1. PRINCIPLES OF STERILISATION AND DISINFECTION
A Presentation By
Ms R.Venkatajothi, MSc., MPhil, PhD
Senior Lecturer
Department of Microbiology
Faculty of Medicine
AIMST UNIVERSITY

2. OBJECTIVES The objectives of this lecture are to
Define Sterilization, Disinfection and Antisepsis.
Describe the physical and chemical methods of sterilization and disinfection commonly used in the microbiology laboratory and the hospital.
Explain the importance of Quality control.

3. INTRODUCTION STERILIZATION:-
Microorganisms cause contamination, infection and decay, it becomes necessary to remove or destroy them from materials or from areas. This is the object of Sterilization.
STERILIZATION:-
Sterilization is define as the process by which an article, surface or medium is free of all living microorganisms either in the vegetative or spore state.

4. DISINFECTION:-
Disinfection means the destruction or removal of all pathogenic organisms or organisms capable of giving rise to infection. Disinfection can be done by chemicals.
ANTISEPSIS:- Reduction of or inhibition of microbes found on living tissue.
ANTISEPTICS:-
Chemical disinfectants which can be safely applied to the skin or mucous membrane and are used to prevent infection by inhibiting the growth of bacteria are called antiseptics.

5. DIFFERENCES BETWEEN ANTISEPTICS AND DISINFECTION1.
All antiseptics are disinfectants.
All disinfectants are not antiseptics. 2.
It can be apply on the live tissues.
It can be applied on inanimate object. 3.
Eg:- Skin or mucous membrane.
Eg:- Surface, lab working tables, floor or material.

6. METHODS OF STERILISATION AND DISINFECTION
The following methods are commonly used in the microbiology laboratory and hospital.
(I) PHYSICAL METHOD
Sunlight, Drying,
Dry heat: Red heat, Flaming, Incineration, Hot air oven.
Moist heat: Pasteurization, Boiling, Autoclave (Steam under pressure).
Filtration and Radiation etc.
(II) CHEMICAL METHOD
Alcohols, Aldehydes, Halogens (Iodine, Chlorine etc), Phenols, Gases and Surface - active agents (soap, detergent) etc.

7. HOT AIR OVEN
This is the most widely used method of Sterilization by Dry heat.
A holding period of 160°C for one hour is used to sterilize glassware, forceps, all-glass syringes, swabs etc.
Glassware should be perfectly dry before being placed in the oven. Test tubes and flasks should be wrapped in paper.
The material should arranged so as to allow free circulation of air in between the objects.

8. Hot air oven continued…..
It should not be overloaded.
It must be fitted with a fan to ensure even distribution of air.
The oven is usually heated by electricity with heating elements in the wall of the chamber.
The oven must be allowed to cool slowly for about two hours before the door is opened, since the glassware may crack due to sudden or uneven cooling.

9. HOT AIR OVEN

10. STERILIZATION BY MOIST HEAT
Moist Heat is divided into 3 methods;
1) Temperature bellow 100°C
Eg: Pasteurisation of milk.
2) Temperature at 100°C
Eg: Boiling of water.
3) Temperature above 100°C (Steam under pressure)
Eg: Autoclave.

11. AUTOCLAVE
In its simplest form, the laboratory autoclave consists of a vertical or horizontal cylinder of gunmetal or stainless steel.
The lid or door is made airtight.
The autoclave has on its lid or upper side a discharge tap for air and a safety valve that can be set to blow off at any desired pressure.
Heating is by gas or electricity.
Sufficient water is put in the cylinder, the material to be sterilized is placed on the tray and the autoclave is heated.
The lid is screwed tight.

12. Autoclave continued …..
The steam pressure rises inside and when it reaches the desired set level, the safety valve opens and the excess steam escapes.
From this point, the holding period is calculated. The holding period is 121° C at 15 pound pressure for 15 minutes.
When the holding period is over, the heater is turned off and the autoclave allowed to cool till the pressure inside is equal to the atmospheric pressure.
The discharge tap is opened slowly and air is let into the autoclave.
The domestic pressure cooker serves as a miniature autoclave and may be used for sterilizing small articles in clinics.

