For: 3rd-year Medical Technology students
Scope: Condensed strictly from the provided lesson materials.
Macroscopic examination: gross appearance and adequacy.
Note: swab/aspirate, stool consistency, blood or mucus, volume, and fluid clarity.
Microscopic examination: specimen quality, cellular response, presumptive organism clues, and need for additional testing.
Example: compare saliva and sputum by quantitating WBCs and epithelial cells.
Culture workup: correlate microscopic findings with colony growth and biochemical tests.
Shape | Description | Examples/arrangements |
|---|---|---|
Coccus | Spherical | Diplococci, tetrads, streptococci, staphylococci, sarcinae |
Bacillus | Rod-shaped | Single rods, diplobacilli, streptobacilli, palisades |
Coccobacillus | Short, almost spherical rod | Haemophilus influenzae |
Vibrio | Comma-shaped | Vibrio cholerae |
Spirillum | Rigid spiral | Spirillum minus |
Spirochete | Flexible spiral with axial filaments | Treponema pallidum |
Other | Pleomorphic or filamentous | Variable shape |
Arrangement mnemonics:
Staph = clusters like grapes.
Strep = chains.
Diplo = pairs.
Tetrad = four; sarcina = eight.
Purpose: differentiates bacteria based mainly on cell-wall structure.
Step | Reagent | Gram-positive | Gram-negative |
|---|---|---|---|
1 | Crystal violet | Purple | Purple |
2 | Gram iodine | Purple | Purple |
3 | Alcohol/acetone | Remains purple | Becomes colorless |
4 | Safranin | Purple | Pink |
Cell-wall comparison
Gram-positive: thick peptidoglycan, lipoteichoic acid, no outer membrane/LPS.
Gram-negative: thin peptidoglycan, periplasmic space, outer membrane, LPS; lipid A contributes to endotoxin.
Procedure essentials: fix smear, crystal violet, iodine, decolorize carefully, counterstain, examine with oil immersion at 1000×.
General rules from the lesson
Most cocci are Gram-positive except Neisseria, Veillonella, and Moraxella.
Most bacilli are Gram-negative; important Gram-positive exceptions listed are Mycobacteria, Corynebacteria, Clostridia, Bacillus, Erysipelothrix, Lactobacillus, Listeria, Nocardia, and Actinomyces.
Stain | Main use/result |
|---|---|
Acid-fast stain | Lipid/wax-rich cell walls; positive = red, negative = blue |
Acridine orange | Gram-positive and Gram-negative bacteria, living or dead |
Calcofluor white | Fungal cultures; bright apple-green/blue-white fluorescence |
Methylene blue | Metachromatic granules |
Lactophenol cotton blue | Medically important fungi in slide culture |
India ink | Negative stain for yeast capsule; dark background with clear halo |
Endospore stain | Malachite green stains endospores; safranin counterstains cells pink/red |
Acid-fast methods: Ziehl-Neelsen, Kinyoun, and auramine-rhodamine. Acid-fast positive organisms retain carbol fuchsin or fluoresce bright orange/yellow, depending on method.
Ocular lens: 10×.
Scanner: 4× objective = 40× total.
LPO: 10× objective = 100× total.
HPO: 40× objective = 400× total.
Oil immersion: 100× objective = 1000× total.
Brightfield: primary routine microscope.
Darkfield: primarily for spirochetes.
Electron microscopy: specialized; transmission EM for ultrastructure, scanning EM for surface form.
Fluorescence microscopy: uses fluorochromes such as acridine orange, auramine-rhodamine, and calcofluor white.
Assess after approximately 18–24 hours when appropriate:
Size: large, medium, small, pinpoint.
Form: punctiform, round, filamentous, irregular.
Margin: entire, curled, wavy, lobate, filamentous.
Elevation: flat, raised, convex/dome, umbilicate, umbonate.
Surface: smooth, concentric, wrinkled, contoured.
Density: transparent, translucent, opaque.
Color/pigment: white, gray, yellow, buff; examples include green metallic Pseudomonas aeruginosa, brick-red Serratia marcescens, purple Chromobacterium violaceum, and brown-black Prevotella melaninogenica.
