Gram positive practical lab 24-25
Page 1: Staphylococcus Species
Major species:
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Page 2: Identification & Classification
Microscopy & Staining
Cultural Characteristics
Biochemical Reactions
Immunological Test
Molecular Identification (DNA)
Page 3: LAB DIAGNOSIS
Specimens depend on infection type:
Suppurative lesion: Pus
Respiratory infection: Sputum
Bacteremia & septicemia: Blood
Food poisoning: Feces, vomit, remains of suspected food
Detection of carriers: Nasal swab
Page 4: Microscopical Examination - Staphylococcus
Characteristics:
Shape: Cocci
Size: Small
Arrangement: Grape-like bunches
Gram Reaction: Positive
Motility: Non-motile
Capsule: Non-capsulated
Spore Formation: Non-spore forming
Page 5: Culture Characteristics
Culture on Nutrient Agar (Pigmentation test):
Staphylococcus aureus: Golden yellow
Staphylococcus saprophyticus/citreus: Lemon yellow
Staphylococcus epidermidis/albus: White
Page 6: Mannitol Salt Agar
Selective and differential medium:
High salt concentration inhibits some microorganisms
Mannitol: Fermented by some, producing acids
Phenol red: Indicator for pH change
Red (basic) ➜ Yellow (acidic)
Staphylococcus aureus: Ferments mannitol ➜ Yellow
Other Staphylococcus species: Red
Page 7: Biochemical Tests - Gelatin Stab
Gelatin liquefaction test:
Gelatin + Organisms ➜ Amino acids
Incubation:
Positive: Gelatin is liquefied
Negative: Gelatin remains solid
Page 8: Biochemical Tests - Catalase Test
Reaction:
2H2O2 ➜ 2 H2O + O2
Procedure:
Cultured Staphylococcus in 1% glucose media
Add H2O2; positive result produces air bubbles indicating catalase presence
Page 9: Streptococcus Species
Major species:
Streptococcus pyogenes
Streptococcus viridans
Streptococcus fecalis
Page 10: Microscopical Examination - Streptococcus
Characteristics:
Shape: Cocci
Size: Small
Arrangement: Chains
Gram Reaction: Positive
Motility: Non-motile
Capsule: Capsulated
Spore Formation: Non-spore forming
Page 11: Nutrient Agar & Blood Agar
Nutrient Agar: Weak growth (Fastidious microorganisms)
Blood Agar:
Enriched and differential media
Hemolysis differentiation:
No/γ Hemolysis: No change
Partial/α Hemolysis: Green discoloration
Complete/β Hemolysis: Clear zone around colonies
Page 12: Hemolysis Types
α Hemolysis: Partial hemolysis (e.g., Streptococcus viridans)
β Hemolysis: Complete hemolysis (e.g., Streptococcus pyogenes)
γ Hemolysis: No hemolysis (e.g., Streptococcus fecalis)
Page 13: Biochemical Tests - Streptococcus
Catalase: Negative
Bile Solubility Test: Negative
Test for lysis resistance by bile
Results: Yellow, turbid remains after bile addition
Optochin Test: Negative
Page 14: Diplococcus - Streptococcus pneumoniae
Characteristics:
Shape: Cocci
Size: Small
Arrangement: Pairs
Gram Reaction: Positive
Motility: Non-motile
Capsule: Capsulated (related to pathogenicity)
Spore Formation: Non-spore forming
Page 15: Culture Media for Diplococcus
Blood Agar: Alpha hemolysis
Chocolate Agar: White growth on brown medium
Preparation: Blood agar heated to 100ºC for 15 mins
Enriched medium features
Page 16: Biochemical Tests - Diplococcus
Bile solubility: Positive
Page 17: Optochin and Sensitivity Tests
Ethylhydrocuprein HCl (1/4000): Positive test; resistant to Optochin indicates Streptococcus viridans, whereas sensitive indicates Streptococcus pneumoniae
Page 18: Bacillus Species
Major Species:
Bacillus anthracis
Bacillus cereus
Bacillus subtilis
Clostridium Species
Page 19: Laboratory Diagnosis for Bacillus cereus
Isolation of strains from suspect food, feces, or vomitus to test enterotoxigenicity via serological or biological tests
Page 20: Microscopic Examination - Bacillus
Characteristics:
Arrangement: Rods in chains
Size: Long, thick
Spore Formation: Spore forming (central-subterminal spores)
Motility: Motile (except Bacillus anthracis)
Gram Reaction: Positive
Capsule: Capsulated
Page 21: Culture Characteristics - Bacillus
Nutrient broth and agar:
Turbidity observed
Growth characteristics on blood agar
Page 22: Biochemical Tests - Bacillus
Starch Hydrolysis Test
Starch + Iodine ➜ Blue color
Starch + Amylase ➜ Maltose
Maltose + Iodine ➜ No blue color
Clear zone formation after starch hydrolysis
Casein + Caseinase ➜ Amino acids
Page 23: Clostridium Species
Major Species:
Clostridium tetani
Clostridium perfringens
Clostridium botulinum
Clostridium difficile
Characteristics:
Strict anaerobes
Page 24: Laboratory Diagnosis - Clostridium
Organisms rarely isolated from wound sites
Clostridium tetani produces a terminal spore, giving it a "tennis racket" appearance
Clostridium perfringens food poisoning diagnosed by detecting toxins in feces
Page 25: Microscopic Examination - Clostridium
Characteristics:
Shape: Rods
Size: Thin
Spore Formation: Spore forming (terminal)
Arrangement: Single, paired, chains
Gram Reaction: Positive
Capsule: Non-capsulated
Motility: Motile
Page 26: Culture Characteristics - Clostridium
Cooked Meat Media: Used to grow anaerobic bacteria
Supernatant observed during incubation
Page 27: Litmus Milk Test
Saccharolytic (e.g., Clostridium perfringens)
Lactose fermentation produces acid and gas, turns litmus pink
Proteolytic (e.g., Clostridium tetani)
Casein digestion produces short peptide chains and ammonia, turning litmus blue
Page 28: Mycobacterium Species
Major Species:
Mycobacterium tuberculosis
Mycobacterium leprae
Diseases: Tuberculosis, Leprosy
Page 29: Microscopic Characters - Mycobacterium
Staining: Acid-fast
Size: Small
Shape: Red curved rods on blue background
Arrangement: Single, pairs, or groups
Page 30: Culture Media - Mycobacterium
Lowenstein-Jensen Medium: Egg-based, selective for Mycobacterium growth
Appearance: Milky or turbid green colonies after 6-8 weeks
Middlebrook Medium: Agar-based, faster growth formulation
Page 31: Biochemical Tests - Mycobacterium
Niacin Test:
Mycobacterium tuberculosis accumulates niacin as a metabolic byproduct
Procedures to detect niacin accumulation confirm species identity.