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Practical Medical Microbiology PHT313

By Dr. Mohamed Al-Agamy Assistant Professor of Microbiology Department of Pharmaceutics & Microbiology College of Pharmacy King Saud University 2010/1431. Practical Medical Microbiology PHT313. Classification of Bacteria. Staphylococci.

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Practical Medical Microbiology PHT313

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  1. By Dr. Mohamed Al-Agamy Assistant Professor of Microbiology Department of Pharmaceutics & Microbiology College of Pharmacy King Saud University 2010/1431 Practical Medical MicrobiologyPHT313

  2. Classification of Bacteria

  3. Staphylococci • Three important species of staphyloccoci have medical importance • S. aureus:pathogenic and commensally found in nose • S. epidermidis: non pathogenic and commensals in skin • S. saprophyticus:causes UTI in sexually active women • Rarely found in healthy humans but is commonly isolated from animals and their carcasses • S. haemolyticus: Frequently found as a commensal organism on humans and animals • It occurs infrequently as a cause of soft-tissue infections, usually in immunocompromised patients

  4. General characteristics Gram Positive Cocci Grape-like Non Motile Non Spore Forming Non Fastidious Facultative Anaerobes Fermentative (O+/F+) Halotolerant Catalase positive Characteristics of S. aureus Production of coagulase Production of phosphatase Production of DNase Ferment Mannitol Gelatin liquefied β-hemolysis on blood agar Acidification & clotting of litmus milk General characteristics

  5. Gram stain of Staphylococci

  6. Antigens Protein A Capsule Adhesins Enzymes Coagulase Lipase Hyaluronidase Staphylokinase Nuclease Toxins α-Toxin β-Toxin δ-Toxin P-V Leukocidin Enterotoxin Exfoliative Toxin Toxic Shock Syndrome Toxin Virulence Factors

  7. Laboratory Diagnosis • I- Specimen: • Pus, Urine, Stool, Blood, CSF • II- Gram Stain: • Gram positive cocci, arranged in cluster • III- Culture: • A. Blood agar (Non-Selective Media) • Colonies of S.aureus are golden yellow and -hemolytic • Colonies of S. epidermidis are non-pigmented and non-hemolytic

  8. Colonies of S. aureus showing beta hemolysis Colonies of s. epidermidis (up) showing porcelin- white colonies as compared to S. aureus (down) the golden appearance of the colonies. This clear distinction in colony color is not seen at all times. Mannitol Salt Agar

  9. 2. Mannitol Salt Agar (MSA) • MSA is selective and differential medium for staphylococci • MSA contains • NaCl (7.5%), as selective agent • Mannitol as a differential agent • Phenol Red (pH indicators) • turns yellow in acidic pH and turns red in alkaline pH • S. aureus ferment mannitol and give yellow colonies • S. epidermidis and S. saprophyticus do not ferment mannitol and appear red on MSA.

  10. IV- Biochemical tests 1. Catalase test H2O2 Catalase H2O + O2 (gas, ↑) Staphylococci • Catalase test is used to distinguished between staphylococci (positive) from streptococci (negative) • Flood the culture with drops of 3% H2O2 • Catalase-positive cultures give air bubble at once • The test should not be done on blood agar because blood itself will produce bubbles

  11. 2- Coagulase Test Principle: • This test is used to differentiate between S. aureus (CPS) & other Staphylococcus species (CNS) • This test is done by tube method or slide method Fibrinogen (Plasma) Coagulase Fibrin (Clot)

  12. The tube coagulase test (Free): • Procedure: • Mix 0.1 ml of culture + 0.5 ml of plasma • Incubate at 37C for 4 h • Observing the tube for clot formation • Any degree of clotting constitutes a positive test • Advantage • More accurate • Disadvantage • Time consumed • The slide coagulase test • Procedure: • Used to detect bound coagulase or clumping factor • Add one drop heavy bacterial suspension and one drop of plasma on slide • Mixing well and observing for clumping within 10 seconds • Advantage • Rapid diagnosis • Disadvantage • Less accurate S. aureus S. epidermidis

  13. 3- Deoxyribonuclease (DNAase) test • Principle: • DNA is hydrolyzed into oligonucleotides by the action of DNase • S. aureus produces DNase while S. epidermidis and most staphylococci have not DNase • DNA is insoluble in acid • Nucleotides are soluble in acid • Procedure & result: • Inoculate DNA agar plate with tested organism in circular motion (Spot) • Incubate at 37C for 24 h • Observe DNase activity by adding 1N HCl to the agar surface, a zone of clearing indicates a positive test • The zone represents the absence of DNA • The medium around colonies not producing DNase remains opaque, which is a reflection of the precipitation of DNA by the added acid.

