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EXERCISE NO. 3
METHOD OF STERILIZATION
(CHEMICAL METHOD)

I. OBJECTIVE :

At the end of the activity, the students should be able to show the
effectiveness of some chemical agents commonly used to kill the bacteria in
various routine procedures in the hospital.

II. INTRODUCTION :

Disinfection plays a very important role in infection control not only in the
hospital but also at home. It can be carried out by a variety of chemical
agents belonging to several classes among which are detergents, phenols,
organic solvents, alcohols, acids, used alkali, salts of heavy metals, oxidizing
agents, coal, tar dyes and alkalizing agents.

Each class of disinfection has its own mode of action. There are 2
common antimicrobial modes of action for disinfectants and antiseptics.

1. They may damage the lipids and/or proteins of the semipermeable

cytoplasmic membrane of microorganisms resulting in leakage of cellular
materials needed to sustain life.

2. They may denature microbial enzymes and other proteins, usually by

disrupting the hydrogen and disulfide bonds that give the protein its three
dimensional functional shape. This blocks metabolism.

The more commonly used disinfectants are as follows

1. Phenol and phenol derivatives

Phenol (50-10%) was the first disinfectant commonly used. However,

because of its toxicity and odor, phenol derivatives are now generally
used. These include orthophenylphenol, hexachlorophene, triclosan,
hexylresorcinol, and chlorhexidine. Orthophenylphenol is the agent
Lysol®.Hexachlorophene in a 3% solution is combined with detergent
and is found in Phisohex®. Triclosan isa phenolic antiseptic very
common in antimicrobial soaps and other products. Hexylresorcinol is in
throat lozenges. A 4% solution of chlorhexidine in isopropyl alcohol and
combined with detergent is a common handwashing agent and surgical
scrub. These agents kill most bacteria, most fungi and some viruses, but
are usually ineffective against endospores. They alter membrane
permeability and denature proteins.
2. Soaps and detergents :

Soaps are only mildly microbicidal. Their use aids in the mechanical
removal of microorganisms by breaking up the oily film on the skin
(emulsification ) and reducing the surface tension of water so it spreads
and penetrates more readily. Some cosmetic soaps contain added
antiseptics to increase antimicrobial activity. Detergents maybe anionic
or cationic. Anionic (negatively charged) detergents such as laundry
powders , mechanically remove microorganisms and other materials
but are not very microbicidal. Cationic (positively charged) detergents
alter membrane permeability and denature proteins. They are
effective against many vegetative bacteria, some fungi, and some
viruses. However, bacterial endospores and certain bacteria such as
Mycobacterium tuberculosis and Pseudomonas species are usually
resistant. They are also inactivated by soaps and organic materials like
excreta. Cationic detergents include the quaternary ammonium
compounds such as benzalkonium chloride and zephiran.

3. Alcohols

70% solutions of ethyl or isopropyl alcohol are effective in killing

vegetative bacteria, enveloped viruses, and fungi. However, they are
usually ineffective against endospores and non-enveloped viruses. Once
they evaporate, their cidal activity will cease. Alcohols denature
membranes and are often combined with other disinfectants, such as
iodine, mercurial, and cationic detergents for increased effectiveness.

4. Acids and alkalis

Acids and alkalis alter membrane permeability and denature

proteins and other molecules. Salts of organic acids, such as calcium
propionate, potassium sorbate, and methylparaben, are commonly used
as food preservatives. Undeccylenic acid (Desenex®) is used for
dermatophyte infections of the skin. An example of an alkali is lye
(sodium hydroxide).

5. Heavy metals

Heavy metals, such as mercury, silver, and copper, denature

proteins. Mercury compounds (mercurochrome, merthiolate) are only
bacteriostatic and are not effective against endospores. Silver nitrate
(1%) is sometimes put in the eyes of newborns to prevent gonococcal
ophthalmia. Copper sulfate is used to combat fubgal diseases of plants
and is also a common algicide. Selinium sulfide kills fungi and their
spores.

