Identifying Bacteria

Identifying Bacteria

  • Accurate bacterial identification is essential in clinical microbiology to guide patient treatment and ensure reliable laboratory results.

  • This lesson reviews:

    • Pure culture isolation

    • Gram stain interpretation

    • Key biochemical tests

    • Manual and automated identification methods used to identify Gram-positive and Gram-negative organisms.

Bacterial Culture Basics

Individual Colony

  • Many colonies can be present on media.

Why Is a Pure Culture Important?

  • A pure culture is required to accurately study the following characteristics:

    • Cultural characteristics: Growth patterns on media.

    • Morphological characteristics: Shape, arrangement, staining.

    • Physiological characteristics: Biochemical reactions, metabolism.

  • Identification results may be inaccurate without a pure culture.

Enterobacteriaceae Overview

  • Characteristics:

    • Gram-negative bacilli

    • Facultative anaerobes

    • Non-spore forming

    • Generally, motile, but some are non-motile.

    • Usually oxidase negative.

    • Ferment sugars, producing lactic acid and various other metabolic end products.

  • Habitat:

    • Many are normal intestinal flora in humans and animals.

    • Others are found in water, soil, plants, or as pathogenic species.

  • Common pathogens include:

    • Salmonella

    • Shigella

    • Escherichia coli (E. Coli is notably important in clinical settings).

Identification Testing of Organisms

Manual Identification Methods

  1. Gram Stain Reaction:

    • Determine if the organism is Gram-positive or Gram-negative, cocci or bacilli, and their arrangement (chains, clusters, pairs).

    • This step is crucial for guiding the appropriate identification pathway.

  2. Growth Characteristics on Primary Media:

    • Observe growth vs. no growth on selective and differential media.

    • Check for lactose fermentation (if applicable) and pigment production.

    • Findings will narrow down possible organism groups.

  3. Colony Characteristics on Blood Agar:

    • Careful macroscopic examination provides clues such as:

      • Hemolysis patterns:

      • Alpha (α)

      • Beta (β)

      • Gamma (none)

      • Color

      • Shape

      • Odor

      • Consistency: (mucoid, dry, smooth, rough)

      • Atmospheric requirements:

      • Aerobic

      • Anaerobic

      • CO₂ enriched.

  • Observation of colony characteristics on selective/differential media:

    • Color (often indicates fermentation of sugar(s) present in the media)

    • Shape of the colony

    • Odor

    • Consistency

    • Presence of H₂S

    • Sheen

Identification of Gram-Positive Bacteria

Catalase Test:

  • This test is utilized to differentiate Staphylococcus species from Streptococcus species.

  • Background:

    • Aerobic organisms use oxygen as the terminal electron acceptor during respiration.

    • By-products include hydrogen peroxide (H₂O₂), which is toxic to bacteria.

    • To protect themselves, some bacteria produce catalase, which breaks down H₂O₂ into water and oxygen.

Method:

  1. Place a small amount of the organism on a glass slide using a plastic loop.

  2. Cover with a cover slip.

  3. Add a drop of 3% hydrogen peroxide under the cover slip.

  4. Bubbles indicate catalase production.

    • Staphylococcus: Catalase positive (bubbles produced).

    • Streptococcus: Catalase negative (no bubbles produced).

Controls:

  • Positive Control: Staphylococcus epidermidis

  • Negative Control: Streptococcus agalactiae

  • Note: Avoid contamination from red blood cells when using Blood Agar, as they contain catalase.

Coagulase Test:

  • This test differentiates Staphylococcus aureus from other Staphylococcus species.

  • Staphylococcus produces the enzyme coagulase, which clots fibrinogen in plasma.

  • Test Method:

    • Can be performed using slide or tube methods.

    • If the slide test is negative, repeat using the tube method.

Slide Method:

  1. Place a drop of rabbit plasma on a glass slide.

  2. Mix a small amount of the bacterial colony in the plasma.

  3. Observe for clumping (indicates coagulase presence).

Tube Method:

  1. Place 0.5 ml of rabbit plasma in a test tube.

  2. Mix a small amount of the bacterial colony.

  3. Incubate at 35°C to 37°C for 4 hours.

  4. Check hourly for clot formation (some may dissolve the clot).

  5. Interpretation: Clot formation indicates positive coagulase.

    • Staphylococcus aureus: Coagulase positive.

    • Other Staphylococcus species: Coagulase negative.

Controls:

  • Positive Control: Staphylococcus aureus

  • Negative Control: Staphylococcus epidermidis

Other Tests for Gram-Positive Organisms:

Antibiotic Disk Testing:

  • Used in identifying certain bacteria.

