Sterilization

Following are the objectives of sterilization and disinfection :

1. To prevent transmission of disease and infection.
2. To prevent contamination and growth by undesirable organisms.
3. To prevent deterioration and spoilage of material by microorganisms.
Microorganisms can be removed, killed or inhibited by physical or chemical processes.

Sterilization :
A process of destroying all forms of microbial life. A sterile object is free from living organism.

Disinfection :
A process of destroying infectious agents only or killing only vegetative forms but not resistant spore organisms.

Antisepsis :
A process that prevents growth or action of either by destroying or by inhibiting growth and metabolic activity.

Sanitizer :
A process that reduces extent of microbial population to safe levels, kills 99.9% of growing bacteria. Usually sanitization term used
for inanimate objects in daily use. Disinfection would produce sanitization.

Antimicrobial agents :
Interferes with growth and action of microorganisms. It may be inhibition of growth (Bacteriostatic) or killing of microorganisms
(Bactericidal). Such agents are used to treat infectious and hence known as Therapeutic agents.

The control of microbial growth is necessary in many practical situations, and significant advances in agriculture, medicine, and food
science have been made through study of this area of microbiology.

"Control of microbial growth", as used here, means to inhibit or prevent growth of microorganisms. This control is affected in two
basic ways: (1) by killing microorganisms or (2) by inhibiting the growth of microorganisms. Control of growth usually involves
the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents which kill cells are called
cidal agents; agents which inhibit the growth of cells (without killing them) are referred to as static agents. Thus, the term
bactericidal refers to killing bacteria, and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria,
a fungicide kills fungi, and so on.

In microbiology, sterilization refers to the complete destruction or elimination of all viable organisms in or on a substance being
sterilized. There are no degrees of sterilization: an object or substance is either sterile or not. Sterilization procedures involve the use
of heat, radiation or chemicals, or physical removal of cells.

DEATH OF BACTERIA :
Irreversible loss of the ability to reproduce is what death of organism means. The cells are killed over a period of time at a constant
exponential rate that is the inverse of exponential growth rate.

Some portion of population dies during any given time. The graph of logarithm of number of survivors v/s time in hours shows that
the death rate is constant. Slope of this curve is a measure of death-rate. All conditions are kept uniform including age or physiological
state of growth. If these conditions are not the same, then there will be a lot of difference in susceptibility of microorganisms to lethal
agents. Both the slope of death curve and its form greatly affected by the cells ion the population. The probability of killing the
organisms is also proportional to concentration of chemical agent or intensity of physical agent. It takes time to kill the population and
if we have many cells, we must treat them for a longer time.

Factors influencing action of chemical agents and microorganisms :

Temperature :
Increase in temperature with chemical agent hastens destruction of bacteria. Small amount of chemical agent at elevated temperature
gives the same result as larger amount of the same at low temperature.

Kind of microorganism ;
Species differ in their susceptibility to different lethal agents. Vegetative cells are more susceptible than spore formers. Bacterial
spores are most resistant of all living organisms in their capacity to survive under adverse environmental conditions.

Physiological state :
Young, actively metabolizing cells are easily destroyed / killed than old cells in the case of those chemical agents which causes
damage through interference with metabolism, non growing cells would not be affected in that case because these cells are not actively
metabolizing. Changes in the nature of cell membranes during aging, affects permeability characteristics of the cell wall which may
account for difference in resistance.

Environment :
Physical and chemical properties of medium carrying the organism (i.e. environment) has a profound influence on rate and efficiency
of destruction of microorganisms by chemical agents; eg. Effect of heat is greater in acidic medium than in alkaline medium,
consistency of medium greatly influences the interaction of chemical agent with the microorganism; for eg. High concentration of
carbohydrates increases the thermal resistance or heat stability of organisms. presence of organic matter reduces the efficiency of
antimicrobial agent by inactivating it or protecting the organism from the chemical agent by masking it. Organic matter added to a
disinfectant may have in anyond of the following outcomes:

1) Combination of disinfectant and organic matter to form a nonbactericidal produce / compound.

