Practical examination
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1 Practical examination
2 I. Sterile media
3 1. Bouillon, 2. Slant agar, tube agar
4 4. Enrichment media: meat bouillon
5 3., 5., 6.: Agar, blood agar and chocolate agar plates
6 7. Selective and differentiating media c. Eosin methylene blue (EMB) Lactose + colonies Lactose - colonies
7 7. Selective and differentiating media g. TCBS Vibrio cholerae
8 Transport swabs (e.g. Stuart) 8. Transport media
9 9. Blood culture
10 10. Virus transport medium
11 II. Cultures
12 Staphylococcus aureus and S. epidermidis on agar plate and blood agar plate
13 4. S. epidermidis (S) and S. saprophyticus (R) with novobiocin disc
14 5. Streptococcus pyogenes on blood agar Pinpoint colonies, strong β-hemolysis!
15 6. Str. mitis on blood and chocolate plates
16 7. Streptococcus pneumoniae on blood agar plate (optochin S)
17 8. Haemophilus influenzae on chocolate agar Morning dew colonies, running nose smell!
18 9/12. E. coli and Klebsiella on agar plate
19 9/12. E. coli and Klebsiella on EMB plate
20 10. Proteus on agar and blood agar (swarming!)
21 11. Pseudomonas on agar plate Green pigment!!! Pleasant odour Mucoid colonies
22 13. Salmonella on bismuth sulphite medium
23 14. E. coli and Salmonella on brillant green medium
24 15. E. coli and Shigella on DC medium
25 16. Faeces of patient with dysentery on DC medium (E. coli + Shigella)
26 18. Urease test NH 2 -CO-NH 2 (ureum) CO 2 + NH 3 (ammonia) Christensen medium: indicator (phenol red) urease +: purple urease -: citric yellow Urease +: Proteus, Klebsiella, Helicobacter
27 19. Corynebacterium on Clauberg and Löffler medium Clauberg Löffler
28 20. Mycobacterium tuberculosis on Löwenstein-Jensen medium
29 21. Leptospira in Korthof medium
30 22. Bacillus cereus on agar and blood agar plate
31 Clostridium tetani and C. perfringens in Holman and thioglycolate media
32 GasPak
33 III. Reactions and methods
34 1. Elek test Aim: test for toxin production Filter paper with antitoxin Precipitation arch Corynebacterium
35 2. Widal-type reaction Gruber-Widal: abdominal typhus (Salmonella) Weil-Felix: epidemic typhus (Rickettsia) Wright: Brucella / Francisella With Proteus OX19 antigen! titre: the smallest amount of antibody, which can cause positive reaction!!!
36 3. Wasserman test for syphilis (CF) Positivity: no hemolysis 1 2 2: 4-fold increase in titre compared to 1 - indicates infection
37 Paul-Bunnell test Mononucleosis infectiosa EBV: P-B positive CMV: P-B negative Patient s antibodies agglutinate sheep rbc
38 3. Hemagglutination inhibition (HI) (Rubella)
39 4. Hemagglutination (HA) and -inhibition (HAI) hemagglutination hemagglutination inhibition CF test hemolysis (-) no hemolysis (+)
40 5. ELISA tests: HIV-1 and HIV-2
41 5. ELISA tests: Hepatitis Anti-HAV (competitive ELISA) Anti-HCV (indirect ELISA) HBsAg (sandwich ELISA) Anti-HBsAg (competitive ELISA)
42 8. Phage typing
43 10. Steps of Gram staining 1/a. Crystal violet 2 1/b. Lugol solution 1 2. Differentiation with 96 % ethanol 3. Washing with water 4. Safranine 1 5. Washing 6. Drying 7. Microscopic examination (immersion)
44 11. Steps of Ziehl-Neelsen staining acid fast staining Carbol-fuchsin (on filter paper!) heating 3x (until steaming) Differentiation: acidic alcohol 3% HCl, 96% ethanol acid fast thorough water washing counter staining 1-2 methylene blue or malachite green careful rinsing, drying with filter paper
45 Antibiotic susceptibility test with disc diffusion ( antibiogram )
46 IV. Microscopic preparations
47 1.a Staphylococcus (Gram +)
48 1.b Streptococcus pyogenes (Gram +)
49 1.c Streptococcus pneumoniae (G +)
50 1.d Neisseria (Gram -)
51 1.e E. coli (Gram -)
52 1.f Haemophilus influenzae (Gram -)
53 1.g Bacillus (Gram + spore forming)
54 1.h Clostridium tetani and perfringens (Gram + spore forming)
55 1.i Corynebacterium - Neisser staining
56 2.a Neisseria gonorrheae in pus (methylene blue staining)
57 2.b Candida albicans from culture
58 3. Silver impregnation: Leptospira
59 4. Ziehl-Neelsen staining: Mycobacterium tuberculosis
60 5.a Plasmodium falciparum in thin blood film (Giemsa staining)
61 5.b Trypanosoma brucei in thin blood film (Giemsa staining)
62 6. Anorectal cellulose-tape preparation (Enterobius)
63 VI. Tests performed by the students
64 Catalase test Catalase +: Staphylococci Catalase -: Streptococci performance: adding a few drops of H 2 O 2 to the culture, strong bubbling if positive H 2 O 2 H 2 O + O 2
65 Clumping test - Coagulase test Coagulase +: Staphylococcus aureus Coagulase -: all other Staphylococci ( CNS ) Staphaurex : latex agglutination performance: mixing bacteria with fibrinogenbound latex particles on microscope slide clumping
66 Oxidase test Oxidase +: Pseudomonas, Neisseria, Vibrio, Campylobacter purpose: detection of cytochrome-oxidase enzyme diagnostic use: differentiation of obligate aerobes performance: filter paper on microscope slide add oxidase reagent (= parephenylene-diamine derivative) add bacteria positivity: pink / purple colour + -
67 Indol test triptophane indol (triptophanase enzyme) Indol +: E. coli amylalcoholic (=organic) reagent indol is dissolved here after mixing intensive red ring on top (alcohol is lighter then water!) can be performed in ureumindol tube
68 6. Slide agglutination with E. coli Bacterium suspension (control) NEGATIVE POSITIVE White particles, clear solution
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