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Culturing Microorganisms

The document discusses the preparation of culture media for growing microorganisms. It defines culture media as an aqueous solution containing necessary nutrients. Media can be classified by physical state (liquid, semisolid, solid), chemical composition (synthetic or non-synthetic), and function (general-purpose, enrichment, selective, differential). The document provides detailed instructions on sterilizing equipment and materials, calculating amounts of media needed, mixing and sterilizing the media, pouring plates, and proper disposal. Safety procedures are also outlined.

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216 views26 pages

Culturing Microorganisms

The document discusses the preparation of culture media for growing microorganisms. It defines culture media as an aqueous solution containing necessary nutrients. Media can be classified by physical state (liquid, semisolid, solid), chemical composition (synthetic or non-synthetic), and function (general-purpose, enrichment, selective, differential). The document provides detailed instructions on sterilizing equipment and materials, calculating amounts of media needed, mixing and sterilizing the media, pouring plates, and proper disposal. Safety procedures are also outlined.

Uploaded by

Insatiable Clee
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Microbiology and

Parasitology
Session 3
Culturing
Microorganisms

Jolee Ann J. Huliganga

J.A. Huliganga Notre Dame of Marbel University


Objective/s
• At the end of the lesson, students must be able to:
1. State the six basic components that are found in all bacteriological
media.
2. Explain the difference between a defined and complex medium.
3. Explain the difference between differential and selective media.
4. Prepare and sterilize a complex agar medium.

J.A. Huliganga Notre Dame of Marbel University


Culture Media
• Culture Medium
• is a basically an aqueous solution to which all the necessary
nutrients have been added.

• Classification according to primary levels:


• Physical state
• Chemical composition
• Functional type

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Physical State
• Liquid Media – water-based solution
that do not solidify at temperature
above freezing point.
• Commonly termed broths, milk, or
infusions.

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Physical State
• Semisolid Media - exhibit a clotlike
consistency at ordinary room
temperature.
• contain an amount of solidifying agent
(agar or gelatin) which thickens them
but does not produce a firm substance.

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Physical State
• Solid Media - these provide a firm surface on which cells can form
discrete colonies and are advantageous for culturing and isolating
bacteria and fungi.
• Solid media come in two forms: liquefiable or reversible solid media and
nonliquefiable or non-reversible solid media.

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Chemical Composition
• Synthetic- chemically defined
• Composed of pure organic and inorganic compounds.
• Non-synthetic – contain at least one ingredient that is not
chemically defined.
• Not a simple compound, pure compound.
• Most of these substance are extracts of animals, plants, or yeasts.

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Functional Types
• General-purpose Media
• contain a mixture of nutrients –supports growth of
pathogens and non-pathogens alike.
• Grow as broad a spectrum of microbes as possible.

Nutrient Broth
Nutrient (beef extract and
Agar (beef peptone) dissolved in
water
extract and
peptone)

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Functional Types
• Enrichment Media
• Increase number of desired microorganisms.
• Contains organic substances such as blood, serum, hemoglobin, or special
growth factors.

Blood Agar (gen. nutrients w/ 5% blood)

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Functional Types
• Selective Media
• Contains one or more agents – inhibit growth of certain microbe/s
• Encourage only certain microbes to grow.
• Samples from feces, saliva, skin, water, and soil.
MacConkey Agar (MAC) Eosin Methylene Blue (EMB)

J.A. Huliganga Notre Dame of Marbel University


Culture Media: Functional Types
• Differential Media
• Allows several types of microorganism and are designed to display visible
differences among those microbes.
• Shows variations in colony, size or in color, media color changes or gas
bubbles and precipitates.
MacConkey Agar (MAC) Eosin Methylene Blue (EMB)

J.A. Huliganga Notre Dame of Marbel University


Media Preparation

J.A. Huliganga Notre Dame of Marbel University


Sterilization of equipment and materials
• Medium (media, plural): a nutrient
blend used to support microbial
growth.
• ❖ Autoclave: The principle of
sterilization in an autoclave is that
steam under pressure is used to
produce a temperature of 121ºC
which if held for 15 minutes all
microorganisms including bacterial
endospores will be destroyed.

