# 10 Embedding procedures

   

Light microscopy:

Please note: Many of the chemicals listed here are potentially carcinogenic and or flammable. Extreme caution must be exercised when handling them. Use a fume hood where possible.

For normal (routine microscopy) , FAA is used routinely as the fixative. It is important to note that FAA will not preserve cytoplasmic detail very well. After cutting the specimen or specimens, transfer immediately  to FAA (SASS 1958) and fix under low vacuum for 24 hours, after which the tissue pieces should be diced into smaller, more manageable pieces, and dehydrate these through an an alcohol series. Other methods using acrolein (tear gas) are detailed in Feder and O'Brien's (1968) paper, but these are not recommended as routine procedures unless adequate ventilation and a fume cupboard are available.

Embedment is in in paraffin wax to which 20% (w/w) of ceresin (beeswax) can be added to give more body. Sections are routinely cut on a rotary microtome at 12mm.

Basic Microtechnique for light microscopy:

 It is imperative that the material to be sectioned is supported by a suitable medium during the sectioning process, in order to obtain relatively thin (10-15µm) serial sections .  There are several alternative support mediums, each of which have their problems and difficulties associated with their use.  Wax infiltration is the easiest method.  Paraffin wax is a common embedding medium that is relatively easy to use.  The tissue is infiltrated into the paraffin wax mixture (via a series of ethyl-butyl alcohol mixtures) liquid paraffin and then into the wax medium.  Various suitable waxes are commercially available.  The trend nowadays is to use a monomer wax which is usually a compound of purified paraffin and plastic polymers, in which is incorporated dimethyl sulphoxide (DMSO) for rapid tissue infiltration.  The material is then sectioned with a microtome, sections collected on glass slides and then processed further.  Steps involved in this process are as follows:

  1.  Collect plant material that you wish to section and cut into smaller pieces as, and if necessary, preferably with a sharp razor blade. 

  2. Immerse in the killing and fixing agent.  

  3. Dehydrate the specimen, through a series of alcohols - this involves using a graded series of ethyl alcohol: Tertiary butyl alcohol (TBA), and the substitution of TBA by liquid paraffin. 

  4. The liquid paraffin is then substituted with paraffin wax (in oven) and after a suitable infiltration period, the infiltrated specimens are poured into moulds, which, when set, are affixed to microtome chucks.            

  5. Trim top and bottom faces of the square and orientate the block, block, so that when you section, a ribbon is formed.

  6. Section the tissues into very thin slices, usually done with a microtome. 

  7. De-wax, and rehydrate carefully. 

  8. Stain the sections and mount in mounting medium and cover with a cover glass.

  Subdividing material for processing  

Some preliminary remarks concerning the action of reagents in the preservation of tissues will aid us in understanding the killing and fixing process.  The reagents, which are commonly used to kill cells, are toxic to protoplasm.  Remember this when you use fixatives during the laboratory sessions in order to stop the life processes as quickly as possible, the killing fluid must be able to reach the cytoplasm as quickly as possible.  Most reagents penetrate the cuticle very slowly, but penetration is much more rapid through cut surfaces.  It is therefore necessary to cut the organ or plant part that we wish to make permanent preparations of into the smallest pieces that will show the necessary relationship of parts.  Subdivision of soft material is best done with the aid of a sharp razor blade.  Be careful of exerting too much pressure on the organ or plant part that you wish to cut up, as this will result in distortion and rupture the more delicate parts.  

Killing and preserving cell contents  

This is one of the most critical steps in tissue processing.  This process should be accomplished with the minimum disturbance of the protoplasmic organization within the cells, and a minimum disturbance and distortion of the cellular arrangement.  In addition, to killing the protoplasm, the process must be able to fix the undistorted structure and further, be able to render the mass of material firm enough to withstand handling.  Thus, the requirements for a good preservation are as follows:

  1. Kill the protoplast without distortion. 

  2.  Preserve to fix the fine detail.

  3. Hardening the material.

  There are comparatively few fundamental formulae in the literature, and many variations of these, to suit special requirements.  The formulae used consist of ingredients in such proportions that there is a balance between the respective shrinking and swelling actions of the ingredients themselves.

