Practical Microbiology I (2023/2024) - BUC

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Badr University in Cairo

2024

BUC

Prof. Dr. Haneya Anani

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microbiology gram-positive bacteria laboratory identification practical microbiology

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This is a practical guide for Microbiology I, covering the identification of Gram-positive bacteria. The document includes lab procedures, learning outcomes, and tables of contents for different lab sessions.

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Practical Guide in Technical Aspects of Microbiology I Course Code: MIB2117 Identifications of Gram-positive Bacteria School of Applied Health Science Level 2/ S 1 Academic year: 2023 /2024 Prof. Dr. H...

Practical Guide in Technical Aspects of Microbiology I Course Code: MIB2117 Identifications of Gram-positive Bacteria School of Applied Health Science Level 2/ S 1 Academic year: 2023 /2024 Prof. Dr. Haneya Anani Medical Microbiology and Immunology Faculty of Medicine 1 5 Marks Name of student ……………………………………………………... ID…………………………………………………………………………….. Section group ………………………………………………………….. Level………………………………………………………………………… Semester ………………………………………………………………… Prof. Haneya Anani 2 Table of contents Lab. No. 1 laboratory identification of Staphylococci Lab. No. 2 laboratory identification of Streptococci Lab. No. 3 Laboratory identification of Neisseria infection Lab. No. 4 Laboratory identification of Corynebacterium diphtheriae & Diphtheroid Lab. No. 5 Laboratory identification of Mycobacterium tuberculosis infection Lab. No. 6 Laboratory identification of Bacillus anthracis Lab. No. 7 Laboratory identification of anaerobic bacteria Lab. No. 8 Laboratory identification of Listeria & Haemophilus influenzae Prof. Haneya Anani 3 Write the bacterial cell shape & Gram stain reaction? Objective lens X Ocular lens = total magnification What is the total magnification of our light microscopes if 100 X, 10 X and 40 X are being used ? Low power = Name the medium. High power = Oil immersionProf.lens = Haneya Anani Lab. No. 1 laboratory identification of Staphylococci Learning Outcomes 1. Know the morphology and culture characters of Staph. aureus 2. Know the laboratory identification of staphylococcal infections Prof. Haneya Anani 5 laboratory identification of Staphylococcus aureus Specimen : according to site of infection Direct smear stained with Gram stain shows Gram-positive cocci arranged in clusters among the pus cells. Culture the samples on 1. Blood agar to test hemolytic reactions 2. Nutrient agar shows endopigment production Golden yellow pigment ( Staph. aureus ) Lemon yellow pigment ( Staph. saprophyticus) White pigment ( Staph. epidermidis) Colonies are identified by Gram stained film shows Gram-positive cocci arranged in clusters. Confirmatory identification by catalase test and coagulase test and fermentation of mannitol Staph. aureus is : Positive catalase test Positive coagulase test Positive DNase test They produce yellow color on mannitol salt agar Yellow colonies of Staphylococcus aureus White pigment Golden yellow pigment Lemon yellow pigment Flashcard of catalase test  Catalase test : it is used to differentiate between streptococci (catalase-negative) from staphylococci (catalase positive).  The test is performed by 3% or 10 % hydrogen peroxide to a colony on an agar plate or slant  Catalase-positive cultures produce bubbles  It should be performed by a wooden or plastic sticks, never done by metal loop ( may give false positive results).  Avoid picking pieces of the blood or chocolate agar during isolation of the colony ( may give false positive results). Catalase test is done from nutrient agar Tube coagulase test Aim of experiment : this test for the bacteria's ability to clot blood plasma using the enzyme coagulase ( convert fibrinogen to fibrin) Name of reagent: rabbit or human plasma Principle of coagulase Test: Coagulase is an enzyme-like protein and causes plasma to clot by converting fibrinogen to fibrin. Staphylococcus aureus produces two forms of coagulase: bound and free. Membrane bound coagulase (clumping factor) can be detected by clumping of bacterial cells in the slide coagulase test Extracellular free coagulase can be detected by change the plasma into clot in the tube coagulase test. Tube coagulase test 1. Dilute the plasma 1: 5 in saline ( mix 0.2 ml of plasma with 1.8 ml of saline 2. Take 3 small test tubes and label as T (Test), P (Positive control) and N (Negative control). Positive control is S. aureus and Negative control is coagulase negative isolate. 3. Pipette 0.5 ml of the diluted plasma into each tube. 4. Add 5 drops (0.1 ml) of the Test organisms or broth culture to the tube labelled “T”, 5 drops of positive control ( of Staph aureus to the tube labelled “P” and 5 drops of coagulase negative isolate to the tube labelled “N”. 5. After mixing, incubate the three tubes at 35-37C. 6. Examine for clotting at 30 minutes intervals for up to 6 hours. 7. Any degree of clotting in coagulase test is considered positive, the tube can be left at room temperature for 24 hours following incubation for 2-6 hours before reporting the result. Positive: St. aureus Negative : Coagulase negative staph Slide coagulase test 1. Place a drop of saline on each end of a slide 2. With the bacterial loop emulsify a portion of the isolated colony in each drop to make two thick suspensions. 3. Add a drop of undiluted human or rabbit plasma to one drop of suspension and mix gently. 4. Observe the clumping of the organisms within 10 seconds. 