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Control of Microorganisms
Definition of Terms
Sterilization

the process by which all living cells (pathogenic or not), viable spores, viruses,
and viroids are either destroyed or removed from an object or habitat

process of sterilization in the laboratory = autoclave

Cleaning

the removal of dirt or visible soil from objects and surfaces

Normally accomplished manually or mechanically using water with

detergents or enzymatic products

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 Decontamination

the process of removing or neutralizing contaminants that have accumulated

on personnel and equipment

one of the most critical aspects of health and safety especially for
hazardous sites

pandemic

chemical decontamination

neutralize hazardous chemicals before they can cause harm

biological decontamination

example sa pic

radiological decontamination

nerve agent decontamination

Nerve agents are highly poisonous chemicals that work by preventing

the nervous system from working properly.

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 Disinfection

describes a process that eliminates many or all pathogenic

microorganisms, except bacterial spores on inanimate objects

Disinfectants

agents, usually chemicals, used to carry out disinfection and are normally
used only on inanimate objects

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 Sanitation

the microbial population is reduced to levels that are considered safe by

public health standards

reduced lang cuz microbes are present everywhere (air, environment)

on the preparation of products meant to be consumed

food

the focal point of public health policy

ex. pagkuha ng basura everyday

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 Antisepsis

prevention of infection or sepsis and is accomplished with antiseptics

Antiseptic

chemical agents applied to living tissue to prevent infection by killing or

inhibiting pathogen growth

living tissue or sa body ginagamit

They also reduce the total microbial population

common antiseptic solutions for cleaning wounds include

iodine

hydrogen peroxide

alcohol

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 Germicide

kills pathogens but not necessarily endospores

general term for agents used to kill germs — bacteria, viruses, fungi

specific = virucide, bactericide, fungicide, sporicide, tuberculocide

-cidal = kills

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 Bacteriostatic and Fungistatic

they do not kill, but they prevent the growth

-static = prevents or stops the growth

erythromycin

stops the production of protein in ribosomes

di na magrereplicate ung organism

Conditions Influencing the Effectiveness of

Antimicrobial Agents

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 Population Composition

endospores are more resistant than vegetative forms

dependent on the type of cell or capability of the organism

endospores are more resistant to harsh environmental conditions

vegetative forms (actively dividing) die more quickly

Concentration or intensity

generally, the higher the concentration, the better

exception for alcohol

70% alcohol vs 90-99% alcohol

alcohol at high concentrations precipitates more readily

Use of the more concentrated solutions (99%) will result in almost

immediate coagulation (turning into a solid or semi-solid state) of
surface or cell wall proteins and prevent the passage of the
alcohol into the cell.

When the outer membrane is coagulated, it protects the virus or

bacteria from letting through the isopropyl. Thus the stronger solution
of isopropyl is creating protection for the germ from the antiseptic
properties of isopropyl, rendering the virus or bacteria more

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 resilient against the isopropyl alcohol. To put it simply, higher
concentrations cause an external injury that forms a protective wall
and shields the organism.

Furthermore, 99% isopropanol evaporates very quickly which does

not allow it to penetrate cell walls and kill bacteria, and therefore isn’t as
good for disinfecting surfaces. In other words, it breaks down the
outside of the cell before it can penetrate the pathogen

alcohol at 70% nakakapasok sa microorganism

Coagulation of surface proteins proceeds at a slower pace, thereby

allowing the alcohol to enter the cell.

70% alcohol, being a dilution of absolute alcohol, contains water which

is essential in the denaturing process of proteins.

Due to the concentration difference of water and alcohol on either side

of the cell wall, 70% alcohol enters the cell to denature both
enzymatic and structural proteins. This increases the potency of its
antimicrobial properties.

Duration of Exposure

generally, longer contact time = more effective

ex. binababad ang chlorine

exceptions: volatile substances — substances that evaporate easily

alcohol = nag-eevaporate agad

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 Temperature and pH

higher temperature = more effective

more acidic = more effective

~in terms of killing or the efficacy of the antimicrobial

Local Environment

working area

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 equipment should be cleaned first to reduce microbial load before
applying any disinfectant

Methods of Sterilization

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 Physical Methods

Heat

Thermal Death Point (TDP)

the lowest temperature at which all the microorganisms in a particular

liquid suspension will be killed in 10 mins.

