This document discusses various chemical methods used for sterilization. It defines key terms like sterilization, disinfection, and antisepsis. It describes ideal properties of chemical disinfectants and their modes of action. Several chemical agents are explained in detail, including their spectrum of activity, concentrations used, limitations, and common applications. Physical methods of sterilization are also briefly mentioned.

1. CHEMICAL METHODS
PRAVEG GUPTA

3. WHY NEEDED?
 Microorganisms are ubiquitous.
 Since they cause
contamination, infection and
decay, it becomes necessary to
remove or destroy them from
materials or from areas.
 This is the object of sterilization.

4.  The process of sterilization is used
 in microbiology for preventing contamination by
extraneous organisms,
 in surgery for maintaining asepsis,
 in food and drug manufacture for ensuring safety from
contaminating organisms, and
 in many other situations.
 The methods of sterilization employed depend on
 the purpose for which it is carried out,
 the material which has to be sterilized and
 the nature of the microorganisms that are to be removed or
destroyed.

5. DEFINITIONS
 Sterilization – process by which an article, surface or
medium is freed of all living microorganisms either in the
vegetative or spore state.
 Disinfection – destruction or removal of all pathogenic
organisms. (sanitization w.r.t. Food processing and
catering)

6.  Antisepsis – prevention of infection, usually by inhibiting
the growth of bacteria in wounds or tissues.
 Antiseptics – chemical disinfectants which can be safely
applied to skin or mucous membrane and are used to
prevent infection by inhibiting growth of bacteria.
 Bactericidal – agent which can kill bacteria.
 Bacteriostatic – agent which only prevents multiplication
of bacteria.

7. PHYSICAL AGENTS
 Sunlight
 Drying
 Dry heat – flaming, incineration, hot air.
 Moist heat – pasteurization, boiling, steam
under normal pressure, steam under pressure.
 Filtration – candles, asbestos
pads, membranes.
 Radiation
 Ultrasonic and sonic vibrations.

8. CHEMICAL AGENTS
 Alcohols – ethyl, isopropyl, trichlorobutanol
 Aldehydes – formaldehyde, glutaraldehyde
 Dyes
 Halogens
 Phenols
 Surface active agents
 Metallic salts
 Gases – ethylene oxide, formaldehyde, betapropiolactone.

9. IDEAL CHEMICAL DISNIFECTANT
 An ideal chemical antiseptic or disinfectant should have the
following properties:
 Wide spectrum of activity
 Active in the presence of organic matter
 Effective in acid as well as alkaline media
 Speedy action
 High penetrating power
 Stable
 Compatible with other antiseptics and disinfectants
 Not corrode metals
 Not cause local irritation or sensitization
 Not interfere with healing
 Not be toxic if absorbed into circulation
 Cheap and easily available
 Safe and easy to use

10. MODES OF ACTION OF CHEMICAL DISINFECTANTS
 Protein coagulation
 Disruption of cell membrane resulting in
exposure, damage or loss of the contents.
 Removal of free sulphydryl groups essential for the
functioning of the enzymes.
 Substrate competition – a compound resembling the
essential substrate of the enzyme diverts or misleads the
enzymes necessary for the metabolism of the cell and
causes cell death.

11. ALCOHOLS
 Ethanol (80% v/v ethyl alcohol)
or 2-propanol (60-70% v/v iso-
propyl alcohol) solutions are
used to disinfect skin and
decontaminate clean
surfaces.
 Spectrum: Effective against
fungi, vegetative
bacteria, Mycobacterium species
and some lipid-containing
viruses.

12. Limitation: Not effective against spores.
Concentration: Most effective at 70% in
water.
Caution: Do not use near flames due to
flammability.
May swell rubber or harden plastics.

14. ALDEHYDES
 FORMALDELYDE: Precautions are required when
handling formaldehyde
 Formalin is 37% w/v formaldehyde gas in water.
 Spectrum: Active against most microorganisms.
 Bactericidal, sporicidal, virucidal.
 13% v/v formalin is a good decontaminant (but has an
irritating odour).
 8% v/v formalin in 80% v/v alcohol is effective against
vegetative bacteria, spores and viruses.
 Does not corrode stainless steel.

15.  Uses:
 Formaldehyde is used to preserve anatomical
specimens, and for destroying anthrax spores in hair
and wool.
 10% formalin containing 0.5% sodium tetraborate is used
to sterilize clean metal instruments.
 Formaldehyde gas is used to sterilize instruments, heat
sensitive catheters and for fumigation of wards, sick
rooms and laboratories.
 It can also be used for clothing, bedding, furniture and
books.
 To disinfect equipment such as centrifuges or biosafety
cabinets.

