Microparasitology assignment

1) STERLIZATION

Sterilization is defined as the process where all the living microorganisms, including bacterial spores are
killed.

Sterilization can be achieved by physical, chemical and physiochemical means. Chemicals used as
sterilizing agents are called chemisterilants.

DISINFECTION

Disinfection is the process of elimination of most pathogenic microorganisms (excluding bacterial

spores) on inanimate objects. Disinfection can be achieved by physical or chemical methods. Chemicals
used in disinfection are called disinfectants. Different disinfectants have different target ranges, not all
disinfectants can kill all microorganisms. Some methods of disinfection such as filtration do not kill
bacteria, they separate them out.

ANTISEPSIS

Antisepsis is the use of chemicals (antiseptics) to make skin or mucus membranes devoid of pathogenic
microorganisms.

BACTERICIDAL

Bactericidal is that chemical that can kill or inactivate bacteria. Such chemicals may be called variously
depending on the spectrum of activity, such asbactericidal,virucidal,fungicidal, microbicidal, sporicidal,
tuberculocidal or germicidal.

BACTERIOSTATIC

A bacteriostatic agent or bacteriostat, abbreviated Bstatic, is a biological or chemical agent that

stops bacteria from reproducing, while not necessarily killing them otherwise. Depending on their
application, bacteriostatic antibiotics, disinfectants, antiseptics and preservatives can be
distinguished.

Bacteriostasis is a condition where the multiplication of the bacteria is inhibited without killing them.

SPORICIDAL
An agent that destroys bacterial and mold spores. Because spores are more difficult to kill than vegetative
cells, a sporicide also acts as a sterilizing agent.

FUNGICIDAL

Fungicidal is a substance that destroys or inhibits the growth of fungi. Fungicides are biocidal chemical
compounds or biological organisms used to kill parasitic fungi or their spores. A fungistatic inhibits
their growth.
VIRICIDAL

A viricidal is any physical or chemical agent that deactivates or destroys viruses. This differs from an
antiviral drug, which inhibits the proliferation of the virus. Virucides are usually labeled with
instructions for safe, effective use

2) PHYSICAL METHODS OF STERLIZATION

Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in
it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries, the
sunlight is more effective in killing germs due to combination of ultraviolet rays and heat. By
killing bacteria suspended in water, sunlight provides natural method of disinfection of water
bodies such as tanks and lakes. Sunlight is not sporicidal, hence it does not sterilize.

Heat: Heat is considered to be most reliable method of sterilization of articles that can withstand
heat. Heat acts by oxidative effects as well as denaturation and coagulation of proteins. Those
articles that cannot withstand high temperatures can still be sterilized at lower temperature by
prolonging the duration of exposure.

DRY HEAT:

Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing
spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple
method for effective sterilization of such articles, but is limited to those articles that can be
heated to redness in flame.

Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times. Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short exposure.

This method too is limited to those articles that can be exposed to flame. Cracking of the
glassware may occur.

Incineration: This is a method of destroying contaminated material by burning them in

incinerator. Articles such as soiled dressings; animal carcasses, pathological material and
bedding etc should be subjected to incineration. This technique results in the loss of the article,
hence is suitable only for those articles that have to be disposed. Burning of polystyrene
materials emits dense smoke, and hence they should not be incinerated.

Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed
to high temperature (160o C) for duration of one hour in an electrically heated oven. Since air is
poor conductor of heat, even distribution of heat throughout the chamber is achieved by a fan.
The heat is transferred to the article by radiation, conduction and convection. The oven should
be fitted with a thermostat control, temperature indicator, meshed shelves and must have
adequate insulation.
 Infra red rays: Infrared rays bring about sterilization by generation of heat. Articles to be
sterilized are placed in a moving conveyer belt and passed through a tunnel that is heated by
infrared radiators to a temperature of 180oC. The articles are exposed to that temperature for a
period of 7.5 minutes. Articles sterilized included metallic instruments and glassware. It is
mainly used in central sterile supply department. It requires special equipments,hence is not
applicable in diagnostic laboratory.

MOIST HEAT:

Moist heat acts by coagulation and denaturation of proteins. At temperature below 100oC:

Pasteurization: This process was originally employed by Louis Pasteur. Currently this procedure
is employed in food and dairy industry. There are two methods of pasteurization, the holder
method (heated at 63oC for 30 minutes) and flash method (heated at 72oC for 15 seconds)
followed by quickly cooling to 13oC. Other pasteurization methods include Ultra-High
Temperature (UHT), 140oC for 15 sec and 149oC for 0.5 sec. This method is suitable to destroy
most milk borne pathogens like Salmonella, Mycobacteria, Streptococci, Staphylococci and
Brucella, however Coxiella may survive pasteurization. Efficacy is tested by phosphatase test
and methylene blue test.

Vaccine bath: The contaminating bacteria in a vaccine preparation can be inactivated by heating in a
water bath at 60oC for one hour. Only vegetative bacteria are killed and spores survive.

