Labquality External Quality Assessment Programmes General Bacteriology 1 4/2010

Labquality

External Quality Assessment

Programmes

General Bacteriology 1

4/2010

Photos and text:

Markku Koskela, M.D., Ph.D.

Clinical microbiology specialist

Oulu, Finland

Sample 13/2010

Cerebrospinal fluid

Meningitis in an adult patient

Finding:

Streptococcus pneumoniae ie

pneumococcus as the pathogen

Lab exercises:

Isolation and identification of the pathogen.

Antimicrobial susceptibility tests.

Sample 13/2010. Pneumococcus

Growth on sheep blood agar and on chocolated blood agar in 5% CO₂, at +35 °C after 24 hours. -> An alpha-haemolytic streptococcus

Sample 13/2010. Pneumococcus

Pneumococcus: sensitivity to optochin as a diagnostic test.

Gram+diplococcus ”Lancet shaped”

Bile solubility: positive Negat

control Test

Sample 14/2010

Pus from leg ulcer of an old farmer

Expected findings:

Normal bacterial flora of the skin containing

group B beta-haemolytic streptococcus (GBS)

ie Streptococcus agalactiae as the pathogen.

Lab exercises:

Sample 14/2010

Primary culture

Growth on sheep blood agar and on chocolated blood agar in 5% CO₂, at +35°C after 24 hours.

Sample 14/2010

Primary culture

Growth on sheep blood agar and on chocolated blood agar in 5% CO₂, at +35°C after 48 hours.

Sample 14/2010

GBS ie Streptococcus agalactiae

Pure growth on sheep blood agar plate after 24 hours.

Catalase: negat Gram+diplococcus or cocci in chains

Streptococcal latex-test.

Sample 14/2010

Coagulase negative staphylococcus

(Staphylococcus haemolyticus)

Staphylococcal latex-coagulase teast: 1. Negat (S haemolyticus) 2. Posit (S aureus)

Pure growth on sheep blood agar plate after 48 hours.

Flat, glistening, white and slightly beta-haemolytic colonies

resempling (?) S aureus.

Sample 15/2010

Pus from frontal sinus of a 37-year-old male

Findings:

Fusobacterium necrophorum and

Prevotella melaninogenica, as pathogens.

In addition, Streptococcus viridans, representing the

normal flora of the upper respiratory tract. Copathogen?

Lab exercises:

Sample 15/2010 Primary aerobic culture

Growth on sheep blood agar and on chocolated blood agar in 5% CO₂, at +35 °C after 24 hours. -> An alpha-haemolytic streptococcus

Sample 15/2010. Primary anaerobic culture on

anaerobic blood agar plate (FAA)

48 h,

at +35°C.

A large inhibition zone around a metronidazole disk caused by anaerobes. Some anaerobes are producing a brown pigment.

Inhibition of streptococcal growth around an

aminoglycoside disk.

Anaerobes are growing close to the disk.

Sample 15/2010. Primary anaerobic culture

A brown, flat, glistening colony sensitive to metronidazole -> An anaerobe?

A gray, flat, matt colony sensitive to metronidazole ->An anaerobe?

Single colonies on anaerobic FAA plate.

A white colony: most probably a facultative anaerobic streptococcus.

Sample 15/2010. Primary anaerobic culture

Additional incubation of FAA-plate up to

5

days

A brown, flat and glistening colony (Prevotella sp)

A gray, flat and matt colony (Fusobacterium sp)

Sample 15/2010.

Prevotella melaninogenica

Anaerobic growth on Kanamycin-vankomycin-laked blood-agar (KVLB)

2-5 days

Pigment production not as strong as on FAA plate

Sample 15/2010.

Primary anaerobic culture:

EYA-plate

Lipase positive colonies on Egg yolk agar (EYA). (Fusobacterium)

2 days

Sample 15/2010. Aerotolenrance test (

48

h, +35°C)

1 (Fusobacterium sp) and 2 (Prevotella sp) are growing only

anaerobically. Streptococcus viridans (3) is facultatively anaerobic following that it is growing on all the three plates and in all the three athmospheres. 2 3 Aerobic growth on blood agar Microaerophilic growth on chocolated blood agar in 5% CO₂ Anaerobic growth on FAA-plate 1

Sample 15/2010.

Fusobacterium necrophorum

Anaerobic identification test (A I):

Sample 15/2010.

Fusobacterium necrophorum

Anaerobic growth on FAA plate, 72 h. Flat, matt, haemolytic colonies.

Positive lipase reaction slowly within (3) to 5 days

Sample 15/2010.

Fusobacterium necrophorum

Gramnegative rod Rapid indole

Sample 15/2010.

Fusobacterium necrophorum

- - - , - - - , - - - , + - - , - - - , - - +

Sample 15/2010.

Fusobacterium necrophorum

Rapid ID 32 A: 0000600000 Fusobaterium necrophorum 71%

- - - , - - - , - - - , - - - , - -

-Sample 15/2010.

Prevotella melaninogenica

FAA 72 h: Colonies with a brown pigmentation

KVLB 72 h: Slowly pigmented colonies

Sample 15/2010.

Prevotella melaninogenica

Anaerobic identification test (A I):

Sample 15/2010.

Prevotella melaninogenica

Gramnegative rod Rapid indole

Sample 15/2010.

Prevotella melaninogenica

- - + , + + - , + + + , + + - , - - + , - -

Sample 15/2010.

Prevotella melaninogenica

Rapid ID 32A: 4701050222 Prevotella melaninogenica (67,5 %)

- - + , + + + , - - - , + - - , - +

-Sample 15/2010.

Streptococcus viridans

An alpha-haemolytic streptococcus resistant to optochin.

Sample 16/2010

Amnionic fluid

Finding:

Listeria monocytogenes, as the pathogen

Lab exercises:

Sample 16/2010 Primary aerobic culture

Growth on sheep blood agar plate on 5% CO₂, at +35°C, 24 to 48 h. -> Middle-sized, beta-haemolytic, convex and glistening colonies which are catalase positive.

1 2

Catalase test:

1. Listeria monocyt. Posit 2. Negative control (Group B

beta-haemolytic

Sample 16/2010: Primary aerobic culture

Growth on chocolated blood agar plate in 5% CO₂ at +35°C within 24 to 48 hours.

->Middle-sized, convex and glistening colonies.

Sample 16/2010: Primary aerobic culture on

selective Palcam agar plate

Aerobic growth on a listeria-selective Palcam agar plate at +35°C within 24 to 48 hours -> Graygreen, esculin positive colonies.

Sample 16/2010.

Listeria monocytogenes

”Umbrella motility” at +30°C

Sample 16/2010.

Listeria monocytogenes

+ - - ,+ + +, + - -, - + - , + - -, - - -, +

RapID CB Plus: 1712101 Listeria monocytogenes (42%)

Listeria innocua (37%)

The strain is beta-haemolytic -> Listeria monocytogenes

Kata-laasi

Sample 16/2010.

Listeria monocytogenes

- + + , + - + , + - ,