2) Combination of the disinfectant with organic material to form precipitate, thus forming a physical barrier between disinfectant and
the microorganism.
3) Accumulation of organic matter on microbial cell surface to provide a coating / covering which will impair the contact disinfectant
and the microbial cell. If components like serum or yeast extract are added to system more disinfectant will be required for the same
effect.

Medium of growth of microorganisms is very important, because one medium may support the growth of a particular type of
microorganism while another medium may not support the growth of the same type. So death may be consequence of growth medium.
Bacteriostasis may be mistaken for bacterial action. Some compounds are bactericidal at higher concentrations. It may be necessary to
add compounds which will neutralize the cidal or static effect of disinfectant in the medium otherwise there will be no growth.

MODE OF ACTION OF ANTIMICROBIAL AGENTS :

It is important to predict the conditions under which the antimicrobial agent will function most effectively, also the kind of organisms
against which it will be effective. Certain enzymes, cytoplasmic membrane, cell wall- there are many possible sites of damage to the
cell.

1. Damage to cell wall :

Cell walls of Gram positive bacteria are attacked by lysozyme found in tears, leukocytes, mucos secretions. Enzymes produced by
several bacterial species attack the cell-wall is followed by lysis. Some agents inhibit the formation of cell-wall material resulting in
protoplast formation eg. Penicillin
.
2. Alteration to cell permeability :
The antimicrobial activity of phenolic compounds, synthetic detergents, soaps and quaternary ammonium compounds is attributable to
their effect on cell permeability. These destroy the selective permeability of the membrane permitting leakage of cellular constituents
eg. Leakage of phosphate and nitrogen components from the cell.

3. Alteration of protein and nucleic acid molecules :

Some substances that denature the proteins or nucleic acid may irreparably damage the cell. High temperature and high concentration
of some chemicals can bring about irreversible coagulation of these vital protoplasmic constituents.

4. Inhibition of enzyme action :

Each of hundred of different enzymes in the cell is a target for an inhibitor. Many agents affect enzymes in the energy-yielding
reactions like the glycolytic systems, the Kreb’s tricarboxylic acid cycle and the cytochrome system, eg. Cyanide inhibits cytochrome
oxidase, fluoride inhibits glycolysis and trivalent arsenic compounds block the tricarboxylic acid cycle. Dinitrophenol uncouples
oxidative phosphorylation. Strong oxidizing agents e.g. halogens and H2O2 may damage cellular constituents to such an extent that
they can no longer perform normal metabolic functions e.g. the activity of many enzymes depend upon one of their components, a
sulfhadryl group –SH. An oxidizing agent may alter this chemical arrangement and inactivate the enzyme.
Inactivatin of enzyme may result from combination with metallizations silver, copper, mercury.

5. Antimetabolites :
They act by interference in a specific biosynthesis. E.g. inhibition of folic acid sysnthesis by sulfanilamide. One of the compounds of
folic acid is p-aminobenzoic acid whose chemical structure is similar to that of sulfanilamide competes with PABA for enzyme
surface, thereby preventing synthesis of folic acid.

Many other synthetic processes can be interrupted by compounds structurally related to but slightly different that natural metabolite.
Such a substance is known as antimetabolite.

6. Inhibition of nucleic acid synthesis :

Certain chemicals and naturally occurring substances are powerful inhibitors of RNA and DNA synthesis. They are :
1. Compounds that interfere with the formation of building blocks of nucleic acids viz. purines and pyrimidnes.
2. Compounds that interfere with the polymerization of nucleotides into nucleic acid.
Interference with their formation and function would seriously impair the cell.