Serum bath: The contaminating bacteria in a serum preparation can be inactivated by heating in
a water bath at 56oC for one hour on several successive days. Proteins in the serum will
coagulate at higher temperature. Only vegetative bacteria are killed and spores survive.

Inspissation: This is a technique to solidify as well as disinfect egg and serum containing media.
The medium containing serum or egg are placed in the slopes of an inspissator and heated at 80-
85oC for 30 minutes on three successive days. On the first day, the vegetative bacteria would die
and those spores that germinate by next day are then killed the following day. The process depends
on germination of spores in between inspissation. If the spores fail to germinate then this technique
cannot be considered sterilization.

At temperature 100oC: Boiling: Boiling water (100oC) kills most vegetative bacteria and viruses
immediately. Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant.
Some bacterial spores are resistant to boiling and survive; hence this is not a substitute for
sterilization. The killing activity can be enhanced by addition of 2% sodium bicarbonate. When
absolute sterility is not required, certain metal articles and glasswares can be disinfected by
placing them in boiling water for 10-20 minutes. The lid of the boiler must not be opened during
the period.

Steam at 100oC: Instead of keeping the articles in boiling water, they are subjected to free steam
at 100oC. Traditionally Arnold’s and Koch’s steamers were used. An autoclave (with discharge
tap open) can also serve the same purpose. A steamer is a metal cabinet with perforated trays
to hold the articles and a conical lid. The bottom of steamer is filled with water and heated. The
steam that is generated sterilizes the articles when exposed for a period of 90 minutes. Media
 such as TCBS, DCA and selenite broth are sterilized by steaming. Sugar and gelatin in medium
may get decomposed on autoclaving, hence they are exposed to free steaming for 20 minutes
for three successive days. This process is known as tyndallisation (after John Tyndall) or
fractional sterilization or intermittent sterilization. The vegetative bacteria are killed in the first
exposure and the spores that germinate by next day are killed in subsequent days. The success
of process depends on the germination of spores.

RADIATION:

Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy rays
with poor penetrative power while ionizing rays are high-energy rays with good penetrative
power. Since radiation does not generate heat, it is termed "cold sterilization". In some parts of
Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent.

FILTRATION:

Filtration does not kill microbes, it separates them out. Membrane filters with pore sizes
between 0.2-0.45 µm are commonly used to remove particles from solutions that can't be
autoclaved. It is used to remove microbes from heat labile liquids such as serum, antibiotic
solutions, sugar solutions, urea solution. Various applications of filtration include removing
bacteria from ingredients of culture media, preparing suspensions of viruses and phages free of
bacteria, measuring sizes of viruses, separating toxins from culture filtrates, counting bacteria,
clarifying fluids and purifying hydatid fluid. Filtration is aided by using either positive or negative
pressure using vacuum pumps.

SONIC AND ULTRASONIC VIBRATIONS: Sound waves of frequency >20,000 cycle/second kills
bacteria and some viruses on exposing for one hour. Microwaves are not particularly
antimicrobial in themselves, rather the killing effect of microwaves are largely due to the heat
that they generate. High frequency sound waves disrupt cells. They are used to clean and
disinfect instruments as well as to reduce microbial load. This method is not reliable since many
viruses and phages are not affected by these waves.

3) CHEMICAL METHODS OF DISINFECTION:

Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Some
chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can
sterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucus
membranes are called antiseptics.

An ideal antiseptic or disinfectant should have following properties:

ƒ Should have wide spectrum of activity

ƒ Should be able to destroy microbes within practical period of time

ƒ Should be active in the presence of organic matter

ƒ Should make effective contact and be wettable

ƒ Should be active in any pH

ƒ Should be stable

ƒ Should have long shelf life

ƒ Should be speedy

ƒ Should have high penetrating power

ƒ Should be non-toxic, non-allergenic, non-irritative or non-corrosive

ƒ Should not have bad odour

ƒ Should not leave non-volatile residue or stain

ƒ Efficacy should not be lost on reasonable dilution

ƒ Should not be expensive and must be available easily

Such an ideal disinfectant is not yet available. 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.

Classification of disinfectants:

1. Based on consistency

a. Liquid (E.g., Alcohols, Phenols)

b. Gaseous (Formaldehyde vapor, Ethylene oxide)

2. Based on spectrum of activity

a. High level

b. Intermediate level

c. Low level

3. Based on mechanism of action

a. Action on membrane (E.g., Alcohol, detergent)

b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)

ALCOHOLS:

Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.

Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol

Application: A 70% aqueous solution is more effective at killing microbes than absolute alcohols.
70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to ethanol. It
can also be used to disinfect surfaces. It is used to disinfect clinical thermometers. Methyl alcohol
kills fungal spores, hence is useful in disinfecting inoculation hoods.

Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

ALDEHYDES:

Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably
damages nucleic acids. It kills all microorganisms, including spores.