  14. 4- Novobiocin Sensitivity • Novobiocin resistance is intrinsic to S. saprophyticus but uncommon in other clinically important species. • A simple disk diffusion test for estimating novobiocin susceptibility is used to distinguish S. saprophyticus from other clinically species • Inoculated overnight culture on Mueller-Hinton agar or MSA • Add novobiocin disk on inoculated plate • Incubate at 37C overnight

  15. Differentiation between Staphylococcus species

  16. Preparation of smear Solid culture Liquid culture Distribute culture in slide Air dry Heat fix Ready to stain Gram Stain Primary Dye (C.V.) Mordant (iodine) Decolorizer (Alcohol) Counterstain (Safranin) All applied for 1 min After each step wash with water Blot dry Add one drop of immersion oil Examine under oil immersion lens Preparation of Smear and Staining

  17. Practical Work • Gram stain • Catalase test • Mannitol fermentation on MSA • DNAase Test • Tube Coagulase Test (Demo) • Novobiocin sensitivity (Demo)

  18. General Characteristics of Streptococci Gram positive cocci Chains or pairs Usually capsulated Non motile Non spore forming Facultative anaerobes Fastidious Fermentative (O+/F+) Catalase negative (Staphylococci are catalase positive) Streptococci

  19. Classification of Streptococci • Streptococci can be classified according to: • Oxygen requirements • Anaerobic (Peptostreptococcus) • Aerobic or facultative anaerobic (Streptococcus) • Hemolysis on Blood Agar (BA) • Serology (Lanciefield Classification)

  20. Classification Based on Hemolysis • Hemolysis on blood agar • -hemolysis • Partial hemolysis • Green discoloration around the colonies • e.g. non-groupable streptococci • S. pneumoniae & S. viridans • -hemolysis • Complete hemolysis • Clear zone of hemolysis around the colonies • e.g.Group A & B • S. pyogenes & S. agalactiae) • -hemolysis • No lysis • e.g. Group D • Enterococcus spp -hemolysis -hemolysis -hemolysis

  21. Serology: Lancefield Classification • Streptococci classified into many groups from A-K & H-U • One or more species per group • Classification based on C- carbohydrate antigen of cell wall • Groupable streptococci • A, B and D (more frequent) • C, G and F (Less frequent) • Non-groupable streptococci • S. pneumoniae (pneumonia) • viridans streptococci • e.g. S. mutans • Causing dental carries

  22. The following tests can be used to differentiate between -hemolytic streptococci Lancefield Classification Bacitracin susceptibility Test Specific for S. pyogenes (Group A) CAMP test Specific for S. agalactiae (Group B) Differentiation between -hemolytic streptococci

  23. Bacitracin sensitivity Test • Principle: • This test is used for presumptive identification of gp A • To distinguish between S. pyogenes (susceptible to B) & non group A such as S. agalactiae (Resistant to B) • Bacitracin will inhibit the growth of gp A Strep. pyogenes giving zone of inhibition around the disk • Procedure: • Inoculate BAP with heavy suspension of tested organism • Bacitracin disk (0.04 U) is applied to inoculated BAP • After incubation, any zone of inhibition around the disk is considered as susceptible

  24. CAMP test • Principle: • Group B streptococci produce extracellular protein (CAMP factor) • CAMP act synergistically with staph. -lysin to cause lysis of RBCs • Procedure: • Single streak of Streptococcus to be tested and a Staph. aureus are made perpendicular to each other • 3-5 mm distance was left between two streaks • After incubation, a positive result appear as an arrowhead shaped zone of complete hemolysis • S. agalactiae is CAMP test positive while non gp B streptococci are negative

  25. CAMP test

  26. Differentiation between -hemolytic streptococci • The following definitive tests used to differentiate between S. pneumoniae & viridans streptococci • Optochin Test • Bile Solubility Test • Inulin Fermentation

  27. Optochin Susceptibility Test • Principle: • Optochin (OP) test is presumptive test that is used to identify S. pneumoniae • S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml) giving a inhibition zone ≥14 mm in diameter. • Procedure: • Blood agar plate is inoculated with organism to be tested • OP disk is placed on the center of inoculated BAP • After incubation at 37oC for 18 hrs, accurately measure the diameter of the inhibition zone by the ruler • ≥14 mm zone of inhibition around the disk is considered as positive and ≤13 mm is considered negative • S. pneumoniae is sensitive (S) while S. viridans is resistant (R)

  28. Optochin Susceptibility Test Optochin resistant S. viridans Optochin susceptible S. pneumoniae

  29. Bile Solubility test • Principle: • S. pneumoniae produce a self-lysing enzyme to inhibit the growth • The presence of bile salt accelerate this process • Procedure: • Add 10 parts (10 ml) of the broth culture of the organism to be tested to one part (1 ml) of 2% Na deoxycholate (bile) into the test tube • Negative control is made by adding saline instead of bile • Incubate at 37oC for 15 min • Record the result after 15 min

  30. Bile Solubility test • Results: • Positive test appears as clearing in the presence of bile while negative test appears as turbid • S. pneumoniae soluble in bile whereas S. viridans insoluble