Consistency: creamy (S. aureus), sticky (Neisseria), brittle (Nocardia), dry (Streptococci), dry/waxy (diphtheroids).
Odor: use only approved laboratory procedures; examples in the lesson include old-sock odor (S. aureus) and fruity/grape odor (P. aeruginosa).
Swarming: hazy blanket of growth, classically Proteus.
Hemolysis on blood agar
Type | Appearance | Lesson examples |
|---|---|---|
α | Incomplete; greenish discoloration | S. pneumoniae |
β | Complete clearing | S. pyogenes, S. agalactiae, Listeria monocytogenes |
γ | No hemolysis | No effect |
α′ | Inner α zone and outer β zone | Pattern described in lesson |
Terms:
Culture: growth of microorganisms in a medium.
Inoculate/plant/cultivate: introduce specimen or organism into medium.
Transplant/subculture: transfer growth to fresh medium.
Classifications
Physical state: liquid, semisolid, solid. Solid media may be liquefiable or non-liquefiable.
Composition: synthetic/chemically defined, complex/non-synthetic, tissue culture.
Distribution: plated or tubed.
Function/use: supportive, enriched, enrichment broth, selective, differential, transport, susceptibility, biochemical.
Medium | Main use/principle | Key result |
|---|---|---|
Blood agar | Enriched isolation; observe hemolysis | α, β, or γ patterns |
Chocolate agar | Fastidious and non-fastidious organisms | Enriched medium |
MacConkey agar | Selective/differential for Gram-negative enteric bacilli | Lactose fermenter = pink/red; non-fermenter = colorless |
EMB | Selective/differential for Gram-negative enteric bacilli | E. coli = green metallic sheen; other coliforms = purple |
Hektoen enteric agar | Enteric Gram-negative bacilli | Salmonella = green with black center; Shigella = green; coliforms = yellow-orange |
Salmonella-Shigella agar | Selective for Salmonella/Shigella | Salmonella = colorless with black center; Shigella = colorless |
Mannitol salt agar | Selective for staphylococci; 7.5% NaCl | S. aureus ferments mannitol, yellow halo; many CoNS remain pink/red |
Thayer-Martin | Selective for Neisseria gonorrhoeae | Vancomycin inhibits Gram-positive; colistin inhibits Gram-negative competitors; nystatin inhibits yeast |
Modified TM | TM plus trimethoprim; suppresses Proteus swarming | Selective for pathogenic Neisseria |
Martin-Lewis | Selective Neisseria medium | Uses vancomycin, colistin, trimethoprim lactate, anisomycin |
New York City agar | Selective Neisseria medium | Uses vancomycin, colistin, trimethoprim lactate, amphotericin B |
Lowenstein-Jensen | MTB medium | Malachite green inhibitor |
Mueller tellurite | Corynebacterium diphtheriae | Potassium tellurite inhibitor |
Stuart/Amies/Cary-Blair/Transgrow | Transport media | Preserve specimen during transport |
Mueller-Hinton agar | AST for most bacteria | Standard disk diffusion medium |
HTM or MH with chocolate base | AST for Haemophilus spp. | Supports fastidious organism |
Middlebrook 7H10/7H11 | AST for mycobacteria | Specialized medium |
Primary plating: choose media based on specimen and likely pathogens; use nonselective, enriched, selective, or differential media as appropriate.
Quadrant streak: sterilize and cool loop between sectors to dilute inoculum and obtain isolated colonies.
Semiquantitative streak:
1+ = growth in first quadrant.
2+ = growth through second quadrant.
3+ = growth through third quadrant.
4+ = growth through fourth quadrant/heavy growth.
Quantitative culture: calibrated loops, commonly 0.01 or 0.001 mL, streaked down the center.
Specimen preparation: direct inoculation for pus, urine, sputum, and sterile body fluids; concentrate large fluid volumes by centrifugation or filtration when appropriate; send one swab for smear and one for culture; homogenize tissue before culture.
Incubation: commonly 35–37°C for bacteria; 28–30°C for fungi. Many cultures are held 48–72 hours; some require 5–7 days.
Definitions
Antimicrobial: chemical compound that kills or suppresses microorganisms.
