Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

The overall goal of this procedure is to serotype isolates of streptococcus pneumoniae using antisera attached to latex particles. The first step in S stereotyping, a pneumococcal isolate is to test it against the negative control latex reagent. The second step of the procedure is to test the pneumococcal isolate with the latex pool reagents until a positive reaction is observed.

The third step is to test the isolate with the groups and types present in the positive pool using the antier manufacturer's key to select the appropriate reagents to test. The final step is testing the isolate with any factor latex reagents required, and then to interpret the results using the antisera manufacturer's key to determine the final serotype. The main advantage of this technique over other methods like the que test, is that it doesn't require the use of microscope and it's relatively simple and quick to perform.

Demonstrating this procedure will be Belinda Research assistant from our laboratory. First label, a two milliliter micro fuge tube. Choose one with a round bottom to help prevent particles from settling during the antibody attachment stage.

Using a 1000 microliter pipette add 975 microliters of glycine buffered saline to the tube. Then add 25 microliters of the pneumococcal antiserum needed for the latex reagent that is being prepared. Invert this five times to mix next dilute 120 microliters of polystyrene latex particles in 1080 microliters of sterile physiological saline.

Invert this five times to mix pipette 1000 microliters of the latex suspension into the 1000 microliters of antiserum solution. Invert this five times to mix and then transfer to a 37 degree Celsius incubator and load onto a slowly rotating wheel. Let the mixture incubate for two hours after the incubation, spin down the particles at 1, 100 times G for 15 minutes.

Remove the supernatant with a 1000 microliter pipette. Discard the supernatant and resuspend the pellet in two milliliters of sterile saline. Now centrifuge the mixture again, discard the supernatant and resuspend the washed pellet in one milliliter of glycine buffered saline.

Transfer the suspension to a five milliliter screw cap tube and add one more milliliter of glycine buffered saline. Now add two milliliters of glycine buffered saline with 0.2%BSA. Then add 40 microliters of 10%sodium azide.

As a preservative, be sure to adhere to the material data sheet. Safety precautions when using sodium azide. Next, consult the antibody manufacturer's key to determine the proper quality control isolates to test against the reagent.

Then test the reagent Using these positive and negative control serotype isolates Quality control testing is very important. The process ensures that a correctly reacting reagent has been produced, which is a vital control step. Store the reagents that have passed quality control at four degrees Celsius.

Begin with preparing the cultures. Using a sterile loop, select a single colony and inoculate one third of a non-selective blood agar plate. Then streak the plate to obtain single colonies.

Incubate the plate overnight at 37 degrees Celsius in an atmosphere of 5%carbon dioxide. The next day before SYP begins. Remove the latex reagents from the fridge and allow them to come to room temperature.

Remove the plate from the incubator. The culture must be pure and growth must be confluent or nearly confluent. Over one third of the plate, invert the negative control latex reagent several times.

Remove 15 microliters and pipette onto a labeled microscope slide. Then using a sterile one microliter loop, make a sweep of the culture until the center of the loop is approximately half full. Gently smear the contents of the loaded loop onto the slide close to the drop of negative control reagent.

The inoculum should be visible on the glass. Then using the loop, mix the inoculum into the latex reagent drop. Try not to spread the drop across the slide.

Lift the slide both ends and gently rock it back and forth. Keep the liquid rotating slowly and try to keep the drop from spreading across the slide. Continue rocking the slide for one minute and then read the result.

Results are red against a dark background. The drop of negative control reagents should be unchanged with no agglutination or clearing. Once it is established that there is no reaction with a negative control reagent, start testing each of the pool latex reagents until a positive reaction is obtained.

Use the same procedure and a new sterile loop for each reagent. Usually three pools are tested on each slide. Rock the slide for one minute before assessing a reaction.

A positive latex reaction occurs when specific antibodies attached to the latex particles bind to the capsule of the pneumococcus and a gluten nation of labeled particles ensues. This is generally accompanied by some clearing of the background suspension. A negative reaction is characterized by the latex reagent suspension remaining smooth and white with no agglutination or clearing.

Occasionally, some probable negative reactions display weaker agglutination around the edge of the drop, not accompanied by clearing of the background suspension. Other probable negative reactions appear stringy with no background clearing in either of these two ambiguous cases. Repeat testing or try a different s stereotyping method.

Once a positive reaction is obtained, use the manufacturer's key to determine which groups or types are in that pool mixture. Then test the groups or types that make up this pool with the isolate until a positive reaction is observed. If a type is identified, then no further testing is needed.

However, if a group is identified, then refer to the manufacturer's key to find out which factors must be tested to identify the serotype. When the factor test results are determined, again, refer to the key to determine the final serotype. Once mastered, this simple and inexpensive technique can be used to serotype.

A pneumococcal is isolate in 10 minutes if performed correctly. We're conducting this procedure. It's important to remember to use fresh sterile loops and pipette tips for each of the tests so that you don't cross contaminate either your tests or your reagents.

After watching this video, you should have a good idea of how to prepare and use latex reagents for s stereotyping. The pneumococcus.