  31. Differentiation between -hemolytic streptococci Differentiation between -hemolytic streptococci

  32. Outline of differentiation between Gram-Positive cocci e.g. S. epidermidis

  33. Practical Work • Gram stain of Streptococcus species • Hemolysis on blood agar (S. pyogenes, S. pneumoniae and Enterococcus faecalis) • Bacitracin susceptibility test (S. pyogenes and S. agalactiae) • CAMP test (S. agalactiae and S. pyogenes ) • Optochin susceptibility test (S. pneumoniae and S. viridans) • Bile solubility test (demo)

  34. Aerobic Spore Forming Bacillus sppClassification of Bacteria

  35. Aerobic Spore Forming Bacillus spp Bacillus species Pathogenic Bacillus anthracis Bacillus cereus Non-pathogenic Bacillus subtilis

  36. Bacillus species General Characteristics Very large Gram positive bacilli 1-1.2 µm in width x 3-5µm in length Arranged in long chains Motile exceptB. anthracis Spore forming (outside the host) Spores are central and oval Capsulated (inside the host) Non fastidious Aerobic Fermentative i.e. O+/F+ Catalase positive Natural Habitats It is found in soil habitats

  37. Anthrax Anthrax is caused by B. anthracis Types of Anthrax Cutanoues Anthrax (Malignant Pustule) (20% fatal) Intestinal Anthrax Pneumonic Anthrax (Woolsorters disease) Virulence factors Poly-D-glutamyl Capsule Mediates the invasive stage of the infection Antiphagocytic Anthrax Exotoxins Mediates the toxigenic stage

  38. Bacillus cereus • B. cereus is a normal inhabitant of soil • Isolated from foods (Grains and spices) • B. cereus causes food poisoning • B. cereus deposits its spores in food • Bacteria germinates in food & begin releasing their exotoxins • Spores are not killed during cooking • The following table differentiates between Bacillus sp.

  39. Identification of Bacillus Spp. • Specimen • Pastular exudates in malignant pustule • Sputum in pneumonic anthrax • Stool in intestinal anthrax (also in food poisoning by B. cereus) • Stool specimen is emulsified and heated to 80 C to kill non spore forming microorganism • Morphology • Macroscopical (Cultural characteristics) • Microscopical (Gram Stain, Spore Stain)

  40. Cultural Characteristics • Grow on nutrient Agar • On ordinary medium • Grow aerobically at 37C with characteristic mucoid or smooth colonies, which indicates the pathogencity of organism (presence of capsule) • Rough colonies are relatively avirulent • Stab culture on gelatin medium results in inverted fire tree appearance. • Growth on Blood Agar • Bacillusanthracis colonies are non hemolytic • B. cereus colonies are β-hemolytic • B. subtilis colonies are β-hemolytic

  41. Microscopical examination • Gram Stain, Capsule stain and motility • Gram positive bacilli • Found in chains • B. anthracis is not motile • B. cereus is motile • B. anthracis is capsulated inside the host

  42. SporeStain • Bacillus spores are oval & central • By spore staining (Malachite green & safranin), the spore appears green while the vegetative cells appear red.

  43. Biochemical tests 1- Catalase Test Broth Cultutre & H2O2 on the slide • All Bacillus species are catalase positive • Remember: staphylococci are catalase positive H2O2 added on culture grown on nutrient agar

  44. 2- Starch Hydrolysis (Amylase Activity) • Principle • Starch + Iodineblue color • Glucose + Iodine No reaction • Nutrient Agar containing 1% Starch + M.O Glucose • Procedure • Inoculate nutrient agar plate containing 1% Starch with the M.O. • Incubate the plate at 37 for overnight • After incubation, flood the plate with Iodine solution • Result • Activity of amylase is indicated by a clear zone around the growth while the rest of the plate gives blue color after addition of iodine solution Amylase Iodine Appearance of colorless zone around the growth

  45. Spore Stain Procedure • Make a heat fixed smear of Bacillus • Place the slide on the slide rack • Cover the smear with malachite green stain • Apply heat for 3-5 min without boiling and drying of the slide • Wash the slide gently in running water about 20 S • Counterstain with safranin for one minute • Gently rinse with water • Gently blot the slide dry, no rubbing, and let it air dry and examine with oil immersion optics. • Observe red vegetative cells and sporangia, and green endospores and free spores

  46. Practical Work • Gram Stain • Spore Stain • Catalase Test • Starch hydrolysis

  47. Clostridia General Characteristics of Clostridia Large Gram positive Straight or slightly curved rods with slightly rounded ends Anaerobic Spore bearing Fermentative, or proteolytic or both Catalase and oxidase are negative Natural Habitats Their habitats are soils and animal & human gut which invade the blood and tissue when host die and initiate the decomposition of the corpse (dead body)

  48. Clostridium Diseases Their pathogenesis by producing potent exotoxins and enzymes which attack the neurons pathways Rapid diagnosis is crucial or patient will die

  49. Clostridium tetanicausing tetanus General characteristics of Cl. tetani Gram positive, straight, slender rod with rounded ends All species form endospore Spores are terminal drumstick with a large round end) Fermentative Obligate anaerobe Motile by peritrichous flagella Grows well in cooked meat broth and produces a thin spreading film when grown on enriched blood agar Spores are highly resistant to adverse conditions

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