Antibiotic: natural, semisynthetic, or synthetic antimicrobial molecule.
Bacteriostatic: inhibits growth.
Bactericidal: kills bacteria.
Intrinsic resistance: naturally present.
Acquired resistance: obtained from exogenous DNA.
MIC: lowest concentration inhibiting visible growth.
MBC/MLC: lowest concentration killing the organism.
Major targets
Target | Examples from lesson |
|---|---|
Cell-wall synthesis | β-lactams, vancomycin, bacitracin, D-cycloserine |
Cell membrane | Polymyxin B, colistin; daptomycin for Gram-positive infections |
30S ribosome | Aminoglycosides, tetracyclines, tigecycline |
50S ribosome | Macrolides, clindamycin, chloramphenicol, linezolid, streptogramins |
Folate synthesis | Sulfonamides, trimethoprim; TMP-SMX gives synergy |
DNA synthesis | Quinolones/fluoroquinolones; target DNA gyrase/topoisomerase |
RNA synthesis | Rifamycins; inhibit DNA-dependent RNA polymerase |
Mycolic-acid synthesis | Rifampin, isoniazid, pyrazinamide, ethambutol, streptomycin |
β-lactams: the β-lactam ring binds PBPs/transpeptidases and inhibits peptidoglycan cross-linking. Gram-negative organisms require passage through porins. Classes include penicillins, cephalosporins, carbapenems, and monobactams.
Folate pathway: sulfonamides inhibit dihydropteroate synthetase; trimethoprim inhibits dihydrofolate reductase. Sequential blockade produces synergy in TMP-SMX.
Kirby-Bauer disk diffusion essentials
Standard inoculum: 0.5 McFarland ≈ 1.5 × 10⁸ CFU/mL.
Medium: Mueller-Hinton agar.
pH: 7.2–7.4.
Depth: 3–5 mm.
Incubation: 35–37°C, 16–18 hours, aerobic, no CO₂.
Interpret the zone diameter using the applicable standardized breakpoint.
Other AST methods: dilution tests, E-test, automated systems, and modified methods for slow-growing or fastidious organisms.
Gram-positive cocci in pairs, tetrads, or grape-like clusters.
Nonmotile; non-spore-forming.
Facultative anaerobes.
Catalase-positive; oxidase-negative.
Tolerate/grow in 7.5–10% NaCl.
Staphylococcus aureus
S. epidermidis
S. saprophyticus
S. lugdunensis
S. haemolyticus
Factor | Key function/association |
|---|---|
Enterotoxins A–E, G–J | Heat-stable; food poisoning, especially A, B, D; B/C may cause enterocolitis |
TSST-1 | Superantigen; toxic shock syndrome |
Exfoliative toxin | Epidermolysis; scalded skin syndrome and bullous impetigo |
α-hemolysin | Damages RBCs, platelets, macrophages |
β-hemolysin | Sphingomyelinase C; disrupts RBC membranes |
γ-hemolysin/PVL | Leukocyte toxicity; PVL associated with severe skin disease/necrotizing pneumonia |
Coagulase | Converts fibrinogen to fibrin clot |
Staphylokinase | Dissolves fibrin clots |
Hyaluronidase | Spreading factor; hydrolyzes hyaluronic acid |
Lipase | Hydrolyzes lipids; supports skin infection/boils |
β-lactamase | Cleaves β-lactam ring of penicillins |
Protein A | Binds Fc portion of IgG and interferes with opsonization |
PBP2′ | Altered PBP associated with resistance to some β-lactams |
Clinical infections: folliculitis, furuncles, carbuncles, wound/skin infections, scalded skin syndrome, toxic shock syndrome, and toxic epidermal necrolysis as listed in the lesson.
MRSA: associated with the mecA gene and altered PBP2′. The lesson identifies vancomycin as first-line treatment.
S. epidermidis: biofilm/slime; prosthetic-device infection, hospital-acquired UTI, prosthetic-valve endocarditis.
S. saprophyticus: adherence to urogenital epithelium; UTI in sexually active young females and older women with indwelling catheters.
S. lugdunensis: mecA gene listed; associated with endocarditis and UTI.
S. haemolyticus: vancomycin resistance listed; associated with endocarditis and UTI.
Specimens: aspirates or swabs.
