold change
In its death rate.
• B. subtilis has Z value of 6.5˚

f- value
• Used in the food industry, also called “Unit of Lethality” has been
devised called as F- Value
• This can be defined as the equivalent in minutes of 121˚C of all heat
consider with respect to its capacity to destroy spores or vegetative cell of
a particular organisms
• The F value for a process is the number of minutes required to kill a
known population of microorganisms in a given food under specified
conditions.
• This value is also used to calculate the probable number of bacterias
remaining after the process in food.

Q10 Value or Temperature Coefficient
• It is defined as the increase in rate of reaction or killing rate of a
sterilization process brought after increasing the temp. by 10°C.

InactivationFactor:
• It is the degree to which the viable population of organisms is
reduced by applying a sterilization process.
• It is obtained by dividing the initial viable count by final
viable count.
• Inactivation factor (IF) = 10 t/D
• Where, t = exposure time in minutes ,
• D = Decimal reduction time for the same temperature and
conditions.

STERILITY INDICATORS
• Changing appearances in colour or pattern, the sterilization indicators visually
show if cleaning conditions are passing or procedures have been completed.
• • Eliminating any confusion or possibility instruments will not be sterile,
indicators are used routinely in clinical and research environments where
contamination elimination is crucial.
• With the temperature resistance required to endure the purification, the
sterilization indicators are available in different forms such as tapes,
ampoules, and sticks.
• Monitoring of sterilization process can be achieved by the use of
1. Physical Indicators
2. Chemical Indicators
3. Biological Indicators

1. Physical Indicators
•Monitoring physical indicators involves observing the gauges
or displays on the sterilizer and recording the time,
temperature, and pressure associated with each sterilization
cycle for each load.
• Some sterilizers have recording devices that print out these
parameters.
•Correct readings do not guaranty sterilization, but incorrect
readings can be the first indication of a problem with the
sterilization cycle and suggest the load may not be sterile.

i) Moist heat Indicator:
A Master Process Record (MPR) is prepared as part of the validation procedure for
a particular autoclave
The MPR should be checked at annual intervals and whenever significant changes
occur in the BPR(Batch Production Records)when compared with the MPR.
Microprocessor-controlled sterilization cycles are now a part of modern
autoclaves.
ii) Dry heat:
in dry sterilization processes, a temperature record chart is made of each
sterilization cycle and is compared against a master temperature record.
iii) Radio sterilization:
A plastic dosimeter gives an accurate measure of the radiation does absorbed and
is considered to be the best technique currently available for the radio sterilization
process.

iv) Gaseous methods:
For gaseous sterilization procedures, elevated temperatures are monitored
for each sterilization cycle by temperature probes and routine leak test are
performed to ensure gas-tight seals. Gas concentration is measured
independently of pressure rise, often by reference to the weight of gas used.
Pressure and humidity measurements are record.
v) Filtration:
Bubble point pressure test is a technique employed for determining the pore
size of filters and may also be used to check the integrity of certain types of
filter devices immediately after use. The principle of the is that the filter is
soaked in an appropriate fluid and pressure is applied to the filter. The
pressure difference when the first bubble of air breaks away from the filter
is equivalent to the maximum pore size. When the air pressure is further
increased slowly, there is general eruption of bubbles over the entire
surface. The pressure diffference is equivalent to the mean pore size.

2.CHEMICAL INDICATORS
•Chemical indicators use sensitive chemicals to assess critical
variables (e.g., time, temperature, or steam saturation) during
a sterilization cycle.
•They are applied either to the outside or placed on the inside
of each instrument unit (e.g., packs, peel pouches, containers,
etc…
•They do not prove that sterilization has been achieved, but
they can provide an early indication of a problem and where
in the sterilization process the problem might exist.

I) Browne’s tubes:
Most commonly used chemical indicator for heat process
Contains small sealed coloured tubes having a reaction mixture and an indicator
Expose to high temperature resulting in the change of colour of the
indicators.(Red to green)
II) WITTNESS TUBES
• Consist of single crystalline substances of known melting point contained in
glass tubes
Ex: Sulphur(115°C), Succinic anhydride(120°C), Benzoic acid(121°C), etc.
• A dye may be included to show more clearly that the crystals have melted.
• Indicates that a certain temperature has been reached.

III)Heat Sensitive Tape:
• It is an adhesive tape used in autoclaving to indicate a specific
temperature.
• Autoclave tape works by changing color after exposure to
temperatures commonly used in sterilization processes, typically
121°C in a steam autoclave.
• Small strips of the tape are applied to the items before they are placed
into the autoclave.
• The tape is similar to masking tape but slightly more adhesive, to
allow it to adhere under the hot, moist conditions of the autoclave.
• One such tape has diagonal markings containing an ink which changes
colour (usually beige to black) upon heating.

IV) Royce Sachet:
• It's a chemical indicator used for “Ethylene Oxide Sterilization”.
• Its a polyethylene sachet containing magnesium chloride and bromophenol blue
indicator.
• Ethylene oxide penetrates polyethylene bags and reacts with the contents of the
sachet.
• At a given concentration - time exposure the color of the mixture changes from
yellow to purple due to formation of “Ethylene chlorohydrin”.
V) Chemical Dosimeter:
• It is the best technique available to measure radiation dose absorbed during
sterilization by radiation.
• The radio sensitive materials are impregnated in a plastic container and changes
color from yellow to red on exposure to the radiations.

3.BIOLOGICAL INDICATORS
•Biological indicators (BIs), or spore tests, assess directly the
killing of known highly resistant, non pathogenic bacterial
spores.
•Geobacillus stearothermophilus (G. stearothermophilus) spores
test steam and unsaturated chemical vapor sterilizers.
•Bacillus atrophaeus (B. atrophaeus) spores test dry heat
sterilizers.
•Bacterial spores in the test products are more resistant and are
present in greater numbers than common microbial
contaminants found on patient-care items.

Sterilization Process Species Used
Autoclaving at 121°C Bacillus stearothermophilus
Clostridium sporogenes
Dry heat at 160°C Bacillus subtilis var.niger
Ethylene Oxide Bacillus subtilis var.niger
Ionizing radiations Bacillus pumilus
Membrane filter (0.45 micrometer) Serratia marcescens
Membrane filter (0.22 micrometer) Pseudomonas diminuta

THANK YOU