The document discusses different types of culture media used in microbiology, including solid, semi-solid, and liquid media. Liquid culture media is described in more detail. It can be used to grow large amounts of bacteria for downstream applications. Nutrient broth is provided as a common example of a liquid medium, and steps are outlined for preparing and inoculating nutrient broth with bacteria from a solid culture. Tips for properly transferring liquids between sterile containers using pipettes are also presented.

1. Microbiology Lab
Prepared by: Kazhaleh Mohammadi
Knowledge University
2022-2023
Culture Media
Liquid Culture Media

2. Types of Media in Microbiology
• Many bacteria, yeasts, and molds that occur in nature can also be grown in
the laboratory. In addition to water, the culture media used for this
purpose have to contain the nutrients the microorganisms need for
growth, in particular;
• an energy source (typically sugars)
• useable sources of the elements needed for biosynthesis and metabolism
(especially nitrogen, sulfur, phosphorous)

3. Types of Culture Media
• SOLID CULTURE MEDIA
• SEMI-SOLID CULTURE MEDIA
• LIQUID CULTURE MEDIA

4. Types of Culture Media
Solid Media – contains 2% agar
• Colony morphology, pigmentation, hemolysis.
• Eg: Nutrient agar, Blood agar
Semi-Solid media – 0.5% agar.
• Eg: Motility medium
Liquid Media – no agar.
• For inoculum preparation, Blood culture, for the isolation of pathogens
from a mixture.
• Eg: Nutrient broth

5. LIQUID CULTURE MEDIA
One method of bacterial culture is liquid culture, in which the desired bacteria are
suspended in a liquid nutrient medium, such as Luria Broth, in an upright flask. This
allows a scientist to grow up large amounts of bacteria for a variety of downstream
applications.
Liquid cultures are ideal for the preparation of an antimicrobial assay in which the
experimenter inoculates liquid broth with bacteria and lets it grow overnight (they may
use a shaker for uniform growth). Then they would take aliquots of the sample to test
for the antimicrobial activity of a specific drug or protein (antimicrobial peptides).

6. Liquid media: These are available for use in test tubes, bottles, or flasks.
Liquid media are sometimes referred to as “broths” (e.g nutrient broth). In a
liquid medium, bacteria grow uniformly producing general turbidity. Certain
aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known
to grow as a thin film called ‘surface pellicle’ on the surface of the
undisturbed broth. Long chains of Streptococci when grown in liquid media
tend to entangle and settle to the bottom forming granular deposits. Liquid
media tend to be used when a large number of bacteria have to be grown.
LIQUID CULTURE MEDIA

7. Liquid cultures are ideal for the preparation of an antimicrobial assay
in which the experimenter inoculates liquid broth with bacteria and lets
it grow overnight (they may use a shaker for uniform growth). Then
they would take aliquots of the sample to test for the antimicrobial
activity of a specific drug or protein (antimicrobial peptides).
LIQUID CULTURE MEDIA

8. What is Nutrient Broth?
Basically, the Nutrient Broth is the nutrient agar that lacks of the
solidifying agent, agar powder. They remain in liquid form at room
temperature and are usually used to maintain the stocks of
microorganisms. In general, they are used to grow fastidious
organisms. Also, you can enrich your nutrient broth with blood,
serum, sugars… etc for special purposes.

10. Pre-sterilized glass or plastic graduated pipettes are
used to transfer specific volumes of sterile liquids
accurately. It is important that you learn how to use
these tools correctly, since it may be necessary to
transfer sterile and sometimes contaminated liquids
among various bottles and tubes.
LIQUID CULTURE MEDIA

11. How to prepare Nutrient Broth?
1) Add 13g of nutrient broth powder in 1L of distilled water.
2) Mix and dissolve them completely.
3) Pour them into the final containers (eg. conical flask)
4) Sterilize by autoclaving at 121°C for 15 minutes.

12. Transferring bacteria to a Nutrient Broth
1) Take up the inoculating loop by the handle, and hold the wire on the flame of a
Bunsen burner until it glows red then cool it.
2) Pick up the solid culture with your left hand. Still holding the loop like a pencil,
3) Pass the neck of the open tube rapidly through the Bunsen flame two or three
times (do not overheat; if it is glass, it could crack or burn you later; if it is
plastic, it could melt). This flaming sterilizes the air in and around the mouth of
the tube.
4) Insert the loop into the open plate. Touch the loop (not the handle) to culture.

13. 5) Insert the loop into the tube and down into the broth. Gently rub the loop
against the wall of the tube (don't agitate or splash the broth), making sure the
liquid does not touch the loop handle.
6) As you withdraw the loop, touch it to the inside wall of the tube (not the tube's
mouth) to remove excess fluid from it. Pull it out without touching it, again
flame the tube neck, replace the closure, and put the tube back in the rack.
7) Now carefully flame the loop, and be sure all of the wire is sterilized. When the
wire has cooled, the loop can be placed on the bench top.
8) Label the tube you have just inoculated with your name and the date.
Transferring bacteria to a Nutrient Broth

14. Some Tips To Remember
• The pipette and the media are sterile; there should never be any
direct contact with your hands, skin, or lab surfaces.
• Caps or lids on tubes or bottles should never be set down on lab
surfaces.
• Tubes or bottles should be held at an angle during the transfer
process, to minimize the potential for airborne contaminants to make
their way into the opening.
• Passing the opening of the tube or bottle briefly through a flame
before and after the transfer process will discourage airborne
contaminants from getting into the sterile liquid.