Aseptic technique, culturing and preservation by Likhith K

Aseptic Methods of Inoculation,
Achievements and Maintenance
BY
LIKHITH K
BiSEP – 2021
Dept of Biotechnology
St Aloysius College
Mangaluru, Karnataka

What is aseptic technique ?
Objective
How can microorganism be killed?
Concepts in maintaining sterile conditions
Instrument used in sterilization
Flaming of inoculation loop
Chemical sterilization
Preview to maintenance
Common methods of isolation
Preservation
Methods of preservation
Conclusion
Reference
Contents

What is Aseptic Technique?
Aseptic technique is a method of compete elimination
of microorganism, used in laboratories or clinical
setting to prevent the contamination or growth of
unwanted microorganism.

Objectives
The goal is to prevent contamination of what you are
working on, whether it is someone’s wound or a
bacterial culture that is of interest.
Microorganisms are everywhere! In the environment
and in and on your body, Therefore, aseptic technique
takes vigilance as bacteria and other microbes may be
present on your work bench, floating in the air currents,
etc.
Proper aseptic technique can prevent contamination
from any source.

How can microorganisms be killed?
Principle of sterilization :
Denaturation of proteins
Interruption of DNA synthesis/repair
Disruption of cell membranes

How can we Minimize contamination
One way to minimize contamination is to not perform
your techniques in drafty areas, this lessens the chance
of air-born microorganisms contaminating your culture.
Also you should always make sure that the surface that
you are working on has been disinfected, eliminating
another potential source of contamination.
Any materials that will contact your experiments should
be sterilized by autoclaving and flaming.

Concepts in maintaining sterile conditions
Sterilization : It is a process by which an article,
surface or medium is made free of all microorganisms
either in vegetative or spore form.
Disinfection : Destruction of all pathogens or
organisms capable of producing infections but not
necessarily spores. All organisms may not be killed but
the number is reduced to a level that is no longer
harmful to health.

Antiseptics : Chemical disinfectants which can safely
be applied to living tissues and are used to prevent
infection by inhibiting the growth of microorganisms.
Asepsis : Technique by which the occurrence of
infection into an uninfected tissue is prevented.

Instruments used in sterilization
Physical Method of Sterilization
Principle Instruments used
1 Dry Heat Hot Air Oven
2 Moist Heat Autoclave
3 Radiation Gamma-ray Chamber

Hot air oven
 Sterilization by dry heat is performed by conduction. The
temperature is consumed by the surface of the objects, then
moves towards the core of the object, coating by coating.
The whole object will ultimately attain the temperature
needed for sterilization to take place(150 to 180C).
 Dry heat causes most of the injury by oxidizing particles.
The primary cell components are damaged and the organism
dies. The temperature is kept for about an hour to eliminate
the most ambitious of the resistant spores.

Autoclave
 The basic principle of steam sterilization, as accomplished in
an autoclave, is to expose each item to direct steam contact at
the required temperature and pressure for the specified time.
 Thus, there are four parameters of steam sterilization: steam,
pressure, temperature, and time. The ideal steam for
sterilization is dry saturated steam and entrained water
(dryness fraction ≥97%).
 Pressure serves as a means to obtain the high temperatures
necessary to quickly kill microorganisms. Specific
temperatures must be obtained to ensure the microbicidal
activity. The two common steam-sterilizing temperatures are
121°C (250°F) and 132°C (270°F).

 These temperatures (and other high temperatures)must be
maintained for a minimal time to kill microorganisms.
Recognized minimum exposure periods for sterilization of
wrapped healthcare supplies are 30 minutes at 121°C (250°F) in
a gravity displacement sterilizer or 4 minutes at 132°C (270°F)
in a pre vacuum sterilizer.
 At constant temperatures, sterilization times vary depending on
the type of item (e.g., metal versus rubber, plastic, items with
lumens), whether the item is wrapped or unwrapped, and the
sterilizer type.
 Moist heat destroys microorganisms by the irreversible
coagulation and denaturation of enzymes and structural proteins.
In support of this fact, it has been found that the presence of
moisture significantly affects the coagulation temperature of
proteins and the temperature at which microorganisms are
destroyed.

Gamma ray chamber
 The gamma irradiation process uses Cobalt 60 radiation to kill
microorganisms on a variety of different products in a specially
designed cell.
 Gamma radiation is generated by the decay of the radioisotope
Cobalt 60, with the resultant high energy photons being an
effective sterilant.
 A key characteristic of gamma irradiation is the high penetration
capability, which allows for delivery of target radiation dose to
areas of products that may be higher in density.
 The unit of absorbed dose is kiloGray, expressed as kGy. Delivery
and absorption of dose by product is determined by product
density, packaging size, dose rate, exposure time and facility
design.