16. Metal instruments, biosafety
cabinets, clothing, bedding, furnitur
e and books

17. FUMIGATION TECHNIQUE
 After sealing the windows and other outlets, formaldehyde
gas is generated by adding 250g of KMnO4 to 500 ml
formalin for every 1000 cu.ft of room volume.
 Caution: The reaction produces considerable heat, and so
heat resistant vessels should be used.
 After starting generation of formaldehyde vapour, the
doors should be sealed and left unopened for 48 hours.
 Caution: Formaldehyde can react with free chlorine to
produce toxic gas. Remove hypochlorite solutions and
hydrochloric acid from spaces to be decontaminated.
 Irritant vapours are released hence Neutralise with
ammonia following decontamination.

18. FUMIGATION

19. GLUTARALDEHYDE:
 Concentration: Glutaraldehyde is commercially available
as 2% w/v aqueous solution which must be made alkaline
to "activate" (e.g. by addition of 0.3% sodium bicarbonate).
 A 2% glutaraldehyde solution, for at least 10 hours, can be
used to sterilize heat labile items.
 Spectrum: Active against vegetative bacteria, spores, fungi
and many viruses.
 Note: Also available in stable glycocomplexed form which
does not require addition of alkaline buffer.

20. Use: for instruments such as
cystoscopes, bronchoscopes, corrugated rubber anesthetic
tubes, face masks, endotracheal tubes, metal
instruments, polythene tubing.

21.  Caution: Glutaraldehyde is known to cause dermatitis and
asthma. Less irritating than formaldehyde.
 Glutaraldehyde should not be used in an area with
little or no ventilation.
 Eye protection, a plastic apron, and gloves must be
worn
 Should be stored away from heat sources and in
containers with close-fitting lids.
 The length of time that glutaraldehyde solutions can
be used varies but they are usually good for up to 14
days.
 Solutions should be replaced any time they become
cloudy.

22. Glutaraldehyde
Advantages
 Numerous use studies published
 Relatively inexpensive
 Excellent materials compatibility
Disadvantages:
 Pungent and irritating odor
 Relatively slow mycobactericidal activity
 Coagulate blood and fix tissues to surfaces

23. CHLORINE COMPOUNDS
 Generally used in the form of sodium hypochlorite.
 Effective against a wide variety of microorganisms
(vegetative bacteria and viruses). Preferred disinfectant
for HIV and hepatitis viruses.
 Concentration: Use at 0.1% as a general disinfectant.
 Effective between a pH range of 6-8.

24.  Limitations:
 Less suitable in the presence of organic matter (such as
blood). Concentration must be increased to retain action
(0.5%).
 Strength decreases on standing (make fresh solutions
daily).
 High concentrations corrode metal surfaces, and
bleach and damage fabrics.

25.  HYPOCHLORITE:
 These are inexpensive, broad spectrum chlorine releasing
disinfectants of choice against viruses, including hepatitis
B virus.
 Aqueous solution of sodium hypochlorite (5.25%) is called
household bleach.
 It is used in a concentration of 0.2-1% depending upon the
circumstances.
 Caution: hypochlorites are inactivated by organic
matter. They corrode metals, hence contact of the
chemical with the metallic instruments and equipment
must be avoided.

26.  In case of heavy soilage eg
blood spillage, a
concentration of 10000
ppm (1:5 dilution of
household bleach) of
available chlorine is
recommended.
 Uses: it has great
widespread use as a
laboratory disinfectant
on surfaces of bench and in
discard spots.

28. IODINE COMPOUNDS
 Iodine is used in aqueous or alcoholic solution.
 Rapidly effective against most microorganisms.
 Concentration: Usually diluted to 1% w/v free
iodine, optimum pH neutral to acid.
 Caution: Not suitable in the presence of organic matter.
 Stains skin and may cause irritation.
 Decomposes when heated above 40ºC.
 Do not use on aluminium or copper.

29.  Method of use: Dilute in
alcohol for washing
hands, or use as a
sporicide.
 Prepare dilutions daily.
 Use: Most commonly used
for skin disinfection and
decontaminating clean
surfaces.

30.  Iodophores:
 They have largely replaced the aqueous and tincture forms
of iodine since the side effects like staining and irritation
are far less in iodophores than in aqueous or tincture
iodine.
 Iodophores are chemical complexes with iodine bound to a
carrier such as polyvinylpyrolidone (povidone, PVP) or
ethoxylated nonionic detergents.
 Free microbicidal iodine is gradually released from these
compounds.

