Preparing and Sterilizing media

Preparing and Sterilizing Media

Media
The many types of media used in microbiology vary according to the needs of the particular
organisms to be studied. A distinction between complex and defined media is sometimes made.

Complex media contain all necessary ingredients for growth of a microorganism, but they are in
crude form, that is, not all the components of the media or their exact quantities are known. Many
components in complex media are acid or enzymatic digests of various plant tissues, meats, casein,
and yeast cells that provide rich sources of polypeptides, amino acids, vitamins, or minerals.
Examples of such components of media are peptones or tryptones, which are enzymatic
hydrolysates of animal protein, or yeast extract, which is autolyzed yeast, rich in B-complex
vitamins. Some carbohydrate is usually present in these crude digests, but many complex media
are supplemented with sugar, usually glucose.

Defined media (holidic) provide the nutrients required for growth in the form of relatively pure
chemicals of known concentration. They vary according to the nutritional needs of the
microorganism. The essential components of a medium for growing nonfastidious heterotrophs,
such as Escherichia coli, are inorganic salts and carbon and nitrogen sources. A suitable
defined medium for E. coli might be composed of NH4Cl, MgS04, KH2PO4, Na2HPO4, and
glucose. Other essential elements such as iron, manganese, and copper are usually present, as
contaminants of the chemicals, in sufficient amounts for growth. Glucose provides energy and a
source of carbon, and the ammonium chloride is the nitrogen source. A more fastidious
heterotroph, lacking the synthesizing capabilities of E. coli, might require the addition of various
vitamins, amino acids, purines, and pyrimidines.

Culture media must be adjusted to a suitable pH. This is usually done before sterilization.

Culture media are usually sterilized by heating in an autoclave at 121°C under 15 pounds of steam
pressure for 15 to 30 minutes.

ADJUSTING THE pH
Most dehydrated media are sold with their pH pre-adjusted according to the requirements of the
microorganisms it will be used for. These medium also contains buffering agents that are used to
maintain the pH within a desired range. However, before the medium is dispensed and sterilized
the pH should be checked and adjustments made if necessary.

If a standardized pH meter is available, use it to determine the pH of the medium. If the medium is
requires adjustment then use NaOH or HCl to correct the pH (unless otherwise stated). For a
medium whose pH changes during heating, adjustment must be made after sterilization; for this,
sterile acid or alkali is added aseptically.

19
 Preparing culture media

Preparing Culture Media

After completing this exercise you should be able to:
1. Prepare and sterilize nutrient broth and nutrient agar media for the culturing of
microorganisms.
2. Aseptically prepare agar plates and slants.
tubes at an inclined position and allow the
The most common complex medium for agar to cool.
culturing microorganisms is nutrient broth 5. When the agar has cooled, aseptically pour
containing beef extract and peptone. Beef four plates, approximately 15 ml per plate.
extract is prepared by boiling lean beef in water If bubbles appear on the surface of the agar,
and then evaporating the stock to a paste. The remove the lid, and quickly flame the
major constituents are protein degradation surface with an inverted Bunsen burner to
products, organic bases, vitamins, and mineral remove the bubbles.
salts. 6. Incubate your labeled plates and tubes for
48 hours at 30°C.
Laboratory supply firms prepare a dehydrated
broth of 0.3% beef extract and 0.5% peptone 7. At the next laboratory period, observe your
that can be reconstituted with water and plate for contamination. Discard
supplemented with 1.5-2.0% agar if a solid contaminated plates in the biohazard bags
medium is desired. Since such dehydrated provided.
products usually have a preadjusted pH of about 8. Record results on the report sheet.
7, further adjustment should not be necessary.
For broth cultures:
In this exercise, you will prepare and sterilize 1. Put 0.8 g of dehydrated nutrient broth
nutrient broth, agar plates and slants. These will powder in a 250-ml Erlenmeyer flask.
be used for microbial transfers in the next Slowly add 50 ml of distilled water while
laboratory session. swirling the flask gently to dissolve the
powder. The solution should be clear.
PROCEDURE 2. When the nutrient broth is completely
To make plates and slants: dissolved, place 3 - 4 ml in the 5 tubes
1. Put 1.6g of dehydrated nutrient broth provided and sterilize in an autoclave
powder and 2g of agar into a 250-ml (repeat steps 7 and 8).
Erlenmeyer flask. Slowly add 100 ml of
distilled water while swirling the flask
QUESTIONS (15 Marks)
gently to dissolve the powder. The solution
will be slightly cloudy in appearance as agar 1. Why was it unnecessary to adjust the pH of
will not dissolve until it is heated to about the nutrient broth? (2 marks)
100°C. 2. How would you adjust the pH of an agar
2. The agar medium is then sterilized in the medium? (3 marks)
autoclave (see next exercise). 3. Why has it been impossible to grow some
types of microorganisms on a defined
3. After sterilizing the nutrient agar, swirl it medium? (5 marks)
gently to disperse the agar evenly and cool it 4. What are the advantages of complex media in
to about 45°C. the general culturing of microorganisms? (5
4. Four sterile tubes of nutrient agar will be marks)
distributed and these tubes should be used to
make agar slants. To make slants, place the

20
 Preparing culture media

Sterilization Methods
After completing this exercise you should be able to:
1. Understand the effectiveness of the different methods of sterilization
2. List the advantages and disadvantages of moist and dry heat sterilization.

