The Complete Bacterial Identification Cheatsheet: Methods, Techniques & Interpretations

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

  1. Primary stain: Crystal violet (1 minute)
  2. Mordant: Gram’s iodine (1 minute)
  3. Decolorizer: Alcohol/acetone (10-30 seconds)
  4. Counterstain: Safranin (30 seconds)

Interpretation

ResultCell WallAppearanceExamples
Gram-positiveThick peptidoglycan layerPurple/blueStaphylococcus, Streptococcus, Bacillus
Gram-negativeThin peptidoglycan layer with outer membranePink/redEscherichia, Pseudomonas, Salmonella

Common Morphologies

ShapeArrangementExamples
Cocci (spherical)ClustersStaphylococcus aureus
 ChainsStreptococcus pneumoniae
 Pairs (diplococci)Neisseria gonorrhoeae
 TetradsMicrococcus species
Bacilli (rod-shaped)SingleEscherichia coli
 ChainsBacillus cereus
 PalisadesCorynebacterium species
 FilamentsActinomyces species
SpiralComma-shaped (vibrio)Vibrio cholerae
 SpiralHelicobacter pylori
 Corkscrew (spirochete)Treponema pallidum

Special Stains

StainPurposePositive ResultExamples
Acid-fast (Ziehl-Neelsen)Mycobacteria and related organismsRed bacilli on blue backgroundMycobacterium tuberculosis
EndosporeBacterial endosporesGreen spores, red vegetative cellsBacillus, Clostridium
CapsuleCapsular materialClear halo around bacteriaStreptococcus pneumoniae, Klebsiella
FlagellaBacterial flagellaVisible flagella extending from cellsProteus mirabilis
Metachromatic granulesStorage granulesPurple granules within cellsCorynebacterium diphtheriae

Culture-Based Identification

Common Culture Media

General Purpose Media

MediumUseAppearanceExamples
Blood AgarGrowth of fastidious bacteria, hemolysis patternsRed, opaqueβ-hemolysis (S. pyogenes), α-hemolysis (S. pneumoniae)
Chocolate AgarGrowth of extremely fastidious bacteriaBrown, opaqueHaemophilus influenzae, Neisseria species
MacConkey AgarDifferentiation of lactose fermenters, selection for Gram-negativesPink/purple (lactose +), colorless (lactose -)E. coli (pink), Salmonella (colorless)
Mannitol Salt AgarSelection for staphylococciYellow (mannitol +), red (mannitol -)S. aureus (yellow), S. epidermidis (red)
Nutrient AgarGeneral purpose growthVariesMost non-fastidious bacteria

Selective and Differential Media

MediumSelectivityDifferential PropertyExamples
EMB AgarGram-negative enteric bacteriaLactose fermentationE. coli (metallic sheen), Enterobacter (purple)
XLD AgarSalmonella, ShigellaH₂S production, carbohydrate fermentationSalmonella (black center), Shigella (red)
TCBS AgarVibrio speciesSucrose fermentationV. cholerae (yellow), V. parahaemolyticus (green)
CNA AgarGram-positive cocciHemolysisStreptococci, staphylococci
Hektoen EntericSalmonella, ShigellaH₂S, lactose fermentationSalmonella (green with black center), Shigella (green)
Löwenstein-JensenMycobacteriaGrowthM. 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

PatternAppearanceMechanismExamples
Alpha (α)Greenish discoloration around coloniesPartial hemolysis; reduced hemoglobinStreptococcus pneumoniae, viridans streptococci
Beta (β)Clear zone around coloniesComplete hemolysisStreptococcus pyogenes, Staphylococcus aureus
Gamma (γ)No hemolysisNo hemolytic activityEnterococcus faecalis (some strains)

Biochemical Tests

Primary Biochemical Tests

TestPrinciplePositive ResultNegative ResultKey Organisms
CatalaseDetection of catalase enzyme (H₂O₂ → H₂O + O₂)BubblingNo bubbling(+) Staphylococci, (-) Streptococci
OxidaseDetection of cytochrome c oxidasePurple colorNo color change(+) Pseudomonas, Neisseria, (-) Enterobacteriaceae
CoagulaseConverts fibrinogen to fibrinClot formationNo clotting(+) S. aureus, (-) S. epidermidis
IndoleTryptophan to indole conversionRed ring after Kovac’s reagentNo red ring(+) E. coli, (-) Klebsiella
CitrateUtilization of citrate as carbon sourceBlue color (Simmons citrate)No color change (green)(+) Klebsiella, (-) E. coli
UreaseHydrolysis of urea to ammoniaPink-red colorNo color change (yellow)(+) Proteus, Helicobacter, (-) E. coli
H₂S ProductionHydrogen sulfide productionBlack precipitateNo black precipitate(+) Salmonella, (-) E. coli
MotilityBacterial movementDiffuse growth away from stab lineGrowth only along stab line(+) E. coli, (-) Klebsiella

