Laboratory 4:

Media preparation and evaluation:

Why cultivate bacteria? Obtain definitive identification and characterization Determine which bacteria is likely a contaminant or

colonizer Storage of pure cultures

What is culture media

Media that provides a surface, necessary pH, nutrients that bacteria need to live

What am I going to learn from Lab Topic Media preparation & Culture

Use, defined ,complex, enriched selective & differential media to culture microbes.

Learn how to prepare media for bacterial cultivation

Describe colony morphology and its relationship to microbial identification.

Interpret results of microbial growth on various culture media.

Types of bacterial culture media

A. On Consistency There are three physical forms of media Solid, Semisolid , and Liquid

B. On Chemical Composition - Defined , undefined or complex - Simple media, special media(enriched, selective, and deferential ) -Aerobic and anaerobic media - Cell culture for obligate intracellular bacteria (e.g., Chlamydia spp)

Selective MediaMedium Contents Reaction

MacConkey agar

lactose, bile salts, crystal violet,neutral red

Only one sugar Inhibit G+ve organismsTurns red when acid (pH<6.8) is producedNon-lactose fermenters appear colorless or transparent

EMB agar LactoseSucrose

Eosin methylene blue

Strong acid production by organisms such as E. coli results in a metallic green sheen.Weaker fermentation of lactose results in colonies with a pinkish-purple color nonlactose fermenters remain colorless.pH indicatorInhibit G+ve organisms

MSA Manitol salt agar

High salts 7.5% NaCl MannitolpH indicator phenol red

Inhibit most G-ve bacteria and many G+veDifferentiate between salt tolerant bacteria by mannitol fermentation

Lactose Fermentation

Galactoside bond

Glucose + Galactose

Two enzymes are required:1. beta-galactoside permease to transport the disaccharide

through the CW2. Beta galactosidase which hydrolyze the galactoside bond

Question: Why glucose is not included in selective media?

.

2 enzymes needed

Fermented by the EMP pathway

Selective Media

EMB

MacConkey

SS Agar + salmonella

MacConkey + E. coli

American Society for Microbiology © 2012 Manitol salt agar

Streptococci

Streptococci are typically grouped by hemolysis on blood agar plates:

Alpha hemolysis:› occurs when the RBCs are intact, but hemoglobin is

converted to methemoglobin . This causes a greening of the plate.

Beta hemolysis:› True hemolysis due to hemolysin, an erythrocyte lysing

enzyme. › The plate becomes clear where the blood cells have been

lysed. Gamma hemolysis , there is actually no hemolysis.

American Society for Microbiology © 2012

Swarming by Proteus vulgaris

1. Read the label on a bottle of dehydrated agar. It specifies the amount of dehydrated powder required to make 1 liter (1,000 ml) of medium. Calculate the amount needed for 0.2 liter and weigh out this quantity2. Place 200 ml of distilled water in an Erlenmeyer flask. Add the weighed, dehydrated agar while stirring with a glass rod to prevent lumping.3. Set the flask on a tripod. Using a Bunsen flame, slowly bring the rehydrated agar to a boil. Stir often.4. When the agar mixture is completely dissolved, remove the flask from the flame ,close it with the aluminum foil plug or cap, and give it to the instructor to be sterilized in the autoclave5. When the flask of sterilized agar is returned to you, allow it to cool to about 50°C . pour the melted, sterile agar into a series of petri dishes. The petri dish tops are lifted with the left hand, and the bottoms are filled to about one-third capacity with melted agar When the plates are cool (agar solidified), invert them to prevent condensing moisture from accumulating on the agar surfaces.

Procedure

Escherichia coli

Staphylococcus aureus

Proteus mirabilis

Staphylococcus aureus

Escherichia coli

Staphylococcus epidermidis

Staphylococcus aureus , Bacillus subtilis and Enterococcus faecalis

What to Do

References - American Society for Microbiology © 2012

- Difco manual , (1998 ) .(11 edition )

- Harley−Prescott. (2002).Laboratory exercise in microbiology

- Winn, Washington C.Koneman, Elmer W.. (Eds.) (2006) Koneman's color atlas and textbook of diagnostic microbiology /Philadelphia : Lippincott Williams & Wilkins