One of the most useful simple killing and preserving fluids is known as FAA, and is represented by the following formulae:

  Table 1: Composition of FAA

 

Ethyl Alcohol

50cc

Glacial acetic acid

 5cc

Formaldehyde (37-40%)

10cc

Water

35cc

 

 

 

Pieces of thin leaf material are killed and hardened in about 12 hours.  Thick leaves or small stems require about 24hrs.  Woody twigs should be kept in FAA for about a week before continuing with processing.  Note: Propionic acid may also be used, in which case, the formula is designated as FPA.  Other well known killing and fixing fluids are chromo-acetic, and Flemmings.

This series of operations removes water from the fixed and hardened tissues.  The removal of water is a necessary preliminary step to the infiltration of the specimen into a matrix that is not soluble in water.  Complete removal of water ensures adhesion of the matrix to the external and internal surfaces of sells and tissues.  The process consists of treating the tissue with a series of solution, which contain an increasing proportion of the dehydrating agent, and progressively less water.  Some workers regard Tertiary butyl alcohol (TBA) as being an ideal dehydrating agent.  The cost is high, but the results are well worth it! 

During this laboratory course, we will use TBA, in a TBA series, for dehydrating your specimens.  You will note that TBA 1 contains 10% TBA, and TBA 7 is 100%.

 

  Table 2: The TBA series  

TBA

95% ethyl alcohol

Absolute ethyl alcohol

TBA

Water

Paraffin oil

1

50

0

10

40

0

2

50

0

25

30

0

3

50

0

35

15

0

4

50

0

50

0

0

5

-

25

75

0

0

6

-

-

50

0

50

7

-

-

0

0

100

(volumes per 100 ml)

Please note: Tissue segments need to be in the mixtures listed above for between 6 to 12h, with preferably two changes during that time.

Step 6 is essential to ensure that the wax (added in step 10) replaces TBA effectively, thus supporting the specimen.

Begin infiltration with paraffin after step 7.

 Step 8 consists of paraffin oil or liquid paraffin.

Step 9  50% liquid paraffin:50% wax

Step 10  wax.

  When the tissue has been infiltrated with paraffin wax in an embedding oven, the wax effectively supports the tissues.  Infiltration consists of dissolving the wax in the solvent containing the tissues, gradually increasing the concentration of the wax and decreasing the concentration of the solvent.  This process is carried out in an embedding oven, usually at 50 to 70c, depending on the melting point of the wax used.  Infiltration with wax as stated, the wax matrix serves to support the tissues against the impact of the knife. It is therefore very important that the infiltration procedure is carried out properly and carefully! 

Step 8: Liquid paraffin: The specimen should be left in liquid paraffin on a warming tray, until they have sunk to the bottom of the specimen tube.  This may take two or more hours, depending on the hardness and size of the specimen that you are embedding. 

Step 9: Impregnation with wax: Half fill a specimen tube with melted paraffin wax and leave until the top solidifies.  Pour the specimen onto the wax (remember, the specimen is now in liquid paraffin).  Place the specimen vial in the embedding oven.  The specimen will slowly sink to the bottom of the vial as the wax melts.  When the specimen has been at the bottom of the specimen tube for at least one hour, decant the wax-liquid paraffin mixture into the container that is provided, and replace with pure wax.  Leave in the oven overnight, to drive off any remaining solvents.  WARNING: DO NOT OPEN THE OVEN MORE THAN NECESSARY OTHERWISE IT WILL COOL AND THE WAX WILL SOLIDIFY. 