6. Chlorine

Chlorine gas reacts with water to form hypochlorite ions, which in

turn denature microbial enzymes. Chlorine is used in the chlorination
drinking water, swimming pools and sewage. Sodium hypochlorite is the
 active agent in household bleach. Calcium hypochlorite, sodium
hypochlorite and chloramines (chlorine plus ammonia) are used to
sanitize glassware, eating utensils, dairy and food processing equipment,
and hemodialysis systems.

7. Iodine and Iodophores

Iodine also denatures microbial proteins and is usually dissolved in

an alcohol solution to produce a tincture. Iodophores are a combination of
iodine and an anionic detergent ( such as polyvinylpyrolidone) tat reduces
surface tension and slowly releases the iodine. Iodophores are less
irritating than iodine and do not stain. They are generally effective against
vegetative bacteria. Mycobacterium tuberculosis, fungi, some viruses, and
some endospores. An example is Betadine.

8. Aldehydes

Aldehydes, such as formaldehyde and glutaraldehyde, denature

microbial proteins. Formalin (37% aqueous solution of formaldehyde
gas) is extremely active and kills most forms of microbial life. It is used in
embalming, preserving biological specimens, and in preparing vaccines.
Alkaline glutaraldehyde (Cidex®) , acid glutaraldehyde (Sonacide®) , and
glutaradehyde phenate solutions (Sporicidin®) kill vegetative bacteria in
10-30 minutes and endospores in about 4 hours. A 10 hour exposure to a
2% glutaraldehyde solution can be used for cold sterilization of
materials.

9. Ethylene oxide gas

Ethyline oxide is one of the very few chemicals that can be relied
upon for sterilization (after 4-12 hours exposure) . Since it is explosive,
it is usually mixed with inert gases such as freon or carbon dioxide.
Gaseous chemosterilizers, using ethylene oxide , are commonly used
to sterilize heat sensitive items such as plastic syringes, petri plates,
textile sutures, artificial heart valves, heart-lung machines, and
mattresses . Ethylene oxide has a very penetrating power and
denatures microbial proteins. Vapors are toxic to the skin, eyes, and
mucous membranes and are also carcinogenic.

In this exercise, the lethal action of each agent will be evaluated by

determining the number of bacteria that survive after a brief exposure to
the agent.

III. MATERIALS :
Group 1 - 1 : 1000 solution of Zephiran (cationic detergent)
Group 2 - 1 : 1000 solution of merthiolate
Group 3 - 5% Phenol
Group 4 – 70% Alcohol
Group 5 – Povidone iodine
Group 6 – Cidex (hexachlorophene)
 Each group will have :
1 nutrient agar plate
4 test tubes with 1 ml sterile water
1 tube containing heavy suspension of S. aureus
1 tube containing dilute suspension of S. aureus
2 ml sterile pipette
1 test tube rack
1 alcohol lamp
1 inoculating loop
1 aspirator

IV. PROCEDURE :
1. Divide the nutrient agar plates into 4 quadrants. Label the quadrants I, II,
III, IV. Indicate the chemical agent to be used.
2. For each of the chemical agent to be used , prepare
4 tubes containing sterile water – label the tubes 1,2,3,4
1 tube containing heavy suspension of S. aureus (Tube H)
1 tube containing dilute suspension of S. aureus ( Tube D)
3. To the tube containing the dilute solution of bacteria (Tube D) do the ffg :
a. Transfer a loopful of suspension to Tube 1. Mix by gently shaking the
tube. Flame sterilize the wire loop and streak a loop full from Tube 1 on
quadrant 1 of the agar plate.
b. Pipet 0.3 ml of the chemical disinfectant and add to the Tube D. Mix .
after 1 minute transfer a loop full from Tube D to Tube 2. Mix gently.
Flame sterilize the wire loop and streak a loop full from Tube 2 to
quadrant II.
4. To the tube containing the heavy solution of bacteria (Tube H), do the ffg :
a. Add 0.3 ml of the chemical disinfectant to the tube containing heavy
solution or Tube H and mix well. After 1 minute, transfer a loop full
from Tube to Tube 3 and mix well. Flame sterilize the wire loop and
streak a loop full from tube 1 to quadrant III.
b. After 3 minutes of adding the chemical disinfectant, transfer again a
loop full from Tube H to Tube 4 and mix well. Flame the wire loop and
streak a loop full from tube 4 to Quadrant 4.
5. Invert and label all plates and incubate for 18-24 hours. Follow up and
record all results.
6. Observe the growth in all quadrants and record the results in the table
below.

