Download Practical General Microbiology Lab

Isolation and Identification
of
Staphylococci
Gram Stain
Gram Positive coccus
Catalase
+
_
Streptococci
Oxidase
+
_
+
Staphylococci
Micrococci
Coagulase
_
Staphylococcus epidermidis
Staphylococcus aureus
CNS
Staphylococcus saprophyticus
Gram positive cocci
Catalase +ve
Oxidase -ve
Coagulase
_
+
Staphylococcus aureus
Staphylococcus epidermidis
CNS
Staphylococcus saprophyticus
Negative
Positive
Positive
Negative
Slide method
Tube method
Coagulase test
Non-hemolytic Staphylococcus species: Staphylococcus epidermidis
Staphylococcus saprophyticus: non-hemolytic, bright white, creamy colonies
Strains of Staphylococcus aureus produce a golden yellow pigment
Strains of Staphylococcus aureus not a golden yellow pigment producer
MANNITOL SALT AGAR ( MSA )
INGREDIENTS
Peptone.
Beef Extract.
D-Mannitol .............. 1.0%.
Sodium Chloride ...... 7.5%.
Agar ......................... 1.5%.
Phenol Red. AS PH INDICATOR
Final pH 7.4 ± 0.2 at 25°C.
PRINCIPLE AND RESULTS
Mannitol Salt Agar is a nutritive medium due to its content of
peptones and beef extract, which supply essential growth
factors, such as nitrogen, carbon, sulfur and trace nutrients.
The 7.5% concentration of sodium chloride results in the
inhibition of bacterial organisms other than staphylococci.
Mannitol fermentation, as indicated by a change in the phenol
red indicator, aids in the differentiation of staphylococcal
species.
Mannitol Salt agar
Staph. epidermidis
Staph. aureus
Mannitol Salt agar
Coagulase Negative Staph
Staph
saprophyticus
Staph
epidermidis
Novobiocin resistance Test:
Procedure:
1. Inoculate blood agar plate with the test organism.
2. Aseptically apply Novobiocin disc onto
the center of the streaked area.
3. Incubate the plate at 37oC for 24 hrs.
NB
Novobiocin test
A novobiocin disk will be placed on the plate, Novobiocin is an antibiotic that many
Staphylococcus strains are sensitive to with the exception of one ,
Staph. saprophyticus that is risist to novobiocin antibiotic.
Staphylococcus epidermidis Growing
on Blood Agar
Note there is no hemolysis (gamma reaction) on the blood agar and the
organism is sensitive to the antibiotic novobiocin as shown by the zone of
inhibition.
Staphylococcus saprophyticus Growing
on Blood Agar
Note there is no hemolysis (gamma reaction) on the blood agar
and the organism is resistant to the antibiotic novobiocin.
Staphylococcus aureus Growing on
Blood Agar
Note beta hemolysis (complete lysis of the red blood cells around the
colonies; see arrows) on the blood agar and the organism is sensitive to
the antibiotic novobiocin.
DNase TEST AGAR
Media ingredients
Enzymatic Digest of Casein.................1.5% Deoxyribonucleic Acid.............0.2%
Enzymatic Digest of Animal Tissue.....0.5% Agar........................................1.5%
Sodium Chloride...................................0.5% Final pH: 7.3 ± 0.2 at 25°C
Principles of the Procedure
The nitrogen, vitamin, and carbon sources are provided by Enzymatic Digest of
Casein and Enzymatic Digest of Animal Tissue. Sodium Chloride provides
essential ions while maintaining osmotic balance. Deoxyribonucleic Acid
enables the detection of DNase that depolymerize DNA. Agar is the solidifying
agent.
Test Procedure
1.
2.
3.
4.
Inoculate plates by spotting or streaking a heavy inoculum of test organism.
Incubate plates at 35 ± 2°C for 18 – 24 hours and up to 48 hours.
Flood plates with 1 N HCl.
Observe for clearing around the spot or streak. Record results.
Results
A zone of clearing around the spot or streak indicates DNase activity.
Staphylococcus aureus Growing on
DNase Agar
Note there is breakdown of the DNA in the agar. There is a clear zone (arrow) around
the bacterial growth where there is no longer any DNA left in the agar to precipitate
out of solution after the 1N HCL was added.
Staphylococcus epidermidis Growing
on DNase Agar
Note there is no breakdown of the DNA in the agar. After adding the 1N HCl, the entire
plate turned cloudy as the DNA precipitated out of solution. There is no clear zone
around the bacterial growth.
DNase Test Agar with Methyl Green
Methyl green forms a complex with intact (polymerized) DNA to form the green
color of the medium. DNase activity depolymerizes the DNA, breaking down the
methyl green-DNA complex, which results in the formation of colorless zones
around colonies of the test organism. A negative test is indicated by the
absence of a colorless zone around the colonies.
DNase Test Agar with Toluidine Blue
Toluidine blue forms a complex with intact (polymerized) DNA. In the intact
DNA complex, the toluidine blue has the normal blue color. Dnase activity
depolymerizes the DNA, breaking down the dye-DNA complex. In the presence
of nucleotides produced from the DNase depolymerization, the dye takes on its
metachromatic color, forming pink to red zones around bacterial growth. A
negative test is indicated when the medium remains blue.
Protein A Latex Test
L
Protein A is found
L
IgG
S
S
S
L
S
Fc
on the cell
surface of about 95
% of human strains
of S. aureus and
has the ability to
bind the Fc portion
of immunoglobulin
G (IgG)
L
L
S
S
Protein A
S=S.aureus with Protein A
L=Latex particle
S
25
Staphyloslide™ Latex Test for Staphylococcus aureus
Latex Test consists of latex particles coated with human fibrinogen and IgG. On
mixing the latex reagent with colonies of staphylococci which have clumping
factor or Protein A present, cross-linking will occur giving visible agglutination of
the latex particles.
Such agglutination will occur notably with S. aureus.
If neither clumping factor nor Protein A are present, no agglutination will occur
and the result will be regarded as negative.
The End