CONTROL BY PHYSICAL AGENTS :

High temperature with high humidity is one of the most effective methods of killing microorganisms. Moist heat kills microorganisms
by coagulating their proteins and is a rapid and effective heat at 120 C. While about 2 hour exposure dry heat at the same
temperature is required. Vegetative cells are much more sensitive than spores. Cells of most bacteria are killed in 5-10 min by moist
heat at 50-60 C, their spores are killed in the same time but at 70-80 C. Most spores are killed above 100 C.

Thermal death point is the lowest temperature at which a suspension of bacteria is killed within 10 minutes. TDT is the shortest
period of time required to kill a suspension of bacteria at a prescribed temperature and under specific conditions. Canning industry
carries out extensive studies on those factors to establish satisfactory processing temperature for the preparation of canned food
maintained at constant defined level.

Moist heat :
Heat in the form of saturated steam under pressure is the most practical and dependable agent for sterilization. Steam under pressure
provides temperature above those obtainable by boiling. It has the advantage of rapid heating, penetration and moisture in abundance
which facilitates the coagulation of proteins. Sterilization using moist heat as the lethal agent is accomplished using instruments like
autoclave etc.

Autoclave :
It is a apparatus which has double jacketed wall filled with steam and maintained at a predetermined temperature and pressure for any
period of time. Air in autoclave should be replaced by steam as it reduces the temperature attained within the chamber. It is not the
pressure that kills the organisms but the high temperature of steam that is actually causing lethal irreparable injuries to the cell
subsequently leading to the killing of that cell.

Usually a pressure of 5 psi (pounds per square inch) at 109 C is used for substances immiscible in water, normally 15 psi at 121.5
C for 20 minutes for routine materials such as nutrient media etc. hence.

Some substances are altered or destroyed by extensive heat treatment of steam under pressure. If they can withstand the temperature of
free-flowing steam, it is possible to sterilize them by Fractional Sterilization (Tyndallization). This method involves heating the
material to be sterilized for 3 consecutive days with a proper incubation period in between two consecutive sterilization days. Heat-
resistant spores germinate during the incubation period. On subsequent exposure to heat the vegetative cells will be destroyed. An
apparatus known as Arnold Sterilizer is used for this purpose.

Boiling water cannot be used in the laboratory as a method of sterilization. Pasteurization of milk, cream and alcoholic beverages is
done by subjecting them to a controlled heat-treatment which kills microorganisms of certain types but not all types. Pasteurized milk
is not sterile milk. The temperature selected for pasteurization is based on the TDT curve of the most resistant type of pathoges to be
destroyed by this process in the concerned substance to be sterilized.

Dry heat :
This technique is used for sterilization of glassware, oils, dry powders. For this purpose a oven at a temperature of 180 C is used
and this temperature is maintained for 30 minutes. This is done in situations where moist heat is undesirable or unlikely that steam
under pressure will make direct or complete contact with the material to be sterilized may be due to its high viscosity.
Actual burning i.e. incineration is done to accomplish destruction of carcases, infested laboratory animals and other infected material
to be disposed off.

Heat: most important and widely used. For sterilization one must consider the type of heat, and most importantly, the time of
application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric
of all cells so their destruction guarantees sterility.

Incineration: burns organisms and physically destroys them. Used for needles, inoculating wires, glassware, etc. and objects not
destroyed in the incineration process.

Boiling: 100o for 30 minutes. Kills everything except some endospores. To kill endospores, and therefore sterilize a solution, very
long (>6 hours) boiling, or intermittent boiling is required.

Low temperature :
At low temperatures growth and metabolism of the organism ceases or slows down. It is useful for preservation of cultures because
microorganisms survive extreme cold conditions for e.g. –4to7 C in freezer. Many bacteria and viruses can be maintained
successfully in a deep-freeze unit at temperature of –20 to -70 C. Liquid nitrogen at a temperature of -196 C is used for
preserving cultures of many viruses and microorganisms. The initial chilling kills a certain fraction of population but survivors remain
viable for long periods. So low temperature cannot be used depended upon for disinfection or sterilization. It is only microbistatic.

