Step-by-step Instructions for Staining and Studying Plant Chromosomes

Introduction Equipment Step-by-Step Formulas

Introduction

My purpose here is to share, in as much detail as possible, how one can obtain chromosomes for study in plants.  It is a detailed complement to the pictorial demonstration. There is nothing "fancy" or "high-tech" here.  The broad outlines of the technique are shown with pictures in order to give you a feel for the simplicity of the method.  All of the materials can be purchased from Carolina Biological Supply Company

Modern biology has gone way past this kind of methodology. What follows is very simplistic and it works.  The chromosome number of a species is the first item of information which gives us the beginnings of the genetic basis for that taxon.  There is nothing wrong with techniques which are tried and true.  

You have two parts of the plant to choose from:  root tips or flower buds.  You will have more immediate success with root tips since they are readily available most of the time. On the technique page I mention normally how these are readily available after transplanting a specimen from its natural habitat into greenhouse conditions.  

The advantage of using flower buds is that you are counting chromosomes in meiosis, and when you see them they are pairing.  Since you see chromosomes pairs, there are less to count and less to draw.  The problem is that in using flower buds, there is too much "trial and error."  Since meiosis occurs very early in flower formation, it will take more time to locate cells in meiosis, and you will need to be very apt at dissection since the buds will be very small.  Root tips provide information quickly, but you are looking at cells in mitosis.  Chromosome are not pairing.  You will need to count each one individually.  This gives you more to draw.

The microscope I use came to me from the National Science Foundation because of a grant I wrote in 1981.  I was advised by the peer reviewers of that grant to include a drawing tube as an accessory.   They said it was preferable to a camera, which I had requested.  They gave me good advice.  The advice was not designed to save NSF money as the drawing tube was much more expensive than a camera.  The idea of the drawing tube (sometimes called camera lucida) is that you can focus up and down to obtain chromosomes in different planes of vision.  A typical camera gives a two-dimensional view in a picture.  A drawing tube gives you an opportunity to draw in three dimensions, which is more realistic.  

The technique which follows will take you through the entire process.  There is one important point:  when you do find a mitotic cell with an excellent grouping of chromosomes to count, make a drawing first.  Draw what you see and make sure you got all of the chromosomes.  Then you count what you draw.  It would be good, if possible, to check this with other cells showing similar views.  Ideally it is also a good idea to take another sample from another member of the same species and count that.  This will insure that the number is correct.  

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Biology 207 Students:  as you read through your first Web assignment, you will be told to go to my sabbatical research on Trillium and that link will be here.

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Basic Equipment

1.    Forceps - stainless steel, fine pointed. - this is for handling the root
       tips and flower buds.
2.    Cover slip forceps.  These have a flat edge for handling cover slips.
3.    Dissecting needles - these are helpful in macerating the tissue.
4.    Eyedroppers to apply the stain onto the root tip.
5.    Microscope slides - these can be any issue, but it is important that they
       be cleaned prior to use.  This is true even if they come to you precleaned.
       What I do is take some 95% alcohol and add some 1N Hydrochloric acid
       to it like I am putting cream into coffee.  The slide is then put into that
       solution and wiped dry with a soft cloth.  A baby diaper is perfect for this. 
       Baby diapers still exist...you just need to look for them at a place like
       Kmart, etc.
6.    Cover slips - these are to be as thin as possible, and are glass.  #1 or #0 thickness is recommended.
7.    A square Coplin jar.  This will allow slide and cover slip to be separated from each other. 
8.    A small round dish, like a Syracuse dish or small watch glass, or what was
       once called a Wheaton Dish.  These are small dishes to handle the cover
       slips in.  You will need five of these for the dehydration series.
9.    Several small jars for collecting the root tips.
10.   Various vials and jars for keeping solutions.
11.   The solutions you need are (formulas to make most of these will follow below):

        a.    Aceto-orcein stain
        b.    Carnoy's fixative
        c.    Mixture of ethyl alcohol:glacial acetic acid in
               proportions of 1:1 and 9:1
        d.    95% alcohol
        e.    1N Hydrochloric acid
        f.`    10% solution of Glacial Acetic acid in 
                distilled or deionized water
        f.    Mayer's Albumen
        g.    Euparal - slide mountant

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Step by Step

This technique is modified from the basic method explained by Barbara McClintock (Stain Technology 4:53-56; 1929).