5. No plasma is added to the second suspension to differentiate any granular appearance of the organism from true coagulase clumping. Flashcard of Mannitol salt agar Selective medium mean  Mannitol salt agar is selective and differential medium contain medium for Staphylococci group inhibitors that allow Selective by 7.5% sodium chloride (NaCl) growth of some bacteria  Differential by mannitol sugar but inhibit growth of  pH indicator is phenol red at pH 7.3 others  Staphylococcus aureus ferment mannitol produces yellow ( pH acidic)  Coagulase negative staph. does not ferment mannitol and the color remain ( red) Deoxyribonuclease (DNase) test Deoxyribonuclease (DNase) test is used to determine the ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy for growth. Requirements: Media: DNase Agar or DNase agar with indicator. Reagent: Hcl Procedure Use a heavy inoculum and draw a line from the edge to the center of the plate Incubate the plate at 37°C for 18-24hr. DNase enzyme hydrolyze DNA changed into nucleotides which dissolved in HCl It is used to differentiate S. aureus (DNase +ve) Clear zone around the from other Staphylococci that do not produce growth line such enzyme. Staph. epidermidis and Staph. saprophyticus 1. Staph epidermidis produce white pigment on nutrient agar 2. Staph saprophyticus produce lemon pigment on nutrient agar 3. They are Catalase positive Coagulase negative Mannite non-fermenter Non-haemolytic on blood agar Staph saprophyticus differentiated from Staph epidermidis by novobiocin test Staph.epidermidis is sensitive Staph saprophyticus is resistant Staphylococcus saprophyticus is considered an opportunistic pathogen to which sensitivity should be performed under the following conditions: Female in the childbearing period No other pathogenic growth is present in culture. High count. Micrococcus Common saprophyte in air, water and soil. Gram + ve cocci found in tetrads than clusters. Catalase + ve & coagulase – ve. May give lemon yellow colonies. Acts on glucose oxidatively. Gram positive cocci ( tetrad Micrococcus is similar to Staph. arrangement) epidermidis. It can be differentiate between Staph. epidermidis and Micrococcus by Bacitracin test (0.04- 0.05 IU) Micrococcus is sensitive. Staph epidermidis is resistant Micrococcus colonies on nutrient agar Summary of Identification of Staphylococcus aureus Gram stained film of Staphylococci in pus Beta hemolysis on Staphylococci in culture blood agar Golden yellow colonies of Staph Coagulase test aureus on N agar Catalase test Summary of Identification of Staphylococcus epidermidis Culture of Staphylococcus Staphylococci in culture epidermidis on blood agar, No haemolysis White colonies of Staph epidermidis on N agar Catalase test Coagulase negative Identification of Staphylococcus saprophyticus Novobiocin disc test showing resistance of Staphylococcus No hemolysis on blood agar saprophyticus Coagulase negative Catalase test Staphylococci in culture Laboratory demonstrations Staphylococci Catalase test ( photo) Gram stained film of staphylococci in pus Gram stained film of staphylococci in culture Blood agar showing beta hemolysis Nutrient agar showing endopigment production of staphylococci Tube coagulase test Slide coagulase test ( photo) Non-hemolytic blood agar Culture of Staph on mannitol salt agar Photo of DNase test Phage typing (photo) Complete the chart Staphylococci A. On microscopic appearance shows Gram positive cocci B. Write the results of catalase and coagulase test A. Name the test in (No. 4)……………………………………………. B. Identify the organism arrangement in ( No.3)……………………… C. Name the type of hemolysis……………………………………… D. What is the organism?.............................................................................. Recurrent surgical wound infection was found in a surgical ward at one of the hospitals during post- operative treatment of admitted patients. The patient complain of fever, bone pain, redness, and pus at the site of the incision. Bacteriological examination of a wound swab revealed β -haemolytic Gram positive cocci arranged in grapes. A. Mention the organism that caused the problem. ……………………………………………… B. What is the specific test utilized for its identification based on plasma coagulation? …………………………………………………….. Prof. Haneya Anani 24 The organism isolated form skin lesions from patient with skin scalded syndrome that showed a colonies surrounded by a zone of beta-hemolysis on a blood agar plate. A Gram stained smear showed Gram- positive cocci arranged in clusters. Coagulase positive. A.What is the causative organism? …………………………………………………… B. What is the selective medium used for isolation? ………………………………………………… Prof. Haneya Anani 25 Coagulase test : A. Coagulase is an enzyme that can coagulate human or rabbit ……………………………………………………………………... B. It is used to differentiate between …………………………....and ……………………………………………………………………………………………… ………………………………………………………………………………….. Prof. Haneya Anani 26 The organism isolated from honey crusted skin lesions A Gram stained smear from lesion showed Gram- positive cocci arranged in clusters, coagulase positive. The organism is Staph.aureus. A. What is the other organism that produce β- haemolysis ? ………………………………………………………. …………………………………………………….. B. What is the test can differentiate between them? ……………………………………………………. Prof. Haneya Anani 27 Lab. No. 2 laboratory identification of Streptococcus pyogenes, Streptococcus agalactia and Streptococcus faecalis Learning Outcomes 1. Know the morphology and culture characters of Strept. Pyogenes 2. Know the morphology and culture character of Strept faecalies 3. Know the laboratory identification of Strept pyogenes & S. faecalies Prof. Haneya Anani 28 Streptococci Streptococci are Gram-positive spherical bacteria arranged in chain. Aerobic Streptococci classified according to haemolysis on blood agar : β-haemolytic streptococci that produce complete lysis of red blood cells with release of haemoglobin Alpha-haemolytic streptococci that cause incomplete lysis of red blood cell with formation of green pigments Non-hemolytic streptococci Pathogenic species: Streptococcus pyogenes (β- haemolytic streptococci Group A) Strept. agalactia (β- haemolytic streptococci Group B) Streptococcus faecalies Specimens: Sputum, pus, blood, cerebrospinal fluid Acute & Subacute bacterial endocarditis : blood culture Post-streptococcal infection ( rheumatic fever) :ASO test Direct gram stained film showing gram positive cocci arranged in chains among pus cells Culture on Best growth is achieved on blood agar & chocolate agar. Blood agar: β- (complete) hemolysis = clear zone. Biochemical identification 1. Catalase negative 2. Bacitracin sensitivity : sensitive Identification of Streptococcus pyogenes Streptococcus in pus β- haemolysis on blood agar Catalase test Bacitracin sensitivity : sensitive to Streptococcus in culture bacitracin Laboratory diagnosis of Strept. agalactia There are members of the normal flora of female genital tract Diseases: neonatal meningitis, pneumonia Specimen: CSF and sputum Direct Gram stained film showing gram positive cocci arranged in chains among pus cells Culture on Blood agar: β- (complete) hemolysis = clear zone. The colonies identified by Gram stained film shows Gram positive cocci arranged in chains and biochemical tests Biochemical identification 1. Catalase negative 2. Bacitracin sensitivity : resistant to bacitracin. 