The Laminar Flow Unit
 A laminar flow unit (or hood) is a sophisticated appliance that can
further help prevent contamination of reagents and biological
cultures. Used correctly, it provides the work space with clean, ultra
filtered air.
 It also keeps room air from entering the work area and
both suspends and removes airborne contaminants introduced into
the work area by personnel.
 The most important part of a laminar flow hood is a high-efficiency
bacteria-retentive filter, i.e., the HEPA (high-efficiency particulate
air) filter.

 A certified HEPA filter must capture a minimum of 99.97% of
dust, pollen, mold, bacteria, and any airborne particles with a size
of >0.3 μm at 85 liters/min.
 Laminar flow hoods are essential components of many bio safety
level (BSL)-2 laboratories, where they help prevent spread of
viruses and some bacteria.

Inoculating an agar slant from an agar plate

Chemical sterilization
Gaseous Sterilization
 Gaseous sterilization involves the process of exposing
equipment or devices to different gases in a closed heated or
pressurized chamber.
 Gaseous sterilization is a more effective technique as gases
can pass through a tiny orifice and provide more effective
results.
 Besides, gases are commonly used along with heat treatment
which also facilitates the functioning of the gases.
 However, there is an issue of release of some toxic gases
during the process which needs to be removed regularly from
the system.

 The mechanism of action is different for different types of gases.
 Some of the common gases used for gaseous sterilization are
explained below:
 a)Ethylene oxide
 Ethylene oxide (EO) gas is a common gas used for chemical
treatment applied to sterilize, pasteurize, or disinfect different
types of equipment and surfaces because of its wide range of
compatibility with different materials.
 EO treatment often replaces other sterilization techniques like
heat, radiation, and even chemicals in cases where the objects are
sensitive to these techniques.
 The mechanism of antimicrobial action of this gas is assumed to
be through the alkylation of sulphydryl, amino, hydroxyl, and
carboxyl groups on proteins and imino groups of nucleic acids.

b) Formaldehyde
 Formaldehyde is another important highly reactive gas which is
used for sterilization.
 This gas is obtained by heating formalin (37%w/v) to a
temperature of 70-80°C.
 It possesses broad-spectrum biocidal activity and has found
application in the sterilization of reusable surgical instruments,
specific medical, diagnostic and electrical equipment, and the
surface sterilization of powders.
 Formaldehyde doesn’t have the same penetrating power of
ethylene oxide but works on the same principle of modification
of protein and nucleic acid.
 Formaldehyde can generally be detected by smell at
concentrations lower than those permitted in the atmosphere and
thus can be detected during leakage or other such accidents.

c) Nitrogen dioxide (NO2)
 Nitrogen dioxide is a rapid and effective sterilant that can
be used for the removal of common bacteria, fungi, and
even spores.
 NO2 has a low boiling point (20°C) which allows a high
vapor pressure at standard temperature.
 This property of NO2 enables the use of the gas at standard
temperature and pressure.
 The biocidal action of this gas involves the degradation of
DNA by the nitration of phosphate backbone, which results
in lethal effects on the exposed organism as it absorbs NO2.
 An advantage of this gas is that no condensation of the gas
occurs on the surface of the devices because of the low
level of gas used and the high vapor pressure. This avoids
the need for direct aeration after the process of sterilization.

d) Ozone
 Ozone is a highly reactive industrial gas that is commonly used
to sterilize air and water and as a disinfectant for surfaces.
 Ozone is a potent oxidizing property that is capable of destroying
a wide range of organisms including prions, without the use of
hazardous chemicals as ozone is usually generated from medical-
grade oxygen.
 Similarly, the high reactivity of ozone allows the removal of
waste ozone by converting the ozone into oxygen by passing it
through a simple catalyst.
 However, because ozone is an unstable and reactive gas, it has to
be produced on-site, which limits the use of ozone in different
settings.
 It is also very hazardous and thus only be used at a concentration
of 5ppm, which is 160 times less than that of ethylene oxide.

Liquid Sterilization
 Liquid sterilization is the process of sterilization which involves
the submerging of equipment in the liquid sterilant to kill all
viable microorganisms and their spores.
 Although liquid sterilization is not as effective as gaseous
sterilization, it is appropriate in conditions where a low level of
contamination is present.
 Different liquid chemicals used for liquid sterilization includes the
following:
a) Hydrogen peroxide
 Hydrogen peroxide is a liquid chemical sterilizing agent which is
a strong oxidant and can destroy a wide range of microorganisms.
 It is useful in the sterilization of heat or temperature-sensitive
equipment like endoscopes. In medical applications, a higher
concentration (35-90%) is used.
 H2O2 has a short sterilization cycle time as these cycles are as
short as 28 minutes where ethylene oxide has cycles that as long
as 10-12 hours.

