Introduction: The Importance of Bacterial Identification
Accurate bacterial identification is foundational to microbiology, infectious disease diagnosis, research, and public health monitoring. This cheatsheet provides a comprehensive reference for laboratory techniques used to identify and characterize bacteria, from traditional culture-based methods to cutting-edge molecular approaches. Understanding these methods allows for precise diagnosis, appropriate treatment selection, effective infection control, and insights into bacterial ecology and evolution.
Gram Staining and Morphology
Gram Stain Procedure
- Primary stain: Crystal violet (1 minute)
- Mordant: Gram’s iodine (1 minute)
- Decolorizer: Alcohol/acetone (10-30 seconds)
- Counterstain: Safranin (30 seconds)
Interpretation
Result | Cell Wall | Appearance | Examples |
---|---|---|---|
Gram-positive | Thick peptidoglycan layer | Purple/blue | Staphylococcus, Streptococcus, Bacillus |
Gram-negative | Thin peptidoglycan layer with outer membrane | Pink/red | Escherichia, Pseudomonas, Salmonella |
Common Morphologies
Shape | Arrangement | Examples |
---|---|---|
Cocci (spherical) | Clusters | Staphylococcus aureus |
Chains | Streptococcus pneumoniae | |
Pairs (diplococci) | Neisseria gonorrhoeae | |
Tetrads | Micrococcus species | |
Bacilli (rod-shaped) | Single | Escherichia coli |
Chains | Bacillus cereus | |
Palisades | Corynebacterium species | |
Filaments | Actinomyces species | |
Spiral | Comma-shaped (vibrio) | Vibrio cholerae |
Spiral | Helicobacter pylori | |
Corkscrew (spirochete) | Treponema pallidum |
Special Stains
Stain | Purpose | Positive Result | Examples |
---|---|---|---|
Acid-fast (Ziehl-Neelsen) | Mycobacteria and related organisms | Red bacilli on blue background | Mycobacterium tuberculosis |
Endospore | Bacterial endospores | Green spores, red vegetative cells | Bacillus, Clostridium |
Capsule | Capsular material | Clear halo around bacteria | Streptococcus pneumoniae, Klebsiella |
Flagella | Bacterial flagella | Visible flagella extending from cells | Proteus mirabilis |
Metachromatic granules | Storage granules | Purple granules within cells | Corynebacterium diphtheriae |
Culture-Based Identification
Common Culture Media
General Purpose Media
Medium | Use | Appearance | Examples |
---|---|---|---|
Blood Agar | Growth of fastidious bacteria, hemolysis patterns | Red, opaque | β-hemolysis (S. pyogenes), α-hemolysis (S. pneumoniae) |
Chocolate Agar | Growth of extremely fastidious bacteria | Brown, opaque | Haemophilus influenzae, Neisseria species |
MacConkey Agar | Differentiation of lactose fermenters, selection for Gram-negatives | Pink/purple (lactose +), colorless (lactose -) | E. coli (pink), Salmonella (colorless) |
Mannitol Salt Agar | Selection for staphylococci | Yellow (mannitol +), red (mannitol -) | S. aureus (yellow), S. epidermidis (red) |
Nutrient Agar | General purpose growth | Varies | Most non-fastidious bacteria |
Selective and Differential Media
Medium | Selectivity | Differential Property | Examples |
---|---|---|---|
EMB Agar | Gram-negative enteric bacteria | Lactose fermentation | E. coli (metallic sheen), Enterobacter (purple) |
XLD Agar | Salmonella, Shigella | H₂S production, carbohydrate fermentation | Salmonella (black center), Shigella (red) |
TCBS Agar | Vibrio species | Sucrose fermentation | V. cholerae (yellow), V. parahaemolyticus (green) |
CNA Agar | Gram-positive cocci | Hemolysis | Streptococci, staphylococci |
Hektoen Enteric | Salmonella, Shigella | H₂S, lactose fermentation | Salmonella (green with black center), Shigella (green) |
Löwenstein-Jensen | Mycobacteria | Growth | M. tuberculosis (rough, buff colonies) |
Colonial Morphology Characteristics
- Size: Pinpoint, small, moderate, large
- Shape: Circular, irregular, filamentous, rhizoid
- Margin: Entire, undulate, lobate, filamentous