Desiccation :
It causes absolute cessation of metabolic activity. The death rate depends on kind of organic, material in or on which the organisms are
dried, completeness of drying procedure and the physical conditions to which the dried organisms are exposed. E.g. light, temperature
humidity. Gram negative cocci e.g. Gonococci, are very sensitive to desiccation and so they die in hours on desiccation, Streptococci
are more resistant and so they survive for weeks and Tubercle bacilli dried in sputum remain viable for a still longer period.

Radiation :
Gamma rays and X-rays which have energy of more than about 10ev are called Ionizing radiations because they have enough energy
to pull electrons away from molecules and ionize them. When such radiations passes through cells it creates free hydrogen and
hydroxyl radical and some peroxides which in turn can cause different kinds of intracellular damage. U.V. light does not ionize, it is
absorbed quite specifically by different chemical species that can engage in a variety of chemical reactions not possible for unexcited
molecules. Organisms may be subjected to acoustic radiation (sound waves). Ionizing radiation is also used to sterilize biological
materials. This method is called Cold Sterilization because ionizing radiations produced relatively little heat in the material being
irradiated. Thus it is possible to sterilize heat-sensitive substances by radiations and such techniques are being developed in the food
and pharmaceutical industries.

Ultraviolet light :
Wavelength around 2650A has the bactericidal activity. Although the radiant energy of sunlight, is partly composed of UV light,
most of the shorter wavelength of this are filtered by the earth’s atmosphere is restricted to the span from about 2870 to 3900A .
Many lamps are available which emit a high concentration of UV light in the most effective region 2600 to 2700A . UV light has
very little ability to penetrate matter. UV light is absorbed by many cellular materials but most significantly by the nucleic acids where
it does the greatest damage. The absorption and subsequent reactions are predominantly in the pyrimidines of the nucleotide bases
which result in killing of cells.

X-Rays:
These are lethal to microbes. They have considerable energy and penetration power. They are expensive to produce and difficult to
utilize efficiently since radiations are given off in all directions from their point of origin. They have been used to produce microbial
used in genetic engineering experiments.

Gamma-rays :
These are high energy radiations emitted from radioactive isotopes such as 60 C. they are similar to X-rays butof shorter
wavelength. They have great penetration power and are lethal to all life including microbes. They are used for sterilization of materials
of considerable thickness or volume.

Cathode rays (electron-bean radiation) :

When a high voltage potential is established between a cathode and an anode in an evacuation tube called cathode tube which is a
special type of equipment used to produce electrons of high intensities and these electrons are accelerated to high velocities. They are
used for the sterilization of surgical materials drugs and other material. The material can be sterilized after it has been packaged and at
room temperature.

Filtration :
Biological fluids like serum, enzyes and some vitamins and antibiotics are heat-labile, Bacteriological filters are made of different
materials, eg. Asbestos in Seitz filter, diatomaceous earth in Berkefeld filter, porcelain in the chamberland Pasteur filter. Sintered glass
disks in other filters. The pore diameter in the filters may be one to several micrometers. Porosity alone is not the only factor
preventing the passage of organisms. other factors such as electric charge of the filter, the overall electric charge carried by the
organism and the nature of fluid being filtered have a strong bearing on the efficiency of filtration. Membrane or molecular filters are
composed of biologically inert material like cellulose acetate esters. They are prepared as circular membranes of 150um. thickness and
contain millions of microscopic pores of uniform diameter. Pores may be .005u to 1u. 0.01 to 10um. Negative pressure to filter flask
or positive pressure to above the fluid in the filter chamber is applied to force the fluid through polycarbonate 10um thick film in
which minute holes have been created after bobarding the film with charged particles. High flow rate, low toxicity, chemical inertness,
resistance to damage by most biological fluids and chemicals make them highly advantageous high efficiency particulate air )HEPA)
filters deliver clean sterile air cubicles or rooms, this type of air filtration and bacteria-free air for specific requirements such as in
laboratories, pharmaceutical industries specially dealing with intravenous preparations and some operation theatres.

