Bacterial Identification: Staphylococcus and Streptococcus Species Examination

Bacterial Identification: Staphylococcus and Streptococcus Species

Background: Streptococcus pyogenes

  • Classification: Group A streptococcus (GAS).
  • Morphology:
    • Gram-positive.
    • Nonmotile.
    • Non-spore forming coccus.
    • Occurs in chains or pairs of cells.
  • Metabolism:
    • Fermentative.
    • Catalase-negative.
    • Aerotolerant anaerobe (facultative anaerobe).
  • Growth Requirements: Requires enriched medium containing blood.
  • Distinguishing Features:
    • Typically has a capsule composed of hyaluronic acid.
    • Exhibits beta (clear) hemolysis on blood agar.
  • Clinical Significance (Acute Infections):
    • Pharyngitis: Commonly known as "strep throat."
    • Scarlet Fever: Characterized by a rash.
    • Impetigo: Infection of the superficial layers of the skin.
    • Cellulitis: Infection of the deep layers of the skin.
  • Clinical Significance (Invasive, Toxigenic Infections):
    • Necrotizing fasciitis.
    • Myositis.
    • Streptococcal toxic shock syndrome.
  • Clinical Significance (Immune-mediated Post-streptococcal Sequelae): These can develop following acute infections caused by S. pyogenes.
    • Acute rheumatic fever.
    • Acute glomerulonephritis.

Hemolysis on Blood Agar

  • Classification Basis: The type of hemolytic reaction displayed on blood agar is a long-standing method for classifying streptococci.
  • Types of Hemolysis:
    • Beta-hemolysis (β\beta):
      • Associated with complete lysis of red blood cells (RBCs) surrounding the colony.
      • Results in a clear zone around the colony.
    • Alpha-hemolysis (α\alpha):
      • Associated with partial lysis of RBCs.
      • Often described as "green" hemolysis due to the reduction of red cell hemoglobin.
    • Gamma-hemolysis (γ\gamma):
      • Termed for nonhemolytic colonies.
      • No lysis of RBCs and no color change.
  • Factors Affecting Hemolysis: The specific hemolytic reaction can be influenced by:
    • The species of the red blood cells used in the agar.
    • The age of the red blood cells.
    • Other properties of the base medium.
  • Hemolysis Patterns in Specific Streptococci:
    • Group A streptococci (S. pyogenes): Almost always beta-hemolytic.
    • Related Group B streptococci: Can display alpha, beta, or gamma hemolysis.
    • Most strains of S. pneumoniae: Typically alpha-hemolytic, but can cause beta-hemolysis under anaerobic incubation conditions.
  • Application: Hemolysis type is widely used in rapid screens for the identification of Streptococcus pyogenes and Streptococcus pneumoniae.

Isolating Streptococcus from the Pharynx (Procedure)

  1. Patient Selection: One group member volunteers as the patient, preferably someone without a strong gag reflex for this test.
  2. Materials: Obtain a sterile swab and a tongue depressor.
  3. Swab Collection: Carefully swab the patient's tonsil, ensuring not to touch the cheek or tongue. If the swab touches the cheek or tongue, discard it and obtain a new sterile swab.
  4. Plate Inoculation: Swab the collected sample all across the agar surface of a blood agar plate.
  5. Swab Disposal: Discard the used swab in a biohazard container.
  6. Plate Labeling: Label the blood agar plate with the following information:
    • "Pharyngeal swab"
    • The patient's name
    • The current date.
  7. Incubation: Incubate the blood agar plate at 37extoC37^ ext{o}C for 24−4824-48 hours.

Pharyngeal Swab Results (Analysis)

  1. Hemolysis Identification: After incubation, identify whether the plate contains alpha (α\alpha), beta (β\beta), or gamma (γ\gamma) hemolytic bacteria.
  2. Documentation: Take a photo of your plate and clearly label the colonies that are exhibiting alpha or beta hemolysis.

