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QUALITY CONTROL
1. Grow an E. coli Quality Control strain for 18-24 hours on a MacConkey Agar at 35-37C with ~5%
CO2 (or in a candle-jar).
2. Observe the MacConkey for specific colony morphology.
3. As a sterility test, incubate an uninoculated plate for 48 hours at 35-37C with ~5% CO2 (or in a
candle-jar).
Passing result:
For example, in MacConkey Agar we try to find, does a gram negative rod is lactose fermenter or not?
In TCBS, we try to identify the isolate on the basis of sucrose fermentation. Vibrio cholerae is able to
ferment Sucrose and gives yellow color in TCBS.
Similarly in MSA, we try to find does a particular organism ferments Mannitol or not?
You must be aware by now; all the staphylococci spp are not pathogenic to human. So, next task of
microbiology laboratory (microbiologist) is differentiating S. aureus from other Staphylococcus spp.
Pathogenic staphylococci, i.e. Staphylococcus aureus is able to ferment Mannitol (It is coagulase
test positive) but others (coagulase negative Staphylococcus) are not. So, if that particular specimen
contains S. aureus, it ferments Mannitol (whenever sugar is fermented acid is produced) and changes
the pH of medium to acidic. As MSA contains phenol red as a pH indicator, at pH levels below 6.9, the
medium is a yellow color. But if Coagulase negative staphylococcus (CONS) grow, they cant ferment
Mannitol, so the color of the media around the bacterial colony does not change to yellow, it appear
pink.
# Note: Staphylococcus saprophyticus (coagulse negative Staphylococcus) may ferment mannitol,
producing yellow halo around colonies in MSA thus resembling with S. aureus.
So, MSA is also a differential medium.
Remember that in the neutral pH (6.9 to 8.4) the color of phenol red is red; while above pH 8.4, the color
of phenol red is pink.
Note: Other commonly used media that contain Phenol red as pH indicator are; TSI Agar, Urea base
agar andXLD Agar.
Expected colony characteristics of organism in Mannitol Salt Agar
1. Escherichia coli: Does not grow
2. Staphylococcus epidermidis: Colorless to pink colonies
3. Staphylococcus aureus: Yellow colonies; may have yellow halo around colonies.
Troubleshooting:
1. When grown on mannitol salt agar some species of Micrococcus (Micrococcus is a normal flora
of human skin, mucosa, and oropharynx), such as M. luteus (yellow) can produce yellow
colonies. M. roseus (red) produces pink colonies on MSA. Find out difference between
Micrococcus and Staphylococcus here
2. Enterococcus faecalis and Enterococcus faecium (most common enterococcal species that has
been isolated from human infections) are salt tolerant bacteria. They can ferment mannitol and
produce lactic acid, producing yellow colored colonies on MSA. Catalase test can help to
differentiate between Enterococcus (-ve) and Staphylococcus (+ve).
NaCl
Agar
Distilled water
Combine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and sterilize by autoclaving.
Procedure for the preparation of Blood Agar
1. Prepare the blood agar base as instructed by the manufacturer. Sterilize by autoclaving at
121C for 15 minutes.
2. Transfer thus prepared blood agar base to a 50C water bath.
3. When the agar base is cooled to 50C, add sterile blood agar aseptically and mix well gently.
Avoid formation of air bubbles. You must have warmed the blood to room temperature at the
time of dispensing to molten agar base.
(Note: If you are planning to prepare a batch of blood agar plates, prepare few blood agar
plates first to ensure that blood is sterile).
4. Dispense 15 ml amounts to sterile petri plates aseptically
5. Label the medium with the date of preparation and give it a batch number (if necessary).
6. Store the plates at 2-8C, preferably in sealed plastic bags to prevent loss of moisture. The shelf
life of thus prepared blood agar is up to four weeks.
Quality control of Blood Agar
1. The pH of the blood agar range from 7.2 to 7.6 at room temperature.
2. Inoculate the plates with 5 hour broth cultures of Streptococcus pyogenes and S.
pneumoniae. Inoculate also a plate withH. influenzae and streak with S. aureus
(i.e. Satellitism Test).
3. Incubate the plates in a carbon dioxide enriched atmosphere at 35-37C overnight.
4. Check for the growth characteristics of each species
1. S. pyogenes: Beta-hemolysis
2. S. pneumoniae: Alpha-hemolysis
3. Satellitism of H. influenzae
If either type of hemolysis is present, then one will observe a zone of hemolysis surrounding a growing
colony.
1. Alpha hemolysis: Partial lysis of the RBC to produce a greenish-gray or brownish discoloration
around the colony. hemolysis is due to the reduction of RBC hemoglobin to methemoglobin in
the medium surrounding the colony. Many of the alpha hemolytic streptococci are part of the
normal body flora. But Streptococcus pneumoniae which is also alpha hemolytic causes serious
pneumonia and other deadly infectious disease.
N. meningitidis and H. influenzae should appear as large, round, smooth, convex, colorless-to
grey, opaque colonies on the CAP with no discoloration of the medium.
S. pneumoniae should appear as small grey to green colonies with a zone of alphahemolysis (only slightly green) on the CAP.