1. Disinfectants and Antisepsis PHR 203 Shahana Sharmin

2. Introduction
Antiseptics and disinfectants are extensively used in hospitals and other health care settings for a variety of topical and hard-surface applications.
In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial(disease,originating in hospital) infections
A wide variety of active chemical agents (biocides) are found in these products, many of which have been used for hundreds of years for antisepsis, disinfection and preservation, including alcohols, phenols, iodine, and chlorine.
Most of these active agents demonstrate broad-spectrum antimicrobial activity;
#disinfectant-are products that are applied to non-living objects to destroy microorganism that are living on the object.
#Antiseptic-which destroy m.o. on living tissue.
#Biocide-which destroy all forms of life,not just m.o.
#sensitizer-simultaneously both clean & disinfectant.

3. Modern Use
Disinfectants and antiseptics are now used extensively in hospitals and other medical treatment centers, where they help to maintain a clean environment that can prevent the spread of bacteria from person to person.
Disinfectants and antiseptics used properly are still able to kill bacterial strains that have become resistant to antibiotic treatment.
In the last 20 years, the use of disinfectants such as anti-bacterial sprays has increased in the home but this may not necessarily be a good thing.
The vast majority of bacteria in our home environment are friendly bacteria and do not cause disease. Using anti-bacterial products all over the home is probably unnecessary.
Cleaning danger areas such as toilets, drains and rubbish bins with a suitable disinfectant or bleach solution is very likely to be sufficient.

4. Disinfectant
Disinfectant may be defined as "a chemical or physical process that kills pathogenic organisms in water, air, on surfaces, floors and non-living objects“

5. Criteria of an ideal an ideal antiseptic or disinfectant:
An ideal antiseptic or disinfectant should have following properties:
Should have wide spectrum of activity
Should be able to destroy microbes within practical period of time
Should be active in the presence of organic matter
Should make effective contact and be wettable
Should be active in any pH
Should be stable

6. Criteria of an ideal antiseptic / disinfectant
Should have long shelf life
Should be speedy
Should have high penetrating power
Should be non-toxic, non-allergenic, non-irritative or non-corrosive
Should not have bad odour
Should not leave non-volatile residue or stain
Efficacy should not be lost on reasonable dilution
Should not be expensive and must be available easily

7. Criteria of an ideal antiseptic / disinfectant
The level of disinfection achieved depends on
contact time,
temperature,
type and concentration of the active ingredient,
the presence of organic matter,
the type and quantum of microbial load.
The chemical disinfectants at working concentrations rapidly lose their strength on standing.

8. Substances are used as disinfectants
A wide range of different substances are used as disinfectants, including – a) alcohols (also used as antiseptics), b) aldehydes, such as ortho-phthalaldehyde, c) chlorine and chlorine compounds as bleaches d) hydrogen peroxide e) iodine (tincture and solution -traditional antiseptic) f) potassium permanganate (solution) g) Phenolic compounds also have anti-bacterial activity and are often used in disinfectants bought for use in the home.

9. Disinfectants use in a) Instruments and Materials
Chemicals used to disinfect certain instruments and materials that cannot be sterilized by heat should use disinfectants instead. Examples of things that need to be chemically disinfected are hospital surgical instruments, bougies(thin,flexible instrument ,used to explore/dilate the passage of the body), cycstoscopes, cataract knives etc. Items like these must be immersed in a germicide.
Most of the chemicals are used in the form of solutions, being dissolved in water or in alcohol. From time to time, new chemical compounds appear on the market and all of them have certain advantages and disadvantages. So be careful in choosing the right chemical disinfectant.

10. b) Skin Application
Disinfectants are used to remove as many resident and transient organisms of the skin as possible. In surgery, the preoperative preparation of the skin is necessary to prevent unnecessary contamination of the surgical wound.
The hands and the arms of the surgical team likewise must be prepared by scrubbing and the application of appropriate disinfectants.

11. Antisepsis
Antiseptic is from the Greek word "anti" meaning "against" and "septikos" meaning of or pertaining to "putrefaction" or "putrid.“ Antiseptics are placed on the skin, usually in an area where the skin has been damaged, such as a cut or a graze (lession). They are also used to clean the skin before intentional cuts are made in surgical operations. Those that kill bacteria outright are described as bactericidal. Other antiseptics, described as bacteriostatic, stop bacteria from growing or dividing, but do not kill them.
Putrid-rot & produce unpleasant smell

12. Common antiseptics used
Common antiseptics used include-
a) alcohol,
b) boric acid,
c) hydrogen peroxide,
d) iodine,
e) carbolic acid and other phenol compounds,
f) chlorohexadine as hand wash and mouth wash
g) sodium chloride,
h) sodium hypochlorite and calcium hypochlorite,
i) essential oils - eucalyptus, methyl salicylate, menthol, thymol (Listerine)
k) silver compounds
l) triclosan

13. Antisepsis applies a) Application to Tissues
Antiseptics are applied to tissues that are or may be the seat of infection, in order to assist them in destroying germs and their products (toxins) rapidly and completely.
When used in this way, the ultimate antiseptic should bring about complete sterilization within its sphere of action without causing any damage to tissue cells.
However, the disadvantage of most antiseptics is that in killing the bacteria, they tend to destroy the tissue cells as well.