3. CAMP test : positive prof. Haneya Anani 32 Identification of Streptococcus agalactia Streptococcus in culture β- haemolysis on blood agar Bacitracin sensitivity : resistant to bacitracin CAMP test It is used to differentiate between Streptococcus agalactiae and other beta haemolytic streptococci Procedure: Strept. agalactia produce CAMP factor that synergistically act with beta lysin of Staph aureus and enhance the lysis of red blood cells while S. pyogenes not produce CAMP factor Result: ❑A is positive (Strept. agalactia ) ❑B is negative (S. pyogenes) S. aureus S. agalactia S.pyogenes prof. Haneya Anani 34 Laboratory identification of Strept. pneumoniae Direct gram stained film showing gram positive diplococci capsulated among pus cells. Capsules appear as unstained halos around the organism. Culture on Blood agar: alpha hemolysis The colonies identified by Gram stained film shows Gram positive diplococci capsulated Biochemical identification 1. Catalase negative 2. Quellung test is positive 3. Tests for differentiation from Strept. viridans Pneumococci in sputum , Capsules appear as unstained halos around the prof. Haneya Anani organism. 35 Pneumococci in sputum Positive Quellung test The test is done by mixing of sputum or suspension of pneumoncoccal culture with Alpha haemolysis on specific antiserum on a blood agar microscopic slide and examined by oil immersion Incubate in a 5-10% CO2 lens shows swollen capsule. or candle – jar at 35˚ C Capsule swelling test ( Quellung reaction) This test is used for identification and typing of pneumococci Method: The test is done by mixing of sputum specimen or suspension of pneumococcal culture with specific antiserum on a microscopic slide and examined by oil immersion lens. If antiserum is specific for the pneumococci, the antibodies precipitated on capsule margin and become visible or swollen prof. Haneya Anani 37 Differentiation between pneumococci and Strept. viridans Inulin fermentation Bile solubility test Optochin sensitivity test The mice die within 1-2 days after pneumococci injection Diseases caused by Streptococcus faecalis urinary tract infection, wound infection, bacteremia, meningitis , osteomyelitis Used as an indicator of fecal pollution of water Laboratory diagnosis of Strept. faecalies Gram positive diplococci Specimens: pus , blood , urine, wound swab, water sample Direct gram stained smear showing Gram positive cocci arranged ovoid pairs Culture on Blood agar shows : (no) hemolysis = no zone. Nutrient agar MacConkey’s agar: pinpoint rose pink colonies Selective medium (Bile esculin agar): hydrolysis of esculin produce black colonies prof. Haneya Anani 39 The colonies identified by Gram stained film shows Gram positive cocci arranged ovoid pairs Biochemical identification Catalase negative Esculin hydrolysis: black colonies PYR test : positive Pinpoint pink colonies of Enterococcus on Enterococcus faecalis MacConkey agar without Enterococcus faecalis produce non-hemolytic bile salts and crystal produce black colonies colonies on blood agar violet. on bile esculin agar PYR hydrolysis test Procedure: 1. Moisten the PYR disk slightly with distilled , DO NOT SATURATE. 2.Select 2-3 well isolated colonies of the bacterium to be tested using a sterile loop and rub into a small area of the PYR disk so that there is a visible paste. 3. Allow test organism to incubate at room temperature on the PYR disk for two min. 4. After incubation period add one drop of PYR reagent. Interpretation : Positive result = within one minute of adding PYR reagent, a dark pink or cherry-red color in the area of the suspected organism will appear. Negative result = no color change after adding reagent. PYR test for Group A streptococci and enterococci. Both are positive for this test (right); left is a negative result Enterococcus faecalis Catalase test is………… Type of hemolysis is on blood agar …………………………………………………. Name of the medium is: Describe the film ………………………………………. …………………………………………………. Laboratory demonstrations Streptococci Gram stained film of streptococci in pus Gram stained film of streptococci in culture Gram stained film of pneumococci Gram stained film of strep. fecalis Blood agar showing beta hemolysis of Strept. Pyogenes Blood agar showing partial hemolysis of Strept. viridans Blood agar showing no hemolysis of Strept. Fecalis Culture of Enterococcus on bile esculin agar Bacitracin sensitivity test ( photo) Inulin fermentation & bile solubility test ( photo) Optochin test ( photo) PYR test (photo) Flashcard of bacitracin sensitivity test The used for ………………………………………………………….. The sensitive organism is…………………………………………… Resistant organism is …………………………………………….. prof. Haneya Anani 44 The film was obtained from rust-colored sputum from a patient who complained of fever and chest pain and was diagnosed with pneumonia. Gram- stained sputum film revealed encapsulated Gram- positive diplococci and many polymorphonuclear leukocytes. A.What is the organism that is causing the problem? …………………………………………………… B. Identify the test. …………………………………………………… 45 Prof. Haneya Anani The film was isolated from a throat swab obtained from a patient who had fever and sore throat and was diagnosed as acute follicular tonsilitis. Gram-stained film revealed Gram-positive cocci in chains as well as many polymorphonuclear leukocytes. A.What organism is the source of the problem? …………………………………………………. What is the type of hemolysis on blood agar? …………………………………………………….. 46 Prof. Haneya Anani Lab. No. 3 Laboratory identification of Neisseria infection Learning Outcomes To know characters of Neisseria species ( commensals and pathogenic) Identify the laboratory diagnosis of Neisseria infections Describe the flashcard of each test used for identifications. Prof. Haneya Anani 47 Neisseria N. gonococcus causes gonorrhea N. Meningitidis is the cause of epidemic cerebrospinal meningitis. Morphology : A gram-negative diplococci kidney shaped non motile All species of Neisseria are oxidase positive. Neisseria meningiditis grow blood agar or chocolate agar or Thayer Martin medium Neisseria gonorrhea grow on chocolate agar and TM medium and does not grow on blood agar It needs 5-10% CO2 or candle jar. Thayer Martin is chocolate agar + growth factors + 3 antibiotics ( Vancomycin to inhibit gram + ve organisms, Colistin for Gram - ve and Nystatin for fungi ) Laboratory diagnosis of Gonorrhea Specimen: 1. Urethral discharge from male 2. Endocervical swab from females 3. Pharyngeal swab in oral gonorrhea Urethral discharge 4. Eye swab in acute conjunctivitis Cervical swab Eye swab