  • Optochin antibiotic disks identify Streptococcus pneumoniae from other alpha-hemolytic Streptococci:

    • S. pneumoniae is optochin susceptible; others are resistant.

Method:

  1. Inoculate a pure culture onto a blood agar plate.

  2. Place an optochin disk on the agar surface.

  3. Incubate at 35°C in 3–5% CO₂.

  4. Examine after 18–24 hours.

Interpretation:

  • Zone of inhibition present = Optochin sensitive (S. pneumoniae).

  • No zone of inhibition = Optochin resistant (other Streptococcus species).

Latex Agglutination Test Kits:

  • Example: Prolex Streptococcal Identification Kit.

  • Purpose: Identifies Lancefield groups:

    • Group A, B, C, D, F, G.

  • Principle: Latex particles coated with antibodies specific to streptococcal antigens agglutinate if the antigen is present.

  • Specimen Required: Pure culture isolate.

EIA Methods (Enzyme Immunoassay):

  • Example: Directigen EZ Group A Strep Test.

  • Purpose: Rapid detection of Group A Streptococcus antigen from throat swabs.

  • Principle: Utilizes antibodies specific to Lancefield Group A antigen.

  • Turnaround Time: Approximately 5 minutes.

  • Clinical Advantage: Rapid diagnosis without culture confirmation in many cases.

Miniaturized Multitest Systems:

  • Example: API STAPH-IDENT.

  • Purpose: Identification of Staphylococcus species.

  • Principle: Features a battery of miniaturized biochemical tests that generate an identification profile.

  • Incubation: 35°C for about 5 hours.

Identification of Gram-Negative Bacteria

General Methodology:

  • Manual biochemical tests are common in smaller laboratories.

  • Serve as backup in larger laboratories using automated systems.

Common Manual Tests:

1. Oxidase Test:

  • Detects presence of cytochrome c oxidase.

  • Interpretation: Purple color change indicates positive; no color change indicates negative.

  • Used to differentiate:

    • Oxidase-negative Enterobacteriaceae.

    • Oxidase-positive non-fermenters (e.g., Pseudomonas aeruginosa).

Controls:

  • Positive Control: Pseudomonas species

  • Negative Control: E. coli

2. Triple Sugar Iron (TSI) Agar:

  • Used to screen Gram-negative enteric pathogens, especially in stool cultures.

  • Composition: Contains glucose, lactose, and sucrose. Phenol red is the pH indicator (uninoculated medium is red).

  • **Interpretation of results:

    • Yellow color change indicates sugar fermentation.

    • Black precipitate indicates H₂S production.

    • Bubbles, cracks indicate gas production.**

How to Inoculate TSI Medium:

  1. Select an isolated colony.

  2. Using an inoculating needle, take half of the colony.

  3. Insert the needle straight down into the TSI medium.

  4. Pull the needle straight up and inoculate the slant using a zig-zag pattern.

TSI Agar Positioning:

  • Glucose = butt of the slant.

  • Sucrose = mid-way up the slant.

  • Lactose = upper part of the slant.

Control Interpretation:

  • Yellow butt/red slant = glucose fermenting organism.

  • Red butt/yellow slant near the top = lactose fermenting organism (rare).

  • Red butt/red slant = non-fermenting organism.

  • Yellow butt/yellow slant = organism ferments all sugars.

  • Black butt/red slant = glucose fermenter producing H₂S.

3. ONPG-Pam-Sulphate Media:

  • Used for screening gram-negative organisms in stool cultures.

  • Tests performed:

    1. Motility: Growth spreads from the stab line; H₂S production can cause blackening.

    2. Beta-galactosidase production: Yellow color indicates positive.

    • Lactose fermenters are beta-galactosidase positive.

    1. Phenylalanine deaminase production: Green color indicates production; some can oxidatively deaminate phenylalanine.

    2. H₂S production: Blackening indicates production.

Miniaturized Multitest Systems (MMS):

  • Quick, easy-to-use, uniform, reliable results, requiring minimal media.

  • These have not made traditional methods obsolete; traditional tube IDs are mandatory for uncertain results and identifying saprophytic organisms.

  • API 20E System: Facilitates 24-hour identification of Enterobacteriaceae and other Gram-negative bacteria.

  • Each strip consists of microtubes with dehydrated substrates for testing enzymatic activity and sugar fermentation.

  • Bacterial suspension added for incubation at 35°C – 37°C.

