2. 2
What is Sterilization and Why Required ?
Sterilization is the process by which all living micro-organisms both
pathogenic and non-pathogenic including spores are killed or eliminated.
“sterilization is essential concept in the preparation of sterile pharmaceutical
products”
Sterilization is required to – 1. Prevent the growth of disease
2. Prevent the spread of disease
3.Prevent double surgeries
4. 4
Difference between DHS and MHS
DHS (Dry Heat Sterilization
Tunnel, Hot air Oven, Incineration, Direct Flaming,
Red Heat are some methods for DHS.
ADVANTAGES :-
1. Suitable for moisture labile articles or substances.
2. Easy penetration of heat.
3. Effective to kill PYROGEN also. (Depyrogenation)
4. Non corrosive for metals and sharp instruments.
5. Compatible for SS articles, glass wares, vials,
ampoules, thermostable substances etc.
DISADVANTAGES :-
6. Time consuming
7. Not suitable for thermolabile substances, porous
load
MHS (Moist Heat Sterilization
AUTOCLAVE
ADVANTAGES:-
1. Effective to kill the vegetative and spores of
bacteria. Less time consuming.
2. More effective due to LATENT HEAT OF
CONDENSATION of steam.
3. Effective for porous load due to penetration
of steam.
DISADVANTAGES:-
4. Not effective for pyrogen.
5. Not suitable for hygroscopic substances or
articles.
6. 6
Autoclaving is a process in which saturated steam under pressure is allowed to
Penetrate through materials for 15 minutes at temperature 121ºC.
T- 121.1
F○ = ∆t ∑ 10 Z
F○ - It is defined as thermal lethality time required to eliminate all microorganisms present in a sample
, by exposing them to a temperature of 121.1ºC and it is expressed in minutes.
∆t – The time interval between two temperature readings
T – The temperature at time t of the product under sterilization
Z – Increase in temperature required to effect a 10 fold variation of D value.
D Value - Time required (t) at a specified temperature to reduce the microbial population drom 100
% to 10 % (1 Log reduction)
121.1- 121.1
F○ = 30 ∑ 10 10 = 30 Minutes
110- 121.1
F○ = 30 ∑ 10 10 = 2.33 Minutes
Now 30 minutes sterilization at 110ºC is lethality equivalent to 2.33 minutes of sterilization at 121.1ºC
7. 7
Why autoclave hold time 30 minutes at 121.4 ºC, If guideline says only 15 minutes ?
Generally we are taking spores of GEOBACILLUS STEAROTHERMOPHILLUS at
concentration of 106
, Manufacturer giving the D value 2.5 Minutes.
LETHAL RATE = D value X Log reduction required
= 2.5 X 6 = 15 Minutes (Approx.)
Now think after 6 log reduction possibility of <1 spore is in substances that’s why we are taking
additional 15 minutes i.e. 30 minutes and that is known as overkill approach.
9. 9
Depyrogenation via tunnel is a process in which hot air is used, passed through
HEPA and having precise temperature control to destroy the pyrogen by
Convection. Tunnel is desined to create an ISO 5 or class A environment.
Tunnel having different zone with specified functionality –
DRYING ZONE - This zone cleans , dries and preheats the glassware.
STERILIZATION ZONE – This zone for depyrogenation
Cooling and Stabilization Zone - Lower the temperature prior to exposing in filling area.
T- 250
Fh = ∆t ∑ 10 Z
T = Assumes 250ºC here
Z = 46.4ºC Approx. 50
300- 250
Fh = 3 ∑ 10 50 = 30 Minutes
Here we again taking over kill approach by keeping the temperature more than 300ºC and
exposure time higher.
10. 10
What is disinfectant, uses, types, mode of actions, validation methods
“Disinfection is the process of destruction or removal of microorganism
and reducing them to level not harmful to health.”
If the object is inanimate (Lifeless), such as working area, dishes, benches etc. the
chemical agent known as disinfectant while object is animate (Living) such as
human body tissue, the chemical is known as antiseptic.
Requirements of disinfectant –
1. Broad spectrum (fungicidal, bactericidal, sporicidal, virocidal etc.}
2. Non toxic
3. Fast acting , odorless, surface compatibility, economical, easy to use
4. Solubility and miscibility, stable on storage
Classification of disinfectant –
5. Acids and alkalis 2. Halogens 3. Heavy Metals 3. Phenols & it’s derivatives
4. Alcohols 5. Aldehydes 6. Quaternary ammonium 7. Detergents & soaps
11. 11
Mode of Action of Disinfectant.
1. Alteration of membrane permeability
2. Damage to protein
3. Rupture of cell membrane
4. Damage to nucleic acid
5. Interfere with metabolic pathway
Factors affecting disinfection
6. Concentration of disinfectant i.e. 70 % IPA
7. Temperature
8. Time of contact
9. pH of environment
10. Surface tension
11. Formulation of disinfectant
12. Chemical structure of disinfectant 8. Types and no. of microorganism present
9. Environmental condition
12. 12
Why using 70% IPA instead 100 % ?
It is the better penetration of 70 % IPA through cell wall of bacteria in comparison to
100 % IPA.
We can understand it by an example –
If we boil the egg at higher temperature then the outer surface of egg will cook while inner part
will not cook while at ambient temperature all the egg will beter cook.
As IPA is volatile in nature hence 70 % IPA is less volatile hence provide more contact time.
13. 13
Validation of Disinfectant (Disinfectant Efficacy Challenge)
1. Tube dilution and agar plate method
2. Cup plate method or cylinder plate method
3. Ditch plate method
4. Gradient plate technique
5. Phenol coefficient method (Rideal – Walker test)
14. 14
Sterilization by Filtration method
Filtration is preferred method of sterilizing heat sensitive liquid and gases with out
exposure to denaturing heat.
Rather than destroying contaminating microorganisms, it simply removes them. It is method of choice for sterilizing
antibiotic solution, toxic chemicals, radioisotopes, vaccine etc. which are all heat sensitive.
WORKING PRINCIPLE :-
1. Filters working by physically trapping particles larger than the pore size by retaining.
2. Smaller particles by electrostatic attraction of the particles to the filters.
3. Filtration of liquids is accomplished by either pulling the solution through the filter via vacuum or pressurizing
the liquids through filter by imposing positive pressure.
4. Filtration of air by using HEPA filters to remove organisms larger than 0.3 micron.
TYPES OF FILTER:-
5. Depth Filter
6. HEPA filter
7. Membrane Filters
15. 15
Beside more advantages and preferred choice for sterilization of heat sensitive
Liquid and suitable method for air sterilization, filtration process having some
Disadvantages –
1. Filter Damage
2. Clogged Pores
3. Material Loss
4. Vacuum Damage
5. Separation of similar size particles
6. Required high differential pressure
7. High Cost
8. Unable to separate endotoxin completely
Due to above disadvantages some major regulatory body does not cleared filtration to the perfect sterilization method
and suggesting for heat treatment or terminal sterilization.
16. 16
ASTM ( American Society for Testing Method) and ATCC (American Type Culture
Collection) both giving different standards to validate and test the filters.
There are 8 basic elements should be considered during filter validation –
1. Integrity Testing – BPT, WIT, Diffusion Test, Forward flow test etc.
2. Fit for use
3. Sterilization
4. Stability
5. Binding
6. Compatibility
7. Extractable / Leachable
8. Retention - Brevundimonas diminuta is commonly used as challenge microorganism to retain
a minimum challenge of 107
bacteria per cm2
of filter area.
