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Microbiology

Conventional Biochemical ID

This section delves into the theory, interpretation, and application of conventional biochemical tests used to identify bacteria, focusing on examples like X and V factor testing and carbohydrate utilization by Neisseria species. These tests provide more definitive identification than rapid tests, often employed after preliminary results are available

Theory: The Science Behind the Biochemical Reactions

  • What are Conventional Biochemical Tests?
    • Conventional biochemical tests are laboratory assays that assess the metabolic capabilities of bacteria
    • They utilize specific substrates to detect the presence or absence of enzymes, the production of metabolic byproducts, or the utilization of specific nutrients
    • They are used for definitive identification of bacterial species, often following presumptive identification based on Gram stain, colony morphology, and rapid tests
  • Why Use Conventional Biochemical Tests?
    • Specificity: Provide more specific identification than rapid tests
    • Reliability: Generally, reliable results with proper technique and QC
    • Versatility: Applicable to a wide range of bacterial species
    • Confirmation: Confirm presumptive identifications and differentiate closely related species
  • Examples of Biochemical Reactions
    • Enzymatic Reactions: Detection of specific enzymes (e.g., urease, catalase, oxidase)
    • Substrate Utilization: Ability to utilize specific substrates (e.g., carbohydrates, amino acids, citrate)
    • Product Formation: Production of specific products (e.g., acid, gas, H₂S) from metabolic pathways
    • Growth Requirements: Assessment of growth requirements (e.g., X and V factors)
  • Factors Influencing Biochemical Test Results
    • Inoculum Size: Proper inoculum size is crucial for accurate results
    • Incubation Time and Temperature: Follow recommended incubation protocols
    • Media Composition: Use appropriate media for each test
    • Reagent Quality and Storage: Ensure reagents are fresh and stored properly
    • Purity of Culture: Use pure cultures to prevent misinterpretation

X and V Factors (for Haemophilus spp.)

  • Principle: Haemophilus species require specific growth factors for growth:
    • X factor: Hemin (heat-stable, porphyrin)
    • V factor: Nicotinamide adenine dinucleotide (NAD, heat-labile)
  • Procedure
    1. Inoculate the organism onto a quadrant plate of agar (e.g., chocolate agar or blood agar)
    2. Place X factor, V factor, and a combination of X and V factors (XV) on the agar
    3. Incubate at 35-37°C in 5-10% CO₂
    4. Observe for growth around the factor disks
  • Interpretation
    • X factor only: Growth only around the X factor disk (H. parainfluenzae)
    • V factor only: Growth only around the V factor disk (H. influenzae, some H. parainfluenzae)
    • XV factor: Growth around the XV factor disk (H. influenzae)
    • No growth: Indicates the organism is not Haemophilus
  • Clinical Significance: Differentiates Haemophilus species based on their growth factor requirements
    • H. influenzae: requires both X and V factors
    • H. parainfluenzae: requires only V factor
    • H. ducreyi: requires X factor only

Neisseria Carbohydrate Utilization

  • Principle: Neisseria species ferment (utilize) specific carbohydrates, producing acid
  • Reagents
    • Cystine-trypticase agar (CTA) base
    • Sterile carbohydrate solutions (glucose, maltose, lactose, sucrose, fructose)
    • pH indicator (phenol red)
  • Procedure
    1. Prepare CTA slants by adding the appropriate carbohydrate solution
    2. Inoculate each slant with the organism
    3. Incubate at 35-37°C in ambient air
    4. Observe for acid production (yellow color)
  • Interpretation
    • Positive: Yellow color change (acid production)
    • Negative: No color change (red or orange)
  • Clinical Significance: Differentiates Neisseria species based on their carbohydrate fermentation patterns
    • N. gonorrhoeae: ferments glucose only
    • N. meningitidis: ferments glucose and maltose
    • N. lactamica: ferments glucose, maltose, and lactose
    • N. cinerea: ferments glucose (weakly)
  • Important Notes
    • Neisseria spp. are fastidious and require specific media
    • Some Neisseria species are slow fermenters, so incubation times might be extended
    • N. gonorrhoeae and N. meningitidis may be isolated from sterile body sites

