Journal of Postgraduate Medicine
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Year : 1982  |  Volume : 28  |  Issue : 2  |  Page : 88-91  

Agar-gel double diffusion for Lancefield grouping of beta haemolytic streptococci.

AD Mondkar, SS Kelkar 

Correspondence Address:
A D Mondkar

How to cite this article:
Mondkar A D, Kelkar S S. Agar-gel double diffusion for Lancefield grouping of beta haemolytic streptococci. J Postgrad Med 1982;28:88-91

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Mondkar A D, Kelkar S S. Agar-gel double diffusion for Lancefield grouping of beta haemolytic streptococci. J Postgrad Med [serial online] 1982 [cited 2023 Jun 6 ];28:88-91
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Progress in science depends on the development of newer and better analytical methods. The last two decades have seen a veritable explosion of newer immunological techniques; there is now a plethora of tests for antigen-antibody reactions. For Lancefield-grouping of streptococci, the relatively crude capillary precipitation method has remained the workhorse. Counterelectrophoresis was applied in this field[1], [2], [11] and we have recently described such a study.[9] Of the gel-precipitation tests, agar gel double diffusion (AGD) is perhaps the simplest. We have earlier found out that thin gels and large wells in AGD increase sensitivity for hepatitis B antigen detection.[5] During the last two years we have had occasion to Lancefield-group many beta haemolytic streptococci. The literature contains only two references to AGD in this field.[6], [12] This short communication. describes our experiences with AGD as compared with the classical capillary precipitation test[7] for Lancefield-grouping of streptococcal isolates.


Streptococcal strains

A total of 500 strains of beta haemolytic streptococci isolated in the Microbiology laboratories of the Grant Medical College, Bombay from various clinical materials, were studied. Isolation was by standard methods.[8]

Streptococcal grouping sera

Antisera to Lancefield-groups A, B, C and G were raised in rabbits.[8] Standard strains and antisera were obtained from the ICMR/WHO, Reference Centre at New Delhi. Each locally-raised antiserum was tested with reference antisera in AGD and was found to show a reaction of identity. AL the locally-raised antisera were tested with acid extracts of other groups of streptococci by the standard capillary precipitation test. This was carried out with a view to rule out the possibility of errors due to cross-reactions. All the sera showed monospecificity to the homologous acid extracts.

Capillary precipitation test

This was performed in capillaries of 2-3 mm diameters.[3] Acid extracts of streptococcal isolates were overlaid on an antiserum column in the capillary and readings taken after two minutes.

Agar gel double diffusion test[5]

The gel was made in barbitone acetate buffer (pH 8.6 molarity 0.05 M) by heat dissolving 90 mg of Difco Bacto agar in 10 ml of buffer. Only one and a half ml of hot molten gel was poured on a microscope slide of 25 x 75 mm. Wells were punched in the gel with 4 mm diameter and with an interwell distance of 2-3 mm, in a five well pattern. The central well was filled with the acid extract and the peripheral ones with antibodies of groups A, B, C and G. The slides were then incubated at 40C. The results were read on a dark-ground viewing box. Observations were made daily for three days. This was with a view to detecting any minute cross-reactions between groups. In actual practice with testing strains, the method was slightly different; the central well was filled with the antibody and the peripheral ones with acid extracts of different strains. This helped in economising antisera. With all the 500 strains tested, the results were obtained within 18 to 24 hours.


[Table 1] gives the results of Lancefield grouping. There was complete conformity with both the methods and neither false positives nor false negatives were observed. Some of the strains showed cross-reactivity by both the methods; however, the standard reference strains did not show any such cross-reactions by both the methods. In this respect AGD was more sensitive. The reactivity was always a strong reaction with one antiserum and weak one with another. The group was decided on the basis of the strong reaction.