Step 10: Embedding: Several methods are available, some may have slight advantages over others.  One may use metal moulds, paper on foil boats or even a watch glass.  These methods will be demonstrated to you during the laboratory period by the instructor.  The important point to note here is that once you commence pouring a mould that contains your specimen, you must work quickly, placing the specimen in the mould before the wax starts to solidify.  If you run into a problem, then it is best to re-melt the mould over a flame, and then carry on with the orientation procedure.  Try and arrange your specimen so that it lies horizontally within the wax mould this will make the orientation and trimming of the block easier.  Once the top of the wax has solidified, lift the mould carefully, and place it in cold water to speed up the solidification process.  Leave in water for about 10 mins.

Remove the mould from your specimen and proceed to trim the block up ready for sectioning.

Step 11: Trimming the wax black: Trim the wax black to a suitable size and shape.  Be careful about orientating the specimen, do you want to cut TS or LS?  Study the illustration in the diagram below and make sure you understand them before proceeding.

The operation of the Microtome.  The operation of the microtome can be learned best by watching the procedure adopted by an experienced worker.  This will be demonstrated to you individually and in groups as and when you are ready to cut your first sections.  Study the diagrams below, to familiarize yourself with what to expect when cutting wax-embedded material.

  Do's and Don'ts

  There are several very important DO'S and DON'TS which must be observed during microtoming.

 

 

DO

DO NOT

Work cleanly - clean up after you have finished, and leave everything neat and tidy - as you would expect to find it.

  Handle the knife with care, nasty accidents may be caused by carelessness.

  Label all material carefully, in pencil!

Leave the apparatus in a mess.

Forget to make notes as to which sections that have finished cutting are to be found on a particular slide.

   

Fixing paraffin sections to the slide

  Now that you have obtained a ribbon you will want to affix sections to slides in order that you may proceed and stain these for yourself.  Paraffin sections, either in the form of a ribbon or as single sections are fastened to a glass slide with an adhesive prior to staining.  Adhesion of the section to the slide is influenced by several factors, the most important of which are as follows:

 The slides must be perfectly clean - wash in alcohol, air dry and wipe with a paper tissue.

The adhesive must be suitable for the particular material.

The sections must be properly flattened by heating.

The adhesive must be left to harden completely, thus making it insoluble in the reagents used during the staining procedure.  Ready-prepared adhesive will be provided during practical classes.

  Staining paraffin sections

  Note; when applying adhesive to the slides, apply very little and spread it evenly over the slide.  If you use excessive adhesive, this will result in a background stain that will appear after your sections have been stained.

Paraffin sections affixed to slides are stained and processed by immersion in reagents in staining jars.  For our purpose, it is a safe practical assumption that the staining of cellular structures is based on the specific affinity between certain dyes and particular cell structures.  As is the case during microtomy, staining requires that you work cleanly and carefully.

Sloppy workmanship will result in poorly stained slides, and further, will result in pollution of the stain sequence for all those students using the sequence after you.  So work carefully!!  Staining requires that the sections be dewaxed, stained and mounted as permanent preparations that can be examined with a microscope.  All the sequences to be followed during staining are very time dependent, and should be followed carefully, varying the time that the sections are in a particular stage of the staining procedure will give varying results.

  Examples of Staining Procedures

  Some of the most commonly-used staining combinations is listed below.

 

1. Cellulose Cell Walls

Solvent

haematoxylon (self-mordanting type)

50% ETOH

Fast Green FCF

95% ETOH

Aniline Blue

50% ETOH

Bismarck Brown Y

70% ETOH

Acid Fuchsin

70% ETOH

2. lignified Cell Walls

 

Safranin

50% ETOH

Crystal Violet

50% ETOH

3. Cutinized Cell Walls

 

Safranin

 

Crystal Violet

 

Erythrosin

95% ETOH

4. Cytoplasm

 

Fast Green FCF

 

Orange G or Gold Orange

100% ETOH

 

Staining chart 1: Safranin-Fast Green

 Staining sections is achieved by using protocols and procedures that have been worked out over time.  Do not try to cut corners do not leave any of these steps out, or else the consequences are simple: Your preparations will simply be a dismal failure and complete waste of time and chemicals.

