Empirical sedimentation times for pollen

 

Summary

When preparing pollen preparations from plants for microscopy, it is often useful to concentrate collected pollen, by centrifugation or by allowing it to settle into a pellet (sedimentation) when centrifugation is not an option. 

The following empirical experiments were conducted to find a more rational basis for sedimentation times when centrifugation is not possible that I had evolved when preparing pollen for microscopy. 

My conclusions from the data shown below are:

• For pollen suspended in water or alcohol (iso-propanol), allow 1 minute of sedimentation for every 1 mm liquid height of the pollen suspension.
• If there is still a turbid supernatant from the first sedimentation of fresh pollen from flowers after the alloted sedimentation time, this can be discarded. If in doubt, collect the supernatant and leave to stand overnight, then check any sediment from the supernatant for presence of pollen.
• Pollen suspended in a 20% dilution of honey in water sediments at least 6 times more slowly. convection movements can have an effect in larger vessels. Therefore allow to sediment in a location at constant temperature overnight. Higher dilutions of honey than 1in 5 will speed up sedimentation.

Method

Preparation of pollen

Flower pollen was collected from ripe anthers of Lily, Magnolia and Alstroemeria by rinsing in a 30 ml sample tube filled with iso-propanol. The pollens were allowed to settle overnight and most of the supernatant removed. The lily pollen was resuspended and allowed to settle several times in isopropanol till the supernatant was only faintly coloured. The abundant pellet was then resuspended in 10 ml ispropanol. The supernatants over the magnolia and alstroemeria pollen pellets pipetted off and the pellets resuspended in 1 ml iso-propanol. 

Honey pollen was collected from 50 g spring honey dissolved with 200 ml tap water. The pollen was allowed to sediment out overnight in a jar. Most of the supernatant was carefully siphoned off, leaving about 25 ml liquid. The sediment at the base of the jar was resuspended the remaining liquid and was trasferred to a 30 ml sample tube and allowed to settle overnight. Most of the supernatant above the pellet was carefully removed and the pellet resuspended in 1 ml iso-propanol.

1 ml of each of the flower pollens and the honey sediment was transferred to separate 1.5 ml eppendorf (centrifugation) tubes and centrifuged for 30 s at 7000 RPM (maximum relative centrifugal force 2680 x g) in a microfuge. The supernatant was carefully pipetted off and the pellets resuspended in 1 ml deionised water. The tubes were again centrifuged at 7000 rpm for 30 seconds, the supernatants pipetted off, and the pellets resuspended in 1 ml fresh deionised water.

Microscopy of samples

Approximately 50 µl to 100 µl of each pollen suspension was applied to the centre of a microscope slide, on a hot plate at hand hot, and spread to a disk of approximately 15 mm diameter and allowed to dry. A drop of molten pollen glycerine jelly with dilute basic fuchsin dye  (Brunel glycerine pollen jelly) was added to the centre of each dried disk and a coverslip carefully lowered onto the drop. The slides were left for 15 mintes on the hot plate to allow the glycerine jelly to spread under the slip and hydrate and stain the pollen.

Samples were then viewed under the microscope, first with a 10 x objective NA 0.17 and then with a 40 x objective, NA 0.65, to check that pollen was present and, in the case of honey, that several different pollen species were present.

Pollen grains were photographed under the 40 x objective using a chinese 5 MPx inspection camera with eyepiece adapter and Toupview software on the PC. 20 µm scale bars were included.

Images with the 40 x objective were taken as a sequence of 30 to 50 steps, focussing from the top of a grain to the equator of the grain. The images were then used to create a photostack using the freeware Picolay. 

Pollen sedimentation from suspension in 1 ml water

To record sedimentation of pollen from suspensions in water, in 1.5 ml eppendorf tubes, the remaining pollen preparations in water were made up to 1 ml in their tubes. Each tube was shaken thoroughly and  then suspended in a jar, with a black card as background, and a lamp shining from one side, and a video recoding started immediately using a smartphone on a stand.

Recording was stopped after 20 minutes and the procedure repeated with the next sample.

The four separate videos were combined using Corel VideoStudio plus.

