Lancefield grouping by latex agglutination. Up: the
extraction enzyme and the A, B, C, D, F, and G group
sera. Down: group D - positive strain (left) and negative
control (right).
- Extraction reagent(s)
- A, B, C, D, F, and G latex reagents
- Reaction cards
- Pasteur pipette
- Inoculating loop
Positive result: agglutination occurs with one grouping reagent (or when one grouping reagent gives a substantially stronger reaction
than the other five), resulting a blue granular precipitate. A weak reaction with a single latex reagent should be repeated using a
heavier inoculum.
Negative result: the mix remain homogenous. Faint traces of granular material should also be considered negative.

False positive reactions due to non-specifically agglutination, may appear when testing organisms like
Escherichia coli, Klebsiella or
False negative results can occur if the quantity of antigen used in extraction is insufficient.
Some streptococcus strains may not be groupable.
The reagents may contains toxic or caustic compounds like sodium azide or thiomersal. Avoid inhalation or swallowing.

- Group A:
Streptococcus pyogenes ATCC 19615
- Group B:
Streptococcus agalactiae  ATCC 12386
- Group C:
Streptococcus dysgalactiae subsp. equisimilis ATCC 12388
- Group D:
Enterococcus faecalis ATCC 19433
- Group F:
Streptococcus spp. ATCC 12392
- Group G:
Streptococcus dysgalactiae subsp. equisimilis ATCC 12394

1. John R. Tagg, Philip A. Wescombe, and Jeremy P. Burton. Chapter 7. Streptococcus: A Brief Update on the Current Taxonomic
Status of the Genus. In: (Eds.) Sampo Lahtinen, Arthur C. Ouwehand, Seppo Salminen, Atte von Wright. Lactic Acid Bacteria:
Microbiological and Functional Aspects, Fourth Edition CRC Press, 13 dec. 2011, 123-133.
2. Prolex (TM) Streptococcal Grouping Latex Kit  - package leaflet.
3. Oxoid Streptococcal Grouping Kit  - package leaflet.
4. CDC Streptococcus Laboratory. Streptococcus general methods. Updated March, 2014. Available online. Accessed December,
Lancefield Streptococcal Grouping
Rebecca Lancefield (1933) showed that the majority of pathogenic streptococci possess specific carbohydrate antigens, which
permit the classification of streptococci into groups. The streptococcal group antigens can be extracted  in soluble form and identified
by precipitation reactions with homologous antisera. The latex agglutination method uses latex particles previously coated with
group-specific antibodies (blue polystyrene latex particles sensitized with purified group specific rabbit immunoglobulins).
1. Allow reagents to reach the room temperature. Re-suspend the latex reagents by
shaking the dropper bottle several times.
2. Extraction. Historically, different procedures for extraction of streptococcal antigens
were used: Initially Lancefield (1933) used acid extracts, later Fuller (1938) used
formamide extracts. Different extraction procedures are currently in use depending on
kit's manufacturer: two extraction reagents and a neutralizing solution (Pro Lab
Diagnostics) or a single enzymatic extraction reagent (Oxoid). Below are 2 examples:
- Oxoid method: rehydrate the extraction reagent using sterile distilled water (12ml),
distribute  about 0.4 ml of enzyme preparation in a test tube. Select 2-5 test colonies
with a bacteriological loop and emulsify in the tube. Incubate 10 minutes at 37 ºC;
after 5 minutes shake the tube vigorously then continue the incubation.
- Pro Lab method: add 1 drop of Extraction Reagent 1 to a tube. Select 1-4
beta-haemolytic colonies using a disposable loop or needle and suspend them in
the Extraction Reagent 1. Add 1 drop of Extraction Reagent 2. Mix the reaction by gently
tapping the tube with a finger for 5-10 seconds. Add 5 drops of Extraction Reagent 3 to
each tube and mix by gently tapping the tube with a finger for 5-10 seconds.
3. Dispense one drop from each latex reagent into the circular rings on the reaction
4. Using a Pasteur pipette, for each test put one drop of extract over each drop of latex
5. Homogenize the latex and the extract with a bacteriological loop or with sticks
provided in the kit, using the complete area of the circles. A new loop / stick should be
used with each test circle.
6. Gently rock the cards allowing the mixture to flow slowly over the entire test ring
area. Agglutination is normally taking place within 30 seconds. After 1 minute, any
reaction should be considered negative.
(c) Costin Stoica
Culture media
Biochemical tests
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