Abstract:
Taxonomic studies of microalgae in many laboratories use drawings, photographs, and type specimens because, continuous maintenance of live specimens is mostly impractical. The most
common and easy-to-handle type-specimens for microalgae are permanent slide preparations.
Although there are established methods for preparing permanent slides for diatoms and green
algae, no such methods have been developed specifically for cyanobacteria. Therefore, the
objective of the present study was to develop a simple method for the preparation of permanent
microscopic slides for freshwater cyanobacteria. Six species were selected to represent three
forms of cyanobacteria, filamentous, unicellular colony-forming and unicellular non-colony forming for the preparation of slides. Two acidic dyes, Fast Green and Brilliant Blue were used
along with Mayer’s egg albumin adhesive agent, fixative, dehydration and clearing agents. In the
procedure with Fast Green, we used acetic acid: 96% ethanol (1:3) as the fixative in combination
with gentle heat fixation. The specimens were dehydrated using a series of acetic acid and 96%
ethanol solutions with different retention times. Finally, cleared with Xylene and mount on a drop
of Canada balsam. This method successfully stained filamentous cyanobacteria. However, it
poorly stained colony forming Microcytis. Apparently, the mucilage of the colony does not allow
penetration of the dye to reach cells that are embedded within the mucilage. When this procedure
was used for Synechococcus, which consists of single cells, those cells were loosely adhered to the
glass slide and easily washed out. Therefore, a modified method was developed with Brilliant Blue
including steps to overcome poor staining of colony forming cyanobacteria and loose adherence
of unicellular non-colony forming cyanobacteria. Specimens were fixed to the glass slide using
gentle heat and dehydrated using a series of 30-70% ethanol with various retention times. This
modified method was able to stain Microcytis, Synechococcus and filamentous cyanobacteria. Slide
preparations were found well preserved in terms of color and cellular structures when observed
after 10 months of storage at room temperature. We recommend the method developed with Fast
Green particularly for filamentous cyanobacteria while the method developed with Brilliant Blue
for all three forms of filamentous, unicellular, and colony-forming cyanobacterial species for the
preparation of permanent slides.