Chemical and gas

Chemicals used for sterilization include the gases ethylene oxide and formaldehyde, and liquids such as glutaraldehyde. Ozone,
hydrogen peroxide and peracetic acid are also examples of chemical sterilization techniques are based on oxidative capabilities of
the chemical.
Ethylene oxide (ETO) is the most commonly used form of chemical sterilization. Due to its low boiling point of 10.4ºC at
atmospheric pressure, EtO) behaves as a gas at room temperature. EtO chemically reacts with amino acids, proteins, and DNA to
prevent microbial reproduction. The sterilization process is carried out in a specialized gas chamber. After sterilization, products are
transferred to an aeration cell, where they remain until the gas disperses and the product is safe to handle.

ETO is used for cellulose and plastics irradiation, usually in hermetically sealed packages. Ethylene oxide can be used with a wide
range of plastics (e.g. petri dishes, pipettes, syringes, medical devices, etc.) and other materials without affecting their integrity.

Ozone sterilization has been recently approved for use in the U.S. It uses oxygen that is subjected to an intense electrical field that
separates oxygen molecules into atomic oxygen, which then combines with other oxygen molecules to form ozone.

Ozone is used as a disinfectant for water and food. It is used in both gas and liquid forms as an antimicrobial agent in the treatment,
storage and processing of foods, including meat, poultry and eggs. Many municipalities use ozone technology to purify their water and
sewage. Los Angeles has one of the largest municipal ozone water treatment plants in the world. Ozone is used to disinfect swimming
pools, and some companies selling bottled water use ozonated water to sterilize containers.

Low Temperature Gas Plasma (LTGP) is used as an alternative to ethylene oxide. It uses a small amount of liquid hydrogen
peroxide (H2O2), which is energized with radio frequency waves into gas plasma. This leads to the generation of free radicals and
other chemical species, which destroy organisms.

Disinfection
Control by chemical agents :
Following are the characteristics of a ideal disinfectant.
1. It should have antimicrobial activity at low concentrations.
2. It should be water soluble preferably.
3. It should be stable at room temperature and no loss of germicidal action of the same should occur on storage.
4. It should be nontoxic to human beings and other animals.
5. It should be Homogeneous IE. Uniform in composition.
6. Many disinfectants have affinity for proteins or other organic matter. When they are used in situations where there is considerable
organic matter besides that of microbial cells, little disinfectant will be available for action against the microorganisms.
7. It should have the desired level of toxicity to microorgs at room or 37 C.
8. It should have good penetration power.
9. It should be noncorrosive or nonstaining.
10. It should have deodorizing ability ie. It should be odorless or have a pleasant smell.
11. It should have detergent activity because cleaning improves disinfection efficiency.
12. It should be easily available.

Decontamination of laboratory benches, furniture, equipment and other materials requires the use of chemical disinfectants. Their
activity is related to the following factors:

* concentration
* pH
* contact time
* humidity
* temperature
* presence of organic matter

Choosing a Disinfectant

Microorganisms present a range of resistances to chemical disinfectants and no single disinfectant is effective in all situations.
Consider the following points when selecting a disinfectant:

* type of microorganisms, numbers and presence of spores

* physical situation (surface type, suspension, etc.)
*contact available between disinfectant and microorganisms
* possible interaction between disinfectant and materials
* contact time allowable
* concentration

Selection of antimicrobial chemical agent :

This depends on the following factors :
1. Nature of material to be treated :
The substance selected must be compatible with the material which it is treated.

2. Type of microorganisms :
Chemical agents are not equally effective against all bacteria, fungi, viruses and other micororgs. Differences exist between Gram
positive and Gram negative organisms exists and their susceptibility to different antimicrobial agents. Difference in their action also
exist between strains of same species the agent selected must be known to be effective against the type of to be destroyed.