Thermal Death Time (TDT)

the minimal length of time for all bacteria in a particular liquid culture
to be killed at a given temperature.

💡 Both TDP and TDT are useful guidelines that indicate the severity of
treatment required to kill a given population of bacteria.

Decimal Reduction Time (DRT)

aka D value

time, in minutes, in which 90% of a population of bacteria at a given

temperature will be killed

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 Hydrated (Wet Methods)

Mechanism of Action (MOA)

coagulation of proteins

cuz proteins are the most important biological molecules like

enzymes that direct biological processes (e.g. cell division)

proteins + heat = coagulation → destroy the chemical property

of the protein and render it inactive

Methods:

+ steaming

Autoclaving

aka as steam under pressure

Conditions = 121°C, 15 psi, 15-20 min

121°C cuz ung spores ng virus namamatay at this temp and

sterilization is the process of eliminating all forms of life

need a pressure to reach 121°C

cuz boiling water is just 100°C and just stays there

15 psi para mareach ang 121°C and hindi magstay sa 100°C

psi = pounds per square inch

Autoclave assessment:

Use of autoclave tapes

like masking tape with white stripes

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 A visible reaction is a change in color or formation of stripes

dapat maging black stripes to confirm that the autoclave

was completed

Probes

devices placed inside the autoclave known as thermocouples

binubutasan isang bote ng drug para don ipapasok ung

thermocouple

thermocouples send signals to a computer to verify the

conditions of the autoclave

done by engineer

Biological indicator

use of bacterial spores like Geobacillus stearothermophilus

spores are put into an ampule and the ampule is put inside
a bottle of the drug

autoclave is complete if namatay ung spores inside

done by microbiologist

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Control of Microorganisms 15
 spores need a longer time and higher temp to be killed

Dry Methods

MOA

oxidation of cell

Toxic products of oxidation proceed cytostatic effects causing

membrane damage and leading to cell death via apoptosis or
necrosis

heating the inoculating loop

Methods

use of hot air oven, flaming, and incineration

when to use:

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 gauze, surgical dressing → disposable or infectious wastes →
incinerate

stable to heat → metals and stainless steel

less expensive and shorter time needed

Low Temperatures

MOA

stop microorganism replication

parang naghihibernate sila

long-term storage

Freezing and Refrigeration

most convenient control technique

Deep Freezing

use Liquid N2

Cryoscopic freezer for the preservation of human cells or tissues

Ultraload Freezers

preserve at -70°C

can reach 496°C

Lyophilization

aka Freeze Drying

preserve large volumes of microorganisms into powdered form

freeze-drying of bacterial cultures stabilizes the cultures for long-term

storage while minimizing the damage that may be caused by strictly
drying the sample. Many microorganisms survive well when
lyophilized and can be easily rehydrated and grown in culture media,
after prolonged periods of time in storage.

also used in the biotechnology and biomedical industries to preserve

vaccines, blood samples, purified proteins, and other biological
materials.

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 Filtration

MOA

physical separation of the organisms from a solution

solutions (liquids), powders (solids), air (gas)

Examples

Membrane Filters

Microfilter

HEPA Filter

Two types of filters

Depth Filter

consist of fibrous or granular materials that have been bonded into a

thick layer filled with twisting channels of smaller diameter

normally nakikita sa loob ng pipes na nagfifill ng drugs

Membrane Filter

are porous membranes about 0.1 mm thick, made of cellulose

acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride,
or some other synthetic material

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 Filtration of Air

Examples

use of surgical masks and cotton plugs

Laminar Flow Biological Safety Cabinets

employ HEPA filters.

High-efficiency particulate air (HEPA) filters remove almost all

microorganisms larger than about 0.3 micrometers in
diameter.

