[​Microbiology Notes​]

[STERILIZATION]
The process of sterilization is mainly classified into Physical and Chemical
Methods.

Physical Methods
They are further divided into three types:
1. Temperature
2. Radiation
3. Filtration

Temperature
- Effect of temperature –
● Temperature above the maximum temperature for an organism generally
has a cidal effect
● Temp below minimum temp generally causes inhibition of metabolism.
● Amount of water present in the environment at any temp also influences
the microorganism.
- Dry heat
● Hot air oven
● Direct flaming / incineration
- Moist heat
● Fractional distillation – steaming, Tydalysation – steam Arnold
● Autoclave – steaming + pressure
● Boiling
● Pasteurization
● Canning
● Hot oil
- Advantages
● Fast, reliable and relatively inexpensive
● Doesn’t introduce any chemicals
● Occurs due to alteration in the enzymes molecules or due to chemical
breakdown of structural molecules.
● Heat drives of water ​ ​ water is required by every organism thus can be
fatal to the organism.
Dry Heat Moist Heat

By oxidizing their chemical constitution By coagulating their proteins. It us much

more rapid and effective.

Do not use water ​ ​ heated air is used - Steam is used

convention currents

Lower penetration ability High penetration ability

More time required Max up to 20 mins required

Hot air oven Autoclave

A 2 ​hrs.​ exposure to dry heat is Spores can be killed with a span of 4-20
required. The temperature must be mins at 120⁰.
around 150⁰.

Difference between Moist and Dry Heat:

Instrument Principle & Working Application

Moist Heat

Autoclave Based on the principle of moist heat Sterilization of

sterilization which makes us of the lab glassware.
coagulation of cellular proteins for
killing. Water boils at 100⁰C at 1atm Pre-disposal
pressure. However, the pressure is treatment
increased up to 2 atm at which water is
boiling t 121​⁰C. (15-20 mins) Used to grow
crystals under
high temp and
pressure.

Fractional Exposure to free-flowing steam at 100​⁰C Used for

Sterilization for 30 mins, for 3 consecutive days sterilization of
with periods of incubation. things which
cannot stand
Called fractional sterilization as high pressures
sterilization was accomplished in such as
fractions. ​ ​ Tydallization germination
seeds.
Pasteurization purpose is to reduce bacterial population To prevent
and destroy organisms which may souring of wine
cause spoilage. It doesn’t affect spores. and beer.

Ex – 65-68​⁰C required to kill To increase shelf

Mycobacterium tuberculosis. life of milk

Dry Heat

Hot Air Oven It makes use of convention currents Sterilization of

in the form of heated air which in glassware.
turn penetrates the cells. The oven
uses conduction to sterilize items, It is also used to
which then absorbs the heat and dry up the
moves it towards the center of the specimens.
item.

Examples of temperature:

Radiation

- Energy transmitted through space in a variety of forms is generally called

radiation.
- They have a dual property:
● Continuous wave phenomenon
● Discontinuous particle phenomenon
- Particularly useful where high temp as a method of sterilization cannot be used.
- Better than filtration as there is no issue of clogging.

Difference between the types of radiation:

Features Ionizing Non-ionizing

Nature of X-rays and gamma rays. Rays The range of electromagnetic

rays having high eV are generally spectrum between 260-270nm
used is the most effective range of
UV used here.

Sources Gamma – isotopes of radioactive Mercury arc lamp/ Xenon arc

cobalt-60 lamp
Mode of Penetrate deep into the They generate thymine dimers
Action specimen to be sterilized and and thus are potent mutagens.
when these pass through the
cells they generate radicals
such as Hydrogen and
Hydroxyl. These radicals cause
intracellular damage.

Penetration More less

ability

Wavelength They are lesser than UV Higher wavelength

Applications 1 heat sensitive pharmaceutical 1 hospital rooms and operation

(vitamins and hormones) theaters
Sterilization
of: 2 plastic materials 2 waste water treatment

3 packaged food and medical 3 food processing areas

devices
4 dairy industries
4 X-rays ​ ​ lab equipment
5 laminar airflow chambers

Advantages 1 high penetration ability Comparatively generation of UV

is reasonable
2 great microbial cidal effect

Limitations Gamma rays 1 lesser penetration

1 safety of workers is a concern 2 only microorganism present

due to usage of radioactive on surface die
isotopes
3 potent mutagens ​
2 disposals of residue carcinogens

X-rays

1 expensive to produce

2 they are given off in all

directions from the point of
origin

Filtration
- They are now used to separate viruses from other organisms.
- Mechanical devices
 - Generally used for small scale purposes
- Very useful in water quality assessment
- Used to sterilize heat sensitive liquids such as serum, vaccines, blood products,
drugs, special nutrients added to culture media, milk, water and beer.