Media: blood agar, colistin-nalidixic acid, phenylethyl alcohol, and mannitol salt agar.
Typical culture: smooth, creamy, white, round colonies; S. aureus may be β-hemolytic and creamy/butter-like. On Loeffler serum slant, S. aureus is golden yellow.
Test | Principle/result |
|---|---|
Catalase | 3% H₂O₂; copious bubbles = positive. Separates Staphylococcus (+) from Streptococcus (−). |
Coagulase | Bound coagulase: slide clumping. Free coagulase: tube clot. Positive supports S. aureus. |
DNase | DNA hydrolysis; clear zone on DNase agar = positive. |
Mannitol salt agar | 7.5% NaCl selects staphylococci; phenol red turns yellow when mannitol is fermented. |
Novobiocin | 5 µg disk; >16 mm susceptible, ≤16 mm resistant in lesson. S. saprophyticus resistant. |
PYR | Bright red = positive; detects pyrrolidonyl arylamidase. |
VP | Red = acetoin present via butanediol pathway. |
Microdase | Dark blue/purple = positive; tests cytochrome c oxidase. |
Lysostaphin | Staphylococcal cell wall is lysed; clearing/reduced turbidity = susceptible. |
O/F glucose | Staphylococcus is fermentative; Micrococcus is oxidative. |
Bacitracin | >10 mm zone = susceptible in lesson. |
Staphylococcus vs Micrococcus
Test | Staphylococcus | Micrococcus |
|---|---|---|
Oxygen use | Facultative anaerobe | Obligate aerobe |
Anaerobic growth | Positive | Negative |
Bacitracin 0.04 U | Susceptible | Resistant |
Modified oxidase | Negative | Positive |
Glucose | Fermenter | Oxidizer |
Lysostaphin | Susceptible | Resistant |
Coagulase-negative staphylococci summary
S. aureus: coagulase +, DNase +, mannitol fermentation +.
S. epidermidis: coagulase −, DNase −, mannitol fermentation −, novobiocin susceptible.
S. saprophyticus: coagulase −, DNase −, mannitol fermentation −, novobiocin resistant.
Gram-positive cocci in pairs or chains.
Nonmotile; non-spore-forming.
Facultative anaerobes.
Catalase-negative.
Hemolysis on blood agar is an important presumptive clue.
Group/species | High-yield association |
|---|---|
Group A: S. pyogenes | Strep throat, pyoderma, scarlet fever, post-streptococcal disease |
Group B: S. agalactiae | Neonatal sepsis, pneumonia, meningitis; adult endometritis/wound infection |
Group C/G | Large-colony β-hemolytic streptococci; may resemble S. pyogenes |
S. pneumoniae | Pneumonia, sinusitis, otitis media, meningitis, bacteremia |
Viridans streptococci | Dental caries; subacute bacterial endocarditis |
Enterococcus | Nosocomial UTI, bacteremia, endocarditis |
Group D non-enterococcus | Bile-esculin positive, but differs from Enterococcus in salt tolerance/PYR |
Hemolysis
α: greenish/incomplete. S. pneumoniae, viridans, some Group D.
β: complete clearing. Groups A, B, C, and some Group D.
γ: no hemolysis. Most Group D.
Virulence factors
M protein: antiphagocytic.
Lipoteichoic acid and Protein F: fibronectin binding/attachment.
Hyaluronic acid capsule: masks antigens.
Hyaluronidase: spreading factor.
DNases A–D.
Streptokinase: activates plasminogen and promotes fibrin breakdown.
Streptolysin O: oxygen-labile, immunogenic, causes subsurface hemolysis; measured by ASO titer.
Streptolysin S: oxygen-stable, non-immunogenic, surface hemolysis.
Streptococcal pyrogenic exotoxins: Spe A associated with scarlet fever.
Disease
Pharyngitis and possible peritonsillar abscess.
Impetigo: superficial, contagious lesions.
Erysipelas: raised, sharply demarcated red lesion.
Cellulitis: deeper dermis/subcutaneous tissue; poorly demarcated.
Scarlet fever: toxin-mediated, sandpaper rash, strawberry tongue.
