PREPARING LIQUID AND SOLID MEDIA AND MAINTAINING
MICROBIAL CULTURES
I. Making Liquid Media:
Liquid media is easy to make. Mix the powder according to the package or bottle instructions
with pure water. Researchers use reverse osmosis or filtered water, but for the classroom even
tapwater works fine. (Note, do not use tapwater if you are preparing a minimal medium,
something used for special applications.) I recommend adding powder to the measured water,
not water to powder (that causes powder to form a sticky residue in your vessel. The mixture can
be mixed by hand stirring with a spoon or rod, or a magnetic stir bar. Heat helps the powder
dissolve faster and can be accomplished with low-medium microwaving or heating low-medium
on a hotplate while stirring.
After the powder is dissolved, autoclave (sterilizes the medium) or microwave to boiling, or boil a
few minutes. Boiling will kill all organisms except endospores which are very resistant. But a lot of
school microbiology can still be done with boiled media, since >90% of the time you will not get
contaminating spores in the types of applications we are discussing.
What type of container should I use for media? Obviously if media will be boiled on a hotplate or
autoclaved, you need to use a glass flask or beaker. As with any boiling liquid, it is important to
keep any cap loose. Plastic can be used if microwaving media. Be sure to use a nice large
container so that liquid only takes up ¼-1/2 th e volume. This really helps prevent liquid from
boiling over, foaming over or blurping out.
When the media has been boiled or autoclaved, allow to cool down with a very loosened cap or
foil cover. When cool, you can tighten the cap and store at RT or in the fridge. Waiting overnight
at RT, you will know if your media is truly sterile or not. If it stays nice and clear, you are fine! If it
gets cloudy, you were not so lucky. Media is like food. Sterile food can sit a long time without
spoiling, but some compounds especially vitamins and things will break down or oxidize with time.
Liquid media has a long shelf life but will degrade over time. I suggest keeping it in fridge if you
can, but on shelf, for simple applications it may be good for up to one year or more! You will
notice darkening as media oxidizes and degrades.
Dispensing: If you have an autoclave, sometimes it is easier to pipette or pour non-sterilized
media into smaller tubes or containers and then autoclave these. If you will be using plastic tubes
you can’t do this and will simply dispense the sterile liquid into the tubes using sterile technique.
Types of media: You will most likely use Nutrient Broth or Tryptic soy Broth. These are both
reasonably priced full spectrum broths that support the growth of lots of microbes. If you want to
encourage more environmental organisms (from soil or pond or seawater, for example) I
recommend that you prepare these at a much lower concentration than the basic recipe. ½
strength is a good starting point for these purposes.
Nutrient agar is basically a mixture of beef extract and peptone 9protein from animas). TS broth
consists of tryptose (protein from soy), dextrose and sodium chloride. IN GENERAL, both can be
used for most of the BSL organisms that you cultivate. Nutrient broth/agar makes more sense for
environmental growth experiments, and TS for organisms associated with humans or animals.
It may seem strange to use beef extract and peptone to isolate environmental organisms. TS
broth/agar was developed to use for pathogens as an alternative to Nutrient broth/agar, since it is
not considered a good idea to use media derived from animal tissue in these clinical cultures
since it interferes with some tests.
WISTR Ruth A. Gyure July 2006 Western CT State University
Of course there are many, many specialized media for lots of purposes, some that are very
selective for particular groups and many have chemical indicators such as ph dyes. As you learn
more you may wish to use some of them for special applications or experiments if your budget
allows.
A source for media, no penalty for small orders:
http://www.alphabiosciences.com
BD (Beckton dickinson) has an outstanding online media manual with a pdf descriptive
attachment for each of the media types they sell
http://www.bd.com/ds/technicalCenter/inserts/difcoBblManual.asp
II. Making Solid media:
Making agar plates is not complicated, but a bit trickier than making liquids. It is best to have
someone give you a demonstration and tips. People have different habits and preferences for
making and pouring plates, and I will tell you my tips and suggestions.
a. Remember that agar comes in many varieties. Noble agar or Bactoagar is basically plain
agar with no nutrients added (noble agar is purer and more expensive). You will note that
agarose is also very pure and used for electrophoresis and other molecular applications.
You will probably purchase agar WITH nutrients. Typically you can purchase media of the
same type in either broth formulation or WITH agar.
b. When media is made as agar, the agar part usually consists of 15g per liter, which makes the
agar 1.5% concentration. This makes the perfect consistency or firmness. Remember never
to dilute the agar concentration as you might for broth, since the plates will be too runny.
Suppose you do wish to make ½ strength nutrient agar. You would need to mix the
ingredients separately by hand. 15g agar as usual per liter, but half the beef extract and
peptone.
c. To prepare agar, mix the powder into the water as if you were making broth, following
instructions on packet or bottle. It is helpful to heat the water up a bit before starting.
d. If using a hotplate with a magnetic stirrer (best), again, make sure to use a very large glass
container only ¼ to ½ filled with liquid. Boiling agar is especially prone to foaming and boiling
over, and it happens in a quick instant as the temperature reaches the turning point. Be
prepared, and also be prepared with some hot mits or gloves nearby to remove it from the
plate immediately at that point. Turning down the heat will not be sufficient. Once boiling is
reached, the media is ready to be autoclaved, or used as it is if you do not have that
capability. x
e. If using the microwave, go slowly. Pay attention to the same rules about size of container vs.
volume of liquid. Heat in increments (full heat is fine). For example, if you are preparing
500ml agar, use at least a 1 liter container. Start perhaps with 2 minutes heat. Swilr to mix.