  • Metabolic end products detected via indicator systems or reagents.

Automation in the Bacteriology Laboratory

Automated Streaking:

  • Isoplater: Automatic Petri dish streaking machine.

    • Loads, streaks, and stacks up to 180 pre-inoculated dishes/hour.

    • Benefits include:

    • Cost savings

    • Increased productivity

    • Improved isolation rates

    • Standardized streaking procedures

    • Enhanced safety through HEPA filtration.

    • Easy to operate and maintain; cost-effective (only consumables are loops).

Automated Organism Identification and Susceptibility Systems:

MicroScan WalkAway System:

  • Combines organism identification (ID) and antimicrobial susceptibility testing (AST) on a single panel.

  • Key Features:

    • Uses microtiter test panels with multiple wells containing:

    • Culture media

    • Biochemical substrates for identification

    • Antibiotics in varying concentrations.

    • Performs direct growth-based susceptibility testing, providing rapid results (approx. 4.5 hours).

    • Susceptibility incubation can extend up to 16 hours for accurate resistance data.

How the System Works:

  1. Organism standardized to a specific concentration.

  2. Suspension inoculated into test panel wells.

  3. Panel incubates with biochemical substrates and antimicrobials.

  4. System measures bacterial growth by observing turbidity changes.

  5. Analyzes reactions using software.

  6. Detects expected biochemical reactions, atypical reactions, and unusual resistance patterns.

VITEK-2:

  • Fully automated system for rapid bacterial identification (ID) and AST.

  • Based on broth microdilution MIC method.

  • MIC (Minimum Inhibitory Concentration): lowest antimicrobial concentration that visibly inhibits organism growth.

  • Continuously monitors growth across well plates.

  • Requires positive control well to fulfill predetermined growth thresholds before interpretation.

  • Employs fluorescence technology for detecting bacterial growth and resistance patterns.

Susceptibility Testing

Overview:

  • Antibiotics have varying efficacy against Gram-positive, Gram-negative, or both (broad-spectrum antibiotics).

Manual Method:

Kirby-Bauer Susceptibility Testing Method:

  • Manual sensitivity testing performed using Mueller-Hinton 11 agar and agar diffusion technique.

  • Standardization Variables:

    • pH: 7.2 to 7.4

    • Media depth: max 4 mm

    • Standardized bacterial inoculum: equivalent to 0.5 McFarland standard

    • Antibiotic concentration on the disc is critical

    • Incubation time: 16-18 hours at 35°C.

Procedure:

  1. Media inoculated with a standardized growth amount.

  2. Antibiotic disks placed on the media.

  3. Antibiotics diffuse onto the media surface.

  4. Incubate at 35°C for 16-18 hours.

  5. Plates examined post-incubation for growth.

  6. Measure zones of inhibition to assess susceptibility.

Inoculation Technique for the Kirby-Bauer Method:

  1. Swab dipped in inoculum; ring it out and swab the entire media.

  2. Place antibiotic disks with sterile tweezers, leaving enough space.

  3. Apply gentle pressure to disks for adherence.

  4. Invert plates (lid down) and incubate.

After Growth Inspection:

  • Plates observed for growth; zone sizes measured for sensitivity assessment.

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time of Flight)

  • Employs mass spectrometry for rapid identification of organisms by analyzing protein profiles.

  • Replaces various traditional biochemical tests, significantly shortening turnaround times (minutes versus 24-48 hours).

Methodology:

  1. Produces charged particles (ions) from analyzed substances.

  2. Mass Analyzer: separates ions based on mass.

  3. Detector: generates a signal from separated ions.

Applications:

  • Rapidly identifies various bacteria and yeast.

  • Provides insights into elemental and isotopic compositions, as well as identifying unknown compounds by mass comparison.

How MALDI-TOF Works:

  • Patient samples are plated and incubated.

  • Technologist examines plates and selects samples.

Identification Process:

  1. Using a toothpick, smear a thin layer on the target plate.

  2. Add matrix (α-cyano-4 hydroxycinnamic acid) and let it dry.

  3. Place the target plate in a vacuum inside the MALDI-TOF system.

  4. A laser releases ionized proteins, sending them through an electric field.

  5. Time of flight measured; larger particles travel slower.

Interpretation:

  • Results obtained include confidence scores (0-3) and color scores (red, yellow, green).

  • A combination of these scores dictates identification accuracy.

Current MALDI-TOF Analyzers in Clinical Use:

  • Vitek- MS® by Biomerieux

  • MALDI BiotyperⓇ by Bruker