3. Environmental conditions :
Temperature, pH, time, concentration of the antimicrobial agent used, presence of organic material.

MAJOR GROUPS OF ANTIMICROBIAL AGENTS :

1. Phynols and phenolic compounds,
2. Alcohols,
3. Halogens,
4. Heavy metals and their compounds,
5. Dyes,
6. Detergents,
7. Quaternary ammonium compounds,
8. Acids and Alkalies,
9. Glutaraldehyde,
10. Gaseous Chemosterilizers (ethylene oxide, B-propiolactone, formaldehyde).

Phenols :
They were used by Lister (1860) for the first time in aseptic surgical techniques. It is the standard antimicrobial agent against which
other disinfectants are compared in evaluating their bacterial activity. Many of the phenol derivatives are more effective than phenol
These compounds denature cell-proteins and damage cell membrane, some like hexylresorcinols greatly reduce surface tension.
Depending upon concentration these substances they are either bactericidal or bacteriostatic. Spores and vegetative cells of some
organisms are more resistant to phenols. Some are highly fungicidal. The antimicrobial actoivity is reduced at alkaline pH and by the
presence of organic matter. Low temperature and presence of so also reduce the antimicrobial activity. 2-5% solution of phenol is
employed to disinfect sputum, urine feces and contaminated utensils. Cresols are several times more germicidal than phenol. Cresols
are not completely soluble in water but form emulsion in liquid soaps and alkalies. They are easily dispensable.

Alcohol :
70% ethyl alcohol is the most effective bactericidal concentration. But it is effective against vegetative or nonsporing cells only.
Spores of B. anthracis have survived in alcohol for 20 hrs and those of B. subtilis for 9 yrs.
Methyl alcohol is less bactericidal than ethyl alcohol, also it is toxic. The higher alcohols i.e. propyl, butyl, amyl are more germicidal
than ethyl alcohol. Propyl and isopropyl alcohols in concentration from 40-80% are useful as skin disinfectants. Alcohols are protein
denaturants. They are also solvents of lipids and can damage cell membrane. They are also dehydrating agents. It is possible that very
high concentration remove so much water from the cell that the alcohol is unable to penetrate. Severe dehydration under these
conditions would result in a bacteriostatic condition. Alcohol has got detergent action.
Alcohol concentration above 60% are effective against viruses, however the effectiveness is influenced by the amount extraneous
protein material which reacts with alcohol and protect the viruses by masking it.

Halogens :
Iodine is the oldest and most effective germicidal agent. It is slightly soluble in water but readily soluble in alcohol or aqueous
solution of NaI or KI. It is used as germicidal agent in a form known as tincture of iodine. Several preparations are available.
2%12+2% NaI diluted in alcohol Iodine is also used in a form Iodophors. It is a mixture of iodine with a surface active agent which
acts as carrier and solubilizer for iodine. Oner such agent is polyvinylpyrrolidone (PVP). Iodine is slowly released from PVP-I
complex. Iodophors have the germicidal activity of iodine and are nonstaining andnon irritant.
Iodine is highly effective bactericidal agent and is effective against all types of bacteria. It is also sporicidal for few types of bacteria.
But the rate which spores are killed is influenced by conditions like amount of organic environmental material and extent of
dehydration. It is fungicidal also and to some extent virucidal. Iodine solutions are used chiefly as skin disinfectant and it is of the best
iodine preparation. These can also be used for other purposes like disinfection of water, air (vapors) and sanitation of food utensils.
The mechanism of iodine action is not clearly understood. It has been suggested that the action may involve the halogenation of
tyrosine units of enzymes and other cellular proteins requiring tyrosine for activity which subsequently cause irreparable damage to
the cell. It is also an oxidizing agent.
Chlorine :
It is used in the form of gas or in chemical combination it is one of the most widely used disinfectant. The compressed gas in the form
of liquid is almost universally employed for the purification of municipal water supplies but is very difficult to handle chlorine safely.