b) Glutaraldehyde
 Glutaraldehyde is an accepted liquid sterilizing agent which
requires comparatively long immersion time. For the removal of
all spores, it requires as long as 22 hours of immersion time.
 The presence of solid particles further increases the immersion
time.
 The penetration power is also meagre as it takes hours to penetrate
a block of tissues.
 The use of glutaraldehyde is thus limited to certain surfaces with
less contamination.
c) Hypochlorite
 Hypochlorite solution, which is also called liquid bleach, is
another liquid chemical that can be used as a disinfectant, even
though sterilization is difficult to obtain with this chemical.
 Submerging devices for a short period in liquid bleach might kill
some pathogenic organisms but to reach sterilization submersion
for 20-24 hours is required.

 It is an oxidizing agent and thus acts by oxidizing organic
compounds which results in the modification of proteins in
microbes which might ultimately lead to death.
 Appropriate concentrations of hypochlorite can be used for the
disinfection of workstations and even surfaces to clean blood spills
and other liquids.

Preview to maintenance
 Microorganisms are generally found in nature (air, soil and
water) as mixed populations.
 Even the diseased parts of plants and animals contain a great
number of microorganisms, which differ markedly from the
microorganisms of other environments.
 To study the specific role played by a specific microorganism in
its environment, one must isolate the same in pure culture.

 The two major steps of obtaining a pure culture are as follows :
 Firstly, the culture has to be diluted until the various individual
microorganisms are separated far apart on agar surface that after
incubation they form visible colonies isolated from the colonies
of other microorganisms.
 Secondly, an isolated colony has to be aseptically picked off the
isolation plate

Dilution
Colonies on plate after
the period of incubation

Selection of
individual colony
from mixed colony
Streak plate
culture of pure
colony

 There are several methods of isolating the pure culture of bacteria,
like, streak plate method, pour plate method, spread plate method
and serial dilution.
Why is pure cultures important ?
Pure cultures are important in microbiology for the following
reasons
 Once purified, the isolated species can then be cultivated with the
knowledge that only the desired microorganism is being grown.
 A pure culture can be correctly identified for accurate studying and
testing and diagnosis in a clinical environment.
 Testing/experimenting with a pure culture ensures that the same
results can be achieved regardless of how many time the test is
repeated.
 Pure culture spontaneous mutation rate is low
 Pure culture clone is 99.999% identical

Common methods of isolation
The process of screening a pure culture by separating one type of
microbes from a mixture is called Isolation.
Some common isolation methods are;
I) Streak plate method
II) Pour plate method-
a) a)Loop dilution technique
b) b) Serial Dilution technique
III) Spread plate method
IV) Micromanipulator method
V) Roll tube method

Isolation of microorganisms from soil steps

Preservation
 To maintain pure culture for extended periods in a viable
conditions, without any genetic change is referred as Preservation.
 The aim of preservation is to stop the cell division at a particular
stage i.e. to stop microbial growth or at least lower the growth rate.
 Due to this toxic chemicals are not accumulated and hence
viability of microorganisms is not affected.

Objectives
 To maintain isolated pure cultures for extended periods in a
viable conditions.
 To avoid the contamination.
 To restrict genetic change (Mutation)

The method of preservation is mainly of two
types-
1. Short term methods
2. Long term methods

Short term methods
 Periodic transfer to fresh media-
 Culture can be maintained by periodically preparing a fresh
culture from the previous stock culture.
 Many of the more common microbes remain viable for several
weeks or months on a medium like Nutrient agar.
 It is an advantageous as it is a simple method and any special
apparatus are not required. However it is easy to recover the
culture.
 The transfer has the disadvantage of failing to prevent changes in
the characteristics of a strain due to development of variants and
mutants and risk of contamination is also more in this process.

Preservation of bacteria using glycerol
 Bacteria can be frozen using 15% glycerol.
 The glycerol is diluted to 30% and an equal amount of glycerol
and culture broth are mixed, dispensed into tubes, and then
frozen at -10˚ C.
 The viability of organisms varied such as Escherichia coli,
Diplococcus pneumonia etc. viable for 5 months, Haemophilus
influnzae viable for 4 months, Neisseria meningtidis for 6 weeks
and Neisseria gonorrhoeae for 3 weeks

Storage by drying method
 Spores of some microbes which are sensitive to freeze- drying, can
be preserved by drying from the liquid state rather than the frozen
state.
 Different procedures of drying methods are as follows:
 Paper disc: A thick suspension of bacteria is placed on sterile discs
of thick absorbent paper, which are then dried over phosphorus
pentoxide in a desiccation under vacuum.
 Gelatin disc: Drops of bacterial suspension in gelatin are placed on
sterile plastic petriplates and then dried off over P2O5 under
vacuum.