Background: Staphylococcus

  • Staphylococcus aureus (commonly "staph"):
    • Prevalence: Approximately 30%30\% of people carry S. aureus in their noses.
    • Pathogenicity: Most of the time, S. aureus does not cause harm. However, it can cause infections, particularly serious or fatal ones in healthcare settings.
    • Healthcare-Associated Infections (HAIs) Caused by S. aureus:
      • Bacteremia or Sepsis: Occurs when bacteria spread to the bloodstream.
      • Pneumonia: Predominantly affects individuals with underlying lung disease, including those on mechanical ventilators.
      • Endocarditis: An infection of the heart valves, which can lead to severe complications such as heart failure or stroke.
      • Osteomyelitis: A bone infection that can result from staph bacteria traveling through the bloodstream or being introduced by direct contact (e.g., following a puncture wound of the foot or intravenous (IV) drug abuse).
    • Source of information: CDC website (http://www.cdc.gov/HAI/organisms/staph.html)

Background: Mannitol Salt Agar Plate (MSA)

  • Formulation: Devised by Chapman.
  • Primary Purpose: For the differentiation of Staphylococcus aureus from coagulase-negative staphylococci, such as Staphylococcus epidermidis.
  • Applications: Used for isolating staphylococci from clinical specimens and from cosmetics.
  • Selective Mechanism - High Salt Concentration:
    • MSA contains a 7.5%7.5\% concentration of sodium chloride (salt).
    • This high salt content results in the partial or complete inhibition of most bacterial organisms other than staphylococci, making the medium selective.
    • Osmotic Phenomenon: In a hypertonic solution (high solute concentration), water rushes out of bacterial cells, causing them to shrink and dehydrate.
    • Practical Example: This osmotic phenomenon has been historically used in manufacturing (e.g., salt-curing pork products like country ham, bacon, pancetta, and prosciutto) to act as a preservative and reduce microbial spoilage.
  • Osmotolerant Bacteria: Unfortunately, some bacteria possess the ability to tolerate high solute environments and grow under these conditions; these are referred to as osmotolerant bacteria.
    • Example: Staphylococcal species are known to be osmotolerant.
  • Differentiative Mechanism - Mannitol Fermentation:
    • MSA includes mannitol as a fermentable carbohydrate and phenol red as a pH indicator.
    • Phenol Red Indicator: Changes color based on pH.
    • Mannitol Fermentation (Positive Result):
      • Staphylococcus aureus ferments mannitol, producing acid byproducts.
      • The acid lowers the pH of the medium, causing the phenol red indicator to change to yellow.
      • Result: Yellow colonies and a surrounding yellow medium.
    • No Mannitol Fermentation (Negative Result):
      • Other staphylococci (e.g., Staphylococcus epidermidis) do not ferment mannitol.
      • No acid is produced, so the phenol red indicator remains red or shows no color change.
      • Result: Red colonies and no color change of the phenol red indicator.
  • Bacteria Used in Lab: In the lab, you will encounter: Staphylococcus aureus and Staphylococcus epidermidis (both osmotolerant), and Micrococcus luteus (a bacterium that is less osmotolerant).

Mannitol Salt Agar: Nasal Swab (Procedure)

  1. Patient Selection: Ask one member of your group to volunteer as the patient.
  2. Plate Labeling: Label the outer edge of the agar side of a Mannitol Salt Agar Plate with:
    • Unknown name
    • Patient's name
    • Class session
    • Date
  3. Swab Collection: Obtain a sterile swab and insert it into the external nares (nostril), moving it in a circular direction 55 times to collect a specimen.
  4. Plate Inoculation: Inoculate the MSA plate with a single streak of the collected bacteria, as demonstrated.
  5. Incubation: Incubate the plate with the agar side facing up at 37extoC37^ ext{o}C for 24−4824-48 hours.

Results: Nasal Swab (Analysis)

  • Documentation: Include a photo of your plate.
  • Interpretation: Identify whether the bacteria collected from the nasal swab fermented mannitol (indicated by a color change).

Identifying an Unknown Organism (General Procedure for Gram Stain)

  1. Slide Preparation: Create a bacterial slide containing Unknown A, B, and C. Ensure the slide is allowed to dry thoroughly before heat-fixing the bacteria.
  2. Gram Stain Performance: Perform a Gram Stain. Adjust the decolorization step based on your results and observations from last week's laboratory session.
  3. Microscopic Examination: View the stained slide at 1000extx1000 ext{x} total magnification (TM) under a microscope and take a photo of your results.

Results: Unknown Organism (Analysis for Gram Stain)

  • Gram Result: Identify whether the bacteria are Gram-positive or Gram-negative.
  • Morphological Differences: Note any differences in the bacteria's morphology, arrangement, or other characteristics observed under the microscope.

