Staphylococci Lab 5 PDF

Lab-5Staphylococci BY DR. SHNYAR HAMID QADIR Staphylococci Group Characteristics • Members of the genus Staphylococcus (staphylococci) are Gram-positive cocci • Arranged in grape-like clusters, some single cells, pairs, and short chains are also seen • They are nonflagellate, nonmotile, and non–sp...

Lab-5Staphylococci BY DR. SHNYAR HAMID QADIR Staphylococci Group Characteristics • Members of the genus Staphylococcus (staphylococci) are Gram-positive cocci • Arranged in grape-like clusters, some single cells, pairs, and short chains are also seen • They are nonflagellate, nonmotile, and non–spore-forming. • Staphylococci grow best aerobically but are facultatively anaerobic • Catalase positive. all three types of staphylococcus contain an enzyme called catalase , if you put H2O2 with all this type of staphylococcus bactrtias on petri dish , the actuall solution with there will be bubble ,because they contain this catalase enzyme which convert H2O2 into water and oxgyn and oxgen allows for that kind bubbling effect, so if the solution bubble it means it is catalase positive . • Most are oxidase negative. • Most produce acid from glucose. • Most are resistant to bacitracin. Staphylococci. Gram stain showing the Gram-positive cocci in clusters resembling bunches of grapes Staphylococcus aureus • S aureus is by far the most virulent • Coagulase distinguishes S aureus from other species coagulase when it is converted to fibrin make everything look k like clump , so the staphylococcus is only one that clump up . convert fibrinogin to fibrin • It is common to lump the other species together as coagulase-negative staphylococci (CoNS). Coagulase-Negative Staphylococci • S epidermidis, • And S saprophyticus Identification of Gram-positive Cocci § By Gram Stain § By Colonial Morphology § Biochemical Identification 1. Gram stain • All staphylococci appear as gram-positive cocci • Arrangment: in irregular, often grape-like clusters. 2- Colonial Morphology colony morphology is the visual culture characteristics of a bacterial colony on an agar plate . • They appear on (blood agar, chocolate agar and nutrient agar) • But not on MacConkey agar S.epidermidis Grey-white colonies S.aureus Golden-yellow colonies 2- Colonial Morphology A. β-hemolysis on blood agar • Based on their ability to hemolyze RBCs • Several species of Gram-positive cocci produce exotoxins called hemolysins, which are able to destroy red blood cells (RBCs) and hemoglobin. • Results in a clearing of the medium around the colonies A. β-hemolysis on blood agar • S aureus produce β-hemolysis B. Novobiocin (NB) disc test • Used to identify the novobiocinresistant Staphylococcus saprophyticus • In this test, a 5 µg disc should produce a zone of clearing 16 mm or more to be considered novobiocin susceptible novobiocin Disk Test :A novobiocin-resistant (R) organism is on the left; a susceptible (S) organism is on the right. Colonial morphology of staphylococci Test Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus hemolysis Usually beta Usually none Usually none pigment Often creamy gold Usually white Usually white Novobiocin test Sensitive Sensitive Resistant 3. Biochemical Test A. Mannitol fermentation on Mannitol Salt agar (MSA) If mannitol is fermented, the acid produced turns the phenol red pH indicator from red (alkaline) to yellow (acid). Test Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Mannitol fermentation Positive Negative Positive Mannitol fermentation on Mannitol Salt agar (MSA) Shows S aureus colonies Shows S epidermidis colonies B. Catalase Test • The catalase test is primarily used to distinguish among Gram-positive cocci: • members of the genus Staphylococcus are catalasepositive • members of the genera Streptococcus are catalasenegative. Test Staphylococcus Staphylococcus Staphylococcus aureus epidermidis saprophyticus Catalase production Positive Positive Positive B. Catalase Test • When hydrogen peroxide is added to a catalasepositive culture, oxygen gas bubbles form immediately . If no bubbles appear, the organism is catalase-negative. • 2H2O2 Catalase 2H2O + O2 (g) B. Catalase Test Catalase Slide Test: Visible bubble production indicates a positive result in the catalase slide test. A catalase-positive organism is on the left. A catalase- negative organism is on the right Catalase Tube Test: The catalase test also may be performed on an agar slant. A catalase-positive organism is on the left. A catalase-negative organism is on the right. C. Production of coagulase • Used to distinguish S aureus from other staphylococci • The coagulase converts soluble fibrinogen in the plasma into insoluble fibrin Test Staphylococcus Staphylococcus Staphylococcus aureus epidermidis saprophyticus Coagulase production Positive Negative Negative Coagulase test Coagulase Tube Test: These coagulase tubes illustrate a coagulase-negative organism, below, and a coagulase-positive organism, above.