Sterile ordinary swab Isolation and identification of Neisseria gonorrhoeae The film is diagnostic in acute Direct smear stained by Gram to case of male gonorrhea show gram negative diplococci intracellular and extracellular Culture on chocolate agar or Thayer- Martin medium , and incubated at 37C in 5-10% CO2 or candle jar for 24 hours Gram stained film shows Gram negative kidney shape diplococci extracellular and mainly intracellular Culture of Neisseria gonorrhoeae on chocolate agar shows overgrowth Culture of Neisseria gonorrhoeae on Thayer Martin medium shows Neisseria only Neisseria gonorrhoeae does not grow on blood agar Neisseria gonorrhoeae on chocolate Neisseria gonorrhoeae on Thayer Martin medium agar Confirmatory identification of Neisseria gonorrhoeae The colonies further identified by 1. Gram stained film shows Gram negative diplococci 2. Biochemical reaction Oxidase positive Fermentation of glucose with production of acid only Slide agglutination by latex agglutination with specific antisera Biochemical identification of Neisseria gonorrhoeae Fermentation of glucose with production of acid only Summary of Identification of Neisseria gonorrhoeae Direct stained smear by Gram to show gram negative diplococci intracellular and extracellular Selective medium is TM medium TM medium : It is chocolate agar + growth factor + 3 antibiotic Incubation condition : 5-10% CO2 at Gram negative cocci mainly intracellular 37C Positive oxidase test -Ve Candle jar 5-10% TM agar Oxidase test Laboratory diagnosis of epidemic cerebrospinal meningitis ❑Laboratory diagnosis of case Specimen 1. CSF 2. Blood ❑Laboratory diagnosis of meningococcal carriers 1. Specimen: Nasopharyngeal swab 2. Culture on chocolate agar or Thayer-Martin medium or blood agar and examined the same in acute cases Nasopharyngeal swab Identification of Neisseria meningitidis Isolation and identification of meningococci 1. CSF sample obtained by lumber puncture through spinal needle 2. Detection of leukocytic count elevated predominantly neutrophils 3. CSF centrifuged 4. Supernatant fluid is examined for detection of capsular polysaccharide antigen by latex agglutination with specific antisera 5. A smear prepared from deposit, and stained with Gram stain 6. Culture of deposit on chocolate agar or Thayer-Martin medium , and blood agar incubated at 37C in 5-10% CO2 or candle jar for 24 hours The colonies further identified by 1. Gram stained film shows Gram negative diplococci are intracellular and extracellular in pus cells 2. Biochemical reaction 3. Oxidase positive 4. Catalase positive 5. Fermentation of glucose and maltose with production of acid only Latex agglutination test Rapid test Direct detection of meningococcal antigen by latex agglutination test for antimicrobial treatment Gram stained film shows Gram negative kidne shape diplococci extracellular and mainly Culture of Neisseria intracellular meningitidis on chocolate agar Flashcard of Oxidase test Types of oxidase reagents: Oxidase test Discs Strips Reagent Oxidase test Procedure: Positive oxidase test in: To take a small portion of a bacterial colony Neisseria, Pseudomonas, (preferably young culture) and rub the V.cholera, some species sample on the commercial oxidase disk or of H.influenza, and other strip. types of bacteria. Don't take a big colony Result: Observe inoculated area of disk for Oxidase test is of value to a color change to purple within 10 seconds spot gonococcal and meningococcus colonies Biochemical identification of Neisseria meningitidis Fermentation of glucose and maltose with production of acid only Type of incubator : CO2 incubator or candle jar 5-10 % Co 2 Laboratory identification of Moraxella catarrhalis It can grow at room temperature & on nutrient agar at 37˚C. Moraxella catarrhalis growing on chocolate agar after 24 hours of incubation Moraxella catarrhalis cause otitis media, sinusitis and pneumonia Specimen: ear discharge 1. Direct microscopy of smear Commensal Neisseria on nutrient agar stained with gram stain showing gram –ve diplococci. 2. Culture on nutrient agar 3. The colonies identified by biochemical reaction No acid production from glucose, maltose, lactose and sucrose or (variable). Gram negative No sugar fermentation diplococci extracellular Laboratory demonstrations Neisseria ❑ Gram stained slide showing neisseria in culture ❑ Gram stained slide showing pathogenic Neisseria in pus ❑ Commensal neisseria on nutrient agar ❑ Chocolate agar ❑ Thayer Martin medium ❑ Nasopharyngeal swab ❑ Candle jar ❑ Oxidase test Is positive direct smear from urethral discharge diagnostic for acute male of Neisseria gonorrhoeae case Answer: …………………………………………………….. A 22 year-old man developed sudden onset of fever, severe headache and stiff neck. He goes to fever hospital, a blood and CSF samples were obtained for investigations. By a stained smear from CSF showed a Gram-negative diplococci intra- and extracellular, oxidase test was positive. the case is diagnosed as cerebrospinal meningitis A.What is the causative organism? ………………………………………………………………………… B.What is the rapid diagnostic test that needed to confirm the diagnosis? …………………………………………………………………………….. Lab. No. 4 laboratory identification of Corynebacterium diphtheriae and Diphtheroids Learning Outcomes 1. To know the morphology and culture characters of C. diphtheriae 2. To know the laboratory identification of Corynebacterium diphtheriae & Diphtheroids Prof. Haneya Anani 62 Corynebacterium diphtheriae Coryn = club The genus corynebacteria includes Pathogenic form; (Corynebacterium diphtheriae) Commensal Corynebacterium in skin & mucous membrane Diphtheroids Corynebacterium diphtheria is the causative organism of diphtheria, skin, wound and eye infection Morphology: Gram-positive bacilli, The organism is beaded due to presence of metachromatic granules (phosphate granules). The methylene blue is the only dye that stain bacilli and leave phosphate granule that appear beaded Non motile, Non capsulated Non-spore forming Arrangement : Chinese letters Cultural characters C. diphtheriae are aerobic bacteria that grow best on loeffler’s serum at 37C / 24 hours laboratory identification of Corynebacterium diphtheriae Diagnosis of carrier: Throat or Nasal swabs are identified the same steps in case. Flashcard of blood tellurite agar It is composed of blood agar + 0.04% potassium tellurite The potassium tellurite used to inhibit oral flora Culture on blood tellurite agar is used for differentiation into biotypes ( Gravis, mitis and intermedius) Corynebacterium