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

2.  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

3. 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

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

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

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

7. Day 3 – Virology IHA

8. Day 2 – Virology PCR

9. 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.

10. 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

11. 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 )

12. Identification gallery

13. 5 2 1 5 7 7 3 - Code n°: 5 215 773 See the index for identifying the strain by using the code. Results of the gallery: Report the results on the identification sheet

14. 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

15. 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