• Disinfection

• is the killing, inhibition or removal of

microorganisms that may cause
diseases.
• primary goal is to reduce pathogens

• Disinfectants – are usually chemical

agents that perform disinfection and
normally used only on inanimate objects.

*A disinfectant does not necessarily sterilize an object for

viable spores and few microorganisms may remain.
• Sanitation
• the process where the microbial
population is reduced to levels that
are considered safe by public health
standards.

• Antisepsis
• is the prevention of infection or
sepsis especially on host tissue
accomplished by chemical agents
called antiseptics that prevent
infection by killing or inhibiting
pathogen growth and reduce total
microbial nutrition.
Concepts in Antimicrobial Control

Processes Agents
 Modes of Action of Antimicrobial Agents
• An antimicrobial agent’s adverse effect on cells = mode (or mechanism) of
action.
• Agents affect one or more cellular targets, inflicting damage progressively
until the cell is no longer able to survive.
• Antimicrobials have a range of cellular targets,
• Agents that are least selective in their targeting = effective against the widest range of
microbes (examples include heat and radiation).
• Selective agents (e.g. drugs) tend to target only a single cellular component and are
much more restricted as to the microbes they are effective against.
• The cellular targets of physical and chemical agents fall into four general
categories: 1. the cell wall, 2. the cytoplasmic membrane, 3. cellular synthetic
processes (DNA, RNA), and 4. proteins.
• 1. Physical Control
• 2. Chemical Control
 Physical Control of Microbes

• Heat
• Radiation
• Filtration
 HEAT
• Heat is the most widely used method of
microbial control. It is used in
combination with water (moist heat) or
as dry heat (oven, flames).
• As a rule, elevated temperatures
(exceeding the maximum growth
temperature) are microbicidal, whereas
lower temperatures (below the
minimum growth temperature) are
microbistatic.
 • Heat is the most widely used method of microbial control. It is used in
combination with water (moist heat) or as dry heat (oven, flames).

Moist Heat Dry Heat

- Occurs in the form of hot water, boiling - Refers to hot air (such as in an oven)
water, or steam (vaporized water). or an open flame.
- In practice, the temperature of moist - In practice, the temperature of dry
heat usually ranges from 60°C to 135°C. heat ranges from 160°C to several
- Although many cellular structures get thousand degrees Celsius.
damaged, the most lethal effect is the - Dry heat dehydrates the cell, removing
coagulation and denaturation of the water necessary for metabolic
proteins, which quickly and permanently reactions, and it also denatures
halts cellular metabolism. proteins
• Bacterial endospores exhibit the greatest resistance and the vegetative forms
of bacteria and fungi are the least resistant to both moist and dry heat.
• Destruction of endospores usually requires temperatures above boiling.
 Susceptibility of Microbes to Heat:
Thermal Death Measurements
• A combination of the two variables, heat and time, constitutes
the thermal death time, or TDT, defined as the shortest length
of time required to kill all test microbes at a specified
temperature.
• The TDT has been experimentally determined for the microbial
species that are common or important contaminants in various heat-
treated materials.
• Another way to compare the susceptibility of microbes to heat
is the thermal death point (TDP), defined as the lowest
temperature required to kill all microbes in a sample in 10mins.
Moist Heat Control Methods
Moist Heat Control Methods
Pasteurization is a process where substances such as milk are
treated with controlled heating at temperatures well below
boiling point.
Milk can be pasteurized in two ways:
• 1) In the older method, the milk is held at
63˚C for 30 minutes.
• 2) Flash Pasteurization
= also called as High-Temperature Short-Term
(HTST) Pasteurization
= here, large amount of substances like milk are
subjected with quick heating to about 72˚C for
15 secs, then rapid cooling.
• 3) Ultrahigh-temperature (UHT)
sterilization
• = milk and milk products are heated at 140 to
150˚C for 1 to 3 seconds
Moist Heat Control Methods
• Moist heat sterilization must be carried out at temperatures
above 100˚C in order to destroy bacterial endospores, and
this requires the use of saturated steam under pressure.
• Steam sterilization is carried out with an autoclave, a device
somewhat like a fancy pressure cooker.
• Water is boiled to produce steam, which is released through the
jacket and into the autoclave’s chamber.
• The air initially present in the chamber is forced out until the
chamber is filled with saturated steam and the outlets are closed.
• Hot, saturated steam continues to enter until the chamber reaches
the desired temperature and pressure, usually 121˚C and 15 pounds
of pressure.
• At this temp. saturated steam destroys all vegetative cells and
endospores in a small volume of liquid within 10 to 12 minutes.
• Treatment is continued for about 15 minutes to provide a margin of
safety.
• Things to remember when autoclaving:
1) All air should be flush out of the chamber or else it will not reach 121˚C.
2) The chamber should not be packed too tightly so that the steam could circulate freely and
contact everything in the autoclave.
3) When a large volume of liquid needs to be sterilized, extended sterilization time will be needed.
(5 L of water = 70 mins)
Dry Heat Control Methods
Dry Heat Control Methods
 Radiation
• UV radiation kills all kinds of microorganisms due to its short
wavelength (approx. 10 to 400 nm) and high energy.
• The most lethal UV radiation has the wavelength of 260 nm, the
wavelength most effectively absorbed by DNA.
Radiation
Radiation
 Filtration
• it is an excellent way to reduce microbial population; rather than
directly destroying contaminating microorganisms, the filter simply
removes them.
Filtration
Filtration
• 1. Physical Control
• 2. Chemical Control
 Chemical Control of Microbes