diphtheriae in culture ( Gram stain) Corynebacterium diphtheriae in culture ( Methylene blue stain) Flashcard of Elek’s test It is used for test toxigenicity of the organism A strip of filter paper saturated with antitoxic serum is embedded in a serum agar plate. The strain tested is streaked at right angle to the filter paper and the plate incubated at 37 oC for 2 days. The antitoxin diffusing from the filter paper strip will form precipitation lines with the toxin diffusing from the toxigenic strain. Absence of precipitation lines indicates that the strain is non toxigenic. Positive test Negative test Summary of Identification of C. diphtheriae Blood agar Loffler’s serum No hemolysis Blood tellurite agar Biotypes of C. diphtheriae on blood C. diphtheriae in Chinese tellurite agar Elek’s test letter appearance Diphtheroids It is normal flora of the mucous membrane of the throat, skin and respiratory tract. Many species have been implicated in opportunistic infections. and have been associated with diseases in immunocompromised patient It is Gram-positive short bacilli, non–spore-forming , Non motile Arrangement : palisade form or in pairs at angles Grow on nutrient agar Diphtheroid in culture Catalase test = positive Gram stain palisade form Catalase test : positive Laboratory demonstrations Corynebacteria Gram stained film of C. diphtheriae Methylene blue stained film of C. diphtheriae Gram stained film of Diphtheroids Blood tellurite agar showing C. diphtheriae Nutrient agar showing Diphtheroids Loffler’s serum slope ( uncultured) Elek’s test (photo) Throat swab Is positive direct smear diagnostic for diphtheria case? Answer: …………………………………………………….. The organism was isolated from patient with acne. Gram- stained smear from discharge the showed Gram-positive bacilli arranged in palisade manner. 1.What is the causative organism? …………………………………………………. 2. What is the result of catalase test? ……………………………………………… 3. What are other infection caused by the organism?.................................................................................................................................................................. 4. What condition considers the organism to be pathogenic?................................................................................ 71 Prof. Haneya Anani What is the differences between C. diphtheriae & Diphtheroids? C. diphtheriae Diphtheroids A. Morphology B. Culture C. Pathogenicity Drawing Diphtheroid in culture Corynebacterium diphtheriae Gram 72stain Prof. Haneya Anani Lab. No. 5 Laboratory identification of Mycobacterium tuberculosis infection Learning Outcomes 1. Know morphology and growth characters of M. tuberculosis 2. Identify laboratory identification of Mycobacterium tuberculosis, Prof. Haneya Anani 73 Mycobacterium tuberculosis Morphology: Mycobacteria are thin straight or curved rods. They cannot be classified as either Gram-positive or Gram-negative. They are classified as acid-fast and alcohol- fast as demonstrated by Ziehl- Neelsen stain due to their impermeability by certain dyes and stains. They appear as thin pink rods single or in small groups. Culture Characteristics: M. tuberculosis grows only on selective medium (Lowenstein- Jensen medium) due to specimen contaminated by many types of flora. It is an obligate aerobe. Slow growth rate 2-8 weeks. Laboratory diagnosis Specimen: sputum not saliva.. 3 morning sputum samples collected on 3 consecuative days should be examined as follows: 1. Direct smears are made from the specimen, and stained with Ziehl- Neelsen stain. 2. Decontamination and concentration of sputum and the deposit is cultured on L-J medium and incubated at 35-37 oC. Growth appears up to 8 weeks. 3. Rapid diagnosis can be done by detection of DNA in the patient’s specimens by PCR 1. Indirect methods for the detection of MTB Tuberculin skin test or PPD test Interferon-Gamma Release Assays (IGRA) In Intestinal tuberculosis specimen is stool examined as sputum In Urinary tuberculosis , specimen is 24 hours urine is collected and examined as sputum. Sputum sample Saliva sample Pus cell Epithelial cell Two specimens were sent to the Microbiology lab for culture. One is saliva, and the other is sputum. Which specimen is appropriate for isolation Mycobacterium tuberculosis?................................................................................. Microscopy and culture (M. tuberculosis stained by Ziehl-Neelsen from sputum specimen) that appears as red-pink long or slightly curved bacilli Non- acid fast appear blue. Acid fast bacilli appear thin pink rod in blue background M.tuberculosis shows buff-colored culiflower colonies on Löwenstein– Jensen medium. Ziehl-Neelsen stain experiment Ziehl-Neelsen stain Type of stain: differential stain it is used to differentiate between acid fast bacilli and non- acid fast bacilli Ziehl-Neelsen’s Stain 1. Flood the slide with carbolfuchsin and heat to steam 2. Fix and prepare the slide with gentle heat 3. Leave for 5 minutes. 4. Rinse with distilled water 5. Decolorize with 3% hydrochloric acid in 95 % ethyl alcohol until red color no longer appears in the washing ( about 2 minutes) 6. Rinse with distilled water 7. Counterstain with methylene blue for 1-2 min. 8. Rinse with distilled water, drain and air dry Note: do not touch the smear with end of the oil dispenser because this could transfer acid fast bacilli to another slide Oil must be wiped from objective lens of microscope Rapid diagnosis by culture on fluid media Mycobacteria grow more rapidly and reliably in a liquid culture medium compared with the solid medium. (M. tuberculosis stained by Ziehl-Neelsen from mycobacterium culture fluid medium) that appears as cord like due to cord factor stick bacilli together Rapid diagnosis Culture on fluid media by Mycobacteria indicator tube BACTEC culture system culture on fluid media containing Culture on media containing C fluorescence sensor, when Labeled palmitic acid. Mycobacteria consume O2 produce The growing bacteria utilize the fluorescence that detected by UV. palmitic acid and released radioactive CO2 detected by machine Time of results is 5-15 days The MGIT tube on the right contains growing mycobacteria and is fluorescent when exposed to UV light. In contrast, the tube on the left contains no mycobacteria. Mycobacteria is fluorescent Mycobacterium leprae Mycobacterium leprae acid fast bacilli It does not grow on artificial media Leprosy is a chronic granuloma characterized by skin infiltration and nerve damage They are similar to