MP title: Development of 2D-Zymogram protocols to detect and identify Stenotrophomonas maltophilia periplasmic proteases and compare their activity at 37°C vs 28°C.
Quick facts on Stenotrophomonas maltophilia (S.maltophilia)
- Previously known as Pseudomonas maltophilia or Xanthomonas maltophilia
- Sole member of the genus, Stenotrophomonas
- Gram-negative bacillus (rod-shaped) which is gram-stained pink
- Aerobic (require oxygen to grow and survive)
- Motile by means of flagella
- Found widely in environment and hospitals
- Infects immunocompromised individuals especially
Why study S.maltophilia ?
- Important nosocomial pathogen
- Highly antibiotics resistant
- Little is known of its virulence factors, genetic structure and pathogenicity
- Ageing population
- Increased use of surgical equipments
Culturing of S.maltophilia
Principle: To allow the growth of S.maltophilia on LB agar plate and to obtain single, isolated colonies of the bacterium.
- S.maltophilia isolates (in vials, frozen state)
- Disposable inoculating loops
- LB agar
- 37°C incubator
- Biosafety cabinet 2 (BSC 2)
- 70% ethanol
- Marker pen
- Biohazard bag
- Appropriate personal protection equipments (e.g. Lab coat, covered shoes and gloves)
- Set up BSC 2.
- Dry LB agar in 37°C incubator.
- Swab vials (containing S.maltophilia isolates), inoculating loops and LB agar with 70% ethanol.
- Place the necessary materials into the BSC 2 and arrange them orderly.
- Label the LB agar plate.
- Streak on the LB agar plate with an inoculating loop (with S.maltophilia).
- Dispose used inoculating loops into biohazard bag.
- Swab the LB agar plate with 70% ethanol before incubating at 37°C overnight.
- Observe for single, isolated colonies.
Explanation of methods
1) To provide a sterile environment for culturing to prevent any contamination. To protect the user from S.maltophilia (safety reason).
2) To prevent the formation of water droplets from condensation as the LB agar are stored in the fridge.
3) Aseptic techniques
4) To facilitate workflow within the BSC 2.
5) To facilitate identification of S.maltophilia isolate.
6) To transfer S.maltophilia onto the LB agar plate.
7) Used inoculating loops are considered biohazard wastes and should be disposed in biohazard bag and autoclave later.
8) To disinfect the outer surface of LB agar plate with 70% ethanol before incubation. Incubation at 37°C overnight allows growth of S.maltophilia optimally.
9) This shows that streaking was done properly (recall streaking techniques from Basic Microbiology) and the colonies will be used for subsequent experiment such as inoculation.
That's all for this week!
Tan Han Yang