Pollen sedimentation from suspension in 1 ml 20% honey in water solution

The supernatants were pipetted off the pollen pellets that had sedimented in water in the previous experiment. The pellets were resuspended 1 ml of a 20% honey in water solution (5 g honey mixed with 20 ml deionised water). The suspension was then photographed at time 0 and at 10 minute intervals for up to an hour.

Pollen sedimentation from the top of a 25 ml water or diluted honey column

Three separate experiments were set up, where a lilly pollen suspension was applied on top of either a water or a diluted 1:5 honey water mix, to see how the lily pollen behaved passing through a liquid column:

1. The pellet from a hydrated lily pollen was mixed with 1 ml vegetable oil and then layered on top of 25 ml of deionised water in a 30 ml tube.
2. 1 ml of lily pollen in water was slightly warmed and gently pipetted on top of a cooled column of 25 ml deionised water in a 30 ml tube.
3. 1 ml of lily pollen in water was layered on top of 25 ml of 1:5 honey - water mix.

Each column was then videod over time, 20 minutes for 1 and 2, 1 hour for 3. The three videos were combined for comparison into one video. 

Pollen sedimentation from alcohol check

For some pollens or samples rinsed in alcohol, there is still visible turbid material throughout the supernatant after an hour's settling.  Examples include pollen collected from wind-borne material, some honeys, some pollens collected from flowers). To determine whether this is pollen or pollen rich, the following procedure was used.

Nine pendulous catkins from Silver Birch were rinsed in approximately 25 ml iso-propanol. Any large particulate material was removed with a seeker. The resultant cloudy solution was allowed to stand for 1 hour. The still turbid supernatant was carefully pipetted off to within 1 ml of the pellet. The pellet was transferred to a 1.5 ml tube. The supernatant was centrifuged in 1.5 ml tubes at 7000 rpm for 1 minute, the now clear supernatants removed, and the remaining pellets resuspended and combined in 1 ml isopropanol in one tube. 

The tubes with 1 ml of the original resuspended pellet, and the 1 ml resuspended pellet obtained from centrifuging the supernatant, were centrifuged again at 7000 rpm for 1 minute. The clear supernatants were pipeeted off and discarded and the pellets resuspended in 1 ml deionised water and again centrifuged at 700 x g for 1 minute. The clear supernatants were pipetted off and discarded and the remaining pellets resuspended in 400 µl deionised water.

100 µl of each resuspended pellet was spread in a 12 mm disk on the centre of a microscope slide on a hot plate (hand hot) and allowed to dry and then mounted in glycerine pollen jelly and used for microscopy as described above.

Results

Pollen samples used

Four different pollen samples used for main experiments: Lily, Magnolia, Altroemeria and Spring Honey. Photostacks. 40 x objective. Scale bars 20 µm.

Microscopy of the original pollen samples used in these experiments showed a wide varety of shapes, sizes and surface structure. The samples from flowers were uniform in type, from large (Lily and Altroemeria, 100 µm long axis) to medium (Magnolia, 40 µm long axis).  The honey pollen was predominantly rape (round, 4 sutures and pores, 20 µm diameter) with a scatter of larger and different pollen species. pollen textures varied from netted (Lily) to spiky (daisy type).

Sedimentation in 1 ml water

The video recording shows that all the pollens had completely sedimented out of the suspension in water after 20 minutes. Some pollen grains adhered to the side of the tubes. The height of the water 1 ml water column was 25 mm.

Sedimentation in 1 ml 20% honey in water

Sedimentation observed at 20 minute intervals up to an hour using pollen suspended in 1.5 ml 20% honey in water as the solution.

Both with the lily pollen and pollen from spring honey, a substantial pellet had formed by 1 hour. With the lily suspension, there was still unsedimented material visible, suggesting that a longer time was required for complete settling into a pellet.

Sedimentation though a column of 25 ml water or 20% honey in water

Where pollen had been layered in oil on top of water, there was a slow release of pollen grains, which sedimented at a fairly constant rate through the water column. However, some particles travelled 10 mm in 10 to 30 seconds, whilst the slower ones migrated at 10 mm in up to 90 seconds. The total water column height was 75 mm, which the fastest particles could cover in under 2 minutes, whereas the slowest ones required more than 11 minutes, beyond recording time.

Where the attempt had been made to layer pollen in warm water on top of a cold water column, there was an uneven flow with an initial bulk of pollen dragging water with it as it descended. The column was practically clear of pollen by 10 minutes recording.

