1. Preparation of Cell Suspension for Typing

1. Using a sterile disposable 1 µl loop, take a small sweep of fresh overnight pure culture of pneumococci grown on solid nonselective horse blood agar and inoculate 100 µl Heart Infusion (HI) broth. The suspension should appear to be just visibly turbid. Gently agitate the tube to emulsify.
   Note: Other media such as Brain Heart Infusion broth may also be suitable. Addition of serum (e.g. a final concentration of 10% (v/v) horse serum) and incubation at 37 °C for approximately 1 hr before use may be used to enhance the reaction.

2. Checking Density of Cell Suspension

The cell suspension prepared in step 1.1 will also be used as the negative control, and should be checked for cell density before adding the typing sera.

1. Using a 1 µl loop, transfer a drop of the inoculum prepared in step 1.1 onto a glass microscope slide.
2. Using a pair of forceps, place a small circular coverslip on the suspension.
3. Using phase-contrast setting, observe the preparation microscopically under 400X magnification, to check the density of the suspension (Figure 1A).
   1. If the inoculum is too heavy (Figure 2A), add more broth to the cell suspension made in step 1.1 to dilute it. Gently mix again.
   2. If the inoculum is too light, for example, there are <50 cells visible in the microscopic field (Figure 2B), add more bacteria to the cell suspension preparation made in step 1.1 and gently mix.
      Note: With experience, the density of the cell suspension can be judged by shaking and holding up to the light, where it should appear to be just visibly turbid.
4. Repeat steps 2.1-2.3 until a suitable number of cells are visible in the microscopic field.
   Note: Glass slides and coverslips present a sharps hazard. After use, slides and coverslips are contaminated with infectious material and must be disposed into a biohazard sharps container.

3. Typing

1. Transfer ~1 µl of the prepared inoculum (e.g. using a 1 µl loop) onto the glass microscope slide. Discard the loop into the biohazard sharps container.
2. Add an equal volume (1 µl loopful) of room-temperature antiserum onto the slide and mix both drops well with the loop.
3. Using a pair of forceps, place a small circular coverslip on the suspension, taking care not to touch the drop. Place the coverslip on the suspension immediately after the addition of antisera to prevent the fluid on the slide from drying out.
4. Using a microscope, observe the suspension under phase-contrast with 400X magnification. A negative Quellung reaction is observed when little or no ‘swelling’ of the pneumococcal cells can be seen at 400X magnification, similar to what is observed in the pneumococcal cell suspension with no antisera added (negative control). A positive Quellung reaction is observed when the cells appear enlarged or 'swollen' and more visible when compared to the cells in the negative control.
   Note: Equivocal reactions should be investigated further, for example by examining the reaction after 5-10 min incubation at room temperature, or by repeating the reaction with more antiserum.
5. Conduct pneumococcal serotyping with successive testing of individual antisera pools until a positive reaction is observed. Alternatively, pools can be applied in ‘rounds’ depending upon local incidence of serotypes.
   Note: Testing in ‘rounds’ ensures that the pools containing antibodies to the most common serotypes are tested first, minimizing effort and cost. Concurrent testing of several pools also provides additional negative controls, which can be useful in interpretation. If all the pools in a particular ‘round’ are negative, test the isolate with subsequent rounds of pools.
6. Test the isolate using the appropriate type or group antiserum contained within the reactive pool. Where appropriate, use factor antisera to further differentiate the serotype.
   Note: Keys to pneumococcal antisera supplied by the manufacturer are used in the selection of appropriate pool, type, group or factor antisera and interpretation of results (including any cross-reactions).
7. If negative results are obtained with all pools, test the isolate with Omniserum reagent (contains antibodies against 91 serotypes²¹). If a positive Quellung reaction is still not observed when testing a pneumococcal isolate with the Omniserum reagent, this may be because the isolate is expressing little or no capsule, or that antibodies against the serotype are not represented in the Omniserum. The latter category may include new serotypes.