1.    Obtain fresh root tips from a plant using forceps.

2.    Place the tip into a small vial of Carnoy's fixative (see formula below).  It
       can remain in this fixative up to 48 hours at room temperature.  As an
       alternative, you could try keeping the fixative at 600 C and letting the root
       tip remain in the chemical for 15 minutes.  This will help to soften the
       tissue.

3.    The tips can be stored for later study in a solution of 70% alcohol (see
       formula below).  You will need to rinse the tip a couple of times in the
       alcohol to be sure all of the acetic acid from the fixative is remove.  Acetic
       acid, if still present, could reduce the stainability of the chromosomes. 
       The 70% Alcohol is kept in a refrigerator.  I have used root tip
        preparations more than one year old with good success.

4.    Place the root tip on a microscope slide.  Add a drop of Carnoys.  Under a
       dissection microscope remove the very end of the tip and keep that
       portion, discarding the rest of the organ.  Using dissecting needles attempt
       to macerate that tissue into small bits.  

5.    Add one drop of Aceto-orcein stain (see formula below).  Using an alcohol
       lamp or Bunsen burner as a source of heat, pass the slide over the heat a
       few times so that the orcein will warm up.  This will speed up the staining
       process and allow some of the Hydrochloric Acid to soften the tissue. 
       Don't let it get so hot that it would cause the stain to boil.

6.    Let the slide sit to cool some.  While that is occurring, take a cover slip
       and smear a small amount of Mayer's albumen onto it.  Pass that over a
       flame in order to dry the albumen.  Then set that cover slip aside face up
       so you will know which side has the albumen on it.

7.    Place the slide on a paper towel.  Using forceps lower the cover slip over
       the preparation albumen side down.  Place the end of the paper towel over
       the cover slip and, with your thumb, press down onto the cover slip as hard
       as you can.  This is the squash process designed to cause cells to separate
       from each other and make the chromosomes more visible.  

       It is at this point that you can take a moment to check the slide under a
       microscope.  It might be that the preparation is not worth keeping. 
       Normally if I find cells in mitosis and make some good countable drawings
       of chromosomes, I do that immediately before making the slide
       permanent.

8.    To make the slide permanent, take the slide-cover slip combination  and
       place it into a Coplin jar containing 10% acetic acid (see formula below). 
       Wait about 15 minutes.  The cover slip should separate from the slide in
       that time.  If it does not, don't force it.  You will have to wait for however
       long it takes for the two to separate.  

9.    Remove the cover slip after it has been separated and pass it through
       these five solutions:  1:1 Ethyl Alcohol-Glacial Acetic acid; 3:1
       Alcohol-Acetic acid; 9:1 Alcohol-Acetic acid; and then two changes of just 95% 
       alcohol.  You need to keep track of which side of the cover slip contains
       the tissue.

10.    While the cover slip is sitting in the last change of alcohol, take the slide
         out of the 10% acetic acid and clean it.  Put a drop of Euparal on it. 
         Then using forceps carefully lower the cover slip over the Euparal.  

11.    Wait about 24 hours for the Euparal to dry.  The slide is now permanent.
         I have slides in my collection which are over 30 years old.  The
         chromosomes have not lost any stain over that period of time.

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Formulas

Before I proceed with the formulas, one comment about alcohol would be appropriate.  The alcohol of choice is ethyl alcohol.  Normally it is denatured, but undenatured will work too.  The problem with the latter is that you will have to jump through several hoops in order to obtain this because undenatured ethyl alcohol is drinkable.  It needs to be kept under lock and key.  Denatured will work with no problems at all.  Other alcohols which will work are propyl and butyl alcohols.  