- Elevation: Flat, raised, convex, umbonate
- Surface: Smooth, rough, mucoid, dry
- Consistency: Butyrous (butter-like), viscous, brittle
- Opacity: Transparent, translucent, opaque
- Pigmentation: Colored (describe) or non-pigmented
- Hemolysis: Alpha (α), beta (β), gamma (γ)
Hemolysis Patterns
Pattern | Appearance | Mechanism | Examples |
---|---|---|---|
Alpha (α) | Greenish discoloration around colonies | Partial hemolysis; reduced hemoglobin | Streptococcus pneumoniae, viridans streptococci |
Beta (β) | Clear zone around colonies | Complete hemolysis | Streptococcus pyogenes, Staphylococcus aureus |
Gamma (γ) | No hemolysis | No hemolytic activity | Enterococcus faecalis (some strains) |
Biochemical Tests
Primary Biochemical Tests
Test | Principle | Positive Result | Negative Result | Key Organisms |
---|---|---|---|---|
Catalase | Detection of catalase enzyme (H₂O₂ → H₂O + O₂) | Bubbling | No bubbling | (+) Staphylococci, (-) Streptococci |
Oxidase | Detection of cytochrome c oxidase | Purple color | No color change | (+) Pseudomonas, Neisseria, (-) Enterobacteriaceae |
Coagulase | Converts fibrinogen to fibrin | Clot formation | No clotting | (+) S. aureus, (-) S. epidermidis |
Indole | Tryptophan to indole conversion | Red ring after Kovac’s reagent | No red ring | (+) E. coli, (-) Klebsiella |
Citrate | Utilization of citrate as carbon source | Blue color (Simmons citrate) | No color change (green) | (+) Klebsiella, (-) E. coli |
Urease | Hydrolysis of urea to ammonia | Pink-red color | No color change (yellow) | (+) Proteus, Helicobacter, (-) E. coli |
H₂S Production | Hydrogen sulfide production | Black precipitate | No black precipitate | (+) Salmonella, (-) E. coli |
Motility | Bacterial movement | Diffuse growth away from stab line | Growth only along stab line | (+) E. coli, (-) Klebsiella |
Carbohydrate Fermentation
Carbohydrate | Key Differentiation | Examples |
---|---|---|
Glucose | Basic energy metabolism | Most bacteria ferment glucose |
Lactose | Enterobacteriaceae differentiation | (+) E. coli, (-) Salmonella |
Sucrose | Species differentiation | (+) Klebsiella, (-) E. coli (variable) |
Mannitol | Staphylococcal differentiation | (+) S. aureus, (-) S. epidermidis |
Sorbitol | E. coli O157:H7 screening | (+) Most E. coli, (-) E. coli O157:H7 |
Commercial Biochemical Systems
System | Format | Applications | Examples |
---|---|---|---|
API 20E | 20 microtubules with dehydrated substrates | Enterobacteriaceae and other Gram-negative rods | Identification of clinical isolates from urine, wound |
VITEK | Automated cards with multiple wells | Broad range of bacteria | High-volume clinical laboratory testing |
BD Phoenix | Automated panels | Identification and susceptibility testing | Hospital clinical microbiology |
MALDI Biotyper | Mass spectrometry | Rapid identification | Clinical and reference laboratories |
Microscan | Manual or automated panels | ID and susceptibility testing | Clinical laboratories |
Metabolic Characteristics of Key Bacteria
Organism | Catalase | Oxidase | Coagulase | Indole | Urease | Citrate | Motility | H₂S |
---|---|---|---|---|---|---|---|---|
Staphylococcus aureus | + | – | + | – | – | – | – | – |
Streptococcus pyogenes | – | – | – | – | – | – | – | – |
Escherichia coli | + | – | – | + | – | – | + | – |
Klebsiella pneumoniae | + | – | – | – | + | + | – | – |
Proteus mirabilis | + | – | – | – | + | +/- | + | + |
Pseudomonas aeruginosa | + | + | – | – | – | + | + | – |
Salmonella enterica | + | – | – | – | – | + | + | + |
Enterococcus faecalis | – | – | – | – | – | – | – | – |
Molecular Identification Methods
PCR-Based Methods
Method | Principle | Applications | Advantages | Limitations |
---|---|---|---|---|
Species-Specific PCR | Amplification of species-specific genes | Rapid identification | Fast, highly specific | Limited to targeted species |
