I. Sterile media - Semmelweis
Transcript of I. Sterile media - Semmelweis
![Page 1: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/1.jpg)
Practical examination
![Page 2: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/2.jpg)
I. Sterile media
![Page 3: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/3.jpg)
1. Bouillon, 2. Slant agar, tube agar
![Page 4: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/4.jpg)
4. Enrichment media: meat bouillon
![Page 5: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/5.jpg)
3., 5., 6.: Agar, blood agar and
chocolate agar plates
![Page 6: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/6.jpg)
7. Selective and differentiating media
c. Eosin methylene blue (EMB)
Lactose + colonies
Lactose - colonies
![Page 7: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/7.jpg)
7. Selective and differentiating media
g. TCBS
Vibrio cholerae
![Page 8: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/8.jpg)
8. Transport media
Transport swabs
(e.g. Stuart)
![Page 9: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/9.jpg)
9. Blood culture
![Page 10: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/10.jpg)
10. Virus transport medium
![Page 11: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/11.jpg)
II. Cultures
![Page 12: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/12.jpg)
1-2-3. Staphylococcus aureus and S.
epidermidis on agar plate and blood
agar plate
![Page 13: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/13.jpg)
4. S. epidermidis (S) and S.
saprophyticus (R) with novobiocin disc
![Page 14: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/14.jpg)
5. Streptococcus pyogenes on
blood agar
Pinpoint colonies,
strong β-hemolysis!
![Page 15: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/15.jpg)
6. Str. mitis on blood and chocolate
plates
![Page 16: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/16.jpg)
7. Streptococcus pneumoniae on
blood agar plate (optochin S)
![Page 17: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/17.jpg)
8. Haemophilus influenzae on
chocolate agar
„Morning dew” colonies,
running nose smell!
![Page 18: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/18.jpg)
9/12. E. coli and Klebsiella on agar plate
![Page 19: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/19.jpg)
9/12. E. coli and Klebsiella on EMB
plate
![Page 20: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/20.jpg)
10. Proteus on agar and blood
agar (swarming!)
![Page 21: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/21.jpg)
11. Pseudomonas on agar plate
• Green pigment!!!
• Pleasant odour
• Mucoid colonies
![Page 22: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/22.jpg)
13. Salmonella on bismuth sulphite
medium
![Page 23: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/23.jpg)
14. E. coli and Salmonella on
brillant green medium
![Page 24: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/24.jpg)
15. E. coli and Shigella on DC medium
![Page 25: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/25.jpg)
16. Faeces of patient with dysentery
on DC medium (E. coli + Shigella)
![Page 26: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/26.jpg)
18. Urease test
• NH2-CO-NH2 (ureum)
CO2 + NH3 (ammonia)
• Christensen medium:
indicator (phenol red)
– urease +: purple
– urease -: citric yellow
Urease +: Proteus, Klebsiella, Helicobacter
![Page 27: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/27.jpg)
19. Corynebacterium on Clauberg
and Löffler medium
Clauberg Löffler
![Page 28: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/28.jpg)
20. Mycobacterium tuberculosis on
Löwenstein-Jensen medium
![Page 29: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/29.jpg)
21. Leptospira in Korthof medium
![Page 30: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/30.jpg)
22. Bacillus cereus on agar and
blood agar plate
![Page 31: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/31.jpg)
23-24. Clostridium tetani and C.
perfringens in Holman and
thioglycolate media
![Page 32: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/32.jpg)
GasPak
![Page 33: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/33.jpg)
III. Reactions and methods
![Page 34: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/34.jpg)
1. Elek test
Filter paper
with antitoxin
Precipitation
arch
Corynebacterium
Aim: test for toxin production
![Page 35: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/35.jpg)
2. Widal-type reaction• Gruber-Widal: abdominal typhus (Salmonella)
• Weil-Felix: epidemic typhus (Rickettsia)
• Wright: Brucella / Francisella
titre: the smallest amount of antibody, which can cause
positive reaction!!!
With
Proteus
OX19
antigen!
