Microbiological Media Preparation
Bacteria and fungi are grown on or in microbiological media of various types. The medium
that is used to culture the microorganism depends on the microorganism that one is trying to
isolate or identify. Different nutrients may be added to the medium, making it higher in
protein or in sugar. Various pH indicators are often added for differentiation of microbes
based on their biochemical reactions: the indicators may turn one color when slightly acidic,
another color when slightly basic. Other added ingredients may be growth factors, NaCl, and
pH buffers which keep the medium from straying too far from neutral as the microbes
metabolize.
In this exercise, you will make all-purpose media called trypticase soy broth and trypticase
soy agar. These 2 media----one a liquid and the other a solid---are the exact same formula
save for the addition of agar agar (really---agar agar), an extract from the cell walls of red
algae.
The old way to make media was by the cookbook method--- adding every ingredient bit by bit.
The only time that is done today is when making a special medium to grow a certain finicky
organism, where particular growth factors, nutrients,
vitamins, and so on, have to be added in certain amounts.
This medium is called a chemically defined medium
(synthetic). Fortunately, the most common bacteria that we
want to grow will do nicely with media that we commonly use
in lab. Some of our media is bought, but most is produced
in the prep area behind the lab. Since this type of medium
has some unknown ingredients, or sometimes unknown
quantities it is called complex media.
It is really very simple to make complex media these days:
 rehydrate the powder form of the medium
 stir and boil the agar medium to get the agar powder
dissolved (if making an agar medium rather than a
broth medium)
 distribute the medium into tubes
 autoclave to sterilize the tube media
 autoclave the agar medium for plate production
and then pour into sterile petri dishes
STERILIZATION AND THE AUTOCLAVE
When microbiological media has been made, it still has to be sterilized because of microbial
contamination from air, glassware, hands, etc. Within a few hours there will be thousands
of bacteria reproducing in the media so it has to be sterilized quickly before the microbes
start using the nutrients up. The sterilization process is a 100% kill, and guarantees that the
medium will stay sterile UNLESS exposed to contaminants by less than adequate aseptic
technique to exposure to air.
Media sterilization is carried out with
the autoclave, basically a huge steam
cooker. Steam enters into a jacket surrounding
the chamber. When the pressure from the
steam is at a certain point in the jacket, a valve
allows the steam to enter the chamber. The
pressure will go up over 15 pounds per square
inch (psi): at this point the timer begins to
count down--- usually for 15 minutes, depending
on the type of media. The high pressure in a
closed container allows the temperature to go
above the highest temperature one could get
by just boiling, around 121 degrees C.
Therefore, the parameters for sterilization with
an autoclave are 121 C at >15 psi for 15
minutes. Fifteen minutes is the thermal death time for most organisms (except some really
hardy sporeformers).
The prepared media is distributed in different ways, depending on the form one is making.
Broths and agar deeps are dispensed into tubes and then sterilized. Agar slant tubes are
sterilized and then the rack is tilted to allow the agar to solidify in a slanted fashion. Agar
medium which will be poured into plates is sterilized in a flask, and then poured afterward.
Not all media or solutions can be sterilized via an autoclave. Certain high-protein solutions
such as urea, vaccines, and serum will denature in the extreme heat, and so they may have
to be filter-sterilized without heat.
OBJECTIVES:
Understand how to make media, how to sterilize it, and how to distribute it in different
formats.
Produce TSA plates, TSA slants, and TSB which will be used in subsequent lab periods.
Understand the basics of an autoclave and how it sterilizes, including parameters.
MATERIALS NEEDED: per table
1 sleeve of plastic petri plates (may need a couple more if you pour light)
1 flask melted TSA (in 50C water bath)
1 spatula 24 medium, nonsterile test tubes
2 plastic weigh boats 1 small test tube rack
1 jar nutrient trypticase soy broth powder 12 yellow caps
1 jar agar powder 12 green caps
1-1 liter Erlenmeyer flask 1 pipet pump
1 magnetic stir bar 1 pipet disposal jar
1 graduated cylinder several nonsterile glass10 ml pipets
THE PROCEDURE: (see diagram below text for the entire production)
1. Your table will pour your plates, using a liquified, sterile agar medium which has
previously been made for you (should be sitting in a water bath). The flasks of melted TSA are
in the water bath which is set to 45. This is to prevent them from solidifying, since agar
solidifies at around 42 C. Remove your flask from the water bath only when you are ready
to pour your plates.