Mannitol Salt Agar: Unknown A, B, or C (Procedure)

  1. Unknown Selection: Select 11 unknown tube from the provided samples to share with your group.
  2. Plate Labeling: Label the outer edge of the agar side of a Mannitol Salt Agar Plate with:
    • Unknown name
    • Your group name
    • Class session
    • Date
  3. Loop Sterilization: Sterilize an inoculating loop using a microincinerator.
  4. Sample Collection: Allow the loop to cool completely, then obtain a loop-full of bacteria from the unknown tube.
  5. Plate Inoculation: Inoculate the MSA plate with a single streak of bacteria, as demonstrated.
  6. Incubation: Incubate the plate with the agar side facing up at 37extoC37^ ext{o}C for 24−4824-48 hours.

Background: DNase Test

  • Purpose: DNase Test Agar is used for differentiating microorganisms based on their deoxyribonuclease (DNase) activity.
  • Historical Context and Significance:
    • 19561956 (Weckman and Catlin): Demonstrated a correlation between increased DNase activity in Staphylococcus aureus and its positive coagulase activity. They proposed that DNase activity could serve as an indicator for identifying potentially pathogenic staphylococci.
    • DiSalvo: Confirmed these results, finding an excellent correlation between the coagulase and DNase activity of staphylococci isolated from clinical specimens.
    • Jeffries, Holtman, and Guse: Incorporated DNA directly into an agar medium to facilitate the study of DNase production by both bacteria and fungi.
  • Components of BD DNase Test Agar and Their Functions:
    • Tryptone: Provides essential nutrients to support bacterial growth.
    • Sodium chloride: Maintains the osmotic balance of the medium.
    • High molecular deoxyribonucleic acid (DNA): This is the substrate for the DNase enzyme. Its presence enables the detection of deoxyribonuclease, an enzyme that depolymerizes DNA.
  • Principle of Detection:
    • After incubation of the medium with the test strain, a reagent is added to visualize DNase activity.
    • The plate is flooded with hydrochloric acid (HCl).
    • Hydrochloric acid precipitates polymerized (intact) DNA, causing the medium to appear opaque.
    • Positive Result: Organisms that produce DNase (DNase-positive) will degrade the DNA in the agar. In these areas, the DNA is depolymerized and will not precipitate with HCl. This results in a clear zone around the growth area.
    • Negative Result: Organisms that do not produce DNase (DNase-negative) will leave the DNA intact. Upon addition of HCl, the DNA precipitates, and no clear zone will be observed around the colonies.
  • Primary Application: While mainly used in the identification of staphylococci, the DNase test can also be applied for detecting DNase activity in other microorganisms.

DNase Test: Unknown A, B, or C (Procedure)

  1. Plate Labeling: Label the outer edge of the agar side of a DNase Test Plate with:
    • Unknown name
    • Your group name
    • Class session
    • Date
  2. Loop Sterilization: Sterilize an inoculating loop using a microincinerator.
  3. Sample Collection: Allow the loop to cool completely, then obtain a loop-full of bacteria from the unknown tube.
  4. Plate Inoculation: Inoculate the DNase Test Plate with a single streak of bacteria.
  5. Loop Sterilization (Post-inoculation): Sterilize the inoculating loop again with the microincinerator and allow it to cool.
  6. Incubation: Incubate the plate with the agar side facing up at 37extoC37^ ext{o}C for 24−4824-48 hours.
  7. Reagent Application: After incubation, flood the entire surface of the DNase Test Agar plate with 1extN1 ext{ N} hydrochloric acid (HCl). Allow the acid to penetrate the whole medium surface for 22 minutes.

Results for DNase Test (Interpretation)

  • DNase Positive Organisms:
    • Examples: Staphylococcus aureus or Serratia marcescens.
    • Will be surrounded by clear zones after HCl application.
    • These clear zones indicate the depolymerization of DNA by the organism's DNase enzyme.
  • DNase Negative Organisms:
    • Will not show any clearing around their colonies.
  • Surrounding Medium: The medium further away from the inoculation band, where no DNase activity occurred, will appear opaque and whitish due to the precipitation of polymerized DNA by the hydrochloric acid.
  • Visual Representation: (Refer to provided images for positive (clear zone) and negative (no clearing) results.)