د.محمد

Staphylococcus

The main species are:Staphylococcus aureus ( golden-yellow colonies).Staphylococcus epidermidis (white colonies).Staphylococcus saprophyticus (lemon –yellow colonies).

Microscopic Examination

Gram-positive cocciArranged in grape-like clustersNon motileNon–spore-formingNon-capsulated

Grow well in most routine media e.g : Nutrient Agar. Colonies are 2-3 mm. in diameter after 24 hrs ,circular , convex ,opaque , smooth ,moist with an entire edge and pigmented. facultatively anaerobic .

golden-yellow colonies

Note beta hemolysis (complete lysis of the red blood cells around the colonies; see arrows) on the blood agar .


Mannitol Salt Agar: this is a selective medium for the isolation of Staphylococci depending on their tolerance to a high conc. Of NaCl. Also differentiates S.aureus from S.epidermidis based on mannitol fermentation .

Mannitol Salt agar

Staphylococcus aureus
Staphylococcus epidermidis

Important characteristic of S.aureus

Comparison
Properties
S. aureus
S. epidermidis
S. saprophyticus
Pigment
Golden yellow
white
citrine
Coagulase
+
-
-
Mannitol
+
-
-
Hemolysin
+
-
-
DNase
+
-
-
Novobiocin sensitivity
sensitive
sensitive
Resistant

Catalase Test

The test is used to differentiation between Staphylococcus ,Micrococci(+)and Streptococci(-). Determines the ability of microbes to degrade hydrogen peroxide (H2O2) by producing the enzyme catalase H2O2 is produced during aerobic respiration; must be degraded catalase 2 H2O2 2 H2O + O2(gas bubbles)

Coagulase test

Coagulase is an enzyme which coagulates citrated human or rabbit plasma. This test distinguishes S.aureus(+) from S.epidermidis(-). Coagulase may be bound (slid method) or free (tube method). Fibrinogen Fibrin (Plasma) (Clot)
Coagulase

Coagulase test

Negative
Positive
Slide method
Negative
Positive
Tube method
Coagulase
Staphylococcus aureus
CNS
Staphylococcus epidermidis
Staphylococcus saprophyticus
+
_

DNase TEST AGAR

DNAse Test: This test used to identify S.aureus DNAse (an enzyme that degrades DNA)
Test Procedure 1. Inoculate plates by streaking a heavy inoculum of test organism.2. Incubate plates at 37 °C for 18 – 24 hours and up to 48 hours. 3. Flood plates with 1 N HCl. 4. Record results. Results DNase hydrolyzes DNA The acid precipitates unhydrolyzed DNA A zone of clearing around the colonies 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.
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.
Staphylococcus epidermidis Growing on DNase Agar

Quiz

Q1: Starting from a fixed smear, specify the main steps of Gram stain in the appropriate order ? Q2:

Staphylococcus aureus Staphylococcus epidermidis