Examples: Formaldehyde, Gluteraldehyde

Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms,
chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. Fumigation is
achieved by boiling formalin, heating paraformaldehyde or treating formalin with potassium
permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate
sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize thermometers,
cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure of at least 3
hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at 40oC for 20
minutes is used to disinfect wool and 0.25% at 60oC for six hours to disinfect animal hair and
bristles.

Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration, leaves
non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde requires alkaline
pH and only those articles that are wettable can be sterilized.

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: Joseph Lister used it to prevent infection of surgical wounds. 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. The corrosive phenolics are used for disinfection
of ward floors, in discarding jars in laboratories and disinfection of bedpans. 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 (eg. Savlon). Chloroxylenols are less irritant and can be used for topical
purposes and are more effective against gram positive bacteria than gram negative bacteria.
Hexachlorophene is chlorinated diphenyl and is much less irritant. It has marked effect over gram
positive bacteria but poor effect over gram negative bacteria, mycobacteria, fungi and viruses.
Triclosan is an organic phenyl ether with good activity against gram positive bacteria and effective
to some extent against many gram negative bacteria including Pseudomonas. It also has fair activity
on fungi and viruses.

Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic soaps.
Chloroxylenol is inactivated by hard water.

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. 10% Povidone Iodine is used undiluted in pre and
postoperative skin disinfection. Chlorine gas is used to bleach

water. Household bleach can be used to disinfect floors. Household bleach used in a stock dilution
of 1:10. In higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium
hypochlorite is used in serology and virology. Used at a dilution of 1:10 in decontamination of
spillage of infectious material. Mercuric chloride is used as a disinfectant.

Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is corrosive
and staining.

Bleach solution is corrosive and will corrode stainless steel surfaces.

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: 1% silver nitrate solution can be applied on eyes as treatment for opthalmia
neonatorum (Crede’s method). This procedure is no longer followed. Silver sulphadiazine is used
topically to help to prevent colonization and infection of burn tissues. Mercurials are active against
viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used in
preservation of serum. Copper salts are used as a fungicide.

Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

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 (or quat). 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.

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.

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.

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.

Efficiency testing is done using Bacillus subtilis var niger.

Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.

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 ketane 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.

4) Factors affecting the efficacy of chemical disinfectant

Concentration of the disinfectant solution

Using an unnecessarily high concentration of disinfectant is not
only wasteful but can cause damage
to human tissue. Traumatized tissue
will be most susceptible to further
infection, particularly for those who are in daily contact with numerous organisms. If the
concentration of disinfectant is too low it will be ineffective.
TIME
Time is closely related to the
necessity for correct concentration.
The important principle of strong
enough for long enough must be
upheld if adequate disinfection is to
take place. About one hour of contact should generally be allowed. It must also be
remembered that Gram-negative organisms take longer to destroy by chemical means
than Gram-positive organisms.
Another important factor to bear
in mind is that mist disinfectants
will be more effective in warm conditions than in cold conditions.
Within the limits of the thermostability of the substance used one should therefore use
hot water for making up the solution. This generally speeds up the chemical reaction
and also lowers surface tension.

Surface tension
This is a vitally important aspect of disinfectants and those with a low surface tension
are of great value. There are many substances such as household detergents which are
used for cleaning purposes by virtue of the fact that they lower surface tension. Other
substances, such as the quaternary ammonium compounds are both disinfectants and
surface tension reducers. Unfortunately many of
these substances are associated with
foaming and the suds cause endless
problems for hospital sanitarians.
Sometimes a surface tension reelimination by thorough washing of
an item is not possible, solid masses,
such as faeces, must therefore be
broken up and the quantity of disinfectant increased to cope with blood or mucus.
Obviously, complete contact between items and the disinfectant is required. Air bubbles
must be
eliminated and objects completely
submerged in the disinfectant solution. Oil or grease may also need to be cleaned off
initially.
Deterioration of solution
Solutions deteriorate and as a general rule they should be replaced daily. They should
never be topped up.
Exposure to other inhibiting factors
One should always be aware of
factors which impair disinfectant
action. For example, cotton wool swabs should not be applied directly to the top of a
bottle of any quaternary ammonium compound — by the time the bottle is half used
the substance will be ineffective.
Number of organisms
In the presence of many microorganisms, such as in feces or sputum , they tend to
clump together and are further protected by the presence of organic material. In such
cases the penetration of the disinfectant is impaired and the exposure time and/or the
quantity of solution must be increased.
Species of organisms
a knowledge of the potential pathogens which may be present in a certain situation
will result in the selection of a more
appropriate disinfecting agent.
Bedpans will probably be contaminated by Gram-negative organisms and using a
substance such as Chloroxyanol for disinfection will be a waste of time and even
dangerous if it is then assumed that the bedpans are free from pathogens.

SUBMITTED BY
VELLORE RAJENDRAN VIMAL
11/24/2020