Practical No.

2
Culture Media and Laboratory
Equipments
Department of Microbiology
College of Medicine
Culture Media
Culture media

Types of culture media

Culture media components

Preparation of culture media

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 Advantages of culture media
 To culture microorganisms.

 For pure culture isolation.

 For storage of stock cultures.

 To observe specific biochemical reactions.

 As transport media to preserve bacteria during transportation to the

laboratory.

 For preparation of antigens (vaccines and diagnostic kits).

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 Knowing the microscopic morphology and staining
characteristics of a microorganism present in a clinical
specimen, the microbiologist can make appropriate
decisions as to how it should be cultivated and what
biologic properties must be demonstrated to identify it
fully.
 A suitable culture medium must be provided, and it must contain the nutrients
essential for the growth of the microorganism to be studied.
 Most media designed for the initial growth and isolation of microorganisms are
rich in protein components derived from animal meats.
 Many bacteria are unable to break down proteins to a utilizable form and must
be provided with extracted or partially degraded protein material (peptides,
proteoses, peptones, amino acids).
 Meat extracts or partially cooked meats are the basic nutrients of many culture
media. Some carbohydrate and mineral salts are usually added as well. Such
basal media may then be supplemented, or enriched, with blood, serum,
vitamins, other carbohydrates and mineral salts, or particular amino acids as
needed or indicated.
 Types of Media used in Bacteriology laboratories:
There are several types of media used in bacteriology labs. Several items or constituents
could be added or removed from the routinely used media according to the purpose for use.
 Media could be classified according to their composition;
1. Natural medium: which contains natural constituents, such as; yeast extract, meat
extract, peptones….etc. where the actual contents and their concentrations are not known.
2. Synthetic medium: This type of medium is prepared manually and the actual contents
and their concentrations are exactly known.
3. Semi-synthetic medium: The contents of this medium are mainly synthetic with known
concentrations, but is supplemented with some natural contents in order to enhance growth
or for certain physiological studies. This is used for isolation as well as for physiological
studies.
 Types of media according to their solidity;
1. Solid media: These types of media contains the actual concentration of agar
(which is 2%) which makes the media solid, therefore encourages the formation of
colonies on its surface and could be used for isolation and purification of mixed
samples or cultures.
2. Liquid media: These types of media do not contain agar at all, and therefore the
media remain in liquid form where the bacteria grow better and faster.
3. Semisolid medium: This type of medium contains half the actual concentration of
agar (1% only), and is used mainly for studying motility of bacteria, since the agar
molecules are not so condensed, therefore it will encourage the motile bacteria to
pass through these molecules and go far from the inoculated area forming what is
called brush –like growth around the stabbed area.
According to solidity or physical properties, media can be
classified three types:
1. solid medium ( 2 % of agar)
agar plate /slant medium
2. semi-solid medium (1 % of agar)
3. liquid medium( do not contain agar)
 Types of media according to their function
1. Isolation medium: This type of medium all nutrients that are required by most bacteria, and
is used for growth of unknown bacteria contains from different samples.
Ex. nutrients agar
2. Selective medium: This type of medium contains certain inhibitors that will inhibit the growth of unwanted
bacteria and do not affect the growth of the desired bacteria which will make the isolation and purification much
easier and faster.
Ex. SS agar
3. Differential medium: In such type of medium, certain chemicals or indicators are added in order to
differentiate between two groups of bacteria grown on this medium without needing to do further test for such
purpose which will save time and efforts.
Ex. MacConkey agar
 Isolation medium
All nutrients used for growth of unknown
bacteria.
e.g. nutrients agar

nutrients agar
Selective Medium
the medium that can prevent the
certain bacterial growth while
permitting others.
e.g. SS agar (Salmonella
Shigella agar)

Salmonella Shigella agar 10

 Differential Medium
Some special substrates and indicators are
added into the media in order to produce a
visual differentiation
e.g. EMB agar (Eosin-methylene blue agar).

Eosin-methylene blue agar

Maintenance medium

Cooked-meat medium is used for the preservation

of anaerobic bacteria.

