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.