tubercle bacilli in shape , they are acid fast by 5 % H2SO4 & alcohol fast Diagnosis of leprosy: Injection of exudate into the foot pads of mice induce characteristic lesion. M. Leprae modified ZN stain Or nasal scraping of skin nodule and stained by modified Ziehl-Neelsen stain stain Saprophytic and atypical Mycobacteria Saprophytic mycobacteria It is environmental acid fast –not alcohol fast bacilli Grow rapidly It gives pigmented colonies Atypical mycobacteria It is potentially pathogen that cause pulmonary infection in immunocompromised patients. Laboratory demonstrations Mycobacteria ZN-stained slide showing M. tuberculosis Lowenstein-Jensen medium Modified ZN- stained film of Mycobacterium leprae ZN stain set Complete the table Typical saprophytic mycobacteria mycobacteria Incubation time Colonies Staining 84 Prof. Haneya Anani Practical activity 1.Lowenstein –Jensen medium is ………………………………………………………………………………………… …………. Function of malachite green dye is………………………………………………. ………………………………………………………………….................................... 3.Characteristic colonies is………………………………………………………… 4. The mycobacterium tuberculosis need selective media due to ………………………………………………………………………………………… ………………………………………………………………………………………… The film was isolated from a patient who had chest discomfort, nocturnal sweats, and was diagnosed with tuberculosis. A microscopic examination of a stained sputum smear revealed pink bacilli on a blue background. A.What is the name of the smear staining? ……………………………………………. B. What is the time required for its growth? ………………………………………………... Lab. No. 6 Laboratory identification of Bacillus anthracis Learning Outcomes Know the laboratory identification of Bacillus anthracis Describe differences between Bacillus anthracis and Anthracoids Prof. Haneya Anani 87 Bacillus group This genus includes large aerobic, Gram positive , spore-forming bacilli that have Pathogenic : Bacillus anthracis, which causes diseases mainly in animals, but it can affect man and lead to anthrax Saprophytic : Anthracoids ( which are widely spread in nature (water, soil, and air). Some strains of Anthracoids are pathogenic such as B. cereus cause food poisoning Laboratory identification of anthrax Specimens: sputum , skin exudates Direct microscopic examination 1.Gram stained smear: Gram positive aerobic bacilli with square ends arranged in chains (bamboo stick appearance) and surrounded by unstained halo capsule, 2.Direct smear stained with polychrome methylene blue; for demonstration of polypeptide capsule: the organism appears blue while the capsule purple or pink. The specimen are culture on : Blood agar: non-haemolytic reaction with Medusa head colonies Gelatin medium produce inverted fire tree appearance (due to slow gelatin liquefiers) maximum liquefication on the surface than at the bottom Laboratory identification of Anthracoids Bacillus cereus is Gram positive bacilli , non-capsulated, motile, spore forming Anthracoids are frequently laboratory contaminants on culture media Specimen for isolation of Anthracoids bench , skin , and shoe swab Culture of swab on : ▪ Nutrient agar: incubated aerobically for 37 C. Anthracoids produce large-white colonies, with a rough surface and irregular fimbriated edge. ▪ Blood agar: β-haemolytic colonies. The colonies are further identified by: Microscopy: Gram stained film: Gram positive bacilli with square ends arranged in chains and contain spores, which appear as unstained oval and central spaces Biochemical reactions: Gelatin liquefication test appear rapidly. Identification of Bacillus anthracis Gram stained film of B. anthracis in Gram stain film of Bacillus anthracis culture. Bamboo stick or square shape showing blue bacilli surrounded by a pink with central spores capsule (MacFaydean reaction of polychrome methylene blue stain) Culture of Bacillus anthracis on blood agar shows non-haemolytic reaction with Medusa head colonies Inverted fire tree on gelatin medium Culture of saprophytic Anthracoids Culture of saprophytic Anthracoids on blood on N. agar agar showing B- haemolysis Central spores of B. anthracis Bacillus stearothermophilus It used as biological indicator to test efficiency of autoclave Laboratory demonstrations Bacillus group Photo of Gram stained film of Bacillus anthracis in culture Photo of Gram stained film of Bacillus anthracis in tissue Photo of medusa head colonies of Bacillus anthracis Photo of inverted fire tree appearance of B. anthracis on gelatin medium Culture of Anthracoids on blood agar Biological indicator of autoclave Enumerate 4 character of Bacillus anthracis ……………………………………………… ………………………………………………. ……………………………………………….. ………………………………………………… …………………………………………………. 1.What is the arrangement of B. anthracis? …………………………………………………………… 2.What does Anthracoids form in the environment ? ………………………………………………………………… …………………………………………………… 96 Prof. Haneya Anani Practical activity What are the differences between B anthracis & Anthracoid ? B. anthracis Anthracoids Capsule Effect on blood agar Gelatin liquefaction Sketch Gram stained film of Anthracoids Prof. Haneya Anani Gram stained film of B.anthracis 97 The film is isolated from red skin lesion with black center surrounded by vesicles. Microscopic examination of stained smear showed capsulated Gram positive spore-forming bacilli arranged in chains. A. What is the effect on blood agar? ……………………………………… B.What is the characteristic appearance of culture on gelatin medium …………………………………………… Prof. Haneya Anani 98 Lab. No. 7 Laboratory identification of anaerobic bacteria Learning Outcomes 1. Know what is anaerobic bacteria? 