16S rRNA PCR | Amplification and sequencing of 16S rRNA gene | Taxonomic classification | Universal bacterial marker | Limited resolution for closely related species |
Multiplex PCR | Multiple primer sets in single reaction | Simultaneous detection of multiple targets | Time and cost effective | Complex optimization |
Real-Time PCR | Fluorescent detection during amplification | Quantification and identification | Rapid, quantitative | Equipment cost |
RAPD | Random amplification of polymorphic DNA | Strain typing | Simple, no sequence knowledge needed | Poor reproducibility |
MLST | Sequencing multiple housekeeping genes | Strain typing, evolutionary studies | Highly discriminatory, standardized | Labor intensive, expensive |
Next-Generation Sequencing
Approach | Description | Applications | Considerations |
---|---|---|---|
Whole Genome Sequencing | Complete bacterial genome sequencing | Detailed genetic analysis, outbreak tracking | Data analysis complexity, cost |
16S Metagenomic Sequencing | Community analysis based on 16S rRNA gene | Microbiome studies, environmental samples | Taxonomic resolution limitations |
Shotgun Metagenomics | Random sequencing of all DNA in sample | Functional and taxonomic profiling | Higher cost, complex analysis |
RNA-Seq | Sequencing of expressed RNA | Gene expression studies | RNA instability, complexity |
Interpretation of Molecular Data
16S rRNA Sequence Analysis:
99% identity: Same species
- 97-99% identity: Same genus
- 95-97% identity: Same family
- <95% identity: Different family or higher taxa
Whole Genome Comparison:
- Average Nucleotide Identity (ANI) >95%: Same species
- Core genome SNP analysis for outbreak investigation
- Accessory genome analysis for strain characterization
Serological Methods
Principle and Applications
- Agglutination: Antibody-mediated clumping of cells or particles
- ELISA: Enzyme-linked detection of specific antibodies or antigens
- Lateral Flow: Rapid chromatographic immunoassays
- Immunofluorescence: Fluorescent antibody-based visualization
Common Serological Tests
Method | Applications | Examples |
---|---|---|
Slide Agglutination | Bacterial typing | Salmonella serotyping |
Latex Agglutination | Rapid identification | S. aureus, Streptococcus grouping |
CAMP Test | Streptococcal identification | Group B Streptococcus |
Quellung Reaction | Encapsulated bacteria | Pneumococcal typing |
Widal Test | Enteric fever diagnosis | Salmonella Typhi |
MALDI-TOF Mass Spectrometry
Principle
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry creates spectra of microbial proteins, primarily ribosomal proteins, that serve as a “fingerprint” for bacterial identification.
Procedure
- Pick isolated colony
- Apply to target plate
- Overlay with matrix solution (e.g., α-cyano-4-hydroxycinnamic acid)
- Allow to dry
- Analyze in mass spectrometer
- Compare spectrum to database for identification
Advantages and Limitations
Advantages:
- Rapid identification (minutes)
- Low cost per test after initial investment
- High accuracy for common species
- Minimal sample preparation
Limitations:
- High instrument cost
- Database limitations for rare organisms
- Difficulty with mixed cultures
- Limited strain typing capabilities
Performance Characteristics
Taxonomic Level | Typical Accuracy |
---|---|
Genus | >95% |
Species (common) | >90% |
Species (rare) | Variable |
Strain typing | Limited |
Automated Systems
Comparison of Major Commercial Systems
System | Technology | Turnaround Time | Advantages | Limitations |
---|---|---|---|---|
VITEK | Colorimetric reactions | 4-18 hours | High throughput, integrated AST | Cost, limited to cultured isolates |
BD Phoenix | Colorimetric and fluorometric | A6-16 hours | Accurate, integrated AST | Cost, maintenance requirements |
MicroScan | Colorimetric broth microdilution | 16-48 hours | Accurate for rare organisms, AST | Longer time to result |
