Counting method

1. Use a microscope slide and cover slip. Make sure that the slide and cover slip are clean.  

2. Use a P200 pipettman with a sterile tip to place 0.1 ml (“100” on pipettman dial) on slide. Lower cover slip carefully until it just touches the surface, then drop cover slip on.  

3. Place slide on stage and focus.  You may need to close one eye and focus carefully with the other, then focus the second eye by rotating the eyepiece only.  This may save you from headaches.

4. Imagine that the entire counting area (open space on slide) is divided in half by a horizontal line across the cover slip (see below).  You want to first count the top half.  Move the stage (and attached slide) so that the view is looking at the top half of the of the cover slip. Start in the left corner of the cover slip and move to the right. 

5. Count!   How much of the slide you count depends on a stepwise set of decisions.  If you have a multi-species mixture, then the decisions have to be applied separately for each species (e.g., you might count one row for Bodo, but the entire slide for rotifers).

 FIRST – if the cell-count seems very high, then move to a haphazard location in the middle of the slide and count the number of protozoa.  If the number is greater than 30, then repeat this 2 more times, then stop and write down the three numbers with “eyepiece” as the method.  If the number is less than 30, then . . . .

SECOND – start your count as in #5 above.  Count one row across the top of the notches.  If the number is greater than 150, then stop and record the number for the row with “row” as the method.  However, if the number is less than 150, then . . .  

 THIRD  -- continue counting the slide, as usual, by moving DOWN to the next row, then down again, etc.  You should do about 5 rows to do the top half of the slide.  Now, if the number is greater than 150, stop and record the number with “half-slide” as the method.  However, if the number is less than 150, then . . .  

 FOURTH – go back to the middle, but now count the bottom half of the slide.  When done, record the number, with “slide” as the method. 

Protocol: adapted from Tom E. Miller Laboratory

PDF of the protocol can be found here.