Heat another minute, swirl, etc. As it gets hotter keep an eye on it and shut the power when
you see it boil. Once boiling is reached, the media is ready to be autoclaved, or used as it is if
you do not have that capability. Once boiling is reached, the media is ready to be autoclaved,
or used as it is if you do not have that capability.
f. Some important notes: First, why boil the agar if it is going into the autoclave anyway?? Well,
sometimes in the autoclave, heating may occur unevenly, and mixing by convection may just
not happen exactly as you would wish. You may end up with a blob of unmelted agar in the
bottom of the flask or bottle. If you premelt before autoclaving you have more oversight of the
process. Also, most of us melt the agar first and mix, because we want to do some
dispensing before the final autoclave. If you separate the agar into portions, it is very
important that it is completely dissolved and mixed homogeneously, which only occurs if you
bring it to boiling. Sometimes you will be tempted to stop before a full boil is reached. DO
NOT BE.
WISTR Ruth A. Gyure July 2006 Western CT State University
g. Whether autoclaved or only boiled, the agar should cool down to at least 55-60 before
pouring plates. Do not let it cool below 45C. Using a water bath is the best way to do this, just
put your flask or bottle in the water bath after boiling or autoclaving and let it sit for awhile. If
you don’t have a waterbath, just let it sit awhile till it cools. The agar is good to pour when you
are able to grasp it with your hand without feeling like you have to put it down instantly. It will
still be too hot to hold continuously. That’s about right. Use a moist cloth or small non-bulky
glove to help you grasp the bottle or flask safely while you pour. You can experiment with
different containers and will find that if the container is too big it will be hard to grasp, and
some containers have lips that pour better than others. I prefer to pour from bottles, so I
always make my media in flasks, then pour it into small/medium size bottles for pouring. I
have some tricks for making pouring even more easy which is important if you have to pour
lots of plates, but that’s something for the in-lab demonstration.
h. Pouring plates is as simple as that, just pouring it into each plate one at a time. As with all
sterile technique, only remove each plate lid just before pouring and then immediately replace
it. Some people like to spread the plates out on a bench as they pour (they cool faster) and
others like to stack them (less condensation under the lid as they cool). In any case, move
the plates around as little as possible after pouring and be very careful. As the agar cools and
begins to solidify, moving the plate roughly will cause ripples, waves and unevenness in the
surface.
i. Plates do take a few hours to completely harden, so be patient. It is fine to leave them out
overnight. If they are truly sterile they will stay sterile, and they will have the opportunity to dry
out a little. Once the plates have hardened, store them in a plastic bag (ideally the plastic
sleeve in which the plates came in the box) that is taped shut so plates don’t dry out. It is best
to store them in fridge, though a few days at RT will probably be okay if they were
autoclaved. Mold is the most common contaminant of home-made plates and it can spread
rapidly. Don’t keep plates around too long and check the bags once in awhile for signs of
contaminants. Some people like to label each plate with some abbreviation (for media type)
but some like to only label the bags. I don’t recommend putting a date on them, since it will
be more important to put a date on the plate when you actually inoculate it. It is a nice idea to
put a date on the bag to remind you when you made the plates and how old they are. Plates
may also be stored in plasticware boxes.
III. Growing and Maintaining Live Bacterial Cultures
There are some detailed references on the RESOURCE page, but here I’ll just list some basics.
When you streak or spread some live bacteria (or yeast) on a plate, incubate it at the appropriate
temperature (whatever the bacterium is known to prefer) and once it has grown up on the plate,
remove it from the incubator. If you do not have an incubator, many cultures can be grown at
normal room temperature but may take a bit longer to grow. If you keep plates at warm
temperatures longer than necessary, it does not help the culture, it encourages contamination
and degeneration of the cells on the plate, and speeds up the drying out process.
Grown cultures can be stored in a plasticware box or plastic bag that is kept closed. Placing a
most piece of paper toweling or sponge inside helps maintain a nice humidity level. When the
paper towel or sponge dries out the plates will begin to dry out too, so either re-wet them, or take
this as a sign that you should sort your plates and either discard or transfer them to fresh plates.
Cultures are often stored in the refrigerator and can also be kept at room temperature for many
weeks if you keep an eye on them and the moisture level. Immediately discard any plate that
starts to show signs of mold growth.
If you are going to store a culture it is a good idea to use your ‘streaking for isolation’ technique to
observe isolated colonies. This is some assurance that the culture has remained pure and
healthy (all the colonies are of the same type). If you start to see different types of colonies or
molds, the culture must be discarded, or you can try to re-purify your stock form a single colony of
the correct type. Remember, you can request a fresh culture from me or order one from Ward’s or
other source. Wards and Carolina Biologicals all sell a variety or microbes either on agar in a
WISTR Ruth A. Gyure July 2006 Western CT State University
tube, or in a freeze dried form. In fairness, you will mostly be very happy with these but I have
had some duds with Wards cultures (no growth from the culture most common). Some
companies like these as well as Fisher still sell BactoDiscs (freeze dried) which are fantastic, but
expensive for a bottle of discs. When you need a new fresh culture, all you do is pop a disc into
fresh broth. Easy!
Another nice source for small-scale micro supplies: Presque Isle Cultures. This is a small family
owned business in Erie, Pennsylvania. They are happy to supply small orders, are very
reasonably priced, and very friendly with customer service.
http://www.picultures.com/
When you really need a true and reliable species or strain, I highly recommend Microbiologics
http://www.microbiologics.com/
or ATTC (American Type Culture Collection).
http://www.atcc.org
ATTC sells some bacteria at education pricing, Still they will be more expensive than the other
sources. For quality when necessary they are worth it.
WISTR Ruth A. Gyure July 2006 Western CT State University