MALDI-TOF MS | Protein fingerprinting | Minutes | Very rapid, low reagent cost | High initial investment |
BioFire FilmArray | Multiplex PCR | 1 hour | Direct from sample, rapid | Limited range of targets, cost per test |
Interpretation Considerations
- Confidence scores and probability
- Need for supplemental testing
- Quality control procedures
- Database updates and maintenance
Antimicrobial Susceptibility Testing
Methods
Method | Format | Reading | Applications |
---|---|---|---|
Disk Diffusion | Paper disks on agar | Zone diameter measurement | Routine testing |
E-test | Gradient strip | MIC at intersection | MIC determination |
Broth Microdilution | Microwell plate | Visual or automated turbidity | MIC determination, automated systems |
Agar Dilution | Series of antibiotic-containing plates | Growth/no growth | Reference method |
Automated Systems | Various | Instrument-dependent | Routine high-volume testing |
Interpretation Standards
- CLSI (Clinical & Laboratory Standards Institute): North American standards
- EUCAST (European Committee on Antimicrobial Susceptibility Testing): European standards
- Categories: Susceptible (S), Intermediate (I), Resistant (R)
Key Resistance Mechanisms
Mechanism | Examples | Detection Methods |
---|---|---|
β-lactamase production | ESBL, AmpC, carbapenemases | Phenotypic tests (e.g., clavulanate synergy) |
Modified target site | MRSA (mecA gene), VRE | Cefoxitin screening, molecular detection |
Efflux pumps | Tetracycline resistance | Phenotypic testing |
Altered permeability | Carbapenem resistance in P. aeruginosa | Combined phenotypic methods |
Enzymatic modification | Aminoglycoside resistance | Susceptibility testing |
Special Detection Tests
Test | Detects | Interpretation |
---|---|---|
MRSA screening | Methicillin-resistant S. aureus | Growth on selective media |
D-test | Inducible clindamycin resistance | D-shaped zone of inhibition |
Modified Hodge Test | Carbapenemase production | Clover-leaf shape at inhibition zone |
Carba NP | Carbapenemase production | Color change (yellow to orange/red) |
Cefoxitin screening | mecA-mediated resistance | Resistance to cefoxitin disc |
Identification Flowcharts for Common Bacteria
Gram-Positive Cocci
Gram-positive cocci
├── Catalase-positive
│ ├── Coagulase-positive → Staphylococcus aureus
│ └── Coagulase-negative
│ ├── Novobiocin-resistant → Staphylococcus saprophyticus
│ └── Novobiocin-sensitive → Staphylococcus epidermidis
└── Catalase-negative
├── PYR-positive
│ ├── LAP-positive → Streptococcus pyogenes (Group A)
│ └── LAP-negative → Enterococcus spp.
└── PYR-negative
├── Bile esculin-positive → Other Enterococcus or Group D Streptococcus
└── Bile esculin-negative
├── Optochin-sensitive → Streptococcus pneumoniae
└── Optochin-resistant → Other streptococci (identify by hemolysis and grouping)
Gram-Negative Rods (Enterobacteriaceae)
Gram-negative rods (oxidase-negative)
├── Lactose-fermenting
│ ├── Indole-positive
│ │ ├── Motile → Escherichia coli
│ │ └── Non-motile → Klebsiella oxytoca
│ └── Indole-negative
│ ├── Citrate-positive, Urease-positive → Klebsiella pneumoniae
│ └── VP-positive, Urease-negative → Enterobacter spp.
└── Non-lactose fermenting
├── H₂S-positive
│ ├── Urease-positive → Proteus spp.
│ └── Urease-negative → Salmonella spp.
└── H₂S-negative
├── Urease-positive → Morganella or Providencia
└── Urease-negative
├── Indole-positive → Shigella spp.
└── Indole-negative → Serratia spp.
Non-Fermenting Gram-Negative Bacteria
Gram-negative rods (oxidase-positive)
├── Motile
│ ├── Pigment production
│ │ ├── Blue-green pigment → Pseudomonas aeruginosa
│ │ └── Yellow-green fluorescent pigment → Other Pseudomonas spp.
│ └── No pigment
│ ├── Growth at 42°C → Pseudomonas aeruginosa
│ └── No growth at 42°C → Other Pseudomonas spp.
└── Non-motile
├── Growth on MacConkey → Acinetobacter spp.
