Faculty of Medicine and Health Sciences

General Microbiology Lab (7105404)

Lab-10
Disinfectant and phenol coefficient

Second Semester 2020-2021

1
Introduction:
in certain situations, it is important to control microbial growth either through their
destruction, elimination or inhibition of their growth.

Examples:

Surgical equipment and injected-medications and fluids must be completely germ-free.

Wounds must be treated with certain chemicals (such as Ethanol and Iodine) that can kill
microbes so as to decrease the possibility of wound infection.

Microbial culture media and equipments used during the culturing process must be germ-
free (to avoid contamination).

Surfaces of non-living objects (benches of microbiology labs, bathrooms and floors) can
be treated with certain chemicals such as Ethanol and Dettol so as to kill most microbes.

Washing of hands with detergents and treatment of water with Chlorine (tap water and
swimming pools).
Introduction Cont----:

Preservation of food by using certain methods that decrease or inhibit microbial growth:
1. Freezing and refrigeration
2. Pasteurization
3. Utilization of food preservatives
4. Dehydration and salting
5. Reducing water availability
Definitions:

Sterilization: it is a process that kills or removes all microbes, including bacterial endospores.

Sterilization can be achieved by either physical (most common) or chemical methods (less
common).

Disinfection: it is a process that kills or remove most microbes.

Disinfection can be achieved mainly by certain chemicals known as disinfectants (such as

Ethanol, Iodine, Phenol, Chlorine…….)

Disinfectants: these are chemical substances that either kill microbes or inhibits their growth.

Note: not all disinfectants can be applied on living tissue, because some of them have harmful
effects

Antiseptic: these are a group of disinfectants that are safe to be applied onto living tissue (such
as skin or external wounds) so as to kill vegetative pathogens in order to avoid infection.

Note: Sepsis means the presence of microbes in tissue

Note: high osmotic pressure reduces water availability needed for microbial survival
and/or replication

Examples: Honey and Jam

5
Sterilization Methods:
Physical Methods (commonly-used):

I- Heat-based sterilization: only heat-stable objects or fluids can be sterilized using this method.
Heat, can be classified into:
A- Dry-heat based sterilization:
1- Flaming: during this process, objects that are being sterilized are exposed to a direct flame
(e.g. Bunsen Burner). Metallic loops and needles used during culturing of bacteria are sterilized
by flame.

2- Hot-air (Hot Air Oven): during this process, objects that are being sterilized are exposed to
hot-air (160° to 180°C for 2 hours). This method can be used to sterilize glass and metallic
objects.
Sterilization Methods Cont ----:
B- Moist-heat based sterilization (Autoclaving): it is a very common sterilization method that
utilizes pressurized hot water vapor, under the following conditions:
 Temperature: 121°C
 Pressure: 15 pounds /square inch
 Time: 15 to 20 minutes
Autoclaving can be used to sterilize glass and metallic objects, water, heat-stable solutions and
culture media, certain plastic objects (made of autoclavable plastic material),biological wastes…
II- Radiation-based sterilization:

This process involves the utilization of high-energy radiations such :

1.Ultraviolet (UV) light

2.Gamma rays (ionizing radiation)
3. X-rays (ionizing radiation)

Exposure of microbes to high levels of these radiation results in massive mutations in their genetic
materials that either lead to their death or inhibition of their replication.

This method is mainly used to sterilize heat-sensitive plastic objects such as plastic Petri dishes and
plastic syringes
III- Filtration-based sterilization: this method is mainly used to sterile heat-sensitive liquids . It
involves the utilization of filters with pores that are smaller than the size of microbes. During this
process, microbes are physically-removed (excluded) while the fluid passes through the pores of the
used filter, so that the filtered liquid becomes germ-free (sterile).
Chemical Methods of Sterilization (less common):
1-Ethylene Oxide:

Ethylene Oxide is a gas that is used in 10% concentration (mixed with Nitrogen gas). It
can kill microbes and their spores by alkylating their proteins and nucleic acids.
It can be used to sterilize heat-sensitive materials such as surgical instruments and plastics.

2- By using Glutaraldehyde: It can kill microbes and their endospores by denaturing their
proteins and nucleic acids.
Disinfection:
As mentioned earlier, disinfection is mainly conducted by chemical agents (disinfectants) that
can either kill microbes or inhibit their growth. Killing of microbes by these agents occurs
through:

 Disruption of their cell membranes (such as detergents , Ethanol, Phenol and its
Phenolics derivatives) (see next slide regarding Phenol and its derivatives).

 Denaturation of their proteins and enzymes (such as Ethanol, Iodine, chlorine, heavy-
metal ions, Hydrogen Peroxide, Formaldehyde, Glutaraldehyde and Phenol and its derivatives
(Phenolics and Bisphenols).

 Induction of mutations in their genetic material (such as formaldehyde,

glutaraldehyde, ethylene oxide and radiations).
Important notes:

1-Ethanol: it is a bactericidal and fungicidal but is ineffective against bacterial

endospores. To be effective, ethanol requires the presence of water so as to
facilitates its penetration through both the microbial cell membrane an the
microbial cytoplasm. Accordingly, 70% ethanol is used for disinfection.

2-Chlorine: Commonly used to kill microbes in municipality water supplies as

well as in swimming pools. It is active against endospores. 

3- Iodine: It is the most effective skin antiseptic used in medical practice.

The bactericidal activity of a chemical can be determined by several methods:

1- Dilution method: the antimicrobial activity of a chemical is determined by exposing a test microbe
to serial dilutions of that chemical and measuring the killing time of each of the used dilutions.

2- Disk-diffusion method: this method is similar to the disk-diffusion method used to assess
antibiotics susceptibility. During this method, the antimicrobial activities of certain chemicals are
determined by measuring the diameter of their inhibition zones.

Phenol coefficient:

Phenol was one of the first chemical used in disinfection. Accordingly, the antimicrobial activity of a
chemical ((used as disinfectants or antiseptics) is assessed in relevant to the antimicrobial activity of
phenol.

Accordingly, Phenol coefficient is a measurement that determines the antimicrobial activity of a

chemical in in comparison of the antimicrobial activity of phenol.

It can be determined by calculating the ratio of the highest dilution of that chemical that kills the test
microbe in 10 min (not in 5 min) to the highest dilution of phenol that kills the tested microbe in the
same time period (10 min).
 Phenol Coefficient

Disinfectants that are more effective than phenol have a coefficient > 1. Those that are less
effective have a coefficient < 1.
Experiment:

The antibacterial activities of several chemicals (disinfectants and antiseptics) will be

determined by the disk-diffusion method and compared to the antibacterial activity of phenol.
Procedure:
Prepare a bacterial suspension in normal saline or broth with turbidity equivalent to that of 0.5
McFarland standard.
By using a sterile cotton swab, inoculate Mueller-Hinton agar plates with the test bacterium
uniformly.
Impregnate sterile circular filter papers with 25 µl to each of the chemical intended to be tested
and phenol (reference disinfectant).
Then, place each of the impregnated filter papers at the center of the plates inoculated with the
test bacterium.
 Incubate the plates for 24h at 37 ºC.
Measure the diameter of the inhibition zones.

Determine the ratio of the diameter of the inhibition zone of each of the tested chemicals to the
diameter of the inhibition zone of phenol.