4rth lab exp media preperations

1. Laboratory 4:
Media preparation and evaluation:

2.  Obtain definitive identification and characterization
 Determine which bacteria is likely a contaminant or
colonizer
 Storage of pure cultures

4.  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.

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

6. 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)

7. Medium Contents Reaction
MacConkey
agar
lactose,
bile salts, crystal violet,
neutral red
Only one sugar
Inhibit G+ve organisms
Turns red when acid (pH<6.8) is produced
Non-lactose fermenters appear colorless or
transparent
EMB agar Lactose
Sucrose
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 indicator
Inhibit G+ve organisms
MSA
Manitol salt
agar
High salts 7.5% NaCl
Mannitol
pH indicator phenol red
Inhibit most G-ve bacteria and many G
+ve
Differentiate between salt tolerant
bacteria by mannitol fermentation

8. Galactoside bond
Glucose + Galactose
Two enzymes are required:
1. beta-galactoside permease to transport the disaccharide
through the CW
2. Beta galactosidase which hydrolyze the galactoside bond
Question: Why glucose is not included in selective media?
.
2 enzymes needed
Fermented by the EMP pathway

9. EMB
MacConkey
SS Agar + salmonella
MacConkey + E. coli
American Society for Microbiology © 2012
Manitol salt agar

10.  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.

11. American Society for Microbiology © 2012
Swarming by Proteus vulgaris

13. 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 quantity
2. 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 autoclave
5. 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.

14. Escherichia coli
Staphylococcus aureus
Proteus mirabilis
Staphylococcus aureus
Escherichia coli
Staphylococcus
epidermidis
Staphylococcus aureus , Bacillus
subtilis and Enterococcus faecalis
What to Do

15. - 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