1.

Seed the whole surface of a blood agar plate with Streptococcus pneumoniaefrom stock and incubate overnight at 37 °C. Other media that allow the growth of the bacterium, like Todd–Hewitt agar plates are also appropriate.

2.

Harvest the bacteria and inoculate 40 ml of buffered Todd–Hewitt broth. Composition: Todd–Hewitt broth supplemented with 2% yeast extract and 20 mM Tris(hydroxymethylaminoethane (Tris, pKa = 8.1). Adjust pH with HCl to 7.8 before sterilization.

3.

Incubate the culture for 6–10 h at 37 °C in an incubator at 200 r.p.m. The viability of the bacteria remains high for this period of time in buffered Todd–Hewitt broth.

4.

Centrifuge culture at 10,000 × g at 6 °C for 5 min in sterile conditions and discard supernatant. From this point, bacteria should always be below 6 °C, until they are frozen at −80 °C.

5.

Wash bacteria twice with cold phosphate buffered saline (PBS) by centrifuging at 10,000 × g at 6 °C for 5 min. Bacterial wash after freezing of the stock results in a higher variability in the number of recovered viable bacteria.

6.

Suspend bacteria in 1200 μl of cold RPMI-1640/15% glycerol.

7.

Freeze single use aliquots at −80 °C. The stock concentration usually is above 109 bacteria/ml. Quantify the colony forming units (CFU) as described below one week after freezing because the bacterial death rate is high in the first days.

Experimental infection of whole blood

A modification of the direct bactericidal test of Lancefield [1] was performed.

8.

Collect blood from healthy donors, following informed consent and approval of the protocol by the institution Clinical Research Ethics Board. Clotting was inhibited by K3-ethylenediaminetetraacetic acid (EDTA). We have not tested other inhibitors.

9.

Dilute biological samples (blood, plasma or serum, 40%) with RPMI-1640 medium (60%) and place 200 μl in 2 ml microtubes.

10.

Infect diluted samples with 103 bacteria/100 μl and incubate at 37 °C in a rotator (20 r.p.m.) for the indicated times. Slightly different rotator speeds do not influence the outcome of the experiment.

11.

After incubation, dilute 10 μl of infected blood in 90 μl of water and lyze cells in an ultrasonic bath for 3 min. We use a 35 kHz ultrasonic bath (half wave mode).

12.

Inoculate 50 μl of the released bacteria in 55 mm diameter agar plates (Todd–Hewitt broth with 0.5% yeast extract and 1.5% agar). After absorption, add an agar overlay (Todd–Hewitt broth containing 0.5% yeast extract/0.75% agar) supplemented with 100 mg/ml 2,3,5,-triphenyltetrazolium chloride, a dye indicative of cellular metabolism that stains the colonies deep red [2]. Enumerate CFU after an overnight incubation. S. pneumoniae colonies are small and difficult to visualize. The indicative dye facilitates the detection and enumeration of colonies.

Development of the method

Medium acidification plays an important role in S. pneumoniae autolysis and therefore we tested whether the addition of biological buffers at pH = 7.8 would diminish or delay the process. We tried three buffers: 3-(N-morpholino)propanesulfonic acid (MOPS, pKa = 7.20); 2(4-(2-hydroxyethyl)-1-piperazine)-ethanesulfonic acid (HEPES, pKa = 7,55) and Tris(hydroxymethylaminoethane (Tris, pKa = 8.1), adjusted with NaOH or HCl. While we found that the former buffers were toxic for the bacteria, Tris–HCl allowed S. pneumoniae growth. When bacteria were inoculated in Todd–Hewitt broth we observed that the medium supplemented with 2% yeast extract allowed more growth than with 0.5%, but in both cases the beginning of the autolysis process occurred immediately after reaching the maximum growth point, at 6 h for this strain (Fig. 1). Todd–Hewitt broth supplemented with 2% yeast extract and, additionally, with 20 mM Tris–HCl also reached a high level of growth at 6 h after inoculation, but in contrast with broth without Tris–HCl the autolysis process was delayed for 6 more hours. Using this combination of yeast extract and Tris–HCl, the maximum growth level was slightly lower than in Todd Hewitt/2% yeast extract without Tris–HCl, but similar to that in Todd Hewitt/0.5% yeast extract. This change in the growth curve allows a wider operational window with a diminished risk of manipulating bacteria that have already begun the autolysis process. Without Tris–HCl the optimum point in the curve growth before the beginning of autolysis may vary depending on the strain, but the addition of the buffer provides the confidence that for several hours many strains will not have suffered the autolysis process, avoiding the characterization of the curve growth for each strain. In all cases Todd–Hewitt modified media should be used within two weeks from preparation.

Thank you very much. I will try.