3.4.1. Bacterial Cell Suspensions Preparation
The suspension of 1 x 109 CFU/mL was serially diluted (1:2, 1:4, 1:8, and 1:16) in round-bottomed microplates (see Note 2).
1. Fresh red blood cells: Blood samples obtained by venipuncture with sterile syringes were collected in sterile screw-top tubes by adding 9 vol of blood to 1 vol of sodium citrate solution (3.8%) (see Notes 4 and 5).
2. Washed red blood cells: The blood sample was centrifuged (10 min, 4500g), and the spent supernatant was discarded. The red cells were washed three times with 3 mL of saline solution and then resuspended in the same solution. Red cell concentration was determined by the microhematocrit method. This suspension was diluted to a 2% (vol/vol) concentration (see Note 6-8).
1. In a round-bottomed microplate, 50 ^L of bacterial cell suspension prepared as described in Subheading 3.4.1. were added with 50 ^L of red blood cell suspension (2%) prepared as described in Subheading 3.4.2. Then they were mixed with a circular movement for 1 min and incubated first at 37°C for 30 min and later at 4°C for 6 h (see Note 9).
2. Controls of bacterial suspension and cell red blood suspension were always prepared (see Note 10).
3. The hemagglutination titer was visually determined, and a magnifying lens was used at the limit points. The results were expressed as the inverse of the highest dilution of bacteria producing agglutination.
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