Application: Putting Knowledge into Practice

  • Quality Control (QC)
    • Control Strains: Use known positive and negative control organisms for each test
    • Frequency: Perform QC at the beginning of each day or whenever a new lot of reagents is used
    • Documentation: Record QC results in a logbook or LIS
  • Procedure
    1. Preliminary Testing: Perform Gram stain, colony morphology, and rapid tests
    2. Media and Reagent Preparation: Prepare media and reagents according to the manufacturer’s instructions
    3. Inoculation: Inoculate the test media with a pure culture of the organism
    4. Incubation: Incubate the tests under the appropriate conditions (temperature, atmosphere)
    5. Observation: Observe the results at the recommended time points
    6. Interpretation: Interpret the results based on the expected reactions (color changes, gas production, growth)
    7. Documentation: Record the results in a lab notebook or LIS
    8. Correlation: Correlate the results with other test results and clinical information to arrive at a definitive identification
    9. Reporting: Report the identification to the clinician
  • Example: Identifying a Neisseria species
    1. Gram Stain: Gram-negative diplococci
    2. Colony Morphology: Small, translucent, non-pigmented colonies on chocolate agar
    3. Oxidase Test: Positive
    4. Catalase Test: Positive
    5. Carbohydrate Utilization (CTA)
      • Glucose positive
      • Maltose negative
      • Lactose negative
      • Sucrose negative
      • Fructose Negative
    6. Interpretation: Presumptive identification of Neisseria gonorrhoeae
    7. Further Testing: Perform antibiotic susceptibility testing
  • Example: Identifying a Haemophilus influenzae
    1. Gram Stain: Small, Gram-negative coccobacilli
    2. Colony Morphology: Small, translucent, non-hemolytic colonies on chocolate agar
    3. X and V Factor Test: Growth around the X and V factor disks
    4. Interpretation: Identification of Haemophilus influenzae
  • Troubleshooting
    • False Positives/Negatives
      • Inoculum Size: Use the recommended inoculum size for each test
      • Reagent Quality: Ensure reagents are fresh and stored properly
      • Cross-Contamination: Maintain strict aseptic technique
      • Incubation Time: Follow the recommended incubation times
    • Weak Reactions
      • Old Cultures: Use fresh cultures
      • Incorrect Procedure: Review the test procedure
      • Incubation Conditions: Ensure proper incubation temperature
    • Contamination: Review aseptic technique

Key Terms

  • Conventional Biochemical Tests: Laboratory assays that assess the metabolic capabilities of bacteria
  • Substrate: A substance that is acted upon by an enzyme
  • Enzyme: A protein that catalyzes a specific biochemical reaction
  • Acid Production: The formation of acid as a result of carbohydrate fermentation
  • Growth Factors: Essential nutrients required for bacterial growth (e.g., X and V factors for Haemophilus)
  • X Factor: Hemin, a growth factor required by Haemophilus
  • V Factor: Nicotinamide adenine dinucleotide (NAD), a growth factor required by Haemophilus
  • Carbohydrate Fermentation: The metabolic process by which bacteria break down carbohydrates
  • Cystine-trypticase agar (CTA): A semisolid medium used for carbohydrate fermentation tests
  • pH Indicator: A substance that changes color in response to changes in pH (e.g., phenol red)
  • Gram Stain: A differential staining technique used to classify bacteria based on their cell wall structure
  • Colony Morphology: The visual characteristics of bacterial colonies on solid media
  • Inoculum: The material used to inoculate a culture medium
  • Quality Control (QC): Procedures used to monitor and ensure the reliability of laboratory testing
  • Control Strains: Known organisms used as positive and negative controls in laboratory tests
  • False Positive: A test result that incorrectly indicates the presence of a substance or organism
  • False Negative: A test result that incorrectly indicates the absence of a substance or organism
  • Fermentation: The metabolic process by which bacteria break down carbohydrates in the absence of oxygen, producing acid and/or gas
  • Oxidase: An enzyme involved in the electron transport chain, often present in aerobic bacteria
  • Catalase: An enzyme that breaks down hydrogen peroxide into water and oxygen
  • Antibiotic Susceptibility Testing: Laboratory tests to determine the effectiveness of antibiotics against a bacterial isolate
  • Pathogenicity: The ability of an organism to cause disease
  • Sterile Body Site: A site in the body that is normally free of microorganisms