Observation of Pneumococcal Phase Variation in Colony Morphology

Materials and Reagents

1. Pipette tips
   
2. Petri dishes (100 mm) (Thermo Fisher Scientific, Thermo Scientific ^TM, catalog number: 263991 )
   
3. 50 ml tubes (Thermo Fisher Scientific, Thermo Scientific ^TM, catalog number: 339652 )
   
4. 0.2 μm filter (Pall, catalog number: 4612 )
   
5. 1.5 ml tubes (Eppendorf, catalog number: 022363204 )
   
6. Spreading rods (Sigma-Aldrich, catalog number: Z376779 )
   
7. S. pneumoniae strain ST556 (Li et al., 2012)
   
8. Todd Hewitt broth (Sigma-Aldrich, catalog number: T1438 )
   
9. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
   
10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    
11. Tryptic soy agar (TSA) (BD, Difco^TM, catalog number: 236950 )
    
12. Catalase from bovine liver (Sigma-Aldrich, catalog number: C9322 )
    
13. Phosphate buffered saline (PBS) (Mediatech, catalog number: 21-040-CV )
    
14. Todd Hewitt broth with yeast extract (THY) medium (see Recipes)
    
15. TSA medium (see Recipes)
    
16. Catalase working solution (see Recipes)
    

Equipment

1. Pipettes
   
2. Dissection microscope (ZEISS, model: Stemi 2000-C )
   
3. 37 °C water bath
   
4. Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: GENESYS^TM 30 )
   
5. 37 °C 5% CO₂ incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heracell^TM 150i )
   
6. Autoclave (SANYO, model: MLS-3780 )
   
7. Analytical balance (Mettler-Toledo International, model: NewClassic ML802 )
   
8. Vortex mixer 
   
9. Centrifuge (Eppendorf, model: 5417 R )
   
10. Class II biological safety cabinet (Thermo Fisher Scientific, Thermo Scientific^TM, model: MSC-Advantage^TM Class II )
    
11. Digital single lens reflex camera (Canon, model: EOS 550D )
    
12. Small mirror with a diameter of ~5 cm
    

Procedure

1. Setup of the microscope (Video 1)
1. Position the dissection microscope ~5 cm off the bench by placing two boxes under both sides of the dissection microscope base.
   
2. Take down the illuminator from the microscope.
   
3. Fix the illuminator to the surface of the bench between the two boxes using a piece of tape, and make the direction of the light source vertically towards the observer.
   
4. Remove the lens of the camera and install the camera on the top of the microscope.
   
5. Remove the object stage that is embedded in the base of the microscope.
   
   
   
   Video 1. Setup of the dissection microscope
   
   
2. Preparation of pneumococcal colonies for observing colony morphology
1. Thaw a frozen stock of pneumococcal strain ST556 in a water bath at 37 °C.
   
2. Inoculate 5 ml THY medium with 50 μl of the thawed frozen stock.
   
3. Incubate the culture in a 5% CO₂ incubator at 37 °C for about 5 h.
   
4. Check the optical density (OD) of the culture at a wavelength of 620 nm using a spectrophotometer until the culture reaches mid-log phase (OD₆₂₀ = 0.4-0.6).
   Note: The phase-variable colonies can be formed by either bacterial cells from fresh cultures or frozen stocks. If you want to use the freshly grown culture as described in 2d to make colonies, proceed to step 2g.
   
5. Mix the bacterial culture with an equal volume of 30% glycerol in THY, and store the stocks at -80 °C for future use.
   
6. Thaw a frozen stock of S. pneumoniae strain ST556 in a water bath at 37 °C.
   
7. Dilute the culture or thawed stock with PBS at 1:10,000 dilution to approximately 10⁴ colony forming unit (CFU)/ml.
   
8. Thaw a frozen stock of catalase working solution at room temperature.
   
9. Mix 50 μl of bacterial diluted stock with 100 μl of the catalase working solution, and spread the mixture on a TSA plate.
   
10. Incubate the plate in the 5% CO₂ incubator at 37 °C for 16 h.
    Note: In this protocol, we only detected the phase variation in colony opacity of pneumococcal strain ST556. Based on our previous observations (Li et al., 2016), 6 out of 8 strains showed opaque and transparent colony phenotypes at the 16-h time point, such as ST556 (serotype 19F), TH2901 (serotype 6B), and TH2835 (serotype 14). However, TIGR4 (serotype 4) and D39 (serotype 2) displayed different colony phenotypes at the 24-h time point. The incubation time of the plates should be adjusted according to the strain you use.
    
3. Observation of pneumococcal colonies (Video 2)
1. Open the lid of the plate (Procedure 2), and put the plate with its open side down on the stage of the dissection microscope.
   
2. Turn on the illuminator, and use a small mirror under the stage of the microscope to reflect the light onto the plate.
   
3. Adjust the intensity of the light and angle of reflected light by the mirror until you can see the colonies.
   
   
   
   Video 2. Observation of pneumococcal colonies
   
   
4. Adjust the magnification of the microscope based on your needs.
   
5. Adjust the focus of the microscope to make your field clear.
   
6. Turn on the camera, and turn off the flash and automatic brightness adjustment system of the camera.
   
7. Fix the angle of the mirror by holding it steadily.
   
8. Photograph the colonies.
   

Data analysis

The representative image (Figure 1) illustrates that pneumococcal strain ST556 is capable of spontaneous phase variation in colony opacity, resulting in the formation of opaque and transparent two types of colony morphologies on the TSA plate supplemented with catalase.


Figure 1. A photograph of the phase variation between opaque and transparent colonies in a clonal population of S. pneumoniae strain ST556. Colonies were produced on a TSA plate supplemented with catalase after 16-h incubation and observed under a dissection microscope as described above. The representative opaque and transparent colonies are indicated by red and blue arrowheads, respectively. The scale bar was shown at the bottom right of this image (Li et al., 2016).

Recipes

1. THY medium
1. Dissolve the powder of Todd Hewitt broth (TH) and yeast extract (Y) in ddH₂O (30 g TH and 5 g Y/L)
   
2. Autoclave the medium at 121 °C for 15 min
   
3. Store the medium at 4 °C
   
2. TSA agar plates
1. Dissolve the TSA powder in ddH₂O (40 g/L)
   
2. Autoclave the medium at 121 °C for 15 min
   
3. Pour 15-20 ml TSA medium into each dish
   
4. After solidification, use plates directly or store at 4 °C
   
3. Catalase working solution (40,000-100,000 U/ml)
1. Weigh 0.2 g catalase using an analytical balance
   
2. Dissolve the catalase from 3a in 10 ml PBS in a 50 ml tube by vortex for 1 min
   
3. Centrifuge the solution at 12,000 x g for 10 min at 4 °C
   
4. Sterilize the supernatant from 3c by filtration using a 0.2 μm filter
   
5. Aliquot the solution into 1.5 ml tubes
   
6. Store the tubes at -80 °C
   Note: Avoid using the freeze-thawed catalase solution.
   

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (No. 31530082; No. 31530082) and the Grand Challenges Exploration of the Bill and Melinda Gates Foundation (No. OPP1021992). We thank Dr. Jeffrey N. Weiser (Department of Microbiology, New York University School of Medicine, New York, USA) for sharing his experience about observation of pneumococcal colony morphology.

References

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