Media Preparation & Sterilization

Culture Media: is a medium (liquid or solid) that contains nutrients to grow

bacteria in vitro. Because sometimes we cannot identify with microscopically
examination directly, and sometimes we do culture for antibiotic sensitivity
testing

A medium is sterilized before usage in the lab.

Sterilization methods include: autoclaving, dry-heat, filtration and UV light.

Note: media are sterilized by autoclaving at 121°C. With the autoclave, all
bacteria, fungi, viruses, and spores are destroyed. Some media can’t be sterilized
by autoclaving because they contain eggs or carbohydrates.

Properties of Media

• Support the growth of the bacteria.

• Should be nutritive (contains the required amount of nutrients).

• Suitable pH (neutral to slightly alkaline 7.3-7.4).

• Suitable temperature and suitable atmosphere. (Bacteria grow at 370C).

There are three physical forms of media:

Solid, broth and semi-solid. (they were divided according to their

consistency)

• Solid (agar): Is Broth plus agar (seaweed).

Are prepared by adding a solidifying agent (agar 1.5 -3%).Prepared mainly in

Petri dishes, but also in tubes and slopes. After growth the bacterial colonies
are visible. e.g. blood agar, chocolate agar, MacConkey agar.

Solid media properties

 Used to isolate pure cultures

1
  Ideal for culture storage
 Helpful in the observation of biochemical reactions
Semi-solid agar (soft agar): Contains small amounts of agar (0.5-0.7%). Used to
check for motility and also used as a transport media for fragile organisms. ex:
SIM for motility.

Agar is:
1. Complex polysaccharide
2. Used as solidifying agent for culture media in Petri plates, slants, and deeps
3. Generally not metabolized by microbes
4. Liquefies at 100°C.
5. Solidifies ~40°C.
Liquid (Broth): Growth of bacteria is shown by turbidity in medium. e.g. Nutrient
broth, Selenite F broth, alkaline peptone water.
Liquid medium is best when you want to rapidly increase the concentration of the
organism or when you want to grow motile cells.

The growth in liquid media: 1.Surface growth pellicle, 2.uniformly turbid,

3. Sediment in bottom
1. 2. 3.

Types of Culture Media

1. Simple (basal, ordinary) culture media: are media that contain the basic
nutrients (growth factors) that support the growth of bacteria without
special nutrients, and they are used as basis of enriched media. E.g.
Nutrient broth, nutrient agar, peptone water. They are for the growth of
non-fastidious organisms like E.coli.
2. Enriched Culture Media: are media that are enriched with: Whole blood
e.g. blood agar. Lysed blood (heated to 80C) e.g. Chocolate agar.

3. Selective Media: it is a mediam, which contains inhibitor substances that

prevent the growth of microorganisms other than the bacteria for which
the media is prepared for. For example:EMB (Eosin Methylene blue

4. Differential Media: it is a mediam, which contains a carbohydrate source

and indicator to differentiate microorganisms based on their ability to
ferment carbohydrate to produce acid end-products which react with the
pH indicator and change its color.

e.g. MacConkey agar, which contains neutral red (pH indicator) and is used to
differentiate lactose fermenter (E. coli )and non-lactose fermenter
(Salmonella).the Organisms ferment lactose and produce acid end-products which
react with the pH indicator neutral red, and produce a pink color. If the bacteria
are non- lactose fermenter it will utilize protein source and produce alkaline
products which react with pH indicator neutral red, and produce a pale yellow
color.
Pouring Agar Plates
When petri plates are needed, the agar deeps are melted either in a
boiling water bath (figure a) or by bringing them to 121°C in an autoclave
for 30 to 60 seconds and then releasing the steam under slow exhaust.
After the agar has melted, the pours are transferred to a 48° to 50°C
water bath and kept there for at least 5 to 10 minutes before use (figure b).
The agar deeps should be cooled to about 50°C before they are used to
minimize the amount of steam condensation on the petri plate lids after
the agar has been poured. Agar does not solidify until its temperature
drops to about 42°C. When the deeps have reached 50°C, one is taken
from the bath and the outside is dried with a paper towel (figure c). Its cap
is removed and the top is briefly flamed using a Bunsen burner (figure d).
The agar is immediately poured into a sterile, dry petri plate bottom in
order to avoid contamination (figure e). Replace the top, allow the agar to
cool and harden, and store the petri plates in an inverted position (figure f)