COMPONENTS

PREPARATION

INOCULATION

Culture Media

Culture Media

a source of energy and certain environmental

conditions in order to grow and produce

bacteria.

Depending on the type and combination of

nutrients, different categories of media can

be made.

Types of Media

Basal or complex media.

Enriched media.

Selective media.

Deferential media.

Common Ingredients

Water: essential for bacterial growth.

Peptone : from hydrolyzed animal or plant protein

Meat extract: provide amino acids, vitamins, minerals.

Yeast extract: used as bacterial growth stimulant.

Mineral salts: essential for bacterial enzyme activity,

Sulfur, phosphorus, iron, potassium.

Carbohydrates: simple or complex sugars as source of

energy and carbon.

Agar

It’s solidifying agent of the medium. A

gelatinous Inert polysaccharide material,

derived from sea-weed or certain marine

algae. It is Resistant to microbial action. Non

toxic to bacteria. Dissolves at 100 c°,

Solidifies when cooled below 45 c°. Use 1-2%

concentration.

Forms of Media

1) Liquid form: called broth. Without agar, used to grow bacteria in a large

quantity.

Growth of bacteria turbidity

No growth clear

2)solid:

plate: used mostly to culture organisms

slant: a tube containing solid media that was left to solidify at an angle.

used to keep the bacteria for long period of time (3 months)

Deep agar : agar solidified at bottom of tube. used to keep the bacteria

for long time.

Semi-solid agar deep: same as agar deep except it contains less agar

(0.5% agar) to allow motility of organisms.

Slant Agar Plate Agar

Deep Agar

Media preparation

Equipment: Media powder. Water (100 ml). Balance. Flask ( larger than

the size of media volume).

Weighing plate. Weighing spatula Cylinder.

Bacti-cinerator. Autovalve. Sterile empty Petri

dishes. Autoclave tape. Aluminum foil.

Procedure

Measure out the required volume of the media, e.g. 40 gm of media for 1000 ml of water, let’s say u want to prepare 100 ml of media

40 1000

!! 100

= 4 gm of media for 100 ml of water.

Put the media powder in the flask.Add the water onto the media.Mix well.

Cover the flask with aluminum foil.Stick the autoclave tape on the flask and use

it for labeling.Sterilize the media in autoclave 15-20min at

12oC°.Leave to cool at room temperature.Pour the media in petri dish.Leave to solidified at RT.

Inoculation

It is streaking bacteria on agar plate, allow the bacteria to grow to produce isolated colonies, and pure culture.

Pure culture: culture containing a single species of organism.Mixed culture: culture containing more than one species of

organism.Contamination culture: a bacterial culture that has acquired

unwanted organisms.

In order to obtain well-isolated colonies, the quadrant streak technique should be used.

Quadrant streak technique

Equipment:

Bacti-cinerator.

Loop.

Subculture media.

Agar media plane.

Marker.

PROCEDURE

Sterile the loop by bacti-cineratoe until it is red then allow to cool.

Take a loopful of bacteria from the subculture media Immediately streak the inoculating loop VERY gently

over a quarter of the plate around 4-5 lines (quadrant 1).

Sterile the loop then allow to cool. Go back to the edge of the area 1, extend the streaks

into the second quarter of the plate (quadrant 2). Sterile the loop then allow to cool. Go back to the edge of the area 2, extend the streaks

into the third quarter of the plate (quadrant 3).

Sterile the loop then allow to cool. Go back to the edge of the area 3, extend the streaks

into the forth quarter of the plate in zig zag lines (quadrant 4).

let the bacteria to grow at 37 C° for 24 hr in the incubator.

BASAL OR COMPLEX MEDIA.

ENRICHED MEDIA.

SELECTIVE MEDIA.

DEFERENTIAL MEDIA.

Types of media

Basal or complex media: Nutrient Agar(NA)

It contains nutrients that

allows non fastidious or

Non pathogenic organisms

To grow.

Notice the shape, margin,

elevation, color, size,

Smell of organism .

Notice pigment production by organism

Enriched Media: Blood Agar (BA)/chocolate agar(Choc)

It contains simple nutrients

and additional requirements

such as blood, serum to

allow fastidious or

pathogenic organisms to

grow.

Selective Media: Mac Conkey agar(MAC)

It contains inhibiting agents that inhibit some organisms and allows others to grow.Inhibiting agents : Bile salts, crystal violet.It inhibits gram positiveorganisms, allows growth ofgram negative organisms.

Differential MediaCysteine Lactose Electrolyte Deficient Agar (CLED)

It contains a sugar and Indicatorif organism ferments sugar an acid is

will change the color of produced, thisindicator.

Indicator: is Bromothymol blue.

Sugar is lactose.

If organism ferments lactose it will give

yellow color, if it does not ferment

lactose no changes occurs, colorless.

Selective and Differential Media: MAC

Sugar: is lactose.

.Indicator: is neutral red

If the organism ferment lactose,

It will give pink color =LF.

If organism does not ferment

Lactose, no change in color,

Colorless= NLF

Selective and Differential Eosin Methylene Blue ( (EMB

As Mac Conkey, inhibits G(+ve)organisms, allows growth ofG(-ve) organisms.

Indicator: is Methylene Blue

And Eosin.

Lactose fermentor organsim

Appears dark purple while

non lactose fermentor appears

colorless.

E.coli produces green

Metallic sheen( LF)

Selective and deferential: Mannitol Salt Agar( MSA)

Selective: as it contains 7.5% salt

Only organisms that can tolerate high salt conc. can grow

Differential :as it contains mannitol sugar and phenol red indicator .

The organism that ferments mannitol produces acid thus color changes to yellow

If organism does not ferment mannitol no change in color

Differential MediaBlood Agar

- Types of hemolysis:

α Hemolysis

β Hemolysis

γ Hemolysis

Types of Hemolysis

α hemolysisIncomplete hemolysisgreenish color around

colonies

Types of hemolysis

β hemolysisComplete hemolysis

Clear area around colonies.

Swarming of Proteus on BA

Swarming appear asspreading rose on BA

plate .