Necrotizing fasciitis.
Streptococcal toxic shock syndrome.
Post-streptococcal sequelae:
Acute rheumatic fever: autoimmune reaction affecting heart/blood vessels.
Acute glomerulonephritis: immune-complex deposition; hematuria, proteinuria, edema, hypertension.
Skin tests
Dick test: susceptibility test for scarlet fever; erythema after erythrogenic toxin.
Schultz-Charlton test: diagnostic test; antitoxin neutralizes rash, producing blanching.
Capsular material is a virulence factor.
Vertical transmission.
Major cause of neonatal meningitis; neonatal pneumonia and sepsis.
Adults: endometritis and wound infections.
Usually β-hemolytic.
Capsular polysaccharide is a major virulence factor; lesson notes approximately 80 serogroups.
α-hemolytic, Gram-positive diplococci; lancet/bullet-shaped.
Capnophilic.
Diseases: lobar pneumonia, sinusitis, otitis media, bacteremia, bacterial meningitis.
Neufeld-Quellung: capsular swelling with specific antisera.
Francis test: detects pneumococcal antibody; skin test listed in lesson.
Small-colony, α-hemolytic isolates.
Extracellular dextran/polysaccharide and adhesins.
Dental caries, bacteremia/septicemia, and subacute bacterial endocarditis.
Enterococcal factors: extracellular surface protein, serine protease, gelatinase, cytolysin, and resistance to antimicrobial agents.
Associated with nosocomial UTI, bacteremia, and endocarditis.
Nutritionally variant streptococci (Abiotrophia) require cysteine and pyridoxine; may show satelliting.
Pediococcus and Leuconostoc are Streptococcus-like organisms; vancomycin resistance helps differentiate Pediococcus from viridans streptococci.
Media: blood agar; blood agar with trimethoprim-sulfamethoxazole for selective isolation of β-hemolytic streptococci.
Test | Positive/high-yield interpretation |
|---|---|
Bacitracin 0.04 U | Any zone: Group A sensitive; Groups B/C/F/G resistant. |
SXT 1.25 µg | Any zone: Group C sensitive; Groups A/B resistant. |
CAMP | Arrowhead-enhanced hemolysis with S. aureus: Group B positive. |
Hippurate hydrolysis | Deep purple: Group B positive. |
PYR | Bright cherry red within 5 min: Group A and Enterococcus positive; Group D non-enterococcus negative. |
LAP | Red: S. pneumoniae, S. pyogenes, Enterococcus, Pediococcus positive. |
Bile esculin | Blackening: Group D positive. |
6.5% NaCl | Turbidity/color change: Enterococcus positive; non-enterococcal Group D negative. |
Optochin | Pneumococcus susceptible: >14 mm with 6-mm disk or >16 mm with 10-mm disk. |
Bile solubility | Colony disintegration: S. pneumoniae positive; viridans intact. |
Pneumococcus vs viridans
Feature | S. pneumoniae | Viridans streptococci |
|---|---|---|
Optochin | Susceptible | Resistant |
Bile solubility | Positive | Negative |
Quellung | Positive | Negative |
Inulin fermentation | Positive | Negative |
Morphology | Lancet-shaped diplococci | Variable chains/pairs |
Enterococcus vs Group D non-enterococcus
Enterococcus: 6.5% NaCl growth +, PYR +.
Group D non-enterococcus: 6.5% NaCl growth −, PYR −.
Gram-negative diplococci, often described as coffee-bean or kidney-bean shaped.
Obligate aerobes; nonmotile; non-spore-forming.
Oxidase-positive.
Catalase-positive except N. elongata.
Capnophilic.
Glucose fermenter as a group characteristic in the lesson.
Virulence factors
Receptors for human transferrin.
Outer membrane proteins:
PorB/Protein I
Opa/Protein II: tight attachment and invasion.
Rmp/Protein III
Pili: initial attachment and movement; types T1–T2 listed.
LOS endotoxin: tissue damage; released in outer-membrane blebs.
IgA protease: supports mucosal infection.
Transmission and clinical disease
Usually sexually acquired, often from an asymptomatic carrier.
