Introduction to Common Instruments in the
Laboratory
Hot Air Oven
Sterilization and aseptic processing are essential practices in a
microbiology laboratory. Hot air ovens are electrical devices which use dry
heat to sterilize. They were originally developed by Pasteur. Dry heat
sterilization is used on equipment that cannot be wet and on material that will
not melt, catch fire, or change form when exposed to high temperatures. Dry
heat takes more time to kill microorganisms than moist heat, but it has certain
advantages such as it does not corrode glassware (petri dishes, flasks,
pipettes, and test tubes), metal instruments and can be used to sterilize
powders (starch, zinc oxide, and sulfadiazine), oils, liquid paraffin, fats,
grease, etc. Dry heat sterilization is
slow and is not useful for heat sensitive materials such as plastic and rubber
items.
(i)
Sterilizing
by dry heat is accomplished by conduction. The heat is absorbed by the outside
surface of the item, then passes towards the center of the item, layer by
layer. The entire item will eventually reach the temperature required for
sterilization to take place.
(ii)
Dry heat
does most of the damage by oxidizing molecules. The essential cell constituents
are destroyed and the organism dies. The temperature is maintained for almost
an hour to kill the most difficult of the resistant spores.
(iii)
Hot air
ovens use extremely high temperatures over several hours to destroy
microorganisms and bacterial spores. Nowadays, ovens based on microwaves are
also sold in the market but generally not much in use in the laboratory.
(iv)
The ovens
generally consist of double chamber, the gap between the two walls is insulated
to keep the heat in and conserve energy. There is double walled insulation with
the inner layer being a poor conductor and outer layer being metallic. The oven
is heated from below by using electric current and the heating elements are
arranged in a manner to heat the inside of the chamber of uniformly.
(v)
The
calibration knob sets the desired temperature. A thermostat controls the
temperature which is displayed by the thermometer. An air circulating fan helps
in uniform distribution of the heat. These are fitted with the adjustable wire
mesh plated trays or aluminium trays and may have an on/off rocker switch, as
well as indicators and controls for temperature and holding time.
(vi)
For sterilization,
the holding time depends upon the temperature. If the temperature of the oven
is 160oC, the holding time should be two hours. At, 180oC,
it should be one hour. The holding time can be little more for better
sterilization.
Precautions
The glass materials should be wiped and dried before keeping inside the
chamber in the oven or else it may break. After the holding time is over, the
glassware should not be taken out immediately. The oven must be allowed to cool
down before the door is opened, since otherwise glassware may crack due to
sudden or uneven cooling. The air within the oven, should be circulated by a
fan to ensure uniform distribution of heat. The articles should be kept
properly arranged so as not to obstruct the flow of the air.
Spores of nontoxigenic strain of Clostridium tetaniare used a microbiological test of dry heat efficiency. Paper strips impregnated with 106spores are used, and after sterilization the spores are inoculated into appropriate media (thioglycollate or cooked meat media) and incubated to test for growth.
Autoclave
Moist heat sterilization uses water to boil
items or steam them to sterilize and doesn't take as long as dry heat
sterilization. Steam sterilization is carried out with an autoclave, a device
like a pressure cooker. Chamberland, in 1884 developed the autoclave which
played a crucial role in the growth of microbiology. Autoclave
enables moist sterilization using steam in a closed system, very similar to the
pressure boiling process. In microbiology laboratory, horizontal type autoclave
is necessary.
Moist heat is thought to kill microorganisms
and even spores effectively by degrading nucleic acids and by denaturing
enzymes and other essential proteins. It
also may disrupt cell membranes.
(i)
The autoclave is usually of pressure cooker
type made up of gun metal sheets supported in an iron case. The sidewalls are
heated by the steam jacket. It is closed by a swing door fastened tightly by
radial belts.
(ii)
It is filled with water which is boiled at
100oC to accumulate steam in the the autoclave’s chamber.
(iii)
The air
initially present in the chamber is forced out until the chamber is filled with
saturated steam and the outlets are closed.
(iv)
Hot,
saturated steam continues to enter until the chamber reaches the desired
temperature and pressure, usually 121°C and 15 pounds of pressure. The pressur
valve regulates pressure.
(v)
At this
temperature, 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 ensure sterility.
(vi)
The chamber
should not be packed too tightly because the steam needs to circulate freely
and contact everything in the autoclave.
(vii)
Bacterial
endospores will be killed only if they are kept at 121°C for 10 to 12 minutes.
(viii)
When a
large volume of liquid must be sterilized, an extended sterilization time will
be needed because it will take longer for the center of the liquid to reach
121°C.
The level
of water should be checked before operating. The air should be completely
evacuated and the steam must have access to the materials to be sterilized.
Cotton or glass beads must be sterilized in a glass container closed with foil.
The heat sensitive substances should not be sterilized by autoclaving. Checking
of completion of autoclaving is possible with the use of indicator strips which
change color after the required time interval at the proper conditions.