14. Chemical disinfectants
Chemical disinfection is used only when it is not feasible to sterilize an article by heat. Chemical disinfectants tend to set or solidify "proteinaceous" material such as blood, and organisms contained within this protein precipitate.
Accordingly, chemical disinfection should not be used for instruments contaminated with blood or tissue fluid.

15. Factors influencing disinfectant actions
1. Cleanliness
The presence of blood, pus, oil or grease interferes with the action of all germicides. The effective use of soap and water for cleansing is a necessary preliminary step to ensure optimal effectiveness of a disinfectant.
2. Time
This factor varies from seconds to hours, depending on the kind of disinfectant, the strength of the disinfecting agent, and the characteristics of the organisms to be eliminated. Vegetative organisms may be destroyed in 30 minutes by some of the chemical disinfectants, whereas no amount of time, hours, or days by the same agent would result in sporicidal action.

16. 3. Concentration
Usually a weak solution is not as effective as a strong solution of the same disinfectant. An exception is ethyl alcohol or isopropyl alcohol; 70% aqueous solution is more germicidal than absolute ethyl alcohol or isopropyl alcohol.
4. Temperature
Usually, room temperature is used. However, most chemical agents are more effective if the temperature is raised.
5. Type of Organism
Some organisms are killed more readily than others. Examples of the resistant forms are the virus of serum hepatitis, tubercle bacillus and certain bacterial spores.

17. Mechanism of action
A battery of techniques are available for studying the mechanisms of action of antiseptics and disinfectants on microorganisms, especially bacteria. These include examination of
uptake , lysis and leakage of intracellular constituents ,
Perturbation(alteration of a system) of cell homeostasis,
effects on model membranes,
inhibition of enzymes, electron transport, and oxidative phosphorylation ,
interaction with macromolecules,
effects on macromolecular biosynthetic processes, and
microscopic examination of biocide-exposed cells.
Additional and useful information can be obtained by calculating concentration exponents (n values) and relating these to membrane activity. Many of these procedures are valuable for detecting and evaluating antiseptics or disinfectants used in combination.

18. Alcohols
Several alcohols have been shown to be effective antimicrobials, ethyl alcohol (ethanol, alcohol), isopropyl alcohol (isopropanol, propan-2-ol) and n-propanol (in particular in Europe) are the most widely used. Alcohols exhibit rapid broad-spectrum antimicrobial activity against vegetative bacteria (including mycobacteria), viruses, and fungi but are not sporicidal. They are known to inhibit sporulation and spore germination, but this effect is reversible. Because of the lack of sporicidal activity, alcohols are not recommended for sterilization but are widely used for both hard-surface disinfection and skin antisepsis.

19. PHENOL:
Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of enzymes.
Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol)
Applications: Phenols are coal-tar derivatives. They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. They are not readily inactivated by organic matter. Chlorhexidine can be used in an isopropanol solution for skin disinfection, or as an aqueous solution for wound irrigation. It is often used as an antiseptic hand wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand and skin preparation and for general skin disinfection. Chlorhexidine gluconate is also mixed with quaternary ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (e.g. Savlon).

20. HALOGENS:
Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine, iodophores)
Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such as Povidone-iodine. Iodophores permit slow release and reduce the irritation of the antiseptic. For hand washing iodophores are diluted in 50% alcohol. Mercuric chloride is used as a disinfectant.
Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is corrosive and staining.

21. HEAVY METALS:
Mode of action: Act by precipitation of proteins and oxidation of sulfydryl groups. They are bacteriostatic.
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (e.g., mercurochrome merthiolate)
Applications: these heavy metals are biocidal.
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

22. SURFACE ACTIVE AGENTS:
Mode of actions: They have the property of concentrating at interfaces between lipid containing membrane of bacterial cell and surrounding aqueous medium. These compounds have long chain hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they contain both of these, they concentrate on the surface of membranes. They disrupt membrane resulting in leakage of cell constituents.
Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents containing negatively charged long chain hydrocarbon are called anionic detergents. These include soaps and bile salts. If the fat-soluble part is made to have a positive charge by combining with a quaternary nitrogen atom, it is called cationic detergents. Cationic detergents are known as quaternary ammonium compounds Cetrimide and benzalkonium chloride act as cationic detergents.
Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals.
Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter. Pseudomonas can metabolise cetrimide, using them as a carbon, nitrogen and energy source.

23. DYES:
Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial nucleic acids.
Examples: Aniline dyes such as crystal violet, malachite green and brilliant green. Acridine dyes such as acriflavin and aminacrine. Acriflavine is a mixture of proflavine and euflavine. Only euflavine has effective antimicrobial properties. A related dye, ethidium bromide, is also germicidal. It intercalates between base pairs in DNA. They are more effective against gram positive bacteria than gram negative bacteria and are more bacteriostatic in action.
Applications: They may be used topically as antiseptics to treat mild burns. They are used as paint on the skin to treat bacterial skin infections. The dyes are used as selective agents in certain selective media.

