Pseudomonas Phenotyping

 

 

Following is a protocol for rapid phenotyping of 1000s of isolates for P. aeruginosa. This can be modified for single isolate phenotyping or for other bacteria.

Bacterial archival: For phenotyping, bacteria are frozen in Cryobank 2D barcoded Matrix storage boxes. They are superior to the Nunc 2D barcoded system. The barcodes are read by a hand held Honeywell 4600G 2D barcode reader. The user created barcodes are created using Label Matrix 8.5 software. The labels are printed using Ga international labels RCL6, RCL-14 or RCL-12, depending on the tube or the petri dish.

Bacteria are also frozen in Eppendorf 2ml deepwell boxes. These deepwell plates have a brightly colored area for labeling and numbering,

Inoculation of bacteria for phenotyping: Thaw a deepwell plate, replica plate (using a V&P pintool) into a new deepwell containing 500ul buffered LB broth. Next day, make a 1:50 dilution of from deepwell grown cultures into a standard microwell plate containing final volume of 200ul minimal media. Use a pintool to transfer the cultures into phenotype plates. We use the manual 96 replicator or the 96 robotic pin tool from V and P Scientific. The robotic pintool is operated by a Perkin Elmer Janus MDT robot. When choosing a pintool, use a pin that will transfer 2ul (Cat# D02301, FP12,which can function as a Steer replicator for MIC determination).

For more phenotypes, read Vasil paper on TAT pathway. PMID: 12034867

LasR isolate (green) next to a spreader (clinical isolates)

Omni plates for phenotyping: Phenotyping are done in Omni plates from Nunc.  Omini plates are the size of 96 well plates but have a single well. Need about 40ml of media per plate.

Data acquisition and processing: Scan the bacterial plates using the transparency option in a high end scanner such as EPSON Photo 4990 and also take photographs using a digital SLR with a Macro lens. Make sure to keep the orientation and the XY coordinates of each plates constant. Most images can be analyzed by using ImagJ. ImagJ algorithms have been developed in our lab for:  Diameter analysis (eg:Colony size, swimming diameter, milk lysis diameter) and pigment color. Pigment color RGB values are converted to more perceptual HSV color mode such that visual scoring can be matched with those from the automated analysis.

For accurate color analysis, you will need to calibrate the scanner. This is a simple process. But an IT8 target from Chromix. I suggest the either Kodak or Fuji IT8 targets. Send the scanned file as a TIFF file  to Chromix. They will create a look up table called “ICC” profile. Whenever you open the files in ImagJ or Photoshop, load the lookup table as well. Your image color will modify according to values stored in the ICC profile.

For high throughput phenotyping, make sure to use barcodes to track source plates:

 

 

Phenotypes for Pseudomonas aeruginosa:

P. aeruginosa clinical isolates in milk agar plate

Milk plates-Protease assay:

  • Agar concentration: 2%
  • Milk concentration: 10%
  • Nutrient base: Nutrient agar
  • Make Milk, 20% in 500ml, autoclave for 15 min at 118C  (100g in 500ml).
  • Autoclave 20g agar and Heart infusion broth (2X) in 500ml with a stirbar for 40 min
  • Cool to 55C, mix both solutions, stir, and pour plates.

Note: Phenotype may show temperature dependency. 10% milk is better than 3% (less background). 2% agar is better than 1.5%, because it stops bacteria from spreading.

Incubate at both 37C and 30C

 


 

Swimming Plates:

P. aeruginosa clinical isolates in 0.45% LB motility agar

  • Agar concentration: 4.25% final
  • Agar: 4.25g
  • Media: Miller broth
  • Water to 1L
  • Need 10 plates per 1 EPIC box

Dry plates in the incubator at least 30 min.

Higher agar concentration allows high density array of strains.

Otherwise, they would grow into each other.

Incubate at both 37C and 30C

Twitching plates:

  • Agar concentration: 1%
  • Agar: 10g
  • Media: LB broth lacking salt
  • Plates: 150mm big petri dishes.
  • This is a difficult phenotype to score.  The colonies on the outer edges (rows A and H, columns 1 and 12 will grow larger). So make sure to inoculate many replicate plates.

Incubate at both 37C and 30C