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A coupe of notes to help with microbiology practical tests.
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Practical exam
1.Culture media
1.1 Blood agar: 6 bacteria
1.2. Chocolate agar: 2 bacteria
1.3. Eosin-methylen blue agar: 4 bacteria
1.4. Nutrient agar: 4 bacteria
1.1. Blood agar
α-hemolysis: not complet (biliverdin green)
β-hemolysis: complet (clear zone)
1.1.1. α-hemolytic streptococci
alfa-hemolytic streptococci (colourless colonies, with green zone)
Differentiation (only theoretical):
Bile Optochin S Mice
pathogenicity
BEA Heat resistance
(56 oC, 30 min)
S. pneumoniae + + + - -
E. faecalis - - - + +
Viridans group - - - - -
1.1.2. β-hemolytic streptococci
β-hemolytic streptococci (colourless colonies, clear zone)
Differentiation (theoretical only):
Group Bacitracin S CAMP test
S. pyogenes A S -
S. agalactiae B R +
1.1.3. Non-hemoytic streptococci
E. faecalis
E. faecalis (colourless, non-hemolytic zone)
1.1.4. S. aureus
1-2 mm diameter, beta-hemolytic zone, yellow-gold pigment.
1.1.5. CNS (coagulase negative staphylococci)
S. epidermidis novobiocin S, S. saprophyticus novobiocin R.
1-2 mm diameter, white (grayish) colonies, without hemolysis
Differentiation of staphylococci and streptococci (theoretical only):
microscopical
picture
katalase
test
pigment
production
hemolysis size of colonies
staphylococci grape-like + + β or - 1-2 mm diameter
streptococci chain/pair - - β, α, or - very small (point)
1.1.6. B. cereus
causative agent of food poisoning
1-2 cm diameter, flat, gray, dry colonies with beta-hemolysis
1.2. Chocolate agar
X (hem) and V (NAD) factors are free. H. influenzae and N. meningitidis, N. gonorrhoeae
may grow only on chocolate medium. On this agar only alfa-hemolysis may observed. (beta
never!)
Theoretical quisteion: satellite phenomenon (H. influenzae may grow on blood agar with S.
aureus) int he beta-hemolytic zone of S. aureus (the X and v factors will be free int he
hemolytic area)
1.2.1. Alfa-hemolytic streptococci
alfa-hemolytic streptococci
1.2.2. H. influenzae
colourless colonies, special smell
1.3. Eosin-methylen blue agar
Lactose positive: blue (pink) colonies
Lactose negative: colourless
1.3.1. E. coli
Dark blue dry colonies with metallic sheen. Lactose positive.
1.3.2. Klebsiella pneumoniae
Blue or pink mucoid colonies (lactose positive).
Differentiation between E. coli and Klebsiella pneumoniae
Indol Methyl Red Voges-Proskauer Citrate Motility
E. coli + + - - +
K. pneumoniae - - + + -
1.3.3. Proteus mirabilis or Proteus vulgaris
colourless (lactose negative colonies), oxidase negative
lactose motility H2S oxidase phenylalanin- deaminase urease
Salmonella - + + - - -
Proteus - + + - + +
1.3.4. Pseudomonas aeruginosa
colourless (lactose negative), oxidase positive
1.4. Nutrient agar
No hemolysis! It is suitable for detection of pigment production, or swarming.
1.4.1. S. aureus
1-2 mm diameter. yellow-gold pigment
1.4.2. Coagulase-negatív staphylococcusok (CNS)
1-2 mm diameter white or gray colonies
1.4.3. Proteus mirabilis or Proteus vulgaris
swarming, very bad smell
1.4.4. Pseudomonas aeruginosa
green pigment (diffusion onto agar), fruity smell
2. Microscopical investigation
Stained preparations should check with immersion oil (white ring).
8 slide: 6 Gram stained, 1 methylen-blue stained, 1 Ziel-Neelsen stained.
Gram positive cocci: 3
• grape like: staphylococcus
• chain: streptococcus
• pair: S. pneumoniae
Gram positive long rods:eg. Bacillus spp.
Gram-negative cocci: eg. Neisseria spp.
Gram-negative short rods: eg. E. coli
staphylococcus (grape like)
streptococcus (chain)
S. pneumoniae (diplococcus)
Ziehl-Neelsen methylen blue
2 Gram-negative (pink) 4 Gram-positive (blue or purple)
2. Helminths
1.: Ascaris lumbricoides (20-25 cm, roundworm, geohelminth, larva migration into the lung)
2. Taenia solium (3-5 m pork tape worm, hermaphroditic, number of the branches of the
uterus < 10)
3. Taenia saginata (7-10 m beef tape worm, hermaphroditic, numebr of the branches of the
uterus > 15)
4. Enterobius vermicularis (1 mm, roundworm pinworm, most frequent in Hungary among
children, perianal itching)
5. Fasciola hepatica (3-5 cm liver fluke, hermaphroditic)
6. Diphyllobotrium latum (13 m fish tape worm, hermaphroditic, 2 intermidiate hosts, loss of
B12 vitamin, megaloblastic anemia, rosette like uterus)
4. Collection of the specimen
Hemoculture bottles
It is important to obtain at least 3 specimens (with at least 30 min. between the specimens).
The specimens should be preferably taken before fever spikes (during chills). If possible,
both aerobic and anaerobic bottles should be used (3x 2 bottles, altogether). The site of
venipuncture and the plug of the bottle containing the medium must be properly disinfected.
The amount of blood injected to the bottle should be about 10 % of the liquid medium
(usually 10 ml/ bottle, for chlidren less, approximately 2-3 ml into specific „Pedi” bottle).
1. 3.
5.
2. 4. 6.
4.
Pedi bottle
Keep the specimen under conditions recommended by the manufacturer of the medium (room
temperature or 37 oC).
Uricult:
A midstream specimen taken preferably in the morning, after thorough celaning of the
external genital area.
Keep and transport it at room temperature (native urine at + 4 oC)
Feces (stool:
Keep and transport it at room temperature.
Pus, bile ear, eye etc. transport medium:
Keep and transport it up to 24-48 hours at room temperature.
Liquor (central spinal fluid, CSF):
Collect the CSF by sterile lumbar punction into sterile tube. The site of lumbar puncture must
be properly disinfected. Keep and transport it up to 1 h at room temperature or 37 oC (except
suspected Listeria monocytogenes, at 4 oC)
Sputum
The sputum taken preferably int he morning after the tooth washing. Keep and transport it up
to 1 h at room temperature.