Pollen layered on top of 20% honey solution also flowed down unevenly into the honey solution. whilst a substantial amount of pollen has pelleted after two and a half minutes, vortices set up in the solution still kept pollen grains in suspension, often flowing back up through the column.

Contents of pellets and turbid supernatants from Silver Birch pollen in alcohol.

Top left, pollen clearly visible in the pellet from the alcohol sedimentation (10x objective, 100 µm scale bar). Top right, the material centrifuged down from the turbid alcohol supernatant. This comprises plant and other non-pollen debris (10x objective, 100 µm scale bar). The bottom image shows the distinctive stained Silver Birch pollen from the pellet in detail, using a 40 x objective (20 µm scale bar).

After one hour settling in isopropanol, all the Silver Birch pollen was in the pellet. The turbid material in the supernatant did not contain any of the pollen. Both the pollen pellet and supernatant contained other debris.

Conclusions & discussion

Pollen grains vary in their size, shape, density and surface structure. This makes it difficult to calculate their theoretical sedimentation rate in water or other solutions. If you have access to a centrifuge, the g forces that can be generated easily pellet biological particles. Practical experience with a variety of pollens collected over the past year has demonstrated that 30 seconds in a microcentrifuge giving about 700 x g in a 1.5 ml microcentrifuge tube is sufficient not only to pellet pollen but any other debris in a pollen suspension, giving a clear supernatant.

However, I've obserrved that if I leave pollen suspensions in water or alcohol to stand for any length of time in 1.5 ml tubes, sedimentation into a pellet becomes visible within minutes. Simillarly, when purifying pollen from honey, I and others have found that leaving a larger volume (100s of ml) of a 20% solution of honey overnight will give a good pollen sediment for microscopy.

If you don't have a centrifuge, simply allowing pollen to settle out will work.

The experiments illustrated here show that allowing a 1 ml suspension of pollen, in water or alcohol in a 1.5 ml microcentrifuge tube, to stand for 20 minutes is more than sufficient to pellet pollen from a variety of sources. With a liquid column height of 25 mm in the 1.5 ml tube, a good approximation is  to allow 1 minute per 1 mm water or alcohol height. 

Sedimentation from pollen in  20% honey in water is at least 3 times slower in 1.5 ml tubes. Whilst most pollen may have sedimented after 1 hour in the 20% honey, leaving for at least several hours would be best. presumably this could be shortened with pollen in more dilute honey solutions.

The attempts to determine a sedimentation rate through water or 20% honey were only partially successful. 

Where the pollen grains emerged gradually from oil over a 75 mm column of water, the results were most reliable. The lily pollen grains, with a hydrated length of 100 µm along the longest axis were visible as small specks, which seemed to travel fairly uniformly downwards. However particle speeds varied from 0.1 mm per second to 1 mm per second. The higher speeds tended to be brighter specks, which might suggest aggregates of several grains. With the other two tries, pollen in water suspension applied either to the top of a water column,  or 20% honey column, the density due to high pollen concentration led to non laminar flow and turbulence. Still, practically all the pollen had sedimented in water after 10 minutes, whilst in 20% honey, it took an hour and there were still visible particles in suspension.

Trying to determine whether all pollen has sedimented from a 25 ml suspension of pollen from flowers in alcohol or water can be complicated by a turbid supernatant. The trial of sedimenting a suspention of Silver Birch pollen in alcohol showed that after standing for 1 hour, the pellet constained the pollen (and other debris from or attached to the wind blown catkins), with no significant amount of pollen in the supernatant, which contained undefined debris.

From these experiments, the following are recommended:

• For pollen suspended in water or alcohol (iso-propanol), allow 1 minute of sedimentation for every 1 mm liquid height of the pollen suspension.
• If there is still a turbid supernatant from the first sedimentation of fresh pollen from flowers after the alloted sedimentation time, this can be discarded. If in doubt, collect the supernatant and leave to stand overnight, then check any sediment from the supernatant for presence of pollen.
• Pollen suspended in a 20% dilution of honey in water sediments at least 6 times more slowly. convection movements can have an effect in larger vessels. Therefore allow to sediment in a location at constant temperature overnight. Higher dilutions of honey than 1in 5 will speed up sedimentation.