J.A. Huliganga Notre Dame of Marbel University


Sterilization of equipment and materials
❖Wire loop: Heat to redness in Bunsen burner flame.
▪ Empty glassware and glass (not plastic!) pipettes and Petri dishes: ▪
• Either, hot air oven, wrapped in either grease proof paper or aluminum and held
at 160-180ºC for 2 hours, allowing additional time for items to come to
temperature (and cool down!).

J.A. Huliganga Notre Dame of Marbel University


Sterilization of equipment and materials
❖Note: Plastic Petri dishes are supplied in already sterilized packs;
packs of sterile plastic pipettes are also available but cost may be a
consideration.
▪ Culture media and solutions: Autoclave/pressure cooker.
▪ Glass spreaders and metal forceps: Flaming in alcohol.

J.A. Huliganga Notre Dame of Marbel University


MEDIA PREPARATION CALCULATION
❑Each plate requires 15-20 mL of agar
❑ Each tube requires 5-10 mL of broth/agar
❑ Follow package instructions for preparation.
Instructions are typically written for 1L (1000mL) of
media. If less is desired calculate the amount needed
as shown:
❑ Using the formula: M1/V1 = M2/V2
❑– ratio and proportion

J.A. Huliganga Notre Dame of Marbel University


• For example: If the instructions state 23g for 1L and 600mL is desired,
use a ratio to calculate the amount needed (in this example 13.8 g is
needed for preparing 600mL):

23g/1000mL = Xg/600mL
23 * 600= 1000X
13800 = 1000X
13800/1000 =X
13.8 g = X

Always prepare media in a beaker with 1/3 of empty space. (i.e.


prepare 600mL of media in a 1000mL beaker)

J.A. Huliganga Notre Dame of Marbel University


EXAMPLE:
• A researcher wants to prepare 20 agar plates of
Nutrient Agar (NA) for his/her experiment, how much
of powdered NA does he/she needs?
• Given: NA- 28g/L

J.A. Huliganga Notre Dame of Marbel University


• Given:
• m1 = 28g, v1 = 1000mL
• 20 agar plates = 20 x 20 mL = 400 mL = v2
• m2 = ?
m1v1 = m2v2
28g/1000mL = m2 /400mL
28 * 400= 1000 m2
11200 = 1000 m2
11200/1000 = m2
11.2 g = m2

J.A. Huliganga Notre Dame of Marbel University


❑Mix and stir with a stirring rod
❑Place in a microwave and heat at 3-5 min intervals
❑Heat again until boiling achieved
❑Cover with cotton plug and foil and secure with autoclave
tape
❑Autoclave for 15-30 minutes at 121oC and 15 psi

J.A. Huliganga Notre Dame of Marbel University


❑Once sterilization is complete, open the autoclave and
remove the beaker of media.
❑Allow media to cool slightly, but not for longer than 10
minutes as the agar may solidify.
❑Pour approximately 10-15mL of agar into the plates,
aseptically.
❑When cooled, store upside-down in plastic bags in the
refrigerator to prevent the agar from drying out

J.A. Huliganga Notre Dame of Marbel University


First Aid
• In case of contact with eyes, immediately wash the eyes with large
amounts of water for 15 minutes, while holding eyelids open. Get
medical attention immediately. If contact lenses are worn remove
them immediately.
• In the event of skin contact, wash the area thoroughly.
• In the event of skin contact with a hot beaker, seek medical attention.
• Report any incident to the laboratory manager.

J.A. Huliganga Notre Dame of Marbel University


Disposal Requirements
• Spills can be cleaned up with a paper towel and
disposed of in the trash. Use caution with hot media.
• Unused plates can be disposed of in the waste to be
autoclaved trash cans.
• See the microbiological waste SOP for further
instruction on waste disposal.

J.A. Huliganga Notre Dame of Marbel University


REMEMBER!!

•Bacteria
Are everywhere! Some are harmless, others are pathogenic.
Contaminants are at higher risks, making your media useless.

For this reason, and to protect against disease,


strict sterile procedures must be used.

J.A. Huliganga Notre Dame of Marbel University


Your media is now READY!

J.A. Huliganga Notre Dame of Marbel University

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