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Counting-eggs-and-larvae

The document outlines various techniques for counting eggs and larvae, including the Displacement Method, Image Analysis, and Direct Counting. Each method has its advantages and challenges, such as accuracy issues with overlapping eggs in Image Analysis and the need for dissociation in Direct Counting. Additionally, the use of the ImageJ™ program for automated counting is described, along with steps for preparing images for analysis.

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24 views

Counting-eggs-and-larvae

The document outlines various techniques for counting eggs and larvae, including the Displacement Method, Image Analysis, and Direct Counting. Each method has its advantages and challenges, such as accuracy issues with overlapping eggs in Image Analysis and the need for dissociation in Direct Counting. Additionally, the use of the ImageJ™ program for automated counting is described, along with steps for preparing images for analysis.

Uploaded by

Zak
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Counting eggs and larvae

Dr Robert Browne, AArk Research Officer, Royal Zoological Society of Antwerp,


[email protected]
Special thanks to Jessica Seratt for her contribution to the computer analysis part of this document.

Techniques used to count eggs include the ‘DISPLACEMENT METHOD’, ‘IMAGE


ANALYSIS’, and ‘DIRECT COUNTING’.

‘DISPLACEMENT METHOD’ This method is fast, yields


immediate results for large numbers of eggs, enables field
counts with replacement of eggs, and is reasonably
accurate. In the displacement method a sub-sample of
several hundred eggs are counted and their volume
measured (usually in a measuring cylinder).

The volume of the total


egg mass (including
the sub-sample) is then
measured and the total
number of eggs
calculated. Inaccuracies with this method can occur
through differences in the gel and egg size between
different parts of the spawn mass. This is particularly
the case in some experimental situations where
spawn masses may be of low quality with variable
gel and egg quality.

SN = sub sample number.


SV = sub sample volume.
TV = total volume.
TN = total number

TN = TV/SV x SN
‘IMAGE ANALYSIS’ counts eggs from an image of the spread eggs against a contrasting
background. Inaccuracies with this method can occur through overlapping eggs making
counting difficult. Overlapping eggs are a particular problem where the eggs are in a
globular mass rather than in strings. To some extent image analysis can also be used
for larvae if they are in shallow trays. The number of eggs can also be counted and
marked off digital images by hand.

The eggs must be separate. Separation of eggs is easier with partial removal of the gel
coat. see “Gel removal and extract”. The use of the ImageJ™ program to automatically
count eggs is described at the end of this document “Image analysis of object counts
with ImageJ™”

ImageJ™ program allows for easy counting of eggs. It automatically counts all objects it
recognizes in the final red image (right).

See at documents end: Image analysis of object counts with ImageJ™


‘DIRECT COUNTING’ with pipettes by an experienced operator produces an exact count
of eggs or larvae. However, direct counting can only be used when the eggs are
dissociated (see Reproduction technology > Gel removal and extract), or when the
larvae have separated from the gel naturally or naturally.

The end of a disposable plastic pipette is cut to leave the maximum open tube. Eggs or
larvae - up to just below the diameter of the tube - can then be aspirated and counted
and placed in a new container. This method is often used to count when separating
larvae from infertile eggs to prevent poor water quality causing mortalities.

Left: A complete disposable pipette


(below) and one with the end removed
(above) with toad eggs (middle left)
and larvae (middle right). Plastic
disposable pipettes are graduated in
diameter at the ends. The pipette is
generally best cut off to give best
adjusted to about 1.5 the diameter of
the eggs or larvae. Toad eggs are
shown in the middle left and larvae in
the middle right.

Right: The 15 eggs can easily be


counted as they are transferred into
a new container. Similarly larvae
or small swim ups can also be
counted by this method.
Left: A petri dish of mainly infertile toad eggs and
some larvae about 48hrs after spawning. As the
larvae develop and hatch they excrete enzymes
that dissolve the egg gel. This also enables the
infertile eggs to release their yolk into the water
creating an ideal environment for bacterial
growth.

Right: To prevent hypoxic and unsanitary


water the larvae must be moved to a new
container with fresh water. This is easily
and quickly accomplished using a
disposable plastic pipette.

Image analysis of object counts with ImageJ™


Special thanks to Jessica Seratt for her wonderful contribution to the computer analysis part of this
document.

Using ADOBE Photoshop™ to get the best image for analysis.


1. Take an image with camera of eggs. White background for black eggs and black
background for white eggs. The eggs must be separate. Separation of eggs is
easier with partial removal of the gel coat. see “Gel removal and extract”
2. Open image in Adobe Photoshop™
3. Image > Mode > Greyscale
4. Image > Adjust > Brightness/Contrast. Adjust the contrast and brightness to
maximize separation of the eggs from the background.
5. Delineate the area with eggs with the Elliptic or Rectangular marque
6. > Cut
7. > Create new file > OK
8. Paste
9. Image > Levels - Adjust to distinguish eggs
10. Filter > Noise > Despeckle
11. Save as tif for analysis in image analysis program.
Analysis of image with Image J™
1. Open in” ImageJ™” 1.33u: Wayne Rasband, National Institutes of Health, USA;
http://rsb.info.nih.gov/ij/
2. process > sharper
3. Process > noise > despeckle
4. Image > adjust threshold – image goes red
5. Analyse > particles
6. Show “masks” , “display results”, “summarise” > OK
7. The “Image J™” program then produces a count of objects and a summary of
average size in pixels, and distribution of size. It also produces image of counted
objects

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