According to the Institute of Environmental Science and Technology

(IEST), there are 6 types – A, B, C, D, E & F.

use negative pressure for filtration

positive pressure

palabas hangin pag binuksan

ayaw papasukin yung mga nasa labas

negative pressure

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 paloob hangin pag binuksan

para di lumabas mga microorganism sa loob

bago lumabas yung hangin, filtered na sya

used in pharmaceutical companies for controlled rooms

may particle counter to check the prevalence of

microorganisms in the room

done by a microbiologist

Class 100

strict number of particles lang

mga biosafety lab level 4

naghahandle ng Ebola viruses, etc

Radiation

MOA

cause a lethal form of mutation

like how UV radiation can cause cancer (mutation of cells)

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 destroy bacterial endospores and vegetative cells, both prokaryotic and
eukaryotic

Gamma radiation from cobalt 60 sources is used in the cold sterilization of

antibiotics, hormones, sutures, and plastic disposable supplies

cold sterilization of antibiotics

for heat-sensitive antibiotics → active ingredient will be inactive if

heated

radiation as an alternative for filtration

Ionizing radiation

gamma rays, X-rays, and high-energy electron beams have a

wavelength shorter than that of nonionizing radiation, less than about
1 nm. Therefore, it carries much more energy

Ionizing sterilization

Sterilization by ionizing radiation, primarily by cobalt 60 gamma rays

or electron accelerators, is a low-temperature sterilization method
that has been used for a number of medical products (e.g., tissue
for transplantation, pharmaceuticals, medical devices).

excellent sterilizing agents cuz they can penetrate deep into the object
being sterilized

UV Light/Lamp

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 used in hospitals — ICUs

reduce the microbial load in the room

Food irradiation can eliminate pathogens such as E. coli, Staphylococcus

aureus, and Campylobacter jejuni

food safety process that uses radiation to kill germs that can cause
food poisoning (foodborne illness).

Chemical Agents

Characteristics to be deemed as a favorable chemical agent

must be effective against a wide variety of infectious agents

pede for bacteria, viruses, fungi

broad spectrum of activity

must be toxic to infectious agents, but not to people or corrosive to

common materials

should be stable upon storage, odorless or with a pleasant odor, soluble in

water and lipids for penetration into microorganisms

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 mask the irritating odor of chemical agents because they usually emit an
irritating odor

low surface tension so it can enter cracks in surfaces

surface tension (ST) is the ability of water or liquid to resist an outside

force

high ST = di sila maghahalo and maglalaban lang sila = di magagamit as an

excellent cleaning agent

soaps are called surfactants

surface active agents

pag oily yung gamit, brinebreak down ng soap yung oils into smaller
particles so that it could be easily washed off

low ST para magahlo sila, enter cracks, breakdown oily films and fatty
materials, and be washed off easily

relatively inexpensive

chlorine

sodium hypochlorite

mas mura kesa sa lysol ^^

Common Disinfectants and Antiseptics (cont. ^)

Phenols and Phenolics

MOA

protein precipitation

First widely used antiseptic and disinfectant from where other disinfectants
are compared to

Used as the standard for determining the disinfectant property of substances

— Phenol coefficient

efficacy of antiseptic/disinfectant vs phenol (1 efficacy ng phenol) →

comparison nila

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 ex. Formalin (formaldehyde) has a phenol coefficient of around 0.3 and
compared to phenol, phenol is still better

Employed by Joseph Lister in 1867 to reduce the risk of infection during

operations

Examples: Cresol, Xylenols, Orthophenol

derivatives of phenol

Biguanides

MOA

primarily affecting bacterial cell membranes

disrupts the cell membrane para maglyse yung organism

breakdown of the cell membrane

They are especially effective against gram (+) and gram (-) bacteria

The best-known biguanide is chlorhexidine, which is frequently used for

microbial control of the skin and mucous membranes

common in mouth wash

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 Alcohols

MOA

protein denaturant

Most widely used disinfectants and antiseptics.

Bactericidal and fungicidal but not sporicidal

Some lipid-containing viruses are also destroyed

The recommended optimum concentration of ethanol is 70%

high concentration of alcohol is irritating sa skin — can dry the skin

Two most popular germicides

Ethanol

Isopropyl alcohol

aka rubbing alcohol

Ethyl and Isopropyl alcohol = are present sa market

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 Halogens

MOA

oxidizing agent

oxidize the cell membrane of microorganisms, which results in a loss of

structure and leads to cell lysis and death.