Carbohydrate Fermentation

CarbohydrateKey DifferentiationExamples
GlucoseBasic energy metabolismMost bacteria ferment glucose
LactoseEnterobacteriaceae differentiation(+) E. coli, (-) Salmonella
SucroseSpecies differentiation(+) Klebsiella, (-) E. coli (variable)
MannitolStaphylococcal differentiation(+) S. aureus, (-) S. epidermidis
SorbitolE. coli O157:H7 screening(+) Most E. coli, (-) E. coli O157:H7

Commercial Biochemical Systems

SystemFormatApplicationsExamples
API 20E20 microtubules with dehydrated substratesEnterobacteriaceae and other Gram-negative rodsIdentification of clinical isolates from urine, wound
VITEKAutomated cards with multiple wellsBroad range of bacteriaHigh-volume clinical laboratory testing
BD PhoenixAutomated panelsIdentification and susceptibility testingHospital clinical microbiology
MALDI BiotyperMass spectrometryRapid identificationClinical and reference laboratories
MicroscanManual or automated panelsID and susceptibility testingClinical laboratories

Metabolic Characteristics of Key Bacteria

OrganismCatalaseOxidaseCoagulaseIndoleUreaseCitrateMotilityH₂S
Staphylococcus aureus++
Streptococcus pyogenes
Escherichia coli+++
Klebsiella pneumoniae+++
Proteus mirabilis+++/-++
Pseudomonas aeruginosa++++
Salmonella enterica++++
Enterococcus faecalis

Molecular Identification Methods

PCR-Based Methods

MethodPrincipleApplicationsAdvantagesLimitations
Species-Specific PCRAmplification of species-specific genesRapid identificationFast, highly specificLimited to targeted species
16S rRNA PCRAmplification and sequencing of 16S rRNA geneTaxonomic classificationUniversal bacterial markerLimited resolution for closely related species
Multiplex PCRMultiple primer sets in single reactionSimultaneous detection of multiple targetsTime and cost effectiveComplex optimization
Real-Time PCRFluorescent detection during amplificationQuantification and identificationRapid, quantitativeEquipment cost
RAPDRandom amplification of polymorphic DNAStrain typingSimple, no sequence knowledge neededPoor reproducibility
MLSTSequencing multiple housekeeping genesStrain typing, evolutionary studiesHighly discriminatory, standardizedLabor intensive, expensive

Next-Generation Sequencing

ApproachDescriptionApplicationsConsiderations
Whole Genome SequencingComplete bacterial genome sequencingDetailed genetic analysis, outbreak trackingData analysis complexity, cost
16S Metagenomic SequencingCommunity analysis based on 16S rRNA geneMicrobiome studies, environmental samplesTaxonomic resolution limitations
Shotgun MetagenomicsRandom sequencing of all DNA in sampleFunctional and taxonomic profilingHigher cost, complex analysis
RNA-SeqSequencing of expressed RNAGene expression studiesRNA 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

MethodApplicationsExamples
Slide AgglutinationBacterial typingSalmonella serotyping
Latex AgglutinationRapid identificationS. aureus, Streptococcus grouping
CAMP TestStreptococcal identificationGroup B Streptococcus
Quellung ReactionEncapsulated bacteriaPneumococcal typing
Widal TestEnteric fever diagnosisSalmonella 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

  1. Pick isolated colony
  2. Apply to target plate
  3. Overlay with matrix solution (e.g., α-cyano-4-hydroxycinnamic acid)
  4. Allow to dry
  5. Analyze in mass spectrometer
  6. 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 LevelTypical Accuracy
Genus>95%
Species (common)>90%
Species (rare)Variable
Strain typingLimited

