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Plant Physiology (Biology 327)
- Dr. Stephen G. Saupe; College of St. Benedict/ St.
John's
University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782;
(320) 363 - 3202, fax;
ssaupe@csbsju.edu |
Monitoring Protoplast Viability with Evan's
Blue and Neutral Red
Question(s): Can Evan's blue and Neutral red be used to examine the protoplasts
for viability? What percent of the protoplasts are viable?
Hypotheses: Viable protoplasts will exclude Evan's blue appearing clear or
yellowish against a blue background. Intact viable protoplasts will accumulate neutral red
and turn red. Most of the protoplasts will be viable.
Protocol:
- Add a drop of neutral red (0.1% in 0.7 M sorbitol) to a drop of the protoplast
suspension. Neutral red readily
passes through many membranes and accumulates in the vacuole. It is best to use a
geranium or other unpigmented leaf
protoplast (i.e., unpigmented red cabbage protoplast) for this exercise.
Describe and sketch (or photograph) the results. Include a caption
and plate magnification.
- Add a drop of Evan's blue (0.5% in 0.7 M sorbitol) to another drop of protoplasts (on a
regular slide). Viable protoplasts will
appear white or yellowish against a blue background. Describe and
sketch (or photograph) the results. Include a caption and plate
magnification.
- Gently vortex the protoplast sample. Prepare another slide with Evan's blue and
protoplasts. Determine the viability (percent of the protoplasts that are viable) of your
sample by randomly counting 50 protoplasts and scoring them for Evan's blue staining.
Record these data in Table 1. Note, count only round, intact protoplasts - ignore
malformed ones. Do your Evan's blue data correlate with your observations of cyclosis?
Table 1: Viability of Red Cabbage Protoplasts determined by
Evan's blue |
|
Sample |
Total |
1 |
2 |
3 |
4 |
5 |
# stained (non-viable) |
|
|
|
|
|
|
# unstained (non-viable) |
|
|
|
|
|
|
% viable |
|
|
|
|
|
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Data, Analysis & Conclusions:
- Describe the effect of adding Neutral red to the protoplast suspension. Offer an
explanation for your observations. Do the results support our
hypothesis?
- Describe the effect of adding Evan's Blue to the protoplast suspension. Offer an
explanation for your observations. Do the results support our
hypothesis?
-
What do you conclude about the viability of the protoplasts? How do the vital stain
data correlate with your observations of cyclosis? Do the results support
our predictions?
Last updated:
01/07/2009 � Copyright by SG
Saupe