Factors affecting filtration:

● Pore size
● Electric current through the filter
● Charge carried by microorganism
● Nature of liquid being filtered, ​ ​ more viscous the liquid longer the time taken.
● When filtration containing fungal mycelium is done clogging is an issue.

Types of Filters:

Features Organic Inorganic Membrane

Chemical Candle filters/ Seitz filter Made of organic

nature Berkfeld/ compounds such
Kieselgurh/ as cellulose acetate
diatomaceous or polycarbonate
earth – it is a form Made of asbestos
of algae whose
outermost layer is
extremely hard Characteristic
and porous. feature – INERT
thus doesn’t react
with the material
to be filtered
Made of silica
material

Constructi Prepared by Purified asbestos is New age filter

on carefully mixing pressed together in manufactured by
purified a disc form and the company called
diatomaceous clamped tightly Millipore.
earth, asbestos and between two
other organic smooth mental
materials and by rims by screw
forming cylinders. clamps. Also called
Molecular filters.

The dried and heat The liquid to be

in a furnace up to filtered is poured
into the funnel like
 2000C to bring all structure and
materials together. drawn through the
asbestos disc by
vacuum.

These filters come in

different pore size.

Chemical Methods
Chemical antimicrobial agents are disinfectants which are generally effective in killing
negative forms of life. These may not be very affective in getting rid of sporulative forms.

Ideal Characteristics of Chemical Antimicrobial agents:

✔ Target specific in nature
✔ Broad spectrum of activity ​ ​ should be able to kill bacteria, fungi, algae etc.
✔ Cost effective
✔ Safe for usage – the handler ​ ​ shouldn’t be a skin irritant or a carcinogen
✔ Long shelf-life
✔ Shouldn’t be corrosive
✔ Detergent property should exist ​ ​ provides a good cleansing ability as it can
affect the Cell membrane of the organism.
✔ Shouldn’t leave behind stains
✔ The agent should be able to dissolve in water so that the ability of the agent to
penetrate the cell increases.

Chemical Agents can be of the following types:

1. Phenols and phenolic group
● Obtained by distillation of coal-tar
● Joseph Lister
2. Alcohols
● Popularly used are ethanol and isopropanol
● Methanol is not used as it is highly poisonous. – fumes may cause
permanent damage to the eyes.
3. Halogens
● Any of the elemets of group VII A of periodic table - fluorine, chlorine,
bromine, iodine and astatine
● exist as diatomic molecules – in free state and as salts – in compounds
4. Heavy Metals
 ● Most of them have some degree of toxicity
5. Aldehydes
● General formula – R-CHO, low mol. Wt. aldehydes are antimicrobial
● Formaldehyde and glutaraldehyde are effective in killing spores -
Sporicidal
6. Quaternary Nitrogen Compounds
● Cationic detergents – have a positively charged Nitrogen ad a long
hydrophobic chain
● Structurally like ammonium chloride
Categor Applicatio
Example Efficiency Mode of Action
y n
Phenols and cresols, Effective as 5% aq Specific mode of action is unknown – agreed Strong surface
Phenolic xylenols soln – destroys that it maybe due to damage to plasma tension
Compoun and vegetative cells membrane. As a result, cell contents leak out reductant and
ds and the microorganism dies. a disinfectant.
bisphenol Endospores show
resistance Another theory is that precipitation of Used as
antimicrobial proteins, inactivation of enzymes, and antiseptic in
activity inc ​ leakage of amino acids from the microbial mouthwash,
modification of cells. soaps and
phenol ring cough drops.

Also found in
detergents

Alochols Ethanol and Effective conc. – Protein denaturants​ - may account for their effectively used
isopropanol 60-80%. It kills antimicrobial activity. to reduce the
vegetative cells microbial
only. Solvents for lipids​ - damage lipid complexes flora of the
in the cell membrane. skin and for
It can damage the
viruses by Dehydrating agents​ - accounts for the relative disinfection of
mechanically ineffectiveness of absolute alcohol on “dry” clinical oral
removing them by cells; it is possible that very high thermometers
detergent action. concentrations remove so much water from .
the cell that the alcohol is unable to
It may also cause penetrate.
surface
dehydration of
 membrane –
alcohol may
penetrate within
the cell –
bacteriostatic in
nature – excess
amount may be
bactericidal in
nature.