Examples: Fluorine (F), chlorine (Cl), and iodine (I)

Zonrox

Iodine

Examples: Tincture of Iodine, povidone-iodine (iodophor)

tincture = may alcohol

Tincture of Iodine = 2% iodine + 50% alcohol solution

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 Betadine ®

most common brand

maraming concentration → sa concentrations lang nagkaiba

betadine feminine wash

betadine wound solution

betadine mouth wash

Chlorine

a usual disinfectant for water supplies and swimming pools.

Sodium hypochlorite

a solution made from reacting chlorine with a sodium hydroxide

solution

30 mins contact time required

compared to phenol vv

advantage disadvantage

price carcinogenic, especially in

high concentrations in
relatively effective
drinking water

long contact time required

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 Heavy Metals

MOA

binding to -SH groups of bacterial enzymes resulting in denaturation

SH group = aka thiol group or a sulfhydryl group and more

traditionally referred to as mercaptans

enzymes = protein = contains monomers of amino acids

enzymes have active sites and most active sites contain sulfur

if nagbind don sa sulfur ung heavy metal = it will render the enzyme
inactive

di na magagamit ng bacteria ung enzyme na yun

Silver, Copper, Zinc, Mercury

Oligodynamic property

few concentrations are highly potent

The ability of very small amounts of heavy metals, especially silver and
copper to exert antimicrobial activity

silver coin itatanim sa agar → lalagyan ng microorganism → incubate →

zone of inhibition present cuz silver in coin prevented the growth of
microorganism

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 Examples

1% Silver nitrate

often added to the eyes of infants to prevent ophthalmic

gonorrhea

aka ophthalmia neonatorum or neonatal gonorrhea

irreversible blindness to the baby

if the mother has gonorrhea, the N. gonorrhoeae could enter the

eyes of the child cuz the child will be delivered thru the birth canal
and cause blindness

Silver Sulfadiazine

used on burns

burn ointment

Zinc undecylenate

used as antifungal

Copper Sulfate

an effective algaecide in lakes and swimming pools

Quaternary Ammonium Compound (Quats)

MOA

Surface-active agents

Precipitate or denature proteins

Cationic Surfactants

benzalkonium chloride, benzethonium chloride, methylbenzethonium

chloride, and cetylpyridinium chloride.

benzethonium chloride

aka hyamine

used to treat minor cuts, scrapes, wounds, or cracked skin

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 antiseptic na nilalagay sa gilid ng nails after mag manicure or
pedicure in case na during the process nasugatan ka

color red = coloring lang un pero colorless talaga

Surface-active agents or Surfactants

MOA

can decrease surface tension among molecules of a liquid.

Include soaps and detergents

Soap breaks the oily film into tiny droplets, a process called
emulsification

emulsifying agent of fats in the body = bile

Examples: sodium lauryl sulfate (SLS), soaps

SLS is present in cosmetic products, especially those that form bubbles

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 Aldehydes

MOA

acts through alkylation of amino-, carboxyl- or hydroxyl group, and

probably damages nucleic acids

alkylation

addition or removal of the alkyl group which damages the

nucleic acid

Sporicidal and can be used as chemical sterilants

Chemical sterilants are used in healthcare to chemically treat surgical

instruments and medical supplies to ensure infectious pathogens are not
transmitted to patients

Examples: formaldehyde and glutaraldehyde

formalin

embalming = high concentration = 37%

antiseptic/disinfectant = 10%

Sterilizing Gas

MOA

alkylation

Ethylene oxide (EtO)

used to sterilize many heat-sensitive items like disposable plastic Petri

dishes, syringes, heart-lung machine components, sutures, and catheters.

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 It is explosive so it is supplied in 10% to 20% concentration mixed with inert
gases — either carbon dioxide or dichlorodifluoromethane

expensive

ginagamit sa laboratories or hospitals

Dyes

Gentian violet

a common treatment for thrush — candidiasis — and some other skin, mouth,
and vaginal infections caused by fungi.

Prontosil

red azo dye also called sulfamidochrysoidine, trade name of the first
synthetic drug used in the treatment of general bacterial infections in
humans

Basic Fuchsin

active ingredient of Carbol-fuchsin solution (Castellani’s paint) for treatment

of fungal infections — ringworm and athlete’s foot

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 Methylene blue

used in simple staining

dye that is also used as a medication for conditions like UTI and an antidote
for cyanide poisoning

It is also used as an antifungal and antiseptic agent for fishes

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