Chemical Agent I II III IV

(Tube D, no (Tube D, (Tube H, (Tube H,
treatment) after 1 after 1 after 3 mins
minute of minute of of the
the agent) the agent) agent)
Zephiran ++++ ++ ++ +
Merthiolate ++++ +++ +++ ++
Phenol ++++ + ++ +
Alcohol ++++ + ++ +
Povidone Iodine ++++ - - -
Hexachlorophene ++++ ++ ++ +
V. QUESTIONS :
1. Differentiate between disinfectant and antiseptic.

Disinfectant eliminates most bacteria. most microbial forms, and some

endospores. It is used on inanimate objects, surfaces, and environment.
Specifically, these disinfectants are categorized as high-level disinfectants.
However, some disinfectants are sporicidal, which are either categorized as
intermediate and low-level disinfectants (Murray et al., 2016). Some
examples of intermediate-level disinfectants include alcohols, iodophor
compounds, and phenolic compounds, while low-level disinfectants include
quaternary ammonium compounds. Disinfectants are considered bactericidal.

On the other hand, an antiseptic is a chemical agent used to reduce and

prevent growth of the microorganism in living tissues. It usually has a lower
concentration of biocide than disinfectants. With this, antiseptics are
considered as bacteriostatic.

2. Which of the chemical agents in the experiment is the most effective ?

The most effective is povidone iodine. This is considered as an iodophor

compound and is the most effective halogen for disinfection. While iodine is
highly reactive, povidone iodine is made stable and can be used in living
tissues. Povidone iodine is iodine complexed with polyvinylpyrrol. It
precipitates proteins and causes oxidation of cellular enzymes needed for
the cell’s function (Murray et al., 2016). It is considered bactericidal,
fungicidal, mycobactericidal, viricidal, and sometimes sporicidal. Due to this
broad spectrum targeting, it is the most effective among the chemicals used.

3. Which of the chemical agents used is sporicidal ?

Iodine is sporicidal which can rapidly penetrate the spore and oxidize
proteins and enzymes. The slow release of iodine also helps with this action.

4. Give the mechanism of action of the disinfectant used.

Write your answers in tabulated form as follows

Chemical Agent Mechanism of Action

Zephiran Is a benzalkonium chloride compound classified as a
quaternary ammonium compound. It denatures cell
membrane and release intracellular components.
Many viruses and bacterial endospores are resistant.
It can be bactericidal at high concentrations (Murray
et al., 2016).

Merthiolate Merthiolate or thiomersal is an alkylmercury

compound, a heavy metal derivative. Mercuric ions
interfere with disulfide linking of sulfhydryl groups by
 combining with these groups. (Riedel et al., 2019)

Phenol Phenols disrupt lipids in the membrane by irreversible

inactivation of membrane-bound oxidases and
dehydrogenases. Thus, damaging the enzymes. This
causes leakage of cell contents.

Alcohol Bactericidal against vegetative bacteria, some fungi,

and viruses. It acts by drawing out water from the cell
and denature proteins (Murray et al., 2016).

Povidone Iodine Kills most vegetative cells and is found to be effective

against spores. It causes denaturation of proteins and
oxidation of enzymes which leads to precipitation of
bacteria.

Hexachlorophene This is a halogenated bisphenol which has activity

against gram-positive bacteria. This disrupts the cell
membrane, thus inhibiting the membrane-bound
electron transport chain.

REFERENCES:

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology (8th
ed.). Elsevier.

Riedel, S., Morse, S. A., Mietzner, T. A., & Miller, S. (2019). Jawetz, Melnick &
Adelberg’s Medical Microbiology (28th ed.). McGraw-Hill Education.