Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

1. Preparation of in-house Latex Reagents

1. Preparation of glycine buffered saline (GBS)
   1. Add 1.5 g glycine, 11.7 g sodium chloride and 100 ml of distilled water to a 200 ml glass beaker.
   2. Mix to dissolve using a magnetic stirrer.
   3. Transfer mixture to a 500 ml measuring cylinder and add distilled water to make a total of 200 ml.
   4. Transfer the solution back to the 200 ml beaker, check the pH and adjust to 8.2 with 5 M sodium hydroxide. NOTE: Sodium hydroxide is corrosive, wear personal protective equipment.
   5. Filter sterilize the solution using 0.22 µM filters and several 60 ml syringes into a 250 ml preautoclaved screw capped glass bottle.
   6. Store at RT.
2. Preparation of GBS with 0.2% bovine serum albumin
   1. Add 0.2 g of bovine serum albumin (BSA) powder and 100 ml of GBS to a 200 ml glass beaker and mix to dissolve using a magnetic stirrer.
   2. Filter sterilize the solution through 0.22 µM filters using several 60 ml syringes into a 250 ml preautoclaved screw capped glass bottle.
   3. Store at 4 °C.
3. Latex reagent preparation
   1. Label a 2 ml round bottom microfuge tube.
      NOTE: round bottom tubes are used to minimize particle settling which may affect antibody attachment.
   2. Using a P1000 pipette with sterile tips add 975 µl of GBS into the microfuge tube, then add 25 µl of appropriate pneumococcal antiserum for the latex reagent being prepared (i.e., pool, group, type or factor). Invert five times to mix.
   3. Dilute polystyrene latex particles 1:10 by adding 120 µl of latex particles to 1,080 µl of sterile physiological saline.
      NOTE: This can be made in larger volumes, but should be made fresh each time the latex reagents are produced.
   4. Add 1,000 µl of the 1:10 latex suspension (prepared in step 1.3.3) to the 1,000 µl 1:40 antiserum suspension (prepared in step 1.3.2). Invert five times to mix.
   5. Incubate at 37 °C on a slowly rotating wheel (e.g., 4 rpm for a wheel of 38.5 cm diameter) for 2 hr.
   6. Centrifuge for 15 min at 1,100 x g. Discard the supernatant and gently resuspend the pellet in 2 ml of sterile saline.
   7. Centrifuge for 15 min at 1,100 x g. Discard the supernatant and add 1 ml of GBS and gently resuspend the pellet.
   8. Pipette the latex suspension into a labeled 5 ml screw capped tube. Add another 1 ml of GBS.
   9. Add 2 ml of GBS containing 0.2% BSA (w/v) (pH 8.2) (prepared in step 1.2). Add 40 µl of 10% (w/v) solution of sodium azide as a preservative.
      NOTE: Sodium azide is hazardous if inhaled, and is a skin and eye irritant. It is desirable to purchase a prepared 10% solution rather than the powdered form, as this minimizes the risk of inhalation exposure. Personal protective equipment (including gloves and splash goggles) should be worn when handling this reagent. Refer to the Material safety data sheet for details.
   10. Store latex reagents at 4 °C.
4. Preparation of negative control latex reagent
   1. Label a 2 ml round bottom microfuge tube.
      NOTE: round bottom tubes are used to minimize particle settling which may affect antibody attachment.
   2. Using a P1000 pipette with sterile tips add 975 µl of GBS into the microfuge tube, then add 25 µl normal rabbit antiserum. Invert five times to mix.
   3. Proceed as in steps 1.3.3 to 1.3.10 above