For determining the efficiency of moist heat sterilization, spores of Bacillus stearothermophilus or Clostridium PA3679 is used as test. The spores of this organism need an exposure
of 12 minutes at 121oC to be killed.
Incubator
Abundant growth of microbes is obtained in
the laboratory by growing them at suitable temperatures. This is done by
inoculating the desired microbe into a suitable culture medium and then
incubating it at the temperature optimum for its growth.
Incubator provides a constant temperature specifically suitable for the
growth of a specific microbe. As most of the microbes pathogenic to man grow
well at body temperature of normal human being (i.e. 37°C), the usual
temperature of incubation is 37°C. The incubator has a thermostat, which
maintains a constant temperature, set according to requirement. Accurate
temperature can be seen on the thermometer fixed on the incubator.
Most of the modern incubators are programmable where the operator sets
the desired temperature and the required period of time. The incubator
automatically maintains it accordingly. Moisture is ensured to retard the
dehydration of the media and thereby, avoid false experimental results.
(i)
An
incubator is made up of double walled chamber adjusted to a desired
temperature. It is done by using an external knob controlling the thermostat
system. The gap between two walls ensures heat conduction.
(ii)
There is
provision for cold/warm water or air to be circulated as a jacket around the
incubator to adjust the temperature. Now sophisticated incubators are available
with humidity and oxygen control systems
(iii)
A
thermometer is present for recording the temperature. The temperature of the
incubator is kept constant due to its control by using thermostat.
(iv)
Temperature
greatly influences the microbial growth. Therefore, the instrument is designed
with controls to allow the desired temperature for growth of microorganisms.
(v)
The
variation in temperature should not be generally more than one degree. The
instrument should be accordingly calibrated.
(vi)
Small
square type incubators are better than large ones. If a temperature lower than
the room temperature is required, the water is circulated around the chamber to
pass through an ice chest.
Precautions
The door of the incubator should be opened only when necessary. If tubes are to be incubated for a long time or at higher temperatures, the medium may become dry due to excessive evaporation. In such cases, the cotton plug should be pushed inside the neck of the tube, The tube should be covered with a rubber cap so as to cover the plug. If petridishes are to be incubated for long time, they may be placed in the moist chamber with a damp sterile cotton wool at the bottom.
Laminar Air Flow Cabinet (Biological Safety Cabinets)
Laminar air hood provides aseptic/sterile conditions in a microbiology
laboratory. It is one of the most important air filtration systems, with
high-efficiency particulate air (HEPA) filters, which remove 99.97% of 0.3µm
particles. Laminar air flow cabinets force air through HEPA filters and pass a
curtain of sterile air across the cabinet opening. This protects a worker from
microorganisms being handled within the cabinet and prevents contamination of
the room. These cabinets are used when working with potentially pathogenic
microorganisms. They are also employed in research labs and industries for
conducting assays, preparing media, examining tissue cultures, etc.
Laminar Flow Cabinets can be tailor made to the specific requirements of the
laboratory and are also ideal for general lab work.
(i)
Laminar air flows provide a clean air
environment and maintain a working area devoid of contaminants. All the culture vessels, test tubes, pipette,
tip boxes, stocks of sterile Eppendorfs should be opened only in the Laminar
Air Flow. Culture Media should be poured inside the laminar
air flow to avoid
contamination and wrong results.
(ii)
Laminar air flow cabinets are normally made
of stainless steel with no gaps or joints thereby preventing the build-up of
bacteria from collecting anywhere in the working zone. They are also known as
clean benches because the air for the working environment is thoroughly cleaned
by the precise filtration process.
(iii)
A laminar
flow chamber ensures sterility by several mechanisms. The UV lamp fitted inside the chamber
sterilizes the chamber before operation. Before use, the platform is cleaned
and disinfected with alcohol. The UV light is switched on for 10 minutes to
sterilize the environment inside the chamber and then switched off. The blower
is switched on while working. HEPA filter provides dust free or microbe-free
air. The inoculating area is sterilized by the flame of a bunsen burner. The
heated air becomes light and moves upwards, thereby preventing the dust
particles from falling on the media during the short opening process.
(iv)
Laminar flow cabinets can be either horizontal
or vertical cabinets.
Horizontal
Laminar Flow Cabinets – Here the direction of air is across the work in a
horizontal direction. The constant flow of filtered air provides material and
product protection.
Vertical
Laminar Flow Cabinets - Here the laminar
the air blows down from the top of the cabinet onto the working area. The air
can leave the working area via holes in the base, hence vertical flow cabinets
can provide greater operator protection when working with potentially hazardous
specimens.
While
using laminar air hood, sterilize the cabinet or the surface of the working
area to avoid any kind of contamination. Wipe with ethanol before and after
each use. Horizontal laminar air flow is designed such that the air flows
directly at the operator. It is useful for tissue or cell culture work.
Vertical Laminar air flow is best for working with microbiological specimens
since the aerosols that are generated in the cabinet are filtered out before
they are released into the surrounding environments. The glass door of the
cabinet should never be opened when the UV light is on, because UV light has detrimental
effect on skin and vision.
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