M BACTE: STAPHYLOCCOCUS

• Staphylococcus


  Spherical cells arranged in irregular clusters
• Micrococcus


  Gram-positive cocci, similar to Staphylococcus
• Sir Alexander Ogston established the causative role of the coccus in abscesses and other suppurative lesions


  1880
• von Recklinghausen first observed Staphylococcus in human pyogenic lesions

  1871
• Staphylococcus


  Name given by Ogston due to the typical occurrence of the cocci "in grape-like clusters" in pus and in cultures
• Common isolates of gram-positive cocci

  • Staphylococcus aureus
  • Staphylococcus epidermidis
  • Staphylococcus saprophyticus
  • Staphylococcus lugdunensis
  • Staphylococcus haemolyticus
• Coagulase-positive staphylococci


  Includes Staphylococcus aureus
• Coagulase-negative staphylococci (CONS)


  Includes Staphylococcus epidermidis and Staphylococcus saprophyticus
• Staphylococci are a normal component of man's indigenous microflora and are carried asymptomatically in a number of body sites
• About 10-30% of healthy persons carry staphylococci in the nose, about 10% in the perineum, and about 5-10% in the vagina (which rises greatly during menses)
• Staphylococci


  • Spherical cells arranged in irregular clusters
  • Appear singly, in pairs, or in short chains
  • Gram-positive
  • Lack spores and flagella
  • May have capsules
  • Catalase-positive
  • Nonmotile, non-spore forming, aerobic or facultatively anaerobic
  • Colonies are medium-sized, cream-colored, white or rarely light gold, and "buttery-looking"
  • Some species are beta-hemolytic
• Staphylococci resemble some members of the family Micrococcaceae, such as the genus Micrococcus
• Other gram-positive cocci occasionally recovered from human clinical specimens

  • Rothia mucilaginosa
  • Aerococcus
  • Alloiococcus otitis
• Coagulase-producing (coagulase-positive) staphylococci


  • Staphylococcus aureus
  • Staphylococcus intermedius
  • Staphylococcus pseudintermedius
  • Staphylococcus hyicus
  • Staphylococcus delphini
  • Staphylococcus lutrae
  • Staphylococcus agnetis
  • Some strains of Staphylococcus schleiferi
• Staphylococcus lugdunensis and Staphylococcus schleiferi are sometimes mistaken for coagulase-positive staphylococci because of the presence of clumping factor
• Majority of clinical staphylococcal isolates identified by the tube coagulase test will be Staphylococcus aureus
• Coagulase-negative staphylococci (CONS or non-Staphylococcus aureus) species subdivided into two groups based on their novobiocin susceptibility pattern

  • Novobiocin susceptible group: Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis subsp. hominis, Staphylococcus lugdunensis, Staphylococcus saccharolyticus, Staphylococcus warneri, and other species
  • Novobiocin resistant group: Staphylococcus cohnii, Staphylococcus kloosii, Staphylococcus saprophyticus, Staphylococcus xylosus
• Microscopic examination of purulent exudates, joint fluids, aspirated secretions, and other body fluids

  • Cocci and PMN easily seen when these sites are infected with staphylococci
  • Culture should be done regardless of the results of the microscopic examination
  • Genus or species cannot be appropriately identified
  • Aspirate is the best sample, a single swab would be less satisfactory for both culture and smear results
• Microscopic appearance of staphylococci

  • Spherical cocci, approximately 1 μm in diameter, arranged in grape-like clusters
  • May also be found singly, in pairs and in short chains of three or four cells
  • Non-spore forming, non-motile and usually non-capsulated
  • Stain readily with aniline dyes and are uniformly gram-positive
• Isolation and identification of staphylococci

  • Grow easily on routine laboratory culture media like sheep blood agar (SBA)
  • For heavily contaminated specimens, selective media like Mannitol Salt Agar (MSA), Columbia Colistin–nalidixic Acid Agar (CNA), or Phenylethyl Alcohol (PEA) agar can be used
  • CHROMagar Staph aureus is a selective and differential medium for isolation and identification of S. aureus
  • CHROMagar MRSA can further classify S. aureus into MRSA or MSSA strains
• Cultural characteristics of staphylococci

  • Produce round, smooth, white, creamy colonies on SBA after 18 to 24 hours of incubation
  • Can produce hemolytic zones around the colonies
  • Rarely exhibit pigment production (yellow) with extended incubation
  • Aerobes and facultative anaerobes, optimum pH is 7.5
  • Small colony variants (SCVs) grow as nonpigmented, nonhemolytic pinpoint-size colonies
• Cultural characteristics of other staphylococcal species

  • Staphylococcus epidermidis: small- to medium-sized, nonhemolytic, gray-to-white colonies

  Staphylococcus saprophyticus: slightly larger colonies, about 50% produce yellow pigment