2. Quality Control (QC) of Latex Reagents

1. Bring latex reagent to RT. Immediately before use, gently mix the latex reagent by inverting the tube several times.
2. Check reagent for autoagglutination by pipetting 15 µl onto a glass microscope slide and proceed as in steps 4.7 and 4.8 below. There should be no agglutination of the latex particles. The reagent should appear white and smooth.
3. Test the reagent against a panel of low passage pneumococcal isolates. Use freshly grown cultures prepared as in step 3. The panel of isolates should include isolates of the ‘target’ serotype (the specific serotype for the reagent being tested) and ‘non-target’ serotypes (isolates of other serotypes that should not normally cross react with the reagent). Include at least one isolate of each target and non-target serotype for each latex reagent undergoing QC. If there is only one target or non-target serotype, test two different isolates of the particular serotype.
   NOTE: The testing panel should include several isolates of each serotype, some of which are uncommon in invasive disease and/or type culture collections. It is preferable to include some carriage isolates, as they are often more demanding to serotype (data not shown), thereby further testing the capacity of the reagents and ensuring that the reagents are likely capable of serotyping both invasive and carriage isolates.
4. Proceed with testing of latex reagents with target and non-target pneumococcal isolates as per the method described in steps 3 and 4 below.
5. Quality control reagents at time of production and then at least annually thereafter.
   NOTE: If a reagent does not pass QC, the batch must be discarded and a new preparation made. In the event of repeat QC failure we recommend the reagents be produced using the alternate methods described in Ortika et al., 2013⁸ and in the discussion below.

3. Preparation of Pneumococcal Cultures for Serotyping

1. Using a sterile loop select a single colony of the pneumococcal isolate and streak this out onto a solid nonselective blood agar so that the primary inoculum covers approximately one third of the plate surface. Streak for single colonies.
   NOTE: Plates prepared from defibrinated horse or sheep blood are suitable; but blood plates prepared with human blood or citrated animal blood are not.
2. Incubate plate O/N at 37 °C in an atmosphere of 5% CO_2.
3. Observe the growth on the plate for purity and assess that there is sufficient growth for serotyping.
   NOTE: A pure culture of the pneumococcal isolate is required for serotyping. In our experience, confluent, or near confluent growth covering approximately one third of the plate surface is usually sufficient to complete serotyping. As pneumococcal cultures may be prone to autolysis, cultures prepared for serotyping should be tested within 24 hr of subculture. Do not take the culture plate out of the incubator more than 15 to 20 min before serotyping commences.

4. Conducting Latex Agglutination Serotyping

1. Remove all latex reagents from refrigerator and allow them to warm to RT for approximately 30 min. Immediately before use, gently invert the tubes to mix the reagents.
2. Label a microscope slide.
3. Place 15 µl of negative control latex reagent onto the microscope slide.
4. Using a sterile disposable 1 µl loop take a sweep of the pneumococcal culture so that the loop is approximately half full.
5. Gently smear the inoculum onto the surface of the slide close to the drop of negative control latex reagent. The inoculum should be visible on the glass slide.
6. Quickly and thoroughly mix the inoculum into the negative control latex reagent using the loop. Ensure that the drop does not spread further than its initial size.
7. Pick up the slide, holding it at both ends. Rock the slide back and forth for 1 min. Take care to keep the liquid moving slowly, and ensure that the size of the drop does not increase.
   NOTE: Alternatively use a plate rocker.
8. Observe for macroscopic agglutination (i.e., by naked eye) and clearing of the suspension against a black background.
9. If the negative control latex reagent shows no agglutination or clearing continue testing the culture, replacing the test latex reagents for the negative control latex reagent in steps 4.1 to 4.8 above. A fresh loop must be used each time. Proceed with testing of the latex reagents starting with the pools.
10. Test each of the 'pool' latex reagents in succession until a positive test is obtained.
    NOTE: The order of testing of the pools may be done in ‘rounds’ to reflect the local incidence of particular serotypes (e.g., by testing for the three most likely pools on the first slide. If a positive reaction is not obtained, then test the next three most likely of the remaining pools on the second slide; and so on until a positive reaction is obtained). This ensures that the most common serotypes are tested first, minimizing the time and reagents needed.
11. Using the antisera manufacturer’s key determine which individual antisera are represented in the positive pool.
12. Test each of the groups or types contained in the positive pool individually to determine the ‘group’ or ‘type’ as in steps 4.1 to 4.8 above.
13. Determine the result by reference to the antisera manufacturer’s key. If a ‘type’ is determined (e.g., serotype 5), then no further testing is needed and this result is recorded.
14. If a ‘group’ is determined (e.g., group 19), then refer to the manufacturers key to determine the factors to be tested. Continue testing with the individual ‘factor’ latex reagents and determine the final serotype (e.g., serotype 19B).