Sterilization, the process of killing all living organisms in or on a material, can be achieved by
exposing the material to lethal physical or chemical agents or, for liquids, by mechanically separating
out the organisms. There are many practical methods for sterilizing materials, and the choice often
depends on convenience and the nature of the materials to be sterilized.

HEAT STERILIZATION
Moist Heat
The most widely used equipment for sterilization is the autoclave, which can sterilize metals,
dressings, glassware, and most, but not all, aqueous media. Autoclaves work like pressure cookers.
Laboratory autoclaves are usually operated at a steam pressure of 15 1b/in2 at a temperature of 121°C.
An autoclave sterilizes most materials in 15-30 minutes, the variation in time being due to the surface-
to-volume ratio of the material being sterilized.

Some Aspects of Pressure-Steam Sterilization

Temperature. Bacterial endospores are the forms of life most
resistant to heat, and a killing temperature can be reached
only if the steam is under pressure. A temperature of 121°C
provides a good margin of safety if it is maintained for the
proper length of time.
Moisture. Coagulation of bacterial protoplasm (proteins,
enzymes, and so on) at moderate temperatures requires
moisture, and as moisture is removed, the temperature
required for coagulation increases rapidly. As steam
becomes superheated, it also becomes drier, so the
temperatures and exposure times required for sterilization
increase to approach those of hot-air sterilization (170°C for
one hour). Thus, excessively heated steam loses some of its
efficiency as a killing agent. In addition, the increased
temperatures can be detrimental to the materials to be
sterilized.
Pressure. Pressure itself has no effect on sterilization in the
ranges used with the autoclave. Pressure is useful only for
heating steam to temperatures above 100°C.
Time. Time is needed for the steam to
penetrate and heat materials to sterilizing Autoclave. (Courtesy of Market Forge Co.)
temperatures. Even when sterilizing
temperatures are attained, the spores (and
vegetative cells) are not all killed at once. The death rate is constant at a given temperature, and for
each unit of time of exposure to a lethal agent, a constant proportion of a given population is killed.
Usually it takes 11-12 minutes at 121°C (moist heat) to kill the endospores of thermophilic bacteria.
For some delicate foods or media it is useful to place the medium in a steam sterilizer for short

21
 Preparing culture media

periods, let it cool for several hours, and replace it in the steam sterilizer (intermittent sterilization).
All the vegetative cells are killed in the first treatment. The spores present germinate between the first
and second treatments.
Entrapped Air. The relatively cool air in the sterilizing chamber is more than twice as heavy as steam
at sterilizing temperature. If not allowed to escape, a stratification results. Since air and steam are
slow to mix, the difference in temperature between the upper and lower layer's can be very great,
hence the need for replacing all the air with steam. Even after mixing, the temperature can be below
that required. The thermometer in the discharge outlet indicates that all the air is exhausted when it
reads 100°C.
Nature of the Load. In general, bulky and more impervious materials require more time. It is
preferable, therefore, to sterilize in the smallest convenient units, for example, in five 1-liter flasks
rather than in one 5-liter flask.

Flasks should be plugged with cotton or capped with paper. If it is necessary to use rubber stoppers,
screw caps, or plastic caps, they should be set in place loosely to allow air to escape, to prevent the
containers from bursting or blowing off the caps as steam is generated, and to allow steam to penetrate
easily.

Dry Heat
Dry heat is used to sterilize glassware, other heat stable solid materials, and a few components or
materials (e.g., powder, oil, rice starch and animal food pellets) that would become unusable if exposed
to steam. These items are sterilized in an oven at 160 to 170°C for 2 to 3 hrs. Microbial death
apparently results from the oxidation of cell constituents and denaturation of protein

GAS STERILIZATION
The recent proliferation of plasticware as disposable syringes, petri dishes, culture tubes, plastic bags,
plastic filtration devices, plastic-backed adhesive bandages, and many other items has hastened the
development of a type of autoclave for heat-labile materials: the gaseous autoclave (Figure below).
Ethylene oxide is the gas most commonly used in such instruments. Economic decisions and the kind
of plastic determine the sterilizing regimens in this predominantly commercial process. For example,
if the humidity is kept between 20 and 50% and the concentration of ethylene oxide is kept to 720
mg/l, then sterilization can be achieved in 4 hours at 54.4°C (130°F) or in 8 hours at 37.7°C (100°F).