Prakash, et al[12] described a similar study, with 138 strains of beta haemolytic streptococci and found AGD to be more sensitive, reproducible, economical, reliable and easy to read. Koshi and others,[6] using this technique, grouped 170 strains of beta haemolytic streptococci for comparison with co-agglutination technique. Both the groups of workers did not observe any cross-reactions. The gel in their studies was one per cent Difco Bacto agar[12] or Noble agar[6] in saline. We have found the composition of the buffer to be important in the design of AGD.[5]

The AGD technique we have used was a sensitive one and the increase of cross reactivity observed was probably because of our preoccupation with the phenomenon. A careful study of the literature does indicate the occurrence of cross-reactivity but no possible explanation is available for it.[4], [10], [13] It would be worthwhile pointing out some innovations in the AGD techniques used by us. The majority of workers use thick gels (1 to 2 mm) made by pouring 3 ml of fluid per slide. Thin gels (0.5 to 1 mm) increased both sensitivity and clarity of the reaction a Attempts at reducing the gel-thickness further leads to a possibility of drying of the gels. Reducing the interwell distance to the practical minimum of 2 to 3 mm and increasing well sizes to the reasonable maximum of 4 mm also improves sensitivity.[5] With our experience of grouping over 500 clinical isolates of streptococci we may point out that reading AGD is very much simpler than the capillary precipitation test. Another notable advantage of AGD was the smaller volumes of reactants, particularly the antisera required. Capillary precipitation required about 50 ul of antiserum per strain to be grouped while AGD requires only one tenth of this volume, that too for four strains to be grouped.


We are grateful to Dr. K. B. Sharma, Professor of Microbiology, Lady Hardinge Medical College, New Delhi, who kindly provided the standard streptococcal strains, antisera and training to one of us in this field.


1Dajani, A. S.: Rapid identification of beta haemolytic streptococci by counter-electrophoresis. J. Immunol., 110: 1702-1705, 1973.
2Edwards, E. A. and Larson, G. L.: Serological grouping of haemolytic streptococci by counterimmuno-electrophoresis. Appl. Microbiol., 26: 899-903, 1973.
3Facklam, R. R.: In "Isolation and Identification of Streptococci". Part If. Published by U.S. Department of Health, Education and Welfare, Atlanta, Georgia. 1976, p. 9.
4Farrell, B. and Amirak, I.: Agglutination grouping of streptococci. Lancet, 2: 1082, 1976.
5Kelkar, S. S., Niphadkar, K. B. and Karve, S. R.: The agar gel double diffusion test for detection of the Australia-antigen. J. Postgrad. Med., 20: 35-40, 1974.
6Koshi, G., Thangavelu, C. P. and Brahmadathan, K. N.: The reliability and rapidity of the coagglutination technic and its comparison with precipitin technic in the grouping of streptococci. Amer. J. Clin. Path., 71: 709-712, 1979.
7Lancefield, R. C.: Micro precipitin technique for classifying streptococci Proc. Soc. Exper. Biol. Med., 38: 473-478, 1938.
8Manual: In "Manual of Reference Procedures in Streptococcal Bacteriology and Serology". Published by WHO South-East Asia Regional Office (SEARO), New Delhi, 1976, pp. 35 and 8-10.
9Mondkar, A. D. and Kelkar, S, S.: Counterimmunoelectrophoresis for Lance field-grouping of streptococci. Ind. J. Med. Res., 68: 16-20, 1978.
10Ottens, H. and Winkler, K. C.: Indifferent and haemolytic streptococci possessing group-antigen F. J. Gen. Microbiol., 28: 181-192, 1962.
11Portas, M. R., Hogan, N. A. and Hill, H. R.: Rapid specific identification of group D streptococci by counterimmunoelectrophoresis. J. Lab. Clin. Med., 88: 339-334, 1976.
12Prakash, K., Chawda, S. and Sharma, K. B.: An evaluation of micro-gel diffusion techniques for grouping of beta haemolytic streptococci. Ind. J. Med. Sci., 26: 578-580, 1972.
13Saslaw, M. S., Saul, M., Jenks, S. A. and Jablon, J. M.: Beta haemolytic streptococci from several sources reactive in both group A and F antisera. J. Bact., 77: 519, 1959.

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