Male: urethritis with burning urination and thick purulent discharge; complications include epididymitis, prostatitis, and periurethral abscess.
Female: cervicitis with mucopurulent discharge, dysuria, and intermenstrual bleeding; complications include salpingitis, tubo-ovarian abscess, PID, infertility, ectopic pregnancy, and Fitz-Hugh-Curtis syndrome.
Both sexes: rectal gonorrhea, pharyngeal gonorrhea, disseminated gonococcal infection.
Newborn: ophthalmia neonatorum.
Virulence factors
Receptors for transferrin, lactoferrin, and hemoglobin.
Pili.
Polysaccharide capsule: serogroups A, B, Y, and W-135 listed.
Outer-membrane proteins.
LOS endotoxin.
IgA protease.
Clinical significance
Asymptomatic carriage.
Invasive disease: meningococcal meningitis and meningococcemia.
Meningococcemia may involve purpura, petechial rash, DIC, and Waterhouse-Friderichsen syndrome.
Upper-respiratory-tract commensal.
Virulence factor listed: attachment to respiratory epithelium.
Causes localized, lower-respiratory-tract, and systemic infections.
Specimens
N. gonorrhoeae: male urethra, female endocervix, rectum, pharynx.
N. meningitidis: CSF, blood, nasopharyngeal swabs/aspirates.
Collection and transport
Direct plating to selective medium or use transport systems such as JEMBEC/Amies with Dacron or rayon swabs.
Incubate at 35°C in 3–5% CO₂.
Direct microscopy
Urogenital specimens may show Gram-negative diplococci, including intracellular organisms.
Colony morphology
N. gonorrhoeae: small, tan, translucent, raised.
N. meningitidis: small, tan, possibly mucoid and convex.
M. catarrhalis: smooth, opaque, gray-white; may be swept intact, producing “hockey-puck” movement.
Presumptive identification
Pathogenic Neisseria and M. catarrhalis: oxidase-positive.
Superoxol test: rapid presumptive test; positive result listed for N. gonorrhoeae.
NAAT: detects gonococcal DNA/RNA from swabs or urine; lesson identifies it as the modern gold standard and notes high sensitivity/specificity, including asymptomatic infection.
CTA sugars use phenol red; acid production turns the medium yellow.
Organism | Glucose | Maltose | Sucrose | Lactose |
|---|---|---|---|---|
Moraxella catarrhalis | − | − | − | − |
N. gonorrhoeae | + | − | − | − |
N. meningitidis | + | + | − | − |
N. subflava | + | + | + | − |
N. lactamica | + | + | − | + |
Memory aid:
Gono = glucose only.
Meningo = maltose too.
Moraxella = none.
Organism | Superoxol 30% H₂O₂ | MTM/ML/NYC | BAP | Glucose | Maltose | Lactose | DNase/TH |
|---|---|---|---|---|---|---|---|
N. gonorrhoeae | + | + | − | + | − | − | − |
N. meningitidis | − | + | + | + | + | − | − |
M. catarrhalis | − | variable | + | − | − | − | + |
Gram-positive cocci in clusters + catalase positive: think Staphylococcus.
Staphylococcus + coagulase positive: think S. aureus.
Coagulase-negative staph + novobiocin resistant: think S. saprophyticus.
Gram-positive cocci in chains + catalase negative: think Streptococcus/Enterococcus.
β-hemolysis + bacitracin sensitive + PYR positive: S. pyogenes.
β-hemolysis + CAMP positive + hippurate positive: S. agalactiae.
α-hemolysis + optochin sensitive + bile soluble: S. pneumoniae.
Group D + bile esculin positive + 6.5% NaCl growth + PYR positive: Enterococcus.
Gram-negative diplococci + oxidase positive: think Neisseria/Moraxella.
Glucose only: N. gonorrhoeae.
Glucose and maltose: N. meningitidis.
No carbohydrate utilization: M. catarrhalis.
Specimen quality → direct microscopy → choose appropriate medium → isolate colonies → assess colony morphology/hemolysis → perform screening tests → confirm with biochemical, antigen, molecular, or susceptibility testing.
Study tip: Cover the result column in each table and recall the organism, principle, and expected reaction before checking your answer.