MICROBIOLOGY LABWORK JPEMS 2012 NANTES Dr C. Bressollette (Virology, Nantes) Dr J. Caillon (Bacteriology, Nantes) Dr M. Eveillard (Bacteriology, Angers) Cécile Peltier (Virology, Nantes)

 Virology –Viral cultures: Observation of a cytopathic effect HSV typing by immunofluorescence –Molecular assay Amplification of a viral gene –Serologic assay: Inhibition of hemagglutination  Bacteriology  Analysis of a bacterial mixture  Gram staining  Bacterial identification  Susceptibility to antibiotics  Analysis of the environment The week schedule

Day 3 – Viral cultures  Observation of non infected adherent cells under the microscope Human fibroblasts Primary cells, limited multiplication in vitro HEP-2: epithelial cells, immortalized cell line

Day 3 – Viral cultures  Observation of infected adherent cells under the microscope Enterovirus Respiratory Syncitial Virus

Day 3 – Viral cultures  Observation of infected adherent cells under the microscope Adenovirus HSV

Day 3 – Viral cultures  Observation of infected adherent cells under the microscope Cytomegalovirus

Day 3 – Virology IHA

Day 2 – Virology PCR

Day 3 - Bacteriology Microbial environment  Air : analysis of the environmental bacteria (1 agar plate / row) –Keep opened a blood agar plate during 1 hour (exposed to the air of the laboratory)  Fingers : analysis of the cutaneous flora (1 plate for each student pair) –Produce fingerprints on the agar medium Group 1 : sterile water Group 2 : after washing your hands Group 3 : after hand rubbing with the alcohol-based gel Group 4 : after washing your hands and then hand rubbing with the alcohol-based gel.

Oral cavity : Make a sample by swabbing the oral cavity (1 sample by student pair) Isolation onto a blood agar plate (quadrant method) Perform a gram staining Environmental sample (by pair) Make a environmental surface sample by swabbing (1 sample by student pair). Isolation on a blood agar plate Microbial environment

Day 3 - Bacteriology ANALYSIS OF A BACTERIAL MIX  Reading the identification gallery –Some biochemical tests are spontaneously colored (it is not necessary to add any reagent). –However, some other tests can be interpreted only after adding a reagent: Test VP : add one drop of the reagent VP1 and one drop of the reagent VP2 (read after 10 min) Test TDA : add one drop of the reagent TDA. Test IND : add one drop of the reagent IND (James )

Identification gallery

Code n°: See the index for identifying the strain by using the code. Results of the gallery: Report the results on the identification sheet

Inhibition diameters Mueller- Hinton agar Antibiotic disks Antibiogram The inhibition diameter of the bacterial growth corresponds to the MIC and determine the susceptibility or the resistance to the antibiotic

Interpretation of the antibiograms  The interpretation of the antibiograms is performed by using charts. –Correspondence between the inhibition diameter and the susceptibility or the resistance to the antibiotic –Definition of the critical diameters D and d If diameter ≥ D : Susceptible strain If diameter < d : Resistant strain If diameter is comprised between D et d : intermediate susceptibility