Ethylene oxide and other gases are increasingly important as sterilizing agents. Although many lab-
oratories do not have the equipment necessary to demonstrate gaseous sterilization, you should be
aware of its use and advantages. The use of ethylene oxide vapors under pressure, in special
equipment resembling a modified autoclave, is becoming a common method of "cold" sterilization.
Ethylene oxide is very toxic to viral particles, bacterial and fungal cells, and the most heat-resistant
bacterial endospores. As a sterilizing agent, it is easy to handle with proper equipment and is relatively
inexpensive. Unlike most toxic chemicals, it is relatively noncorrosive and nondeleterious to materials
being sterilized. Residual amounts of it are easily removed by aeration.
Although ethylene gas itself is flammable, 10% ethylene oxide with 90% carbon dioxide or mixtures
with freon are effective sterilizing agents that are nonflammable and nonexplosive. Its disadvantages
include long exposure periods for sterilization (several hours), reactivity with components of media
and certain types of plastics, and, as mentioned, the residue of ethylene oxide that remains after
sterilization, which must be removed by aeration or by allowing the sterilized material to stand.

22
 Preparing culture media

RADIATION
A few commercial processes use radiation for cold sterilization of certain materials (for example,
pharmaceuticals). Irradiation, is the use of high-energy ionizing radiation, which includes gamma
rays from a cobalt-60 or cesium-139 source and cathode rays from electron generators and
accelerators. irradiation by ultraviolet light is not a satisfactory means of sterilization because of
the low penetrating power of the wavelengths of the ultraviolet portion of the spectrum.

Membrane-filter apparatus. A: Example of a

reusable apparatus. B: Example of a disposable plastic
apparatus.

23
 Preparing culture media

Swinney syringe filters. A: Reusable filter

assembled and attached to syringe. B: Reusable
filter component parts. C: Disposable filters.

FILTRATION
The principal method of sterilizing liquids that contain
heat-sensitive components such as vitamins, serum
proteins, and antibiotics is by filtration. Historically,
microbiologists sterilized heat-labile media
components by passing them through autoclave-
sterilized filters made from diatomaceous earth,
asbestos fibers, or sintered glass. Since these types of
filters are hard to clean and have other disadvantages,
they have been replaced by disposable cellulose
acetate or polycarbonate membrane filters held in
stainless steel, glass, plastic, and disposable plastic
funnels (see figure on previous page). Suction can be
used to draw the liquid through the filter. Another
popular filtering device is the Swinney filter which is
used to sterile-filter small amounts of heat-labile
liquids such as antibiotics and serum components as
they are added aseptically to cultures or media. A pore
size of 0.45 μm is conventionally used for
sterilization. Marine microbiologists have found that many marine bacteria can pass through this
pore size, so they use filters with a pore size of only 0.2μm.

24
 Preparing culture media

PROCEDURE
Autoclaves vary greatly from model to model and among manufacturers. Pay careful attention to the
directions given you by your instructor.
Operation of the Pressure-Steam Sterilizer, or Autoclave
1. Ensure the autoclave has in the required volume of distilled water.
2. Load the autoclave with the tubes of nutrient broth and agar, and the flask of nutrient agar
prepared in the previous exercise.
3. Close and lock the door
4. Set the timer for 15 mins and stay and ensure the temperature and pressure gauge is rising and
steam is not escaping from the seals.
Autoclaves for routine laboratory sterilization are usually set for 15 pounds pressure, giving a
temperature of 121°C (250°F).
This is suitable for sterilization if maintained for 15 minutes. When the temperature reaches
121°C, begin timing sterilization.
Note that the thermometer measures the temperature of the steam in the discharge line. If air
has not been completely removed from the autoclave, even though the pressure gauge
indicates 15 pounds pressure, the temperature will not reach 121°C. Therefore, it is essential
to measure sterilization time from the moment the thermometer reaches 121°C rather than
from the time 15 pounds pressure is indicated.
7. The autoclave will automatically shut itself off. Once the pressure gauge indicates there is no
more steam pressure, you may open the door and remove the load. If the materials can be
damaged by prolonged heating and evaporation, cool them promptly

Note: If the materials that were sterilized did not contain liquid, but only if they did not, you
can open the operating valve to release the pressure more rapidly. If liquids are in the chamber,
sudden release of pressure causes them to boil up in the containers, wetting the plugs, and
blowing them out. It is, therefore, necessary always to let the pressure fall gradually when
liquids are being sterilized. Most modern autoclaves, many of which are entirely automatic, are
equipped with automatic locking devices on the doors that do not permit the doors to be opened
until the pressure has lessened

QUESTIONS (25 Marks)

1. What is the theory upon which intermittent sterilization is based? (10 marks)
2. Is dry or moist heat more efficient as a sterilizing agent? Why? (10 marks)
3. What is the temperature in degrees F when it is 170°C? (5 marks)

References
Seeley, H. W., VanDemark, P. J. and Lee, J. J., (1998) Microbes in Action: A laboratory manual of
microbiology, fourth edition, W. H. Freeman and Co., New York. ISBN: 0716721007
Wistreich, G. A., (2003) Microbiology Laboratory: Fundamentals and Applications, second
edition, Pearson Education Inc, Upper SaddleRiver, N.J. ISBN: 0130100749

25
Preparing culture media

26
 Preparing culture media

Name:______________________________________ Stream:_______________________

Lab Partner:_________________________________ Date:__________________________

Preparing Culture Media

Complete table with the results observed. (10 Marks)

Number of contaminated
items

Slants

Plates

Broth

27
28