Culture Media COMPONENTS PREPARATION INOCULATION Culture Media a

Culture Media COMPONENTS PREPARATION INOCULATION

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. Ex: NA. Enriched media. Ex: blood Agar, chocolate Agar. Selective media. Ex: MAC. Differential media. Ex: CLED.

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.

Forms of Media 2) Solid form (with agar): Plate: used mostly to culture organisms to get isolated colony.

Forms of Media Slant: a tube containing solid media that was left to solidify at an angle. Used to keep the bacteria for a long period of time (3 months)

Forms of Media Deep agar : agar solidified at bottom of tube. Used to keep the bacteria for a long time.

Forms of Media Semi-solid deep agar: same as deep agar except it contains less agar (0. 5% agar) to allow motility of organisms.

Media preparation Equipment: § Media powder. § Water (100 ml). § Balance. § Flask ( larger than the size of media volume). § Weighing plate. § Weighing spatula § Cylinder. § Bacti-cinerator. § Autoclave. § 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. Sterilize the media in autoclave 15 -20 min at 121 °C Leave to cool at room temperature. Pour the media in petri dish after labeling. Leave to solidified at RT. Preparation of blood Agar & chocolate Agar

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 § § § § Sterilize the loop by bacti-cinerator 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). Sterilize 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). Sterilize 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.

Lab 8 Types of media BASAL OR COMPLEX MEDIA. ENRICHED MEDIA. SELECTIVE MEDIA. DEFERENTIAL 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

Klebsiella. sp. Bacillus

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 positive organisms, allows growth of gram negative organisms.

Differential Media Cysteine Lactose Electrolyte Deficient Agar (CLED) It contains a sugar and Indicator if organism ferments sugar an acid is produced, this will change the color of indicator. 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 of G(-ve) organisms. Indicator and inhibitors: 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 (colorless)

Staph - Staph epidermidis - Staph saprophyticus Gram +ve Strept Gram –ve - Staph aureus - Alpha haemolytic ex: strept pneumonae ex: strept viridans - Beta haemolytic ex: strept pyogenes (group. A) ex: strept agalactae (group B) - Non haemolytic LF ex: E coli , Klebsiella NLF ex: Salmonella , Shigella, Proteous

Differential Media Blood Agar - Types of hemolysis: α Hemolysis β Hemolysis γ Hemolysis

Types of Hemolysis α hemolysis Incomplete hemolysis greenish color around colonies

Types of hemolysis β hemolysis Complete hemolysis Clear area around colonies.

Swarming of Proteus on BA Swarming appear as spreading rose on BA and NA plates. Bad smell CLED inhibit swarming.

Different bacteria on different media Staph. Aureous NA BA MAC CLED EMB MSA Growth-β H - Growth LF - Salt tolerant (yellow) Manitol fermentor (G +ve) Staph. Epi (yellow) Growth (G +ve) Strep. Group A Growth - Non H Growth LF - Salt tolerant (yellow) Manitol non fermentor (colorless) - - Growth-β H - - - Growth LF Non H (pink) (yellow) LF-green metallic sheen (G +ve) E. Coli (G -ve) Proteous (G -ve) Growthswarming Growth swarming Non H Growth NLF- Growth NLF (Colorless) no swarming - -