└── Poor growth on MacConkey
├── Resistant to polymyxin → Stenotrophomonas maltophilia
└── Sensitive to polymyxin → Burkholderia cepacia complex
Critical Pathogens and Their Key Identification Features
High-Priority Pathogens
Organism | Key Features | Selective Media | Confirmatory Tests |
---|---|---|---|
Staphylococcus aureus | Gram-positive cocci in clusters, β-hemolytic | Mannitol salt agar | Coagulase, PBP2a (MRSA) |
Streptococcus pyogenes (Group A) | Gram-positive cocci in chains, β-hemolytic | Blood agar | PYR, bacitracin sensitivity |
Streptococcus pneumoniae | Gram-positive diplococci, α-hemolytic | Blood agar | Optochin sensitivity, bile solubility |
Enterococcus species | Gram-positive cocci in pairs/chains | Bile esculin agar | PYR, growth in 6.5% NaCl |
Escherichia coli | Gram-negative rod, lactose fermenter | MacConkey, EMB | Indole, lactose fermentation |
Klebsiella pneumoniae | Gram-negative encapsulated rod | MacConkey | Mucoid colonies, citrate, urease |
Pseudomonas aeruginosa | Gram-negative rod, oxidase positive | Cetrimide agar | Oxidase, pyocyanin, growth at 42°C |
Salmonella species | Gram-negative rod, non-lactose fermenter | XLD, Hektoen | H₂S, TSI pattern, serotyping |
Neisseria meningitidis | Gram-negative diplococci | Chocolate, Modified Thayer-Martin | Oxidase, carbohydrate fermentation |
Clostridium difficile | Gram-positive spore-forming rod | CCFA | GDH, toxin detection, NAAT |
Mycobacterium tuberculosis | Acid-fast bacilli | Löwenstein-Jensen | Acid-fast stain, molecular methods |
Bioterrorism Agents
Organism | Key Features | Safety Considerations | Confirmatory Testing |
---|---|---|---|
Bacillus anthracis | Large Gram-positive rod, non-hemolytic | BSL-3 practices | Capsule, motility (non-motile), susceptibility to gamma phage |
Yersinia pestis | Gram-negative coccobacillus, bipolar staining | BSL-3 practices | F1 antigen, biochemical tests, PCR |
Francisella tularensis | Tiny Gram-negative coccobacillus, poor staining | BSL-3 practices | Growth on BCYE/chocolate, PCR |
Brucella species | Small Gram-negative coccobacilli | BSL-3 practices | Urease, oxidase, serology, PCR |
Troubleshooting and Quality Control
Common Identification Problems
Problem | Possible Causes | Solutions |
---|---|---|
No growth on media | Fastidious organism, incorrect conditions | Use enriched media, adjust atmosphere, check temperature |
Mixed culture | Specimen contamination, incomplete isolation | Re-isolate, use selective media |
Ambiguous biochemical results | Uncommon strain, weakly reactive | Extended incubation, molecular methods |
Discrepant results between methods | Method limitations, database issues | Additional confirmatory tests, reference laboratory |
Unusual resistance pattern | New resistance mechanism, contamination | Repeat testing, molecular confirmation |
Quality Control Recommendations
- Frequency: Daily or with each batch of tests
- Control Organisms: ATCC reference strains appropriate for each test
- Common QC Strains:
- E. coli ATCC 25922
- S. aureus ATCC 25923
- P. aeruginosa ATCC 27853
- E. faecalis ATCC 29212
- S. pneumoniae ATCC 49619
Documentation Requirements
- Control results
- Lot numbers and expiration dates
- Environmental conditions
- Technician identification
- Corrective actions for failed QC
- Instrument maintenance records
Resources for Further Learning
Reference Texts
- “Manual of Clinical Microbiology” (ASM Press)
- “Bailey & Scott’s Diagnostic Microbiology”
- “Color Atlas and Textbook of Diagnostic Microbiology” (Koneman)
- “Bergey’s Manual of Systematic Bacteriology”
Online Resources
- American Society for Microbiology (ASM): www.asm.org
- Centers for Disease Control and Prevention (CDC): www.cdc.gov
- Public Health England UK Standards for Microbiology: www.gov.uk/government/collections/standards-for-microbiology-investigations-smi
Professional Organizations
- American Society for Microbiology (ASM)
- Association of Public Health Laboratories (APHL)
- Clinical and Laboratory Standards Institute (CLSI)
- European Society of Clinical Microbiology and Infectious Diseases (ESCMID)