2. Set the petri dishes out, RIGHT SIDE UP (small side down), tops covering the dishes.
When you are ready to pour your plates, obtain your flask of agar and pour the plates.
Depending on whether you pour `heavy` or pour `light', you may use anywhere between 18-
22 petri dishes. Cover the bottom of each plate approximately 2/3 of the way across, put on
the lid and gently rotate the plate to distribute the agar across the entire dish bottom. Allow
all plates to stand until they are completely solidified. Once they have solidified, place them
on the tray at the instructor's table. Agar plates are always placed UPSIDE DOWN (bottom
dish with agar on top), definitely so when you are incubating your cultures.
3. Why are agar plates incubated upside down? Two reasons:
 Although there may be air contaminants in the incubator, it will be more difficult for
them to get onto plates, and then move UP onto the agar to settle since gravity is
working against this movement.
 Often you will see a bit of water condensation on the petri dish cover. The water
molecules are cohesive and tend to run together. If the plate is sitting right side up,
the water droplets can fall onto the agar, creating a kind of little lake on the agar
surface, messing up the plates. Upside down plates prevents the condensation from
dropping on the agar surface.
4. Begin making the TSB (broth) by pouring
500ml of distilled water into a 1L flask. Put in the
stir bar and turn on the stir plate so that the
surface is just disturbed. Add 6.5 grams of the
TSB powder to this flask and allow it to dissolve
(will happen quickly). No heat need be applied at
this stage.
5. Once the powder is dissolved, pipet out twelve
3ml aliquots into tubes and cover each tube with a
green cap.
NOTE: Green caps are always used for TSB.
6. With the remaining solution (about 464ml) still
stirring, add 8 grams of agar powder.
7. The next step will require you to apply heat to the mixture. Before you do this,
however, you should be aware that agar has a strong tendency to boil over when it reaches
100C. Someone in your group should be watching the flask at all times once you see
steam coming off of it. At the first sign that the mix is near boiling, REMOVE it from the
hot plate (paper towels around the flask neck). DO NOT simply turn off the heat, letting
the flask sit there. The metal plate retains a significant amount of heat, and turning off the
heat will not prevent the flask from boiling over. Folded paper towels allow you to grasp
the flask neck tightly, yet not burn your hand.
8. Have you read step 7? Ok, then you can turn on the heat to setting 9 (not High). Make
sure that the magnetic bar is stirring the solution.
9. When the mixture boils and the agar dissolves, it will turn clear, deeper tan. Remove it
from the heat and pipet out twelve 6ml aliquots into medium tubes for slants (they will not be
BE slants until removed from the autoclave and tilted to the side to solidify). Pipet out four
6ml aliquots into medium tubes for agar deeps. Cover the slant tubes with yellow caps and
the deep tubes with black caps. NOTE: from this point on, yellow caps will be used for nutrient
agar slants. Also note: if the agar solidifies in the tip of the pipet, dispose of the pipet in the
pipet jar and get another one. To prevent this from happening, either pipet out all the tubes at
the same time, or leave the pipet in the flask of melted agar.
10. Place all of the tubes you have pipetted out in the plastic autoclave racks on the
instructor's table as well as the remaining of your melted agar. All agar deeps go in one rack,
broths in another rack, etc.
11. Cover the flask with the remaining agar medium (to be sterilized) with a piece of foil and
place in the metal tray on instructor’s table (for sterilization).
12. Dispose of your used pipets in the pipet holder. These glass pipets are reusable, so
don't throw them in the trash.
QUESTIONS:
1. What is a complex medium?
2. Why are pH buffers added to the growth media for microbes?
3. How can the temperature in the autoclave go above boiling temperature of 212 F?
4. Why do you have boil the agar solution BEFORE dispensing it into tubes?
5. At what temperature does agar solidify?
OLANREWAJU ISRAEL.+23470334765