Labquality External Quality Assesment Programmes General Bacteriology 1 1/2010

Labquality External Quality Assesment Programmes General Bacteriology 1 1/2010 Photos and text: Markku Koskela, M.D., Ph.D. Clinical microbiology specialist Oulu, Finland

Sample 1/2010 Pus from an infected wound of a 75 years old male after a broken leg accident Finding: Klebsiella pneumoniae Lab exercises: Isolation and identification of the pathogen. Antimicrobial susceptibility tests.

Sample 1/2010. Klebsiella pneumoniae Growth on sheep blood agar and on chocolated sheep blood agar in 5% CO 2, at +35 C after 24 hours. ->Large and mucoid colonies

Sample 1/2010 K.pneumoniae 1 2 3 4 5 6 Fermentation tests findings: 1) Glucose / gas: + / - 2) Indole - (colorless) 3) motility - (turbidity only in the inoculation area) 4) citrate + (blue) 5) urea + (red) 6) TSI: +/+/- (yellow/ yellow / no gas, H2S -).

Sample 1/2010. K.pneumoniae + - +, - + -, + - -, + - +, + + +, + + +, + + - Oxid API20E 5215773 Klebsiella pneumoniae (97,6%).

Sample 1/2010. Klebsiella pneumoniae Aerobic growth on Cled agar at +35 C after 24 hours. ->Large, mucoid and lactose positive colonies.

Sample 2/2010 Pus from an infected axillary excema Findings: Group A beta-haemolytic streptococcus (GAS) ie. Streptococcus pyogenes and Candida albicans, as the pathogens. A diphtheroid (Corynebacterium amycolatum) as a representative of the normal bacterial flora of the skin. Lab exercises: Isolation and identification of the pathogens.

Sample 2/2010 Pus from an infected axillary eczema Growth on sheep blood agar plate in 5% CO 2 at +35 C, after 24 and 48 hours. 24 h 48 h 1.Beta-haem.streptococcus+++ 2.Something else? 1. Beta-haem.streptococcus 2. White yeast 3. Something more???

Sample 2/2010 Pus from an infected axillary eczema Growth on sheep blood agar in 5% CO 2 at +35 C, after 48 h. 48 h Gram stain of a mixed growth: yeast cells and pseudohyphae, gram positive cocci in chains (staining weakly) and some diphtheroid-like gram positive rods. (Check by 200% magnification)

Sample 2/2010 Pus from an infected axillary eczema Growth an chocolated sheep blood agar at 5% CO 2 in +35 C, after 24 and 48 hours. 24 h 48 h 1. Small and gray streptococcal colonies 2. Graywhite yeast colonies 1.Gray and green (!) streptococcal colonies 2. Graywhite yeast colonies 3. Small diphtheroid colonies??

Näyte 2/2010 Group A beta-haemolytic streptococcus (Streptococcus pyogenes) Streptococcal latex agglutination A + G - Typical beta-haemolytic streptococcal growth on a sheep blood agar plate Gram+cocci in chains

Sample 2/2010 Streptococcus pyogenes Rapid ID32Strep 14032161111 Streptococcus pyogenes (99,9 %)

Sample 2/2010. Candida albicans Germ tube test (serum test), 2 hours at +30 C -> lightmicroscopy of a native sample at 400x magnification: C.albicans posit. vrt. Candida glabrata negat. Colony gram stain: Gram positive yeast cells

Sample 2/2010 Candida albicans Strain identification on a chromogenic agar plate: Chromagar Candida (biomerieux, France) at +35 C within 24 to 48 hours. Dark greenish ( 24 h) -> dark green (48 h) C.albicans colonies

Sample 3/2010 Cardiac pacemaker electrode of a patient with endocarditis Finding: Staphylococcus lugdunensis Lab exercises: Isolation and identification of the pathogen.

Sample 3/2010: Primary culture (Staphylococcus lugdunensis) Growth on sheep blood agar plate in 5% CO 2 at +35 C, after 24 to 48 hours. ->Yellowish (24 h) to straw-coloured (48 h) and beta-haemolytic staphylococcal colonies resembling S.aureus colonies

Sample 3/2010: Primary culture (Staphylococcus lugdunensis) Growth on chocolated blood agar plate in 5% CO 2 at +35 C after 24 to 48 hours. ->Yellowish (24 h) to straw-coloured (48 h) staphylococcal colonies resembling S.aureus colonies

Sample 3/2010. Staphylococcus lugdunensis S.lugdunensis gives a positive reaction in staphylococcal agglutination tests like S.aureus, Staphaurex Remel Europe Ltd, UK S.lugdunensis cannot coagulate rabbit plasma like S.aureus ie. its a coagulase negative staphylococcus! Pasteurex TM Staph Plus, Bio-Rad, France Rabbid plasma coagulation: S lugdun negat S aureus posit

Sample 3/2010. Staphylococcus lugdunensis S aureus S lugdunensis Growth on a S aureus identification agar (SAID, biomerieux, France) within 24 hours at +35 C: S aureus green and S lugdunensis graywhite.

Sample 3/2010. Staphylococcus lugdunensis - + +, + + -, + - -, - + -, + - -, + - +, - - - API20Staph: 6312150 Staphylococcus lugdunensis ( 97 %)

Sample 4/2010 Needle puncture sample from a liver abscess of an immunocompromised patient Finding: Clostridium innocuum Lab exercises: Isolation and identification of the pathogen.

Sample 4/2010. Primary culture (Clostridium innocuum) 1 2 3 Aerotolerans test: 1. blood agar / aerobe: no growth 2. chocolated blood agar / 5% CO 2 : no growth 3. FAA/anaerobe: positive growth and a large inhibition zone around a metronidazole disk.

Sample 4/2010. Clostridium innocuum Growth on anaerobic blood agar (FAA) at +35 C after 48 hours: Entire middle-sized colonies

Sample 4/2010. Clostridium innocuum Rapid tests: Catalase: negat Spot indole: negat Gram-positive rod with terminal spores. (Gram-staining variable!)

Sample 4/2010. Clostridium innocuum Anaerobic identification (A I): Kanamycin R, vankomycin S, colistin R ie. a gram positive anaerobe (Note! It is only moderate sensitive to vancomycin ->It grew well on KVLB-plate.)

Sample 4/2010. Clostridium innocuum Anaerobic growth on egg-yolk-agar (EYA) at +35 C within 1 to 2 days: Lecithinase: negative Lipase: negative

Sample 4/2010. Clostridium innocuum - - -, - - +, - - -, - - -, - + +,+ - - RapID ANA II 040061 Clostridium innocuum (99,4 %)

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