Cooked-meat medium 11
4. Maintenance medium: This type of medium contains certain nutrients that allow slow growth of bacteria,
therefore it will stay longer time before they reach the stationary or the death phase. This type of medium is
used for preservation of bacteria for longer time (1-2 months) before doing the subculture.
e.g. Cooked-meat medium is used for the preservation of anaerobic bacteria.
5. Enriched medium: In this medium certain nutrients or factors are added in order to allow specific
bacteria to grow, because without such factors that bacteria will not grow. Such as the addition of blood,
serum, hemolized blood….etc.
e.g. Blood agar
6. Biochemical test media: There are several types of such media where different biochemical tests are
applied depending on the type of the medium and its contents. Such as the fermentation tests, enzyme
activity, degradation of certain compounds….etc.
7. Antibiotic sensitivity test medium: This type of medium allows the growth of most bacteria and do not
contain any growth inhibitors or materials that may interfere with the action of the antibiotics.

e.g. Müeller-Hinton agar

Enriched Medium
Additional or special nutrients (e.g., serum, growth
factors, trace elements) are added to support some
fastidious bacterial growth.
e.g. blood agar.

Biochemical test:
different compound.
e.g. Simmon citrate agar , Triple sugar iron
slant

Simmon citrate agar Triple sugar iron slant

Antibiotic sensitivity test medium:

Allows the growth of most bacteria .

e.g: Müeller-Hinton agar is most frequently used
in this antibiotic susceptibility test.

Müeller-Hinton agar

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Preparation of Culture Media
Instruments and equipment's
 Instruments and equipment's usually used in bacteriology laboratories;
1. Microscope (with at least 1000X)
2. Incubator
3. Hot air oven
4. Autoclave
5. Anaerobic jar
6. Microflow (laminar flow)
7. Fume hood
8. Hot plate with magnetic stirrer
9. Electronic Balance
10. Top plate balance
11. Shaking Incubator
12. Shaking water bath
13. Bunsen burner
14. Wire loop & wire needle
Equipments

1- Autoclave:- used for sterilization

equipment with high temperature, pressure and steam to sterilize
the culture media and some of metal tools and glass wares.
• Temperature: 121 C
• Pressure: 1 atm. (15 pound/1 itch )
• Time: 10 - 15 min.
• 30 min. (for Petri-Dish cultured media and contaminated tools
& glass wares)
• Sterilization by Autoclave called (Wet heat sterilization).
Bacterial cell is killed by protein denaturation.
2-Hot air oven
Oven: used to sterilize the glass wares and some metal tools by
(Dry heat sterilization).
Temp.: 180
Time: One hour & half.

3-Water bath :- It’s a laboratory equipment made from

a container filled with heated water .
• It is used to incubate samples in water at
a constant temperature over a long period of time.
4-Incubator
used to provide suitable conditions for microorganism
growth temperature , Co2, humidity and oxygen content of
the atmosphere inside.

5- Refrigerator
Used to maintain the sterilized media and broth when not
used to avoid the contamination , and also to preserve the
bacterial culture for long time by preventing the growth at
4C˚.
6-Biosafety cabinet (BSC)
Also called a biological safety cabinet or is an enclosed, ventilated
laboratory workspace for safely working with materials
contaminated with (or potentially contaminated with pathogens).

7- Centrifuge
A machine with a rapidly rotating container that applies
centrifugal force to its contents.
Used for separating substances of different densities.
e.g. to separate red blood cells and other blood components
from whole blood.
Instrument
1. Loop: used for transfer of bacterial cells from
medium to another, sterilized by the flame of burner.

2. Pipette: used for transfer of cultured and uncultured

broth from tube or flask to other, and placed in the
Can. Sterilized in Autoclave.
4 - Petri-Dish(Petri-plate):-
Used for place the solid medium in it.
glass petri-dish used for many times and sterilized by
oven or autoclave while sterilized plastic plates used
for one time.

5 - Spreader: used for spreading bacterial cells on the

surface of solid medium in Petri dish, sterilized by the
flame of burner.
9- Slide
Used for examination of bacterial smear under microscope , it
used for one time.

10- Cover-Slips
Placed on the slide , the bacterial smear may be between the
cover and the slide, it used for one time.

11- Flask
Used for place cultured and uncultured broth in it , sterilized
after plugs with cotton by autoclave.
13- Beaker
Used for graduated the volume of liquids. Sterilized by
oven.

14- Cylinder(Graduated Cylinder)

Used for graduated the volume of liquids, sterilized by
oven.
15- Washing bottle
Used to fill with liquid (specially distilled water)for
washing and homogenizing the glass wares and washing
the slide during staining , don’t need sterilization.

16- pH paper
Used to know the pH of the medium or any liquids
17- Rack
May be wooden , metallic or plastic used to stand and hold
the tube.

18- Burner
May be gaseous or alcoholic , used for sterilized the loop ,
needle and other metallic tools by flame (dry heat
sterilization).
Thank You