PREPARATION OF CULTURE

MEDIA
UNIT II

Asst. Prof. Ruby G. Meim

Asst. Prof. Vivian G. Villegas
Culture Media

- Carbon, Nitrogen, Sulfur, Phosphorus, Hydrogen, Oxygen, and buffers

- Powdered form
- Broth
- Gelatin: first solidifying agent used
- Agar: Red Algae (Rhodophyta)
- Liquifies: 55 – 60C
- Solidifies: 50C or lower
- Melts at 95-100C
Criteria of Culture Media:
1. Sterile
2. Free from inhibitory substances
3. Contain essential nutrients
4. Has adequate moisture, water, salt
5. Has proper reaction/pH (6.5-7.5)
6. Suitable level of oxygen
7. Incubated at proper temperature
Classification of Culture Media
1. Physical state / Consistency
2. Function
3. Composition
4. Use
5. Distribution
According to Physical State
a. Liquid – ex. NB; Thioglycollate
* turbidity
b. Solid – ex. MHA
> 1.5 – 3% (agarose)
c. Semi-solid – ex. SIM Agar
> 0.5 – 1%
d. Biphasic Media – ex. Castaneda
According to Function
a. Simple/Basal/Ordinary
b. Enriched
c. Differential
d. Selective
a. Simple Media

Composition: peptone, beef extract, agar, water

- General cultivation media
- Support the growth of non-fastidious bacteria

Ex. NA, NB
b. Enriched Media

- Contain nutrient supplement

- Used to support growth of fastidious bacteria

Basic medium + enriching substances

ex. NA + blood (or serum, ascitic fluid)
BAP, CAP
c. Differential Media A

- provides distinct colonial appearances of microorganism

- indicator present

A. MSA Manitol Salt Agar (pinkish color) B

- S. aureus (yellow) from S. epidermidis (pinkish)
- indicator: phenol red
B. McConkey Agar
- lactose(pinkish)/non-lactose(colorless)
- indicator: neutral red
C. SCA-Simmon Citrate Agar
- prussian blue/green
-indicator: bromthymol blue
C
d. Selective Media
- Allows growth of specific organisms
- Ex. SSA, EMB
- Inhibitory substances:
alcohol (proteus)
Charcoal hydrate

Dyes (gentian violet, crystal violet) (gr+)

bile salts (Na deoxycholate)

Na azide (gr-)
K tellurite
 According to Use
„Enrichment Medium: ex. alkali peptone water
„Transport Medium: ex. Cary-blair
„Characteristic/Biochemical Characteristic Medium
ex. TSI
TSI
„Motility Medium ex. SIM
SIM
„Antibiotic Susceptibility ex. MHA
„Anaerobic/Reducing Culture Medium
„Pharmaceutical Agar AST
According to Composition

„Chemically Defined/Synthetic
„Complex/Non-chemically defined
„Tissue Culture
According to distribution/ formation
„Plated Medium
„Tubed
„Bottled
„Biphasic
According to Form or Distribution
Plated Media:
A. Simple
B. Radial
C. Clock method
D. Overlap method
According to Form or Distribution
Tubed Media

Inoculating needle Inoculating Loop

Motility test
According to Form or Distribution
Butt / Deep
Butt Slant
Slant
Butt slant
Broth / Liquid Butt

Slant Broth
PROCEDURES HOW TO PREPARE CULTURE
MEDIA
MANNER OF DISPENSING

PLATED TUBED
1. Weigh 1. Weigh
2. Dissolve 2. Dissolve
3. Autoclave 3. Dispense
4. Dispense 4. Autoclave

LAST STEP: FORMING THE AGAR

SAMPLE COMPUTATION:
Prepare 800 mL of nutrient agar:
SAMPLE COMPUTATION Prepare 800 mL of nutrient agar:
FIND X:
23𝑔 𝑥
l a be l
=
1000𝑚𝑙 800𝑚𝑙
23𝑔 (800𝑚𝑙)
𝑥=
1000𝑚𝑙

X= 18.4 grams
 WEIGH:
1. Weigh the desired amount of
dehydrated culture medium
and transfer to a suitable
container.

Ø Use the Erlenmeyer flask for

the plated medium and the
beaker for the tubed medium.
DISSOLVE
2. Transfer the desired amount of powdered culture medium into
the flask/beaker, then add the distilled water for making the
desired volume.
3. Heat and stir until all the dehydrated culture medium go into
the solution. Stop heating as soon as the granules are totally
dissolved and the solution becomes clear.
4. Plug the Erlenmeyer flask with a gauze/aluminum foil. Note
that for the tubed medium, the dissolved medium from the
beaker must be transferred to the appropriate test tubes before
plugging with cotton.
AUTOCLAVE
5. Label the test tubes and
Erlenmeyer flask and place a strip
of autoclave tape on the visible
area of the glassware.

6. Sterilize the culture medium by

autoclaving at 121 C for 15 - 20
minutes at 15/psi.

7. After autoclaving, prepare the

culture medium accordingly.
DISPENSE AND FORM (PLATED MEDIUM)
● Plated Medium: Pour the agar from the Erlenmeyer flask into the petri
dish and allow the agar to solidify on a flat surface.
PLATED MEDIA PREPARATION
Video credits: https://www.youtube.com/watch?v=cneascR3OEc
ØNo bubbles on the surface (if
with bubbles, move a flame on
the surface of the culture
medium)

> Move the plate slightly to check if

the medium has solidified
>PARTLY OPEN LID
- To prevent moisture from
accumulating on the lid

> Store the culture media in

the ref upside down to
prevent condensation /
moisture from dripping down
the surface of the agar
BLOOD AGAR PLATE
● Enriched medium
● Use to grow fastidious
organisms and differentiate
bacteria based on their
hemolytic property.
● Prepared by mixing blood
with a basal medium
(nutrient agar).
BLOOD AGAR PLATE PREPARATION
VIDEO OF BAP
> After autoclaving, lower the temperature of
the medium by letting the side of Erlenmeyer
flask under running water
> After adding the blood, swirl gently to avoid
formation of bubbles
>Observe aseptic technique
>When preparing CAP, same manner of prep
as BAP except that medium temperature is
higher
> We do not heat the culture medium (BAP
and CAP) once the blood is already added.
TUBED CULTURE MEDIUM
● Weigh
● Dissolve
● Dispense
● Autoclave
● Form
FORMING (Tubed media)
a. Slant: Solidify the agar in the test tube in a slanting position. Do not let
the sterile medium touch the cotton when forming.
b. Butt-Slant: Solidify the agar in the test tube in an acute angle slanting
position. Do not let the sterile medium touch the cotton when forming.
c. Butt: Solidify the agar in the test tube in a vertical position.
d. Liquid: Allow the test tube to stay in a vertical position.
HOW TO FORM SLANT TUBE MEDIA
Video credits:
https://www.youtube.com/watch?v=tfI3_9YdMVY
HOW TO PREPARE LIQUID TUBE MEDIUM
Viideo credits:
https://www.youtube.com/watch?v=DyIXxjNbQbU
END OF PRESENTATION