24. HYDROGEN PEROXIDE:
Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA.
Application: It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators. 3%.Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal.
Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic matter drastically reduces its activity.

25. ETHYLENE OXIDE (EO)
Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and hydroxyl- groups.
Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It has a sweet ethereal odor, readily polymerizes and is flammable.
Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is highly flammable, it is usually combined with CO2 (10% CO2+ 90% EO) or dichlorodifluoromethane. It requires presence of humidity. It has good penetration and is well absorbed by porous material. It is used to sterilize heat labile articles such as bedding, textiles, rubber, plastics, syringes, disposable petri dishes, complex apparatus like heart-lung machine, respiratory and dental equipments.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and carcinogenic.

26. BETA-PROPIOLACTONE (BPL):
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl- and hydroxyl- groups.
Properties: It is a colorless liquid with pungent to slightly sweetish smell. It is a condensation product of ketone with formaldehyde.
Application: It is an effective sporicidal agent, and has broad-spectrum activity. 0.2% is used to sterilize biological
products. It is more efficient in fumigation that formaldehyde. It is used to sterilize vaccines, tissue grafts, surgical instruments and enzymes
Disadvantages: It has poor penetrating power and is a carcinogen.

27. TESTING OF DISINFECTANTS:
A disinfectant must be tested to know the required effective dilution, the time taken to effect disinfection and to periodically monitor its activity. As disinfectants are known to lose their activity on standing as well as in the presence of organic matter, their activity must be periodically tested.
Different methods are:
1. Koch’s method
2. Rideal Walker Method
3. Chick Martin test
4. Capacity use dilution test (Kelsey-Sykes test)
5. In-use test

28. Spores of Bacillus anthracis were dried on silk thread and were subjected to action of disinfectants. Later, it was washed and transferred to solid medium.
This method relies on the estimation of phenol coefficient. Phenol coefficient of a
disinfectant is calculated by dividing the dilution of test disinfectant by the dilution of phenol that disinfects under predetermined conditions. Both the phenol and the test disinfectant are diluted from 1/95 to 1/115 and their bactericidal activity is determined against Salmonella typhi suspension. Subcultures are performed from both the test and phenol at intervals of 2.5, 5, 7.5 and 10 minutes. The plates are incubated for hours at 37°C. That dilution of disinfectant which disinfects the suspension in a given time is divided by that dilution of phenol which disinfects the suspension in same time gives its phenol coefficient.

29. Disadvantages of the Rideal-Walker test are: No organic matter is included; the microorganism Salmonella typhi may not be appropriate; the time allowed for disinfection is short; it should be used to evaluate phenolic type disinfectants only.
Chick Martin test: This test also determines the phenol coefficient of the test disinfectant. Unlike in Rideal Walker method where the test is carried out in water, the disinfectants are made to act in the presence of yeast suspension (or 3% dried human feces). Time for subculture is fixed at 30 minutes and the organism used to test efficacy is S.typhi as well as S.aureus. The phenol coefficient is lower than that given by Rideal Walker method.
The classical tests such as Rideal - Walker or Chick - Martin are not practicable.

30. Rideal -Walker Chick-Martin
Volume medium ml ml
Diluent for test disinfectant Water Yeast suspension
Reaction temperature ±0.5ºC ºC
Organism Salmonella typhi Salmonella typhi
Staphylococcus aureus
Sampling times , 5.0, 7.5, 10.0 min min.
Calculation of coefficient Dilution test Mean concentration
killing in 7.5 min of phenol showing
divided by same no growth after 30 min
for phenol divided by same for
test

31. Capacity use dilution test (Kelsey-Sykes test):
Inoculum of four different test organisms, namely Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris are added to the disinfectant in three successive. Dried yeast is included to simulate presence of organic matter. The method can be carried out under 'clean' or 'dirty' conditions. The dilutions of the disinfectant are made in hard water for clean conditions and in yeast suspension for dirty conditions. Test organism alone or with yeast is added at 0,10 and 20 minutes interval. The contact time of disinfectant and test organism is 8 min. The disinfectant is evaluated on its ability to kill microorganisms or lack of it and the result is reported as a pass or a fail and not as a coefficient. The capacity test of Kelsey and Sykes gives a good guideline for the dilution of the preparation to be used. Disadvantage of this test is the fact that it is rather complicated.

32. In-use test:
The routine monitoring of disinfectant in use can be done by the ‘in use’ test of Maurer. This test is intended to estimate the number of living organism in a vessel of disinfectant in actual use.
The disinfectant that is already in use is diluted 1 in 10 by mixing 1 ml of the disinfectant with 9 ml of sterile nutrient broth.
Ten drops of the diluted disinfectant (each 0.02 ml) is placed on two nutrient agar plates. One plate is incubated at 37°C for 3 days while the other is held at room temperature for 7 days. The number of drops that yielded growth is counted after incubation. If there growth in more than five drops on either plate, it represents failure of disinfectant.