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Introduction to Lab 6: Ex. Preparation of Culture Media

Media facts

Preparation

Sterilization

Aseptic technique

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Introduction to Lab 7: Ex. Preparation of Culture Media

The purpose of this exercise is to introduce the concept of growing

microorganisms in the lab in order to be able to study them.

Culture is the term given to microorganisms that are cultivated in

the lab for the purpose of studying them.

Medium is the term given to the combination of ingredients thatwill support the growth and cultivation of microorganisms by

providing all the essential nutrients required for the growth

(that is, multiplication) in order to cultivate these

microorganisms in large numbers to study them.

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Among the different kinds of microorganisms the two groups that

can be grown in cultures are bacteria and fungi.

Algae and protozoa require many different nutrients in minute

quantities that are difficult to anticipate and prepare in the lab.These organisms have different nutritional requirements and

thus various kinds of culture media have been developed.

Primary ingredients required by all living organisms include:a carbon source, water, minerals, and a nitrogen source.

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Living cells need nutrients required for their structure (biosynthesis)

as well as nutrients to provide them with energy to perform all

of their various life processes.

Nutrients are acquired from the environment in which they live intheir natural habitat.

Most of these nutrients are then processed within the cell through a

variety of metabolic pathways to be incorporated in different

ways.The process of building complex structures from simple building

blocks is called anabolism.

The process of breaking up complex materials to harvest the energy

in them is called catabolism.

The ability to use particular compounds is dependent upon the

genetic makeup (DNA) that the cells have.

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Since there are different kinds of organisms that can be grown in

culture media with varying needs, culture media have also

been formulated with different ingredients.

Culture media may be found in one of three states:liquid (called broth)

semi-solid

solid.

Media are solidified by the addition of solidifying agents such as

agar (inert compound).

Varying the concentration of agar will yield varying degrees

of solidification.

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Culture media may be classified as: Synthetic media (Defined)

Complex (Non-synthetic) media

Synthetic media contain only ingredients for which a complete

chemical formula is known.

Complex media contain at least one ingredient for which a chemical

formula is not known (such as milk, egg, malt, animal tissues)

Culture media can also be classified based on the function they

perform in determining various characteristics of organism

that are able to grow on/in them

e.g. Differential, Selective media.

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The ingredients in a medium will affect the chemical nature of the

medium.

This is important because organisms vary in their

requirement for different environments.

One such property is:

pH (which is a measure of the amount of hydrogen ions in

a particular medium).This has to be monitored during the preparation of media since this

will influence the kind of organisms that are able to grow in the

medium.

The pH of the medium will thus determine which organisms are

able to grow on the medium.

For example, fungi prefer acidic media for their growth while bacteria

grow on neutral pH media.

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The primary function of culture media is to be able to grow

particular organisms on/in them.

It is important that these media are devoid of any other living

organisms.This is possible through the process ofsterilization

(a process by which all living organisms and their spore forms

are killed and the medium is made sterile)

Culture media are most commonly sterilized through the process of

autoclaving (using high temperatures that will kill all living

organisms under increased pressure for specified periods of time

in an appliance called the autoclave)

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-3 different culture media will be prepared in the class.

-They will be sterilized in an autoclave and poured into Petri dishes.

-These will then be used for the next exercise to be done in the lab.

The 3 media that are to be prepared are:

Dextrose Starch Agar (DSA)

Malt Agar (MA)

Nutrient Agar (NA)

Be able to assign functions to each of the ingredients for any culture

mediumthat is identify the source of:

carbon, nitrogen, minerals

and any other specific function they may perform(such as solidification)

Most common sources:

- carbon are sugars (carbohydrates)

- nitrogen are proteins (partially degraded

peptone)- animal tissue extracts as a source for minerals

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For example:

Nutrient Agar:

Gelysate peptone- 5.0g (source of carbon and nitrogen);

Beef extract - 3.0g (secondary source of carbon and nitrogen andprimary source of minerals),

Agar -15.0g (solidifying agentif this were not added

medium would be a broth)

Aseptic Technique:

These are various techniques that are used to

minimize the introduction of microorganisms into media

especially during transfer processes, such as

pouring of media into Petri dishes,inoculation of cultures

These techniques include:

cleaning the bench top work areas with disinfectant solution

washing hands before starting workother specific techniques that will be demonstrated in the lab.

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protective clothing

hand washingbench cleaning

loop flaming

pipettors

Aseptic Techniques