Hypochlorites :
Calcium hypochlorite Ca(OCl)2 and sodium hypochlorite NaOCl are widely used. They are in powder or liquid forms and in various
concentrations from 5-70%. CaCl2 is used to sanitized dairy equipment and 1% NaOCl for personal hygiene and household
disinfection.

Chloramines :
They are characterized by the fact that one or more of the hydrogen atoms in an amino group of a compound are replaced by chlorine.
The simplest of all is monochloramine, NH2Cl. Chloramine T and azochloramide have more complex chemical structures.
Chloramines are more stable than the hypochlorites in terms of prolonged released of chlorine.
The germicidal action of chlorine and its compounds comes through the hypochlorous acid which is formed when free chlorine is
added to water.
Cl2 + h2O = HCl + HClO
Hypochlorites and chromides undergo hydrolysis with the formation of hypochlorous acid. The hypochlorous acid is further
decomposed.
HClO = HCl + (O)
The nascent oxygen released in this reaction is a strong oxidizing agent and through its action on cellular constituents microorganisms
are destroyed. Also the chlorine directly combines with proteins of cell membrane and enzymes thereby killing the organisms.

Heavy metals and their compounds :

The most effective among the heavy metals and their compounds are Mercury, Silver and Copper. The ability of extremely small
amount of certain metals, particularly silver to exert a lethal effect upon bacteria is designated oligodynamic action (oligos-small,
dynamic-power), In laboratory if a clean piece of metal is placed on the seeded nutrient agar plate then it shows a clear zone of
inhibition round the metal, after incubation. The concentration of metal involved in this actions in parts per million. The effectiveness
of metal is due to the high affinity of certain cellular proteins for ion, large amounts are accumulated in the cell from a dilute solution.
Oligodynamically active metals, particularly silver have been used in a variety of applications for the purpose of controlling microbial
population. E. g. treatment of water supplies, preparation of aseptic articles etc.
Heavy metal compounds of mercury silver and copper combine with cellular proteins and denature them. HgCl2 combines with the
sulfhydryl group of enzymes.
Salts of heavy metals are also protein precipitant and in high concentration cause the death of a cell.

Dyes :
Usually triphenylmethane and acridine dyes are used. Malachite green, brilliant green and crystal violet are used against Gram positive
cocci at 1:200,000 dilution and against E.coli at 10 times more concentration.
The dyes are effective in the cases where one alkyl group contains 12 or 16 Carbon atoms i.e. efficacy greatly affected by chemical
structure inhibits staphylococcus aureus at 1:1000,000 dilution and E.coli at 1:30,000 dilution. Many of these dyes are used in
selective media which are used in sanitary bacteriology, where detection of E.coli is important. Susceptibility to various dyes is also
used for identification of bacterial. Crystal violet is also used as a fugicide. Mode of action is not yet known, but they may be
interfering with cellular oxidation process.

Acridine :
Acridine dyes include Acriflavine and proflavine. These dys are more effective against Gram positive organisms. they are used for the
treatment of burns and wound and also in ophthalmic applications and bladder irrigation.

Synthetic detergents ;
These are surface tension depressants or wetting agents e.g. soap. But soap is poor detergent/disinfectant. In hard water, surfactants or
synthetic detergents are superior to soap. They don’t precipitate in acid or alkaline water or in hard water. Some of them are highly
bactericidal.
Some of these synthetic detergents ionize with detergent property resident in the action and they are termed as anionic detergents.
Some do no ionize, do not possess weak antimicrobial activity.
Soaps mechanically remove the microorganisms. They reduce surface tension and increase the wetting power of water. The
microorganism become enmeshed in the soap lather and are removed by the water used for rinsing. Cationic detergents are more
germicidal than anionic ones.