Automated Systems

Comparison of Major Commercial Systems

SystemTechnologyTurnaround TimeAdvantagesLimitations
VITEKColorimetric reactions4-18 hoursHigh throughput, integrated ASTCost, limited to cultured isolates
BD PhoenixColorimetric and fluorometricA6-16 hoursAccurate, integrated ASTCost, maintenance requirements
MicroScanColorimetric broth microdilution16-48 hoursAccurate for rare organisms, ASTLonger time to result
MALDI-TOF MSProtein fingerprintingMinutesVery rapid, low reagent costHigh initial investment
BioFire FilmArrayMultiplex PCR1 hourDirect from sample, rapidLimited 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

MethodFormatReadingApplications
Disk DiffusionPaper disks on agarZone diameter measurementRoutine testing
E-testGradient stripMIC at intersectionMIC determination
Broth MicrodilutionMicrowell plateVisual or automated turbidityMIC determination, automated systems
Agar DilutionSeries of antibiotic-containing platesGrowth/no growthReference method
Automated SystemsVariousInstrument-dependentRoutine 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

MechanismExamplesDetection Methods
β-lactamase productionESBL, AmpC, carbapenemasesPhenotypic tests (e.g., clavulanate synergy)
Modified target siteMRSA (mecA gene), VRECefoxitin screening, molecular detection
Efflux pumpsTetracycline resistancePhenotypic testing
Altered permeabilityCarbapenem resistance in P. aeruginosaCombined phenotypic methods
Enzymatic modificationAminoglycoside resistanceSusceptibility testing

Special Detection Tests

TestDetectsInterpretation
MRSA screeningMethicillin-resistant S. aureusGrowth on selective media
D-testInducible clindamycin resistanceD-shaped zone of inhibition
Modified Hodge TestCarbapenemase productionClover-leaf shape at inhibition zone
Carba NPCarbapenemase productionColor change (yellow to orange/red)
Cefoxitin screeningmecA-mediated resistanceResistance 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

OrganismKey FeaturesSelective MediaConfirmatory Tests
Staphylococcus aureusGram-positive cocci in clusters, β-hemolyticMannitol salt agarCoagulase, PBP2a (MRSA)
Streptococcus pyogenes (Group A)Gram-positive cocci in chains, β-hemolyticBlood agarPYR, bacitracin sensitivity
Streptococcus pneumoniaeGram-positive diplococci, α-hemolyticBlood agarOptochin sensitivity, bile solubility
Enterococcus speciesGram-positive cocci in pairs/chainsBile esculin agarPYR, growth in 6.5% NaCl
Escherichia coliGram-negative rod, lactose fermenterMacConkey, EMBIndole, lactose fermentation
Klebsiella pneumoniaeGram-negative encapsulated rodMacConkeyMucoid colonies, citrate, urease
Pseudomonas aeruginosaGram-negative rod, oxidase positiveCetrimide agarOxidase, pyocyanin, growth at 42°C
Salmonella speciesGram-negative rod, non-lactose fermenterXLD, HektoenH₂S, TSI pattern, serotyping
Neisseria meningitidisGram-negative diplococciChocolate, Modified Thayer-MartinOxidase, carbohydrate fermentation
Clostridium difficileGram-positive spore-forming rodCCFAGDH, toxin detection, NAAT
Mycobacterium tuberculosisAcid-fast bacilliLöwenstein-JensenAcid-fast stain, molecular methods

Bioterrorism Agents

OrganismKey FeaturesSafety ConsiderationsConfirmatory Testing
Bacillus anthracisLarge Gram-positive rod, non-hemolyticBSL-3 practicesCapsule, motility (non-motile), susceptibility to gamma phage
Yersinia pestisGram-negative coccobacillus, bipolar stainingBSL-3 practicesF1 antigen, biochemical tests, PCR
Francisella tularensisTiny Gram-negative coccobacillus, poor stainingBSL-3 practicesGrowth on BCYE/chocolate, PCR
Brucella speciesSmall Gram-negative coccobacilliBSL-3 practicesUrease, oxidase, serology, PCR

Troubleshooting and Quality Control

Common Identification Problems

ProblemPossible CausesSolutions
No growth on mediaFastidious organism, incorrect conditionsUse enriched media, adjust atmosphere, check temperature
Mixed cultureSpecimen contamination, incomplete isolationRe-isolate, use selective media
Ambiguous biochemical resultsUncommon strain, weakly reactiveExtended incubation, molecular methods
Discrepant results between methodsMethod limitations, database issuesAdditional confirmatory tests, reference laboratory
Unusual resistance patternNew resistance mechanism, contaminationRepeat 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

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)
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