Aldehydes Formaldehyd 30-40% conc. Both of the commonly used aldehydes, Used in killing
e and formaldehyde and glutaraldehyde, are and
glutaraldeh It is present in all highly reactive molecules that readily preserving
yde three states of combine with vital organic nitrogen biological
matter. compounds such as nucleic acids and specimens.
proteins and inactivate them, probably by
Drawback is that it cross-linking and alkylating molecules. The Can be used in
leaves a stench inactivation of nucleic acids and proteins the ster of
and a stain disrupts the function of cell organelles and, rooms and
as a result, the cells are killed. furniture.
Heavy Mercury Compounds used Antiseptic
metals Copper and are Mercuric ointment.
Silver Chloride,
mercuric oxide, 1% of silver
metaphen and nitrate is
mercresin. added to
ophthalmic
solutions
which is even
Silver nitrate and given for the
silver lactate They bind to the sulfhydryl group of the eyes of
protein and inactive them. infants.

High concentration of metallic salts, Copper

Copper Sulphate – sulphate is
particularly those of mercury, silver and
algicidal and copper coagulate cellular proteins that used in
fungicidal. results in damage or death of the microbial agricultural
cell. fields with a
conc. Of
Metallic salts may also precipate and in high 2ppm to
They are effciet in prevent
concentrations may cause the death of a
the dilution growth of
microbial cell.
1:1000. They are algae.
often limited to
corrsive nature
and toxicity.

Halogens Chlorine highly effective Not clearly understood. Tincture of

bacterial agent iodine is
and is unique in 1 being an oxidizing agent, has property to applied as an
that it is effective irreversibly oxidize and thus inactivate antiseptic in
against all kinds essential metabolic compounds such as households
of bacteria. proteins with sulfhydryl groups. and hospitals
 to disinfect
Sporicidal, 2 may involve its reaction (halogenation) with wounds, cuts,
fungicidal and tyrosine units of enzymes and other cellular and scratches
virusidal in nature proteins that require tyrosine for activity.
– depending upon
the amout
exposed to. Iodophores are
Iodospores - They release iodine slowly into used in
iodine has been the medium and lower the surface tension of hospitals for
complexed with the solution. (nontoxic, non-staining and preoperative
organic odourless) skin
compound to form degerming
iodophore and in
hospitals and
laboratories
for
disinfecting.

Iodine Cl – gaseous form Hypochlorite

or compounds. s: used as
The problem with disinfectant
gaseous form is s, sanitizing
that it is too agents, or
difficult to handle The oxygen released during decomposition of antiseptics.
and require hypochlorous acid (called nascent oxygen) is
special equipment a strong oxidising agent. It acts on cellular Used to sterilize
so it is generally constituents of microorganisms and results municipal
used for large in their death. The killing of microbial cells water
scale purposes. by chlorine and its compounds is also due in supplies.
For small scale part to the direct combination of the cell
operations membranes and enzymes.
chlorinated lime
 [Ca(OCl)​2​] and
hypochlorite Hypochlorites and chloramines, however, Clorine
[NaOCl] can be undergo hydrolysis resulting in the compunds are
used as they are formation of hypochlorous acid. used to
equally effective. disinfect open
wounds.

Quaternary Zephiran Quaternary used for

nitrogen (benzalkoni ammonium sterilization of
compound um compounds are surgical
s chloride), very effective instruments
ceepryn disinfectants and,
(cetylpyridi accordingly, a very
um large number of
chloride) these compounds They are used
and of different as antiseptics
diaparene constitutions have in skin
chloride been synthesized ointments,
and evaluated for lotions and
their ophthalmic
antimicrobial solutions
properties.

used in dairy,
poultry, and
fishing
Quaternary ammonium compounds render industries to
various damaging effects on disinfect
microorganisms. surfaces of
equipment
and the
 environment
They denature proteins, interfere with in general.
glycolysis, and disrupt microbial
membranes.

It has been suggested on the basis of

experimental results that the most likely site
of the damage to the microbial cell is the
plasma membrane because the quaternary
ammonium compounds alter the vital
permeability characteristics of the plasma
membrane.