2. Know the medically important anaerobic bacteria and it is laboratory identification Prof. Haneya Anani 99 Oxygen requirements of bacteria Anaerobic bacteria that grow only in complete absence of oxygen. These are normal flora of human beings and usually present in skin, oral cavity, gastrointestinal tract. Specimen collection for anaerobic cultivation 1-Avoid contamination with normal flora 3- samples is collected by needle aspiration Media for anaerobic culture It should be freshly prepared It should be used within 2 weeks of Robertson’s cooked meat preparation medium It supplemented with special nutrients It contains reducing agents To Heat or steam in water bath at 80C for 2min. when used to expel any dissolved oxygen Anaerobic non-spore forming bacteria Laboratory diagnosis of Actinomycosis Microscopic appearance : Gram positive filamentous bacilli The organism is Actinomyces israelii Duration of culture : 10 days Medium : blood agar or serum glucose agar The characteristic colonies : molar tooth colonies Sulphur granules are crushed between 2 slides to prepare gram stain films that show gram positive mycelia Gram positive branching bacilli of Actinomyces israelii Anaerobic jar Anaerobic spore forming bacteria Clostridium group Clostridia are gram-positive spore forming anaerobic bacteria Natural habitat: – soil, intestinal tract of human and animals – saprophytic in soil and water Medically important colstridia: Cl. tetani causing tetanus Cl. perfringens causing gas gangrene and food poisoning Cl. botulinum causing botulism Cl. difficile casing antibiotics associated diarrhea 10/6/2024 dr. haneya anani 104 Clostridium tetani Morphology: Gram positive bacilli Anaerobic All clostridia is motile except Clostridium perfringens is non-motile Non capsulated They have terminal spores – drum-stick shape. Culture characters Cl. tetani grows in cooked meat medium and blood agar Disease : tetanus 10/6/2024 dr. haneya anani 105 Laboratory diagnosis of tetanus Specimen – deep wound swab Direct smear shows Gram-positive bacilli with drum-stick appearance is only suggestive not diagnostic Cooked meat medium Culture: culture the specimen on Robertson cooked meat medium , incubated overnight at 37 °C then subculture on blood agar and incubated anaerobically at 37 °C Cl. tetani produce swarming and β- haemolysis The colonies identified by motility test ( positive ) Slowly gelatin liquefication Gram stained film demonstrating drum stick spores of Cl. tetani dr. haneya anani 106 Isolation of Clostridium tetani from horse stools sample Culture of the horse stool on: Robertson cooked meat medium , incubated over night at 37 °C and then subculture on Blood agar , incubated under anaerobic condition for 2- 3 days at 37 °C On the blood agar colonies have tendency to swarm over the surface, they are β-haemolysis The colonies identified by Gram stained film shows Gram positive bacilli with terminal round spores; drum stick appearance Isolation of Clostridium tetani Anaerobic jar Clostridium tetani on blood agar showing swarming with β-haemolysis Clostridium perfringens Morphology: Gram positive bacilli Anaerobic Clostridium perfringens is non-motile Non capsulated They have subterminal spores It is normal commensal in human intestine Culture characters Cl. perfringens grows blood agar produce β- haemolysis Disease : gas gangrene 10/6/2024 dr. haneya anani 109 Laboratory diagnosis of gas gangrene Culture of lacerated wound swab on : Robertson cooked meat medium , incubated over night at 37 °C and then subculture on Neomycin blood agar , incubated under anaerobic condition for 2- 3 days at 37 °C Clostridium perfringens produce double zone of β- haemolysis The colonies identified by Gram stained film shows Gram positive bacilli with subterminal spores The colonies identified by Biochemical identifications: Motility is negative Catalase negative Indole negative Gelatin liquefication : Slowly gelatin liquefier Stormy clot formation with litmus milk medium Culture on egg yolk medium shows opaque zones Rapid diagnostic test : Naglar’s reaction is positive Isolation of Clostridium perfringens Anaerobic jar β- haemolysis of Clostridium perfringens Gram positive bacilli with subterminal spores Lecithinase activity of Clostridium perfringens showing opaque zones on egg yolk agar Double zone of β-haemolysis on blood agar Rapid diagnostic test Nagler’s reaction shows Opacity around streak on right. Antibody against α-toxin inhibits activity around left streak. Stormy clot formation Lactose IV) Identification Fermentation Acid Gas Stormy clot Coagulation milk protein Clot Stormy clot reaction by Clostridium perfringens on litmus milk Laboratory demonstrations Clostridium group ❑ Gram stained film of Cl. Tetani in culture ❑ Gram stained film of Cl. Perfringens in culture ❑ Robertson’s cooked meat medium ❑ Anaerobic jar ❑ Naglar’s reaction test (photo) ❑ Stormy clot reaction of Cl. Perfringens ❑ Gram positive branching bacilli of Actinomyces israelii ( photo) Th organism was isolated from deep wound following a car accident. The patient was diagnosed as tetanus. A stained smear from deep wound showed gram positive bacilli with terminal spores. A. What is the causative organism? …………………………………… ………………………………… B. What is the essential medium used for isolation of organism? …………………………………. …………………………………………………… 116 Prof. Haneya Anani What are the differences between Cl. Tetani & Cl. Perfringens ? Cl. Tetani Cl. Perfringens Spore shape Disease Naglar’s reaction Prof. Haneya Anani Questions 1.Is the presence of Gram positive bacilli with drum stick spores in direct wound smear is diagnostic for tetanus ……………………………………………………………………. 2. What is rapid diagnostic test for diagnosis of gas gangrene? …………………………………………………………………………3. What is the characteristic morphology of Cl. perfringens ? ………………………………………………………………………………… 4. What is the characteristic morphology of Cl. tetani? ………………………………………………………………………… 5.Where is the commensal Cl. perfringens found? ………………………………………………………………………………………. 118 Prof. Haneya Anani A 65 year-old woman came to clinic with history of old dental caries and presented by cervicofacial abcess commonly at the angle of the lower jaw, with multiple sinuses. Microscopic examination of pus showed a Gram-positive branching bacilli. A. What is the causative organism? ………………………………….. B. What is the name of device ? ………………………………….. Th organism was isolated from lacerated wound following a car accident. The patient was diagnosed as gas gangrene. A stained smear from wound showed gram positive bacilli with subterminal spores. A. What is the causative organism? ……………………………………… …………………………… B. What is the essential medium used for isolation of organism ? …………………………………. 