Well Slide with Glycerin Phytoplankton Identification and Enumeration Method - Dennis R. Hill – Microbiologist – Des Moines Water Works 

This well slide with glycerin phytoplankton identification and enumeration method was developed to greatly reduce the time and effort in conducting phytoplankton studies.

This method concentrates by evaporation a small aliquot of sample that ends up distributed throughout a thin layer of glycerin held within a 5mm diameter well.  The entire sample aliquot is counted.  This method adapts to conventional style microscopes, but will also work on the inverted styles required by settling methods.

1. Materials
   1. Wells slides.  (Fisher ER-202W)
   2. 1:40 mixture of glycerin and 0.1N Hydrochloric acid.
   3. Slide warmer, heat block, or low wattage hairdryer with low power setting (Conair cordkeeper1875 or comparable).
   4. Microscope, preferably with a 20x objective lens and enhanced optics such as DIC or phase contrast.
   5. Microtiter pipet and tips
   6. Two-key lab counter (Becton Dickinson, Fisher Scientific 02-670-12)
2. Procedure
   1. Add 10ul of a 1:40 mixture of glycerin and 0.1N Hydrochloric acid to a well slide. (A 1:40 ratio is ideal for samples where there are many phytoplankton cells. A 1:60 ratio will work with lesser amounts of cells.) (The glycerin is the final matrix in which the phytoplankton are held. The acid prevents calcium carbonate crystal formation and often improves morphological appearance.) 
   2. Add 50ul of a well mixed sample to the glycerin and HCl primed slide well.
   3. Concentrate the sample through evaporation, which will take about two hours on a 35ºC slide warmer or heat block.  If a hairdryer is used for rapid processing, use the lowest setting, which will evaporate the sample in 20 minutes.  Heating a sample beyond the recommended temperature or time might damage phytoplankton cellular morphology.
   4. When the water has evaporated, the microbes remain uniformly distributed in the thin layer of glycerin, which doesn’t dry.  Do not cover slip the wells.  This provides the best microscopic resolution and prevents the addition of ubiquitous glass fragments that accompany cover slips and contaminate the microscopic field.
3. Microscopic Analysis
   1. Microscopic analysis is best performed using a 20x objective lens, paired with 10x ocular lenses.  Preferably, the microscope will be equipped with DIC (differential interference contrast) optics, or comparable image enhancing optics such as phase contrast, etc.  These criteria will allow ample magnification for viewing details, create the best lighting conditions, (which can otherwise vary because of the refractive index of the glycerol), and present an ideal field size for counting the microorganisms.  The acceptability of different technical microscopic arrangements must be assessed by the scientist making them.  If an inverted microscope is being used, place the slide onto the stage upside down.
   2. Systematically scan the slide to view and count the microorganisms.  Use the mask (slide paint) as a focusing reference.  It will be in the same focal plane as the sample, making it easy to remain focused while studying the slide and thus ensuring no cells are missed due to their presence in a different focal plane.  Furthermore, with this method, the surface of the slide will be discernible even with relatively debris-free samples.  Because each well of the slide is small, assessment of the entire sample aliquot is relatively easy.  A count of the complete well eliminates inaccuracies relating to cell distribution. These aspects of the well slide and glycerin method are what make it superior to settling and chamber-slide methods, where subjective estimates must be made. 
   3. A two-key lab counter may be used, so that easily-managed separate counts of algae and cyanobacteria may be made.
   4. A complete count of each well with an initial aliquot of 0.05ml river or lake water multiplied by twenty will give the total count per milliliter.  Individual cells within a phytoplankton filament or colony are counted, thus an Anabaena trichome might represent thirty cells, etc. 

I developed this well slide and glycerin phytoplankton method several years ago to overcome some of the cumbersome aspects of the conventional settling and chamber-slide methods.  It uses a much smaller and more appropriate volume of water and allows accurate counting of the entire sample that is processed. I have been using it to rapidly assess the various ponds and rivers from which we draw our water for processing.  I have also expanded the method to include centrifuged samples for the assessment of intermediate and final drinking water treatment stages, thus allowing a determination of the phytoplankton removal efficiency of each treatment step. 

I will be glad to discuss the method with anyone, and help you get started using the method. You may contact me at Hill@DMWW.com.

       

See also:

Hill, Dennis R., Basic Microbiology for Drinking Water, 3^rd Edition. 2014. American Water Works Association.

Proceedings from AWWA WQTC14: Taste, Odor, and Toxins: Evaluation and Response to Cyanobacteria Occurrence in Source Water, Well Slide with Glycerin Phytoplankton Identification and Enumeration Method.  Dennis R. Hill, Des Moines Water Works

Hill, Dennis R., Phytoplankton Analysis Guides Source Water Blending, May 2012, Opflow, Ammerican Water Works Association.