• Antimicrobial chemicals occur in the

liquid, gaseous, or even solid state.
• They range from disinfectants and
antiseptics to sterilant and preservatives
• Many solid or gaseous antimicrobial
chemicals are dissolved in water, alcohol,
or a mixture of the two to produce a liquid
solution
• Solutions containing pure water as the
solvent are termed aqueous, whereas
those dissolved in pure alcohol or
water-alcohol mixtures are termed
tinctures.
Chemical Control of Microbes

• Desirable Qualities of Microbicidal

Chemicals
• rapid action even in low concentrations
• solubility in water or alcohol and long-term
stability
• broad-spectrum microbicidal action
without being toxic to human and animal
tissues
• penetration of inanimate surfaces to
sustain a cumulative or persistent action
• resistance to becoming inactivated by
organic matter
• not corrosive and non-staining
• sanitizing and deodorizing properties
• affordability and availability
 • The 3 levels of chemical decontamination procedures
Levels of Effectives of Germicides are high, intermediate, and low.
• High-level germicides kill endospores and, if
properly used, are sterilants.
• Materials that necessitate high-level control are
medical devices—for example, catheters, heart-lung
equipment, and implants—that are not heat-
sterilizable and are intended to enter body tissues
during medical procedures.
• Intermediate-level germicides kill fungal (but
not bacterial) spores, resistant pathogens such
as the tubercle bacillus, and viruses.
• They are used to disinfect items (respiratory
equipment, thermometers) that come into intimate
contact with the mucous membranes but are
noninvasive.
• Low-level germicides eliminate only vegetative
bacteria, vegetative fungal cells, and some
viruses.
• They are used to clean materials such as electrodes,
straps, and pieces of furniture that touch the skin
surfaces but not the mucous membranes.
 Chemical Control of Microbes

Factors Affecting the Germicidal

Activity of Chemicals
• Nature of the microorganisms being
treated
• Nature of the material being treated
• Degree of contamination
• Time of exposure, and
• Strength and chemical action of the
germicide.
Chemical Control of Microbes
• Inactivates enzymes that might
disrupt cell morphology and
toughens cell structures so that
they do not change during staining
and observation.
• A microorganism usually is killed
and attached firmly to the
microscope slide during fixation.
 Two Types

1. Heat Fixation
• bacterial smears are fixed by
gently flame heating an air-dried
film of bacteria.

• it adequately preserves overall

morphology but not structures
within cells.
 Two Types

2. Chemical Fixation
• chemical fixatives penetrate cells and
react with cellular components, usually
proteins and lipids, to render them
inactive, insoluble, and immobile.
• used to protect fine cellular
substructure and the morphology of
larger, more delicate microorganisms.
• common fixative features contain such
components as ethanol, acetic acid,
mercuric chloride, formaldehyde, and
glutaraldehyde.
• Advantages of staining:
• 1. Provides contrast between
the microorganism under
study and the background.
• 2. Allows the study of the
cell’s structure such as
nucleus, vacuoles, cell wall
and other parts of the
microorganism.
• are salts composed of cations and anions
affected by pH
• all dyes have two common features:
• a. They all have chromophore groups, groups
with conjugated double bonds that give the dye
its color.
• b. They can bind with cells by ionic, covalent, or
hydrophobic bonding (+ charged stains binds
with – charged structures).
1. Basic Dyes/ Stains
• methylene blue, basic fuschin, crystal violet,
safranin, malachite green
• have positively charged groups (usually some
form of pentavalent nitrogen) and generally sold
as chloride salts.
• → Basic dyes bind to negatively charged
molecules like nucleic acids and many
proteins.
• → Basic dyes/ stains are usually used to
bacteria since their cell surface is negatively
charged.
• 2. Acid Dyes/ Stains
• eosin, rose bengal and acid fuschin
• possess negatively charged groups such as
carboxyls ( - COOH) and pentahydroxyls ( - OH)
• → Acid dyes/ stains bind to positively charged
cell structures.