31.  Commercial PVP in a
dilution of 1:2 to 1:100 kills
most bacteria including
S.aureus.
 Limitation: may become
contaminated by
organisms like
pseudomonas.
 Uses: iodophors are
widely used for
antisepsis of
skin, mucosa and
wounds. A 2.5%
ophthalmic solution is
an useful prophylactic
against neonatal
conjunctivitis.

32. CHLORHEXIDINE (hibitane)
 Chlorhexidine as chlorhexidine gluconate is dissolved in
70% alcohol.
 Effective against Gram-positive organisms and HIV.
 Active in ph range 5.5 - 8.0.
 Limitation: Not recommended as a general disinfectant.
 Not active against sporulating bacteria or non-lipid-
containing viruses.
 Incompatible with soap and anionic detergents.

33.  Use: savlon
(chlorhexidine+cetrimid
e) is widely used in
burns, wounds, as
bladder irrigant, for
surgical instruments
and pre-operative
disinfection of skin.
 Use as antiseptic. Apply
alcoholic chlorhexidine
to the skin in the event
of accidental
contamination.

34. HYDROGEN PEROXIDE
 A concentration of 3% w/v
generally used for
disinfection.
 Active against a range of
microorganisms.
 Fungi, spores and enteric
viruses require higher
concentration.
 No toxic end-products of
decomposition.

35.  Caution: Do not use on
aluminium, copper, zinc or
brass.
 Mechanism: H2O2 on
decomposition liberates
free hydroxyl radical which
is the active ingredient in
the process.
 Use: H2O2 is applied to
disinfect plastic
implants, contact
lenses, and surgical
prostheses.

36. PHENOLICS
 Synthetic phenolics (clear soluble
fluids) can be used as general
disinfectants in the laboratory.
 Spectrum: Active against bacteria
and lipid-containing viruses.
 Not active against spores and non-
lipid-containing viruses.
 Active in presence of organic matter.
 Use: for disinfecting
floors, walls, benches and other
furniture.

37.  Cresols: (methyl
phenols, lysol)
 Limitation: toxic to skin and
tissues.
 Use: mainly used for
preliminary sterilization of
infected glasswares in
laboratory, disinfection of
excreta, cleaning floors of
wards and operation room
in hospital.
 Chloroxylenol (dimethyl
phenol) is active ingredient
of dettol.

38.  Hexachlorophane: it is
bacteriostatic at very high
dilutions and used in soap and
powder form. It is more effective
against gram positive than gram
negative bacteria and is applied
on skin as prophylaxis against
staphylococcal infection.
 Chlorhexidine: mentioned
previously

39. QUATERNARY AMMONIUM COMPOUNDS
 Quaternary ammonium compounds are positively charged
(cationic) surface-active disinfectants.
 Effective against Gram-positive bacteria and lipid-
containing viruses.
 Not recommended as general disinfectants (they have a
narrow antibacterial spectrum).
 Inactivated by proteins, soap and anionic detergents.
 Eg. Benzalkonium chloride, alkyldimethylbenzyl
ammonium chloride, and cetylpyridinium chloride.
 Used for cleaning of floors of hospitals.

40. ETHYLENE OXIDE
 It is an alkylating agent.
 Exerts lethal effect on proteins of bacteria.
 It is gas at ordinary room temperature and active against all
types of bacteria and spores.
 It has got a good degree of penetration power, even through
plastics.

41. ETO Machine
 Uses: Plastic
goods, polythene tube, artery
and bone
grafts, cystoscopes, vaccines
and culture media can be
sterilized by ethylene oxide.
 These objects are kept in a
cabinet from which air is
removed by a vacuum pump and
then a mixture of ethylene oxide
and carbon dioxide is
introduced in the cabinet.

42. BETAPROPIOLACTONE (BPL)
 It is a condensation product of ketone and formaldehyde
having a boiling point of 163’C.
 It is capable of killing all microorganisms including viruses.
 Uses: Although BPL has a low penetrating power as a
gas, but it is believed to be more efficient for the purpose of
fumigation.
 Its biocidal action is very rapid and only 0.2% BPL is used
for the sterilization of biological products.
 Limitation: unfortunately BPL has carcinogenic effect.

43.  Points to ponder
 Prepare fresh dilutions daily
 Use clean, dry containers
 Use clean water for dilutions
 Use higher concentration or for longer duration if spillage
is heavy.
 Avoid using corrosive disinfectants on metal surfaces.

44. THANK YOU