Quaternary ammonium compounds :

These are cationinc detergents e.g. zephiran, phemerol. They are more inhibitory to Gram positive organisms. They have the ability to
manifest bacteriostatic action far beyond their bactericidal concentration. They are fungicidal and kill the protozoa also. Viruses are
more resistant than bacteria and fungi to these compounds. They are used as skin disinfectants and as sanitizing agents in dairy and
food-processing plants. Their mode of action is not known yet, but may be enzyme inhibition, protein denaturation and disruption of
cell membrane.

Acids and alkalies :

Microorganisms tolerate pH near neutrality. The killing action of mineral acids HCl, H2SO4 is due to hydrogen ion concentration
(dissociation) and final pH. Organic acids ionize to relatively low degree so the antimicrobial action of them must be due to the nature
of molecule. Te disinfectant action of alkalies is dependent on dissociation and the resulting conc of hydroxylions. Ly3e is a
preparation of NaOH which is used as disinfectant. Strong acids and alkalies are sporicidal but they are corrosive. Acids are more
effective than alkalies.

Glyceraldehyde :
It is a saturated dialdehyde. 2 % solution exhibits a wide spectrum of activity. It is effective against vegetative bacteria, fungi spores
and viruses.

Gaseous sterilization :
Many articles cannot be sterilized by heat of liquid chemosterilant. Chemical sterilization by means of a gaseous agent is effective.
The material is exposed to gas in a confined area at room temperature. Main agents are ethylene oxide, B propiolactone and
formaldehyde.

Ethylene oxide :
Below 10.8 C it is liquid, above this temperature is vapourises. Vapours are inflammable so mixture of ethylene oxide 10 to 20 %
with 80 to 90 % CO2, or Feron is used, CO2 or Feron serve as inert diluent which prevent inflammability. It is a unique and powerful
sterilizing agent. Bacterial spores show little resistance to destruction by this agent. It has got very good penetrating power. It passes
through the sterilizes large packets of materials and even certain plastics. It should be used with caution. The concentration of ethylene
oxide and temperature and humidity are critical factors which determine the time required for sterilization. The apparatus used for its
application is an autoclove modified. The variety of material on which it is used includes spices, biological preparation, soil, plastics,
certain medical preparation and contaminated laboratory equipment. It is effective at low temperature and does not damage the
material exposed to it but it is slow in action.
The mode of action is believed to be alkylation reactions with organic compounds such enzymes and other proteins.

Propiolactone :
It is Bactericidal, sporicidal, fungicidal and virucidal also. It is colourless liquid at room temperature with a high boiling point (155
C). It is not flammable, it lacks penetrating power but is very active then ethylene oxide. The usual concentration of ethylene oxide
used is 400 to 800 mg. litre only whereas 2 to 5 mg/litre of Beta-Propiolactone is required.

Formaldehyde :
Formaldehyde is marketed as foamalin which is a aqueous soplution which contains 37 to 40 % formaldehyde. Vaporization of any of
these compounds in an enclosed area for an adequate time will cause sterilization. Humidity and temperature have a pronounded effect
on the microbicidal action of HCHO. Temperature should preferably be room temperature and humidity 60 to 70 %.
This can be done in a variety of ways as follows :
1. Liquid water-soluble substances diluted are dispensed into sterile test tubes to which are added measured amount of test organisms.
At specific interval, a transfer is made from this tube to sterile media that are incubated and observed for growth. This approach can
also be used to determine the number of org.s killed per unit time by performing a plate count at appropriate intervals.

2. The chemical agent is incorporated into a gar medium or broth inoculated with the test-organism and incubated, then observed for
decrease in the extent of growth or complete absence of growth.

3. A plate of agar medium is inoculated with test organism and chemical agent is placed on the medium. Following incubation the
plate is observed for a zone of inhibition around the chemical agent. This is suitable for semi-solid substances.

4. For evaluation of gaseous substances, paper strips impregnated with known number of bacterial spores are exposed to gas under
recommended conditions, after which they are cultured for determining the number of survivors.