 L-drying: Bacteria in small ampoules are dried from the liquid
state using a vacuum pump and desiccant and a water bath to
control the temperature.
 In this suspension of the organisms are dried under vacuum
from the liquid state without freezing taking place.
 Apart from the mentioned methods the organisms are also dried
over Calcium Chloride in vacuum and are stored in the
refrigerator.
 At such conditions the organisms survive for longer period than
the air dried cultures.

Storage by refrigeration
 Culture medium can be successfully stored in refrigerators or cold
rooms, when the temperature is maintained at 4˚C.
 At this temperature range the metabolic activities of microbes
slows down greatly and only small quantity of nutrients will be
utilized.
 This method cannot be used for a very long time because toxic
products get accumulated which can kill the microbes.

Long term methods
Mineral oil or liquid paraffin storage
 In this method sterile liquid paraffin is poured over the slant
culture of microbes and stored upright at room temperature.
 Where as cultures can also be maintained by covering agar slants
by sterile mineral oil which is stored at room temperature or
preferably at 0-5°C.

 It limit the oxygen access that reduces the microorganism’s
metabolism and growth, as well as to cell drying during
preservation.
 The preservation period for bacteria from the genera Azotobacter
and Mycobacterium is from 7-10 years, for Bacillus it is 8-12
years.
Storage in saline suspension:
 Bacterial culture is preserved in 1% salt concentration in screw
caped tubes to prevent evaporation.
 The tubes are stored in room temperature.
 Whenever needed the transfer is made on Agar Slant.

Immersion in distilled water:
 Another inexpensive and low-maintenance method for storing
fungal culture is to immerse them in distilled water.
 Fungi can be stored in this method at 20˚C, survived up to 2-10
years depending upon the species.

Storage in sterile soil
 It is mainly applied for the preservation of sporulating
microorganisms. Fusarium, Penicillium, Alternaria, Rhizopus etc.
proved successful for store in sterile soil.
 Soil storage involves inoculation of 1ml of spore suspension into
soil (autoclaved twice) and incubating at room temperature for 5-
10 days.
 The initial growth period allows the fungus to use the available
moisture and gradually to become dormant.
 The bottles are then stored at refrigerator.
 Viability of organisms found around 70-80 years.

Lyophilization (Freeze–drying)
 It is a vacuum sublimation technique.
 Freeze drying products are hygroscopic and must be protected
from moisture during storage.
 By freezing the cells in a medium that contain a lyo protectant
(usually sucrose) and then pulling the water out using
vacuum(sublimation), cells can be effectively preserved.
 Freezing must be very rapid, with the temperature lowered to
well below 0˚C (as such -20˚C).
 Lyophilized cultures are stored in the dark 4˚C in refrigerators.
 Many microbes preserved by this method have remained viable
and unchanged in their characteristic more than 20 years.

 It is very advantageous as only minimal storage space is required
to preserve.

Cryopreservation
 Cryopreservation (i.e. freezing in liquid nitrogen at -196˚C or in
the gas phase above the liquid nitrogen at -150˚C) helps survival
of pure cultures for long storage time.
 In this method, the microorganisms of culture are rapidly frozen
in liquid nitrogen at -196˚C in the presence of stabilizing agents
such as Glycerol or Dimethyl Sulfoxide (DMSO) that prevent the
cell damage due to formation of ice crystals and promote cell
survival.

By this method species can remain viable for 10-30 years without
undergoing change in their characteristics.

Stored in silica gel
 Microbes can be stored in silica gel powder at low temperature
for a period 1- 2 years.
 The basic principle in this technique is quick desiccation at low
temperature, which allows the cell to remain viable for a long
period of time.
 Some of the species which are preserved on anhydrous silica gel
are such as Saccharomyces cerevisiae, Aspergillus nidulans,
Pseudomonas denitrificans, Escherichia coli etc.

Conclusion
 Many of the microorganisms we will be working with in lab are
known pathogens.
 Proper and appropriate aseptic technique is vitally important for
the safety of all lab personnel; it is also essential for the
successful completion of the lab portion and experiments.
 The skills and awareness you develop practicing aseptic
technique will carry over to your career as a health professional.
 Whichever technique is used for preservation and maintenance
of industrially important organisms it is essential to check the
quality of the preserved organisms stocks.

 Each batch of newly preserved cultures should be routinely
checked to ensure their quality. However preservation is essential
as it reveals great importance in the field of science.
 Preservation helps in research purposes, industry as well as in
biotechnological field.

References
 https://www.ncbi.nlm.nih.gov>articles
 onlinelibrary.wiley.com>doi>full
 https://www.researchgate.net>publication
 https://link.springer.com>chapter
 pubmedcentralcanada.ca>articles>pdf
 www.biologydiscussion.com>maintenance
 Google images

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