There is a new alcohol out which is called "flex."  It is a mixture of more than just alchohol and it works well with standard histological techniques in the animal kingdom.  I don't know if "flex" would work with plant tissue.  The problem with plant tissue is the cell wall.  This makes it hard to "squash" the cell.  The alcohols mentioned other than "flex" are easily obtainable from Carolina Biological Supply.

A second concern relates to the percentages mentioned below.  At least in the case of ethyl alcohol, it comes to you either at 100% or at 95%.  When you open a jar of 100% alcohol it will take in some water from the air and become 95% rather quickly.  Thus a "70% solution" of alcohol is not quite really 70% because you are using 95% alcohol to make the solution.  The process of dehydration so important in making the slide permanent is thus not complete.  There will always be a little water.  I have not noticed this to be a problem in viewing slides which are as much as 35 years old. 

Preparation of Aceto-orcein stain.

There are two stains you can use:  orcein or carmine.  I used carmine first and it worked fine.  You had to use a rusty needle when dissecting the root tip or flower bud in the stain because it acted as a mordant.  The iron would react with the carmine to make the stain hard and tight on the chromosomes.  Later I used orcein simply because it was available to me in my lab during my Ph.d. work.  The following formula applies to either stain.

1.    Mix 45 ml of Glacial Acetic acid with 55 ml. of distilled or deionized water.

2.    Slowly bring it to a boil.  It need not be a "rolling boil." 

3.    Slowly add to it 2 grams of orcein.

4.    Mix as you add it, and let it mix for about 30 minutes if you have one of
       those magnetic mixers.

5.    Place some kind of glass cover over the beaker.  The heat of the acid will
       condense on the glass and then move back down into the mixture.  This
       serves to help in the mixing.

6.    Filter.  You will have to use an aspirator to pull the mixture down through
       the filter.  This will take some time.

7.    Once cooled, the solution is ready for use.  The one other thing you need
       to do is to mix a solution of the stain with 1N hydrochloric acid.  The ratio
       is 9 parts stain to 1 part acid. 

8.      The stain is ready for use.  You might need to re-filter about once a year.

Carnoy's fixative.

This is a mixture of glacial acetic acid and alcohol.  The ratio is 3 parts alcohol to one part acid.  The alcohol can be ethyl, propyl or butyl.  Remember that absolute ethyl alcohol, once opened is no longer absolute.  You can use 95% ethyl alcohol with  no problems at all.  The acid does the killing of the plant cell; the alcohol is the preservative.

1.    The series of Alcohol:Acid mixtures are:

       1 part Ethyl Alcohol to 1 part Glacial Acetic Acid

       3 parts Ethyl Alcohol to 1 part Glacial Acetic Acid

       9 parts Ethyl Alcohol to 1 part Glacial Acetic Acid

2.    The 10% acetic acid is 10 ml. of Glacial Acetic acid and 90 ml. of distilled
       or deionized water.  This is used to separate the slide from the coverslip.

3.     Mayer's Albumen and Euparal are best purchased premixed and ready to
       go.  I am familiar with formulas to make both from scratch, but I never
       tried them.  These are both available from Carolina Biological Supply.

Selected References

The method which is described above is not mine.  I assembled it from various sources during the time of my Ph.D. research.  I have used it more since coming to Bridgewater.  It has produced chromosome counts for over 100 species of plants from Virginia and Bermuda.  There are, however, a couple of books which one might find helpful in the overall technique of working with plant chromosomes.

Fukui, K., and S. Nakayama., Eds.  1996.  Plant Chromosomes:  
     Laboratory Methods.  CRC Press.

Jones, R.N., and G.K. Richards.  1991.  Practical Genetics.  Open University
     Press, Philadelphia.

Sharma, A.K., and A. Sharma.  1965.  Chromosome Techniques, Theory
     and Practice.  Butterworths, London.

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