  Staphylococcus haemolyticus: medium-sized colonies, moderate or weak hemolysis and variable pigment production

  Staphylococcus lugdunensis: often hemolytic and medium sized, small colony variants can occur
• Identification methods for staphylococci

  • Oxidation-fermentation (O/F) reactions in O/F glucose medium

  Microdase Disk (Remel) to rapidly differentiate staphylococci from micrococci

  Molecular testing, plasmid typing, fatty acid analysis, and MALDI-TOF for species and strain identification

  Catalase test to distinguish staphylococci from micrococci

  Coagulase tests (slide and tube) to detect clumping factor and staphylocoagulase
• Ionization time of flight (MALDI-TOF)


  Used for species and strain identification
• Catalase tests


  • Demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2)
  • Used to distinguish Staphylococci spp. and Micrococci spp.
• Normally 3% H2O2 is used for the routine culture while 15% H2O2 is used for detection of catalase in anaerobes
• Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism
• Coagulase tests


  • Clumping factor (cell-bound coagulase) causes agglutination in human, rabbit, or pig plasma
  • Directly converts fibrinogen to fibrin, which precipitates onto the cell surface, causing agglutination
• Coagulase test


  1. Performed on a glass slide using a heavy suspension of organism mixed with saline and a drop of plasma

  2. Glass slide negative result must be followed up by a tube coagulase test
• Tube coagulase method


  • Detects staphylocoagulase, or free coagulase
  • An extracellular molecule that causes a clot to form when bacterial cells are incubated with plasma
  • Reacts with a thermostable, thrombin-like molecule called coagulase-reacting factor (CRF) to form coagulase-CRF complex (resembles thrombin and indirectly converts fibrinogen to fibrin)
• Tube coagulase test


  1. Look for clot formation after 4 hours of incubation at 37° C

  2. If no clot appears, the tube should be left at room temperature and checked the next day to avoid fibrinolysis
• Staphylococcus aureus colonies on nutrient agar

  • 1–3 mm in diameter
  • Smooth glistening surface
  • Entire edge
  • Soft butyrous consistency
  • Opaque, pigmented appearance
  • Most strains produce golden-yellow (aureus) pigment (staphyloxanthin)
  • White-colony strains are fully virulent
  • Pigmentation is characteristic of this species when grown aerobically
  • Pigmentation is enhanced on fatty media, such as tween agar, by prolonged incubation, and by leaving plates at room temperature
  • The pigment is believed to be lipoprotein allied to carotene
• White-colony strains of S. aureus are fully virulent
• Staphylococcus aureus colonies on blood agar


  • Colonies have the same appearances as on nutrient agar, but may be surrounded by a zone of ß-hemolysis
  • Hemolysis is more likely to be present if sheep, human or rabbit blood is used
• Phenolphthalein Phosphate Agar (PPA)


  • An indicator medium and assists in the identification of S. aureus in mixed cultures
  • Colonies are similar to those on nutrient agar
  • Become bright pink when culture plate is inverted over ammonia for a minute or so
• Selective Salt Media


  • Useful for the isolation and enumeration of staphylococci from materials, such as feces, food and dust, likely to contain a predominance of other kinds of bacteria
  • 7–10% of sodium chloride may be added to Nutrient Agar (Salt Agar) or Milk Agar (Salt Milk Agar)
  • Mannitol Salt Agar containing 1% mannitol, 7.5% NaCl, and phenol red in nutrient agar
  • Ludlam's medium containing lithium chloride and tellurite
  • Salt Cooked Meat Broth (10% NaCl)
• Sugar Fermentation


  • Ferments a range of sugars producing acid but no gas
  • Sugar fermentation is of no diagnostic value except for mannitol, which is usually fermented anaerobically by S. aureus but not by other species
• Phosphatase Test


  • Useful screening procedure for differentiating S. aureus from S. epidermidis in mixed cultures
  • S. aureus gives prompt phosphatase reaction, while the latter is usually negative or only weakly positive
  • All strains of S. aureus produce phosphatase which liberates phenolphthalein from sodium phenolphthalein diphosphate
  • S. aureus become bright pink because phenolphthalein is pink in alkaline ph
  • Most other staphylococci form colonies that remain uncolored
• Deoxyribonulease (DNase) Test


  Produces a deoxyribonulease (DNase), and a heat-stable nuclease (thermonuclease)
• Resistance of Staphylococcus aureus


  • S. aureus and the some micrococcaceae are among the hardiest of the non spore forming bacteria
  • They retain their viability for 3–6 months
  • They have been isolated from dried pus after 2–3 months
  • It withstands moist heat at 60°C for 30 min but is killed after 60 min
  • Most strains grow in the presence of 10% NACl
  • These features are of significance in food preservation