![Page 36: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/36.jpg)
3. Wasserman test
for syphilis (CF)Positivity: no
hemolysis
1
2
2: 4-fold
increase in
titre compared
to 1 -
indicates
infection
![Page 37: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/37.jpg)
Paul-Bunnell test
• Mononucleosis infectiosa
• EBV: P-B positive
• CMV: P-B negative
• Patient’s antibodies
agglutinate sheep rbc
![Page 38: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/38.jpg)
3. Hemagglutination inhibition (HI)
(Rubella)
![Page 39: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/39.jpg)
4. Hemagglutination (HA) and -inhibition (HAI)
hemagglutination
hemagglutination
inhibition
hemolysis (-)
no hemolysis (+)
CF test
![Page 40: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/40.jpg)
5. ELISA tests: HIV-1 and HIV-2
![Page 41: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/41.jpg)
5. ELISA tests: Hepatitis
• Anti-HAV
(competitive ELISA)
• Anti-HCV (indirect
ELISA)
• HBsAg (sandwich
ELISA)
• Anti-HBsAg
(competitive ELISA)
![Page 42: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/42.jpg)
8. Phage typing
![Page 43: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/43.jpg)
10. Steps of Gram staining
1/a. Crystal violet 2’
1/b. Lugol solution 1’
4. Safranine 1’
2. Differentiation with 96 % ethanol
3. Washing with water
5. Washing
6. Drying
7. Microscopic examination (immersion)
![Page 44: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/44.jpg)
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
![Page 45: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/45.jpg)
Antibiotic susceptibility test with
disc diffusion (“antibiogram”)
![Page 46: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/46.jpg)
IV. Microscopic preparations
![Page 47: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/47.jpg)
1.a Staphylococcus (Gram +)
![Page 48: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/48.jpg)
1.b Streptococcus pyogenes (Gram +)
![Page 49: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/49.jpg)
1.c Streptococcus pneumoniae (G +)
![Page 50: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/50.jpg)
1.d Neisseria (Gram -)
![Page 51: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/51.jpg)
1.e E. coli (Gram -)
![Page 52: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/52.jpg)
1.f Haemophilus influenzae (Gram -)
![Page 53: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/53.jpg)
1.g Bacillus (Gram + spore forming)
![Page 54: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/54.jpg)
1.h Clostridium tetani and perfringens
(Gram + spore forming)
![Page 55: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/55.jpg)
1.i Corynebacterium - Neisser staining
![Page 56: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/56.jpg)
2.a Neisseria gonorrheae in pus(methylene blue staining)
![Page 57: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/57.jpg)
2.b Candida albicans from culture
![Page 58: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/58.jpg)
3. Silver impregnation: Leptospira
![Page 59: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/59.jpg)
4. Ziehl-Neelsen staining:
Mycobacterium tuberculosis
![Page 60: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/60.jpg)
5.a Plasmodium falciparum in thin
blood film (Giemsa staining)
![Page 61: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/61.jpg)
5.b Trypanosoma brucei in thin
blood film (Giemsa staining)
![Page 62: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/62.jpg)
6. Anorectal cellulose-tape
preparation (Enterobius)
![Page 63: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/63.jpg)
VI. Tests performed by the
students
![Page 64: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/64.jpg)
Catalase test
• performance: adding a few drops of
H2O2 to the culture, strong
bubbling if positive
• H2O2 H2O + O2
Catalase +: Staphylococci
Catalase -: Streptococci
![Page 65: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/65.jpg)
Clumping test - Coagulase test
• “Staphaurex”: latex agglutination
• performance: mixing bacteria with fibrinogen-
bound latex particles on microscope slide
clumping
Coagulase +: Staphylococcus aureus
Coagulase -: all other Staphylococci (“CNS”)
![Page 66: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/66.jpg)
Oxidase test
• performance:
– filter paper on microscope slide
– add oxidase reagent
(= parephenylene-diamine derivative)
– add bacteria
– positivity: pink / purple colour
Oxidase +: Pseudomonas, Neisseria, Vibrio, Campylobacter
• purpose: detection of cytochrome-oxidase enzyme
• diagnostic use: differentiation of obligate aerobes
+ -
![Page 67: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/67.jpg)
Indol test
• triptophane indol
(triptophanase enzyme)
• amylalcoholic (=organic)
reagent indol is dissolved
here after mixing intensive
red ring on top (alcohol is
lighter then water!)
• can be performed in ureum-
indol tube
Indol +: E. coli
![Page 68: I. Sterile media - Semmelweis](https://reader033.fdocuments.in/reader033/viewer/2022042811/6268e110d29e7b69436650db/html5/thumbnails/68.jpg)
6. Slide agglutination with E. coli
Bacterium
suspension
(control)
White
particles,
clear
solution
NEGATIVE POSITIVE