• The pH may alter staining effectiveness since the nature and

degree of the charge on the cell components change with pH
• → Anionic dyes stain best under acidic conditions
• → Basic dyes are most effective at higher pHs
 TECHNIQUES

1. Positive and Negative

Staining
2. Simple and Differential
Staining
a. Acid-Fast Staining
b. Endospore Staining
3. Special Staining
a. Capsular Staining
b. Flagellar Staining
 Positive and Negative Staining
• Positive Staining = involved most procedures
in which the dye actually sticks to the
specimen and gives it color.
• Negative stain = The dye does not stick to
the specimen but settles some distance from
its outer boundary, forming a silhouette.
• Nigrosin (blue-black) and India ink (a black suspension
of carbon particles) are the dyes most commonly used
for negative staining.
• The cells themselves do not stain because these dyes
are negatively charged and are repelled by the negatively
charged surface of the cells.
• The value of negative staining is its simplicity and the
reduced shrinkage or distortion of cells, as the smear
is not heat fixed.
• Negative staining is also used to accentuate the
capsule that surrounds certain bacteria and yeasts.
Simple Staining
• the simplest type of staining in which a
single staining agent is used.
• the value of simple staining lies in its
simplicity and ease of use
• here, the fixed smear is covered with a
single staining agent, excess stain is
washed off with water, and the slide is
blotted dry.
• basic or acid dyes are used for simple
staining frequently to determine the
size, shape and arrangement of
bacteria.
 Differential Staining
• Differential stains use two differently
colored dyes, called the primary dye and
the counterstain, to distinguish between
cell types or parts.
• Differential staining techniques tend to
be more complex and sometimes require
additional chemical reagents to produce
the desired reaction.
• Typical examples include the Gram stain,
the acid-fast, and the endospore stain.
Some staining techniques (endospore,
capsule), which are differential, are also in
the “special” category.
Differential Staining
1) Gram Staining
• developed by Danish Physician Christian Gram
• = it is the most widely employed staining method in
bacteriology.
• = it is a differential staining because it divides bacteria into
two classes – Gram Negative & Gram Positive Bacteria
• General Steps:
• a) Fixation
• b) The smear will be stained with the basic dye crystal violet
or the primary stain.
• c) Followed by treatment with an iodine solution functioning
as mordant (increases the interaction between the stain and
the cell).
• d) Then, the smear is decolorized by washing with ethanol
or acetone.
• e) Finally, the smear is counterstained with a simple, basic
dye different from the crystal violet such as Safranin which
gives pink to red color with gram-negative bacteria.
 • General Steps:
Differential Staining • a) Fixation
• b) The smear will be stained with
the basic dye crystal violet or the
primary stain.
• c) Followed by treatment with an
iodine solution functioning as
mordant (increases the
interaction between the stain and
the cell).
• d) Then, the smear is decolorized
by washing with ethanol or
acetone.
• e) Finally, the smear is
counterstained with a simple,
basic dye different from the
crystal violet such as Safranin
which gives pink to red color with
gram-negative bacteria.
 Differential Staining

2) Acid-fast Staining

• Such staining method uses harsher treatment – the Ziehl-Neelsen Method or

heating with a mixture of basic fushin and phenol.
• Used in staining Mycobacterium tuberculosis and M. leprae which are
responsible for tuberculosis and leprosy.
 Acid-Fast Staining
• In this staining method, once the basic
fuschin has penetrated with the aid of
heat and phenol, the acid-fast cells are
not decolorized by an acid-alcohol wash
and thus, remain red (due to high lipid
concentration on the wall).

• Non-acid-fast bacteria are decolorized by

acid-alcohol and when counterstained
with methylene blue remain blue.
 Endospore Staining
• The endospore stain (spore stain) is
similar to the acid-fast method in that
a dye is forced by heat into resistant
bodies called endospores
• common spore staining process is
known as Schaeffer-Fulton
Procedure:
• In this procedure, a bacterium with
endospore is heated with malachite
green (a very strong stain that can
penetrate endospore).
• Then, the rest of the cell is washed free
of dye with water and is counterstained
with safranin.
 Flagella Staining
• such procedure provide • then, they are stained with
taxonomically valuable information pararosaniline (Leifson Method) or basic
about the presence and distribution fuschin (Gray Method)
pattern of flagella.
• however, flagella is a very thin cell
appendage and can only be seen in
electron microscope.
• to be seen on light microscope:
• the thickness of the flagella is
increased by coating them with
mordant such as tannic acid and
potassium alum