Bacteriology tests

• Gram stain


  A laboratory technique used to classify bacteria into two major groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls
• Staphylococci

  • Round shaped (coccus or spheroid)
  • Gram-positive (purple)
  • Found as single cells, in pairs, or in clusters resembling a bunch of grapes
• The genus name Staphylococcus is derived from Greek terms (staphyle and kokkos) that mean "a bunch of grapes" and "berry", respectively, referring to the characteristic microscopic appearance of these bacteria
• Any area with pus, crusty drainage, or blisters should be cultured. Blood from patients with sepsis, toxic shock syndrome, or pneumonia should be cultured
• Standard microbiological techniques include a positive coagulase test
• S. aureus lyses red blood cells in blood agar plates (hemolytic staphylococci) while S. epidermidis does not (nonhemolytic staphylococci)
• All staphylococci should be further tested to see if the bacteria are resistant to the antibiotic methicillin (and other antibiotics) and thus determine if the organisms are MRSA
• MRSA

  Methicillin Resistant Staphylococcus aureus
• Staphylococcus

  • Round shape (coccus or spheroid shaped)
  • Gram-positive (purple)
  • Found as either single cells, in pairs, or more frequently, in clusters that resemble a bunch of grapes
• Staphylococcus


  Derived from Greek terms (staphyle and kokkos) that mean "a bunch of grapes"
• S. aureus

  • Displays a light to golden yellow pigment
  • Usually beta-hemolytic
• S. epidermidis

  • Has a white pigment
  • Almost always nonhemolytic
• S. saprophyticus

  • Either a bright yellow or white pigment
• Pigmentation is not always a reliable characteristic
• S. aureus colonies


  • Round, convex, and 1-4 mm in diameter with a sharp border
  • Frequently surrounded by zones of clear beta-hemolysis
  • Golden appearance of colonies
• S. epidermidis colonies

  • Showing γ-haemolytic, porcelin-white colonies
• The clear distinction in colony color is not seen at all times
• Catalase

  Enzyme that catalyzes the destruction of hydrogen peroxide
• Organisms capable of producing catalase rapidly degrade hydrogen peroxide
• Catalase has one of the highest turnover numbers compared to all other enzymes
• Catalase production and activity can be detected either by adding the substrate H2O2 to an appropriately incubated (18 to 24 hours) tryptic soy agar slant culture or by slide test
• Organisms which produce the enzyme break down the hydrogen peroxide, and the resulting O2 production produces bubbles in the reagent drop, indicating a positive test
• Organisms lacking the cytochrome system also lack the catalase enzyme and are unable to break down hydrogen peroxide, into O2 and water and are catalase negative
• The catalase test is primarily used to distinguish among Gram-positive cocci. Members of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and Enterococcus are catalase-negative
• Catalase Test (Slide Test)


  1. Transfer a small amount of bacterial colony to a surface of clean, dry glass slide

  2. Place a drop of 3% H2O2 on to the slide and mix

  3. A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling

  4. A negative result is no bubbles or only a few scattered bubbles
• Mannitol Salt Agar (MSA)

  Selective and differential media used for the isolation of Staphylococci from mixed cultures
• MSA Components

  • 7,5% NaCl - selects for species of Staphylococcus
  • Mannitol - alcohol of the carbohydrate mannose, mannitol fermentation produces acid end products which turn the medium yellow
  • Phenol red pH indicator - yellow in acid pH
• On MSA, only pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones
• The reason for this color change is that S. aureus have the ability to ferment the mannitol, producing an acid, which changes the indicator color from red to yellow
• The growth of other types of bacteria is usually inhibited
• This growth differentiates S. aureus from S. epidermidis, which forms colonies with red zones
• On MSA

  • Staphylococcus aureus - colonies surrounded by yellow zones
  • Staphylococcus epidermidis - colonies with red zones
• Coagulase


  Enzyme that clots blood plasma by a mechanism that is similar to normal clotting
• The only significant disease causing bacteria of humans that produce coagulase enzyme are Staphylococcus aureus
• Coagulase


  A virulence factor (disease-causing factor) of Staphylococcus aureus
• Most strains of S.aureus produce one or two types of coagulase; free coagulase and bound coagulase
• Slide Coagulase Test

  1. Divide the slide into two sections, one labeled "test" and the other "control"

  2. Place a small drop of distilled water on each area

  3. Emulsify one or two colonies of Staphylococcus on blood agar plate on each drop

  4. The test suspension is treated with a drop of citrated plasma and mixed well

  5. Clumping of cocci within 5-10 seconds is taken as positive
• Tube Coagulase Test

  1. Three test tubes are taken and labeled "test", "negative control" and "positive control"

  2. Each tube is filled with 1 ml of 1 in 10 diluted rabbit plasma

  3. To the tube labeled test, 0.2 ml of overnight broth culture of test bacteria is added

  4. To the tube labeled positive control, 0.2 ml of overnight broth culture of known S. aureus is added

  5. To the tube labeled negative control, 0.2 ml of sterile broth is added

  6. All the tubes are incubated at 37°C

  7. Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube
• All pathogenic strains of S. aureus are coagulase positive whereas the nonpathogenic species (S. epidermidis) are coagulase negative
• While slide coagulase test is useful in screening, tube coagulase test is useful in confirmation of coagulase test