CULTURE MEDIA PREPARATION

1. Counter-check all computed amounts of media before proceeding to the preparation room to
weigh all the media components needed.
2. Make sure that all media components are available and ready before weighing the
components.
3. Fold a scratch paper/wax paper in a conical/triangular manner and place on top of the
weighing balance, tare the top loading balance then dispense the powder or granules gradually
to prevent over-weighing or over-dispensing of the media.
4. Use a separate spatula for every reagent or media to prevent cross-contamination between
media or reagents. Wash all used spatula before returning them to the drawer.
5. Transfer all weighed media components in a beaker of appropriate volume immediately to
prevent it from caking on the paper. Immediately close the lids of media and reagents as most
culture media and dye powders are highly hygroscopic.
6. Swirl or stir to dissolve all components in distilled water, if the media contains agar, one can
microwave it to boiling to dissolve and homogenize the agar (read the instructions carefully as
some media may only be heated for a brief period of time or may not be heated at all).
7. Transfer the media in a flask of appropriate volume and seal with a cotton plug, paper and
rubber band. Make sure that it is labeled properly with all the information necessary (ie. Initials
or name of media/section/subject) to prevent mix-up or confusion between various media used.
8. The volume of the media should not exceed more than 2/3 of the maximum volume of the
glassware to prevent boiling-over of the media during sterilization or boiling.
9. Sterilize the media using steam under pressure (this may be accomplished using the autoclave
or the pressure cooker). Most media are sterilized at 121ºC 15psi for 15 minutes. However,
some culture media may possess components which may be destroyed by heat so a lower
temperature is employed or the culture media may not be sterilized at all.
10. Most sterile culture media may remain stable and be used for up to two weeks at room
temperature. Media may be refrigerated to prevent excessive moisture loss and maintain its
microbiological stability.
11. Solid media may be re-melted using the microwave. To do this, simply place the media in a
microwave and set to the desired heating time. Watch over the media as it may boil-over the
flask at any one time if left unattended.
12. Once the media is melted, wait for a few seconds to a minute to cool down the media as it may
boil over if agitated immediately after microwaving.
13. Dispense to appropriate containers.

Notes/Hints:
1. To make culture media plates, the media is sterilized, cooled to approximately 45 degrees
Celsius and dispensed into plates. If excessive moisture is formed on the top cover, one may
dry the plates under the laminar flow hood with the lids exposed.
2. To make broth culture media, prepare first the media in bulk then dispense into tubes then
sterilize.
3. To make slants/deeps/stabs, boil or microwave the solid culture media to melt the agar. Once
the media is already homogenized or clear, dispense the appropriate volume to tubes using a
glass syringe. Seal the tubes with either screw-caps or cotton plugs then sterilize. To make
slants, simply place the molten media in a slanted position until the media solidifies.
4. Always use heat-resistant gloves when handling boiling media
5. Some heat-sensitive media components (ie. antibiotics, redox/pH indicators etc) are filter-
sterilized by passing it through a metal-tipped glass syringe connected to a membrane filter.

KR ALVAREZ/2011
DECONTAMINATION

All contaminated lab ware, spent media and microbial cultures are decontaminated after use and
before disposal into the environment. A pressure cooker is a very cheap alternative to accomplish this
task. To do this, sign out the following materials from the preparation room: PC cover, valve, PC
opener, PC basket and plastic tray (to contain all washed glassware).

1. Fill the cooker with tap water enough to reach the base of the basket when loaded.
2. Load the materials to be sterilized or decontaminated. Make sure that screw-caps are
loosened and that autoclavable bags not sealed too tightly around the neck in order to facilitate
faster penetration of steam and equilibration of steam-pressure at all points or areas or items
inside the pressure cooker
3. Cover the pressure cooker so that the arrow markings on the cover and the pot meet.
4. Simultaneously tighten two opposite-facing knobs.
5. Turn on the heat source
6. Build up steam inside the pressure cooker to exhaust atmospheric air for approximately three
minutes
7. Plug/close the steam-exhaust valve to accumulate steam inside the chamber
8. Build up the steam so that the pressure exerted by the steam-compression reaches up to 15
psi (which is approximately equivalent to 121 degrees Celsius). Maintain the steam-pressure at
this level for 15-30 minutes by regulating or adjusting the heat source.
9. Turn off the heat source and let the steam-pressure decrease on its own. DO NOT REMOVE
THE PC VALVE UNTIL THE PRESSURE GAUGE REACHES ZERO AND THE PRESSURE IS
THOROUGHLY EXHAUSTED.
10. Open the pressure cooker by unscrewing the knobs slowly. NEVER ALLOW SKIN TO BE
DIRECTLY IN CONTACT/ON TOP OF THE PRESSURE COOKER UPON OPENING SINCE A
LOT OF STEAM WILL BE LIBERATED UPON INITIAL OPENING OF THE PRESSURE
COOKER
11. Cool the materials first before washing them in the sink as hot non-pyrex glassware may crack
upon contact with cold water.
12. NEVER dispose molten agar and solid wastes through the sink as it may clog pipelines.
13. Routine clean-up of all decontaminated materials will be accomplished per group by rotation.

Notes/Hints:
1. Estimated turn-around time of a decontamination cycle is approximately one to two hours
depending on the load of the pressure cooker chamber and sterilization time employed.
2. A sparker or lighter with a long probe may be useful when igniting the burner.
3. Remember to always TURN OFF the valve of the LPG tank after use.

KR ALVAREZ/2011