25 species of actinomycetes were tested for the occurrence of lectins. Using a battery of normal and desialized erythrocytes, each species was screened for 3 types of lectin activity i.e. surface bound, extracellular and intracellular. As many as 13 species showed one or more types of activity; some of them were characterized with regard to their biological action spectrum and sugar specificity.
Lectins have been reported in almost all groups of organisms ranging from viruses, bacteria and fungi to animals and plants ( MIRELMAN and OFEK 1986). In recent years microbial lectins have attracted attention because they play an important role in mediating various normal and pathological processes in living organisms ( SEQUEIRA 1978, SHARON and LIS 1989). Since actinomycetes, an important group of widely distributed bacteria which also comprises various plant and animal pathogens, have not yet been explored systematically for these compounds, the present investigation was undertaken with the aim of detecting lectins among 25 different species of this bacterial group.
Materials and methods
Preparation of test samples:
The cultures of actinomycetes were procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTEC), Chandigarh, India. Each culture was grown in a specific nutrient broth depending upon its nutritional requirements as specified by IMTEC. The culture was grown for 3 to 6 days depending upon its growth rate and then killed by treating with 1 % formalin for 10 min. The culture broth (free of microbial cells) was used for detecting extracellular lectins after extensive dialysis against 0.01 M phosphate buffered saline (PBS), pH 7.2, for about 3 to 4 days. The washed cell mass was subjected to low speed homogenization to make a uniform suspension. One aliquot was used for the detection of surface bound lectins, the other one was centrifuged at 400xg for 10 min and the pellet was suspended in PBS (1:l v/v). The cells were disintegrated by ultrasonic vibrations at a power of 50 to 60 watts intermittently for 10 min. This suspension was centrifuged at 20,000 x g for 30 rnin at 4 "C and the supernatant thus obtained was used for the determination of intracellular lectin activity. The samples thus prepared were concentrated by dialysis against 5% solution of polyethylene glycol 8000 for 2 to 3 days.
Erythrocyte suspension: Blood samples collected from human and animal sources were mixed with an equal volume of ALSVER'S solution or alternatively with 50 to 100 IU of heparin per ml of blood as anticoagulant. In each case the blood was diluted with PBS and centrifuged for 5 min at 400 x g. The pellet was washed thrice and then resuspended in PBS to yield a 2% suspension (3.5 x 10' cells per ml).
Neuraminidase treatment: One ml of 2% fresh blood suspension was centrifuged and the pellet was resuspended in normal saline, pH 5.0. The suspension was treated with 0.125 units of neuraminidase for 20 min at 37 "C (Warren, 1959). The reaction was stopped by adding 10 ml of PBS and washed three times with the same buffer. The final suspension was reconstituted in PBS.
Agglutination assay in microtitre plates: 50 p1 of test sample and 50 p1 of cell suspension were added to each well. The plates were incubated at 37 "C for 30 min and then stabilized at 4 "C for 2 to 3 hours.