13. AUTOCLAVE

14. FILTRATION Membrane filters
Membrane filters are widely used which is made of cellulose esters or other polymers. Their average pore diameters (APD) is 0.22 mm size.
Filtration helps to remove bacteria from liquids such as sera and solutions of sugars or antibiotics used for preparation of culture media.
Membrane filters

15. RADIATION Applications:-
Two types of radiation are used for Sterilization – Nonionizing & Ionizing.
IONISING RADIATION
NONIONISING RADIATION
Infrared and Ultraviolet rays are of the nonionizing low energy type.
Applications:-
Clean surfaces like inoculation hoods, microbiology & other laboratories, hospital wards and operation theatres etc.
X-rays, Gamma rays & Cosmic rays are highly lethal to all cells including bacteria.
They have very high penetrative power. They damage DNA by various mechanisms.
Applications:-
Commercial plants use gamma radiation for sterilization of disposable items such as plastics syringes, swabs & animal feeds etc.

16. (II) CHEMICAL AGENTS An ideal antiseptic or disinfectant should be:-
Several chemical agents are used as antiseptics and disinfectants used in the microbiology laboratory and hospital. However little is known about the mechanism of action of many of these agents.
An ideal antiseptic or disinfectant should be:-
Have a wide spectrum of activity and must be effective against all microorganisms, that is, bacteria including spores, viruses, protozoa and fungi;
Have speedy action; Have high penetrating power; Be stable.
Not corrode metals; Not cause local irritation or sensitization;
Not interfere with healing; Not be toxic if absorbed into circulation;
Be cheap and easily available; Be safe and easy to use.

17. ALCOHOLS
Ethyl alcohol (ethanol), Isopropyl alcohol and Methyl alcohol are the most frequently used.
They are used as Disinfectants & antiseptics. Their mode of action is denaturing bacterial proteins.
Methyl alcohol is effective against fungal spores and is used for treating cabinets and incubators affected by them.
ALDEHYDES
Formaldehyde is active against the amino group in protein molecule.
It is used to preserve anatomical specimens.
Surfaces which have disinfected by this agent may give off an irritant vapour for some time after disinfection.

18. HALOGENS Iodine is widely used as a skin disinfectant (antiseptic).
It is active against bacteria, fungi and viruses etc. For eg: Betadine.
CHLORINE
Chlorine is used as disinfectants in microbiology laboratories, hospital, water supplies & swimming pools etc.
Chlorine is commonly used as hypochlorites.
Chlorine and hypochlorites are markedly bactericidal.
They have a wide spectrum of action against viruses.

19. PHENOL
Phenol (carbolic acid) is a powerful and good general disinfectants.
Phenol is widely used as disinfectants for various purposes in microbiology laboratories & hospitals. Lysol and cresol are active against a wide range of organisms.
GASES
FORMALDEHYDE GAS
This is widely employed for fumigation of operation theatres and other rooms. After sealing the windows and other outlets, formaldehyde gas is generated and left unopened for 48 hours.

20. Commonly used Chemical Agents in Microbiology laboratory and Hospital
ALCOHOL FORMALDEHYDE
IODINE PHENOL CHLORINE
DETERGENTS
LYSOL

21. IMPORTANCE OF QUALITY CONTROL
Quality control is defined as
a process to identify errors
in a procedure.
This is a reactive process where
errors have already occurred.
Quality control is a measure how
well the measurement
system reproduces the same
result over time and under
varying operating conditions.

22. Importance of quality control continued….
Without quality control, errors in a clinical setting can lead to patient mis-diagnosis, delays in treatment or administration of the wrong treatment and increased costs associated with retesting.
Quality assurance schemes are vital both in a preclinical, clinical settings and in clinical trials.
Quality assurance schemes are essentially procedures designed to ensure that a study being performed is compliant and that data being generated is accurate.
This ensures the reliability of test results, to give the best patient care.

23. Importance of quality control continued….
Example of Quality control:-
Sterilization control for Hot air oven
The spores of Clostridium tetani are used as test of dry heat efficiency.
Paper strips impregnated with spores are placed in envelopes and inserted into suitable packs.
After sterilization, the strips are removed and inoculated into thioglycollate or cooked meat media and incubated for sterility test under strict anaerobic conditions for five days at 37°C. If there is no growth in the culture media, the hot air oven works properly.

24. LEARNING OUTCOMES Define Sterilization, Disinfection and Antisepsis.
At the end of the lecture, student should be able to:
Define Sterilization, Disinfection and Antisepsis.
Describe the various methods of physical and chemical methods of sterilization.
Select the ideal method of sterilization for different types of culture media, laboratory equipments and surgical instruments.
Explain with suitable examples the importance of quality control for sterilization and disinfection.

25. BOOKS FOR REFERENCES
Text book of Microbiology by Ananthanarayan and Paniker’s.
A Text book of Microbiology by P.Chakraborty.
Medical Microbiology by Jawetz, Melnick, & Adelberg's.
Medical Microbiology by Murray.