 An example of a double stain procedure

1

Xylol 1

5min

2

Xylol 2

5 min

3

Xylol/abs. ETOH

3min

4

Abs. ETOH

3 min

5

95% ETOH

3 min

6

70% ETOH

2 min

7

Safranin O

MINIMUM 1 hour

8

70% ETOH

1 min

9

Crystal violet

1 min maximum

10 70% ETOH rinse, 30 sec
11 90% ETOH rinse rinse, 30 sec

12

95% ETOH + Picric acid

‘Dip and drain’

13

95% Ammoniacal alcohol

‘dip and drain’

14

100 % ETOH

2 minutes

15

100 % ETOH

2 minutes

16

Fast Green

30 sec to 1 min maximum

17

Clove oil

30 sec

18

Clove oil: Abs ETOH:Xylol (1:1:1)

‘dip and drain’

19

Xylol  1

1 min

21

Xylol 2

2 min

22

Xylol 3

2 min

23

Mount in DPX/Canada Balsam

 

24

 

Remove from here, but only as coverslips are applied!

 

Many variations exist which are based on the above staining procedure. For example, it can be modified to a triple stain procedure. The choice of the third stain will depend on what feature of the section you wish to enhance or clarify  through the use of a specific counterstain. The example given above makes use of Crystal Violet.  Experience dictates that the best place to add a third stain is between the primary stains. 

 

Staining chart 2: Flemming's triple stain

  Another commonly used staining procedure is a triple stain method.  Flemming's Triple Stain, has application in cytological research, and will ensure that cellular details are more visible than if safranin-fast green were used to stain the sections. Crystal violet will stain.  Details of this procedure are as follows:  

De-wax and dehydrate as in staining schedule 1.  Then follow the procedure detailed below.

 

 

Immerse slide in tap water  5 min

8

Stain: (0,25% ag. crystal violet)

9

Wash in clean tap water 30 sec

10

Stain: 1% iodine + 1% Kl in 70% ETOH 30 sec

11

50% ETOH 30 sec maximum

12

70% ETOH 30 sec maximum

13

+ 1% picric acid in 70% ETOH few sec

14

Ammoniacal 95% ETOH few sec

15

Abs. ETOH, a  few sec

16

1% orange G in clove oil few sec

17

Clove oil few sec

18

Xylene 3 - rinse (if too red return to absolute ETOH and proceed: if too purple, return to clove oil and proceed).

19

Xylene 4

20

Xylene 5 - Remove from here and mount under coverslips

 

For transmission electron microscopy,

The techniques used to prepare (fix) samples varies from laboratory to laboratory, and we have deliberately not included a complete protocol here. Please refer to any one of the many good books on the subject. Better still, consult the nearest electron microscopy facility.

The basic essentials are that tissue pieces must be small, approximately  2 x 2 to 3 x 5 mm pieces at the very largest (then only for soft tissue). Fixation is usually carried out in 3 - 6 % glutaraldehyde in 0.05 M Na-cacodylate buffer either with or without I% tannic acid (TA in 3-6% glutaraldehyde for 2 h) added as a mordant. Paraformaldehyde may be added as a secondary aldehyde fixative, as this ensures good protein and nuclear fixation.

Cold glutaraldehyde/ paraformaldehyde fixation is in all cases carried out for 24 h with at least four changes of fixative. 

Pieces were diced into smaller (2 x 2mm2) squares, rinsed (3 x 20 min) in cold 0.05 M cacodylate, and post-fixed in 2% osmium tetroxide in cacodylate buffer overnight in a refrigerator. 

The post-fixed pieces should be rinsed in cold cacodylate buffer and dehydrated in a graded alcohol series, followed by several changes of propylene oxide. 

Embedment will vary again according to the laboratory procedures adopted

Sectioning is carried out using an ultramicrotome.

Selected Reference:

Feder, N O'Brien, T P  1968. Plant Microtechnique. Some principles and new methods Amer. J. Bot. 55: 123-142