120 Prof. Haneya Anani Lab. No. 8 Laboratory identification of Listeria & Haemophilus Learning Outcomes 1. Know the disease caused by Listeria & Haemophilus 2. Describe the laboratory identification 3. Interpret the results of case study Prof. Haneya Anani 121 Listeria monocytogenes Morphology & Culture ❑ Gram positive bacilli arranged in short chain ❑ Non- capsulated ❑ Motile ❑ Non- spore forming ❑ Listeria is cold-tolerant and can grow Gram positive bacilli in refrigerated drink and food Prof. Haneya Anani 122 Listeria monocytogenes cause Neonatal listeriosis (neonatal bacteremia , sepsis and meningitis) Adult listeriosis: meningitis and gastroenteritis Laboratory identifications : Blood culture CSF culture Grows well on blood agar; colonies produce a narrow zone of hemolysis similar to Group B Streptococcus The colonies identified by Direct smear of specimen shows gram positive bacilli Catalase test : positive They have tumbling motility at 25 C; "umbrella" type Prof. Haneya Anani 123 Differentiating characteristics between L. monocytogenes and Diphtheroids Species Catalase Hemolysis Motility Esculin hydrolysis L. monocytogenes Beta hemolytic Diphtheroids Variable Prof. Haneya Anani 124 Haemophilus influenzae Normal habitat Present in the nasopharynx 75% of healthy children and adult, H. influenza type b (Hib) 3-4% Haemophilus species. Haemophilus influenza : meningitis Haemophilus parainfluenzae : urethritis Haemophilus aegyptius (associated with eye infection Haemophilus ducreyi causes chancroid (sexually transmitted disease) Haemophilus influenzae Pathogenicity : H. influenza is the most common etiologic agent of acute bacterial meningitis young children Haemophilus “ Loves Heme” Cell morphology: H. influenzae are Gram negative coccobacilli that are non motile, non spore forming and have polysaccharide capsule. Haemophilus influenzae Culture and growth characters: H. influenzae is fastidious requires (factor X) and factor V) for growth chocolate agar is a good medium which provide both X and V factors. X factor : hemin V factor: nicotinamide adenine dinucleotide Grow best at 35-37 C , pH 7.6 & 5% CO2 is necessary for growth. Aerobic or facultative anaerobic It does not grow on normal blood agar. Colonies of H. influenzae on chocolate agar (left) H. influenza require nutrient supplementation on chocolate agar ,smooth , Greyish minute colonies and no growth on blood agar ( right) Laboratory diagnosis Specimens are pus, sputum or CSF. 1-Gram staining of CSF commonly reveals pleomorphic, gram-negative coccobacilli 2- Direct detection of H. influenza Type b capsule in specimens either by the o Capsular antigen may be detected in CSF or other body fluids using latex agglutination, ELISA o Detection of capsular polysaccharides (Quellung reaction) Culture; on chocolate agar at in 5% CO2. Colonies are identified by their morphology Satellite phenomena Satellitism is growth of Haemophilus in the vicinity of staphylococcal growth on blood agar Laboratory identification of Haemophilus ducreyi Lab. diagnosis of Chancroid Gram negative coccobacilli school fish appearance 1. Swab from lesion 2. Serum Direct smears made from the lesion, show gram-negative coccobacilli (school fish) Culture on blood agar shows beta hemolysis on rabbit blood, non-hemolytic on horse blood No hemolysis on Culture on chocolate agar horse blood agar Direct immunofluorescence PCR Detection of IgG or IgM antibodies by ELISA is very helpful in diagnosis 131 Prof. Haneya Anani Flat colonies on chocolate agar Prof. Haneya Anani Laboratory identification of Hemophilus ducreyi Swab Gram stained film shows Gram negative coccobacilli ( school No hemolysis on Flat colonies on fish) horse blood agar chocolate agar Serum sample Culture of H. ducreyi on blood agar Indirect immunofluorescence of 132 Gram negative coccobacilli ELISA plate H. ducreyi Laboratory demonstrations Listeria & Haemophilus influenzae ❑Gram stained film of Gram negative coccobacilli ❑Gram stained film of Gram positive bacilli ❑Chocolate agar for Haemophilus influenzae ❑Blood culture bottle ❑Catalase test ❑Motility test ❑Bile esculin hydrolysis ❑Satellitism (photo) Drawing Gram positive bacilli Gram stained film of Clostridium tetani Gram stained film of coccobacilli Satellitism growth of H. influenzae on blood Prof. Haneya Anani agar The film was isolated from patient with neonatal meningitis A. What is the causative organism ? ………………………………………….. B. What is the result of catalase test? 10/6/2024 Prof. Haneya anani 135 Questions 1.What is the name of enriched medium that contain X & V factors? ……………………………………………………………………………… 2.Haemophilus influenzae is the causative organism of ……………… ……………………………………………………………………… 3. Haemophilus influenzae is ……………………………………bacilli 4.What are the direct tests for identification of H. influenzae ? ………………………………………………………………………………… 5.What is the growth result of H. influenzae on blood agar ? ……………………………………………………………………… Prof. Haneya Anani 136 Drawing Actinomyces israelii Stormy clot reaction What are the differences between Listeria monocytogens and Diphtheroids? Listeria monocytogens Diphtheroids Microscopic appearance Blood agar Catalase test Motility test Prof. Haneya Anani 137 Model answer of cases study 1. Surgical wound – Staph aureus 2. Skin scalded syndrome– Staph aureus 3. Impetigo -Staph aureus 4. Pneumonia - pneumococci 5. Tonsilities Strept. pyogenes 6.Meningitis – Neisseria meningitidis 7.Diphtheroid 8.Tuberculosis 9.Cutaneous anthrax 10. Tetanus 11.Actinomycosis 12.Gas gangrene- Cl. perferingens Prof. Haneya Anani 138 References 1 - Jawetz, Melnick, & Adelbery’s Medical Microbiology. Authors: Geo F. Brooks; Janet S. Butel; Stephen A. Morse. Chapter 7. 27th International ed. McJraw-Hill Education 2 - Bauman, Robert, Microbiology with diseases by body system , (2018), 5th ed., Pearson. 3 - Microbiology practical notes (Departmental Book) Authors: Mostafa E, Ayoub A, Nabil Z, Maklad S. Professors of Microbiology , Faculty of Medicine ( Girls) Al- Azhar University. 4. Keith Struthers. Clinical Microbiology. © 2017 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business. International Standard Book Number-13: 978-1-49878689-8 (Pack – Book and E book) 5.Baliey & Scott’s diagnostic microbiology, fifteenth edition. ISBN: 978-0-323- 68105-6. Copyright © 2022 by Elsevier, Inc. International Standard Book Number: 978-0-323-68105-6 Prof. Haneya Anani 139 Date Attendance Lab.1 Lab.2 Lab.3 Lab.4 Lab.5 Lab.6 Lab.7 Lab.8 140 Prof. Haneya Anani Student Reflection The reflection is a self-assessment for your learning activities during the different classes. You are asked to answer the following questions in a narrative manner. 1.What Did I learn? 2.What went well? 3.What went fault? 4.What is your learning plan? ………………………………………………………………………………………………… …………………………………… ………………………………………………………………………………………………… …………………………………………………………….…………………………………… ………………………………………………………………………………………………… ……………………….………………………………………………………………………… …………………………………………………………………………………….…………… ………………………………………………………………………………………………… ……………………………………………….………………………………………………… ………………………………………………………………………………………………… ………….……………………………………………………………………………………… ……………………………………………………………………….………………………… …………………………………………………………………………………………… 141

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