EXP 1: Preparation and sterilization of microbial media

Introduction
Microbiological Culture Media

The nutrient preparations that are used for culturing microorganisms are called media
(singular, medium). Three physical forms are used: liquid, or broth, media; semisolid media; and
solid media. The major difference among these media is that solid and semisolid media contain
a solidifying agent (usually agar), whereas a liquid medium does not. Liquid media, such as
nutrient broth, tryptic soy broth, or brain-heart infusion broth, can be used to propagate large
numbers of microorganisms in fermentation studies and for various biochemical tests.
Semisolid media can also be used in fermentation studies, in determining bacterial motility, and
in promoting anaerobic growth. Solid media, such as nutrient agar or blood agar, are used (1)
for the surface growth of microorganisms in order to observe colony appearance, (2) for pure
culture isolations, (3) for storage of cultures, and (4) to observe specific biochemical reactions.

Microorganisms may be cultured using two different types of media. Chemically defined, or
synthetic, media are composed of known amounts of pure chemicals. Such media are often used
in culturing autotrophic microorganisms such as algae or non-fastidious heterotrophs. In
routine bacteriology laboratory exercises, complex, or non-synthetic, media are employed.
These are composed of complex materials that are rich in vitamins and nutrients. Three of the
most commonly used components are beef extract, yeast extract, and peptones.

Now-a-days, culture media are available commercially as powder; they require only the
addition of water.

Sterilization of Media and Equipment

Sterilization is the process of rendering a medium or material free of all forms of life. There are
three basic ways in which sterilization of media and supplies can be achieved. The most useful
approach is autoclaving, in which items are sterilized by exposure to steam at 121°C and 15 lbs
of pressure for 15 minutes. Under these conditions, microorganisms, even endospores, will not
survive longer than about 12 to 13 minutes. This method is rapid and dependable.

Often, dry glassware such as pipettes and petri plates must be sterilized. Steam tends to etch
glassware and also leaves it damp. Therefore, such items are generally dry-heat sterilized.
Conditions for dry-heat sterilization: 150°C for 1.5 hours, 160°C for 1 hour or 180°C for 30 min.
The oven temperature must not rise above 180°C or any cotton or paper present will char.
Heat-sensitive materials can be sterilized by passing it through a bacteriological filter, which
physically removes bacteria and larger microorganisms from the solution and thereby sterilizes
them without heat. By far, the most useful and popular approach is the use of specially prepared
sterile, cellulose- or polycarbonate-based membranes of the appropriate pore size. Generally,
membranes with 0.22 µm pores are employed in sterilization.

Two other sterilization techniques use ultraviolet radiation and ethylene oxide. Ultraviolet (UV)
radiation around 260 nm is quite lethal to many microorganisms but does not penetrate glass,
dirt films, water, and other substances very effectively. Because of this disadvantage, UV is used
as a sterilizing agent only in a few particular situations. For example, UV lamps are sometimes
placed on the ceilings of rooms or in biological safety cabinets to sterilize the air and any
exposed surfaces.

Many heat-sensitive items such as disposable plastic petri dishes and syringes, sutures, and
catheters are now sterilized with ethylene oxide gas. Ethylene oxide is both microbicidal and
sporicidal and kills by covalently attaching to cell proteins. It is a particularly effective
sterilizing agent because it rapidly penetrates packing materials, even plastic wraps.

Instruments for sterilization

1) Autoclave

Principle:

Boiling point is directly proportional to pressure when the volume is constant. For pressure of
15 lbs temperature of water obtained is 121 °C. If the situation is maintained for 15 min, it is
sufficient to kill vegetative forms and spores of microbes.

Construction:

Moist heat sterilization is done in an autoclave by steam under pressure. Autoclave is a

cylindrical vessel made up of gun metal. It has a lid which can be lifted with screw lamp. Lid is
provided with steam valve. On the rim of the cylinder there is a rubber gas kit which ensures air
tight enclosure. Cylinder contains water upto certain level which can be heated by electrical
means. Above it is a rubber gas kitted perforated diaphragm. To keep the material sterilized,
they are kept on perforated platform. Heat is supplied and water is allowed to boil.

Working:

Steam valve is kept open so that the steam can escape. After sometime the valve is closed,
pressure is increased and when required level is reached the time is noted. Pressure is
maintained for specific time. At the end of specific period, pressure is allowed to drop gently.
Precaution:
1. Safety valve or pressure gauze should be blocked or choked.
2. Petridishes must be placed with lids facing up.
3. All the glassware must be wrapped in a paer and tied with a string.
4. Mouth of glass containing media must be plugged with non-absorbent cotton.
5. All the air must be drawn out before sterilization.

2) Hot Air Oven

Principle:

Mostly hot air sterilization is used for pipettes, test tubes, flasks, etc. Oven is operated at
temperature 150 °C for 1.5 h, at 160 °C for 1 h or at 180 °C for 0.5 h.

These time and temperature are sufficient for destroying the vegetative cells and spores of
microorganisms.

Precaution:
1. While sterilization of glassware they must be clean and dry to avoid contamination after
sterilization, It is necessary to wrap the material with paper and plug the apparatus containing
media.
2. Oven should be loaded only when it is cool and then it should be clean.
3. Temperature should not exceed 180 deg Celsius.
4. Before removing the apparatus, oven should be cooled.
5. Plastic material should not be sterilized by this method.

Experiment 1.

Nutrient medium is a general purpose preparation for culturing microorganisms which are not
nutritionally fastidious.

The broth contains:

3.0 g/L - beef extract
5.0 g/L peptone (a nitrogen source)
15.0 g/L agar powder
1000mL Water

The final pH of media is 7.4.

Sterilization of nutrient agar by autoclaving

Autoclaving is a process that use moist heat and pressure so that all parts of the material to be
sterilized reach 121 degree Celsius for 15 minutes. An autoclave is, in essence, a large pressure
cooker; a chamber which may be sealed off against surrounding air.
Materials for sterilization are placed in the chamber, the door is sealed, and pressurized steam
is forced into the chamber. The incoming steam displaces cooler air through an exhaust valve;
this valve closes when the cell cooler air has been vented.
Steam is continually forced into the chamber until the pressure reaches 103 kPa above
atmospheric pressure (15 lb); at sea level, this pushes the temperature in the chamber to 121
degree Celsius. The high pressure prevents solutions from boiling over at this temperature.
Larger volumes require longer than 15 minutes to heat up to 121 degree Celsius throughout.
After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The
sterilized objects can then be removed.

Material and reagents

Commercial nutrient agar, Balance, Distilled water, Conical Flask, Measuring cylinder,
Autoclaved Petri Plates. Cotton plugs, 70% alcohol, burners

Procedure
1. Appropriate amount of broth (with agar) powder is weighed into Conical flask and dissolve
with distilled water. They are mixed well.
2. The flasks are plugged with cotton and set aside for sterilization.
3. All the media are sterilized at 121 degree celcius for 15 minutes.
4. After autoclaving, the media is removed.
5. The nutrient agar approximately 20ml is poured into the sterile petri dishes aseptically
between the burners.
6. The petridishes are allowed to cool down until the agar solidifies. Further the plates are
incubated at 37oC for 24 hours in an incubator.
7. After 24 hours, the plates are observed for their sterility.
Results

Conclusion: