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.

 

 

Hayfever - Airborne allergens under the microscope

 A day of sneezing and irritated eyes again, now that it has rained in Milton. Here is what I found in the air and put under the microscope - masses of pollen grains, fungal spores and even starch grains! The sample is from a comparable day last week, when I rinsed a roughly 30cm x 30cm (1 square foot) piece of fine netting that had been on the washing line for 18 hours!

The various types are shown with numbered examples on the following figures, using the labelling:

1. Pollen grains from flowering plants stained red and can vary a lot in shape and size
2. Pollen grains from grass - are stained red, plain ovals with a single pore, and can vary in size
3. Fungal spores - generally brown, beige or grey, ranging from lage paddle shapes to small ovals
4. Fragments of fungal hyphae - look like broken brown twigs
5. Starch grains - variable sizes, transparent, circular to oval with a hint of a central marking

From the first picture above you can get an idea of the abundance of pollen and fungal spores in 1/600 of the collected material. The spiky pink pollen comes from a plant in the daisy family.

For us hayfever sufferers, our problem is compounded by the fact that all these very small particles stick quite readily to our hair, faces and clothes. Our symptoms can be triggered if even if just a few become loose when we move indoors or go to bed, hence the advice to wash hair and change outer clothes frequently if you do suffer badly from hayfever. Thank heavens that many of our cars have pollen filters!

I calculated that overall my total sample from a small piece of netting contains about

• 7,200 pollen grains
• 42,000 fungal spores 
• 600 starch grains

Hayfever is when our own immune system becomes sensitive to airborne dust, pollen, spores, hair, for example, treating them as a threat and trying to protect us.  Unfortunately, our eyes and noses are the areas most likely to be exposed and react. What's more, each one of us develops our own typical allerge to one or more of the many different particles in the air.

I can manage with taking hayfever tablets daily during the main hayfever season, and eyedrops when it is particularly bad on the eyes. I only need to avoid going out on particularly bad days. Many others suffer much more seriously and find their lives dramatically impaired. In those cases it is worth seeing if your doctor or health system offer both a service to identify your specific allergens and a possible corresponding desensitisation program. two useful lins in the UK are:

• NHS Hayfever page https://www.nhs.uk/conditions/hay-fever/
• AllergyUK https://www.allergyuk.org/types-of-allergies/hayfever/
• A page from the US NIH on desensitisation https://www.ncbi.nlm.nih.gov/books/NBK279487/

There is also another side to being able to capture and identify pollens and spores - in forensics, when trying to match a person or vehicle to a crime location. Here, the fact that pollen and spores are so pernicious and will be very specific to locations helps, as illustrated in a fantastc book by Patricial Wiltshire 'Traces' - https://www.amazon.co.uk/Traces-forensic-scientist-criminal-investigator/dp/1788700635/ref=tmm_pap_swatch_0?_encoding=UTF8&qid=1688324266&sr=8-1

Airborne particles. I hadn't really realised how abundant they are! My sympathy if you are a hayfever sufferer.

For your interest, below are some more images from the airborne pollen sample.

The caplet shaped pollens are from the same family as Cow Parsley.

The largest pollen grain (1) is similar to Hazel pollen. The grass pollen (2, bottom) has a visible single pore.

The large pollen grain (1) is very characteristic for pines, the two black circles within it are trapped air bubbles. The starch grain (5) is on the large side. Starch in airborne dust most likely comes from decayed plants and very small grains can come from some pollens. The fungal fragments (4) would also have come from dried decaying plants and soil.

 

Reviving UK-DE trade - Some notes on a March 2023 Trade mission

Tower Bridge London at night

Executive Summary

Six German companies specialising in innovative building transformations, modernisations and restoration attended 2 days of talks and bilateral information exchanges in London on 20-21 March before continuing with site visits in the following days. Key messages from both UK and DE side were 1. Shared interests in working and collaboration between companies in the sector. 2. The difficulties in finding skilled craftspersons and apprentices. 3. Cross-border hurdles impacting on collaborations and the need to find suitable solutions to ameliorate them. 4. Deep frustration at the lack of political action in the UK  

The trade mission was a German BMWK Ministry (https://www.bmwk.de/Navigation/EN/Home/home.html) event organised and enabled by Europartnerships (https://www.europartnerships.co.uk/). It was held at both the German Embassy, London and the Institute of Practitioners in Advertising (IPA), both at Belgrave Square.

Day 1. Talks in a secure environment

Hearing that I was the sole person allowed to bring in a laptop for the day's event at the German Embassy, with special dispensation from the Chancellor, I was only mildly disappointed to find out that it was not the German Chancellor, but the almost equally eminent embassy's Chancellor. I am unable for obvious reasons to reveal the other stringent security restrictions and precautions we had to undergo before we could reach our meeting room, but we were in the hands of the extremely professional and courteous and friendly staff throughout. 

We were warmly greeted and had a good outline of the current situation re post Brexit trade and UK politics by the Deputy head of Economics Mariko Higuchi (https://uk.diplo.de/uk-en/01/embassy). The directors of the six german companies were then provided with advice on a wide variety of relevant topics. These ranged from basic cultural differences, via the new tax issues arising through crossborder trade, to the impact of visas, BIM and onsite Health and Safety on doing business in the UK. Key message to emerge, life is simpler if you have partners in both countries.

Compliments to the speakers, Marc Lehnfeld (Director GTAI www.gtai.com), Sven Riemann (Marketing AHK www.ahk-london.co.uk) - a long standing and witty contributor at past events, Martin Werhahn (Tax Services AHK www.ahk-london.co.uk), and Robert Hunt (associate Konduit Ltd www.konduit.uk), who proved himself to be one of the rare breed of  0.5% of british in the UK who could definitely speak German.

Though retired, my role for Europartnerships over the years has been to come in as meeting's chair for the two days, to ensure the fair timing of talks takes us agreeably through to breaks and mealtimes, without bias and on time.

Six different personalilities, skills and businesses

We (the delegates) made our way to the Motel One, Tower Hill, afterwards to meet up at 4:30 to go through the six comapny presentations in a relaxed manner. This is a crucial and really helpful stage for me. It gives me a chance to get to know not only their businesses and objectives, but the different personalities, something cemented in The Dean Swift pub, close to Tower Bridge later that evening. It helps with supporting them on the following day.

I'll try to give some single line summaries of the six different companies and their services:

• Felix Graf of Felix Graf GmbH: A family business with a a strong link with its community and employees, that has grown to specialise in the interior fittings for the hotel and catering industry. www.felixgraf.de
• Lars Krauss of Greengineers: Consultants and specialist planning services to provide tailored sustainable solutions that meet their client's needs. www.greengineers.de/english
• Jörn Brennscheidt of Hokon. Making impossible stairways possible! www.hokon.de
• Thomas Schubert of Ingeneurbüro für Kirchenbau, Glocken und Denkmalpflege. Rebuilding and recovering churches, community buildings and creating welcoming communal places. www.ibkirchenbau.de/about-us
• Christian Schulte of Mühlenhof Restaurierungen GmbH. Sympathetic and authentic restaurations of historic windows, doors, floors, panelling and furniture in collaboration with architects and art historians. www.muehlenhof-restaurierungen.de/en
• Peter Spor of Tischlerei Spor GmbH & Co KG. A traditional joinery and carpentry company whose expertise include dry wall construction and the repair and build of solid wooden floors. www.tischlerei-spor.de/english/fields-of-activity

Most of the companies already have international experience and are english speakers, with several having completed projects in the UK prior to Brexit.

Day 2. Presentations and key issues.

Day 2 was held at the Institue of Practitioners in Advertising (IPA https://ipa.co.uk/), almost opposite to the German Embassy, on the other side of Belgrave Square, a long standing and friendly venue for Europartnerships.

Over 70 UK architects and other companies in the construction and restoration sector had expressed an interest and a substantial number filled the larger conference room upstairs at the IPA. This time it was the turn of Markus Knauf (Head of the Economics Department at the German Embassy https://uk.diplo.de/uk-en/01/embassy) and Mark Dodsworth, director of Europartnerships (https://www.europartnerships.co.uk/)  to welcome the guests, as well as thanking Petra Riemenschneider and team at Europartnerships for their herculean efforts in organising the event.

For once, technology was on our side as the first of the mornings talks, by Marie-Theres Sobik of Thyssen (www.thyssenkrupp-materials.co.uk) on "The vocational training system in Germany" was given remotely using Teams. A training system that had a century of experience behind it, contrasting with the mixed and changing approaches in the UK. Circumstances had prevented Marie-Theres from being able to attend and her physical presence was sorely missed by me and the Europartnerships team. Like Sven mentioned above, she had been a stalwart and bright, constructive presence at many previous events in the past. Afterwards, Michael Schienke of Vorbild Architecture (https://vorbild.co.uk/) and Sven Riemann (AHK www.ahk-london.co.uk) reversed yesterday's role and enlightened the british audience on cultural differences across the channel, revealing that in Germany too, there was a diversity of regional prides, traditions and ways of doing things.

Our six german companies then had their turn in the spotlight. Gradually, the audience came alive with shared experiences and questions not only to the companies, but also about how to overcome some of the hurdles presented since Brexit. After all, the UK had fallen out of the list of top 10 trading partners with Germany. Here, the presentations given to us at the embassy proved invaluable; problems with taxation through the transfer of goods several times back and forth across the UK-EU borders could be overcome to some degree with professional assistance and sometimes even at a final null cost calculation. Building partnerships with one member in the EU and the other in the UK was a good route to take, each familiar with their own ideosyncratic bureaucracies, regulations and regional issues and helping solve the border transitions of goods and services.

What I understood was, that the greatest regret was the loss of access to the Erasmus program, with some UK delegates speaking of the way it had helped their careers. In turn, the German companies had young staff who would leap at the chance to gain experience in the UK. A representative from one of the UK bodies in the industry implored the German Embassy to seek more routes to collaboration in the training of up and coming craftspeople in the construction and in the restoration sectors. Apparently there were promising comments from the government - but at present little action.

We ended the day with a buffet lunch and many individual discussions between attendees and the german company representatives, followed by the longer one-to-ones from pre-arranged appointments.

My personal feeling at the end of the day was, that there were opportunities and an interest in longer term collaboration between the two countries in innovative building transformations, modernisation and restoration, despite the hurdles raised by Brexit. 

From the german companies' point of view, it was an insight into the opportunities that might exist, possible partners to contact when back home, as well as a better knowledge of the potential barriers. Ultimately, it would be their judgement whether it would be worth their time and investment in trying to enter the UK market.

Creamed Honey crystal microscopy using LOCA - Method

Crystals of creamed honey (sourece Honigmanufaktur Meerbusch). 40x objective, industrial microscope camera, transmitted light, crossed polarisation filters and quarter wave plate.

I returned from a family vsit to Germany with a small jar of creamed honey, made by Honigmanufaktur Meerbusch (www.honigmanufaktur-meerbusch.de). Coming to the bottom of the jar, of this very smooth, cream like honey, I looked for the best way to visualise and measure the honey crystals. Through experimentation, a simple method for producing a thin layer of honey crystals and mounting in LOCA  was developed. Samples were best observed between crossed polarisation filters with a quarter wave plate.

Method

I tried several methods to get a thin layer of the sugar crystals.

1. Taking a small drop on a slide, covering with a cover slip and pressing hard. Not very successful,
2. Small drop of honey on a slide mixed with some oil then pressed with a cover slip. Partially successful, giving areas with some thinner layers. see figures 1 & 2
3. Small drop of honey mixed with isopropanol - too viscous too smear.
4. Making a thin streak of honey across the width of a slide and trying to spread it as you would a blood smear, pulling a thin film behind another angled slide. Honey too viscous to be spread.
5. Making a thin streak and  using an angled other slide to smear it thinly. Unsuccessful - the smear was of a viscous thin layer solution wihout many crystals until the place where the dragging slide was pulled off, There a slip of thick crystals remained.
6. The final approach was a modification of method 5. Making a thin streak and  using an angled other slide to smear it thinly at first and then reducing the pressure over a 2 cm stretch of the slide. SUCCESS! there was an area of the smear that was thick enough to leave crystals yet thin enough to have them at almost a single layer. A drop of LOCA was placed on the thin area, a cover slip added and pressed. The slide was set by exposure to a hand held UV torch for 60 seconds and then cleaned according to the method published by Gordon Brown, 2020, creating a permanent slide.

Slides were photographed using a Reichert Zetopan microscope witha 40x objective and a Chinese 5 megapixel objective camera and the software ToupView. Lighting techniques used were standard brightfield, use of crossed polarisation filters without and then with a quarter wave filter. A series of images at different focus through the sample were taken and combined in a focus stack using Picolay.

Results

Method 2. The sample of honey mixed with sunflower oil had areas that were thin enough to see defined crystals. There were planar, needle and fragmentary types. However the sample was still fairly thick.

Figure 1. Creamed honey crystals. Method 2. Mixed with sunflower oil, 40x objective, between crossed polarisation filters. Field of view 250 µm.

Figure 2. Creamed honey crystals. Method 2. Mixed with sunflower oil, 40x objective, between crossed polarisation filters plus quarter wave plate. Field of view 250 µm.

Method 6. Sample spread as a smear of increasing thickness on slide and mounted in LOCA. More parts of the slide could be found where the layer of crystals was thin enough to resolve individual crystals.

The crystals in this cream honey are extrememly thin and show little contrast in transmitted light (figure 3). Greater contrast of the crystals against background is achieved using polarisation. Their thinness is also reflected in the fact that they appear in pale colourless shades between crossed polars (figure 4). The planar like crystals have 120 degree angles reflecting molecular sugar. There are also small needle like crystals. 

Whilst the contrast is less with the inclusion of a half wave plate (figure 5), it does reveal crystals missed under just crossed polars. making this the preferred method for photography.

The images reveal that most of the crystals are smaller than 20 µm in diameter. The result is consistent with the cream like nature of this honey.

Figure 3. Method 6, sample spread and mounted in LOCA. 40 x objective. Normal transmitted light. 

Figure 4.  Creamed honey crystals. Method 6, sample spread and mounted in LOCA. 40 x objective, between crossed polarisation filters.

Figure 5. Creamed honey crystals. Method 6, sample spread and mounted in LOCA. 40 x objective, between crossed polarisation filters plus quarter wave plate.

Discussion

Honey is liquid in the beehive, where it is kept at about 35 degrees C by the bees themselves. When stored at home after purchasing, honey will begin to crystallise from the bottom of the jar upwards if the room temperture is cooler than 35 degrees C. The rate of crystallisation is dependent on the fructose/glucose ratio; more fructose, the slower crystalisation occurs. 

Particle size has an impact of the mouth feel of foods. Larger grains feel coarse and the food 'grainy', whilst particles smaller than 20 µm impart a creamy texture in chocolate, for example. 

Honey with coarse crystals can be liquified again by warming if desired.

On the other hand, a smooth, cream honey can be deliberately created. Liquid honey is seeded with 10% of its volume of existing cream honey or large honey crystals that have been blended into sufficiently small particles. The added crystals are stirred into the liquid honey and the mixture left to stand for a week or more by which time the honey becomes saturated with small crystals and is turned into a cream honey.

Useful references and links

• Important temperatures for beekeeping: https://sheffieldbeekeepers.org.uk/important-temperatures-beekeeping/
• Fact sheet on honey crystalisation: https://www.qsi-q3.com/fact-sheet-crystallization/
• Paper on factors affecting honey crystallisation https://www.researchgate.net/profile/Reshma-Krishnan-3/publication/351418323_Honey_crystallization_Mechanism_evaluation_and_application/links/6099889c299bf1ad8d90881f/Honey-crystallization-Mechanism-evaluation-and-application.pdf
• Creamed honey Wikipedia https://en.wikipedia.org/wiki/Creamed_honey
• Creamed honey -theory and Dyce method https://www.bobsbeekeeping.com.au/image/bee-resources/Creamed-Honey.pdf
• Particle size and grittiness mouth- feel  https://www.jstage.jst.go.jp/article/jhej1987/46/12/46_12_1151/_pdf
• Using LOCA for making permanent slides. Gordon Brown, 2020, 'Living the Vida LOCA', Quekett Bulletin No. 78, pp 18-23. PDF reprints available on request from Gordon Brown, contact detail accessible here https://www.quekett.org/about/contact/gordon-brown 

Feathers under the microscope

The unexpected finding for me personally, on identifying feathers under the microscope, was that it was the downy feathers or down parts of feathers that can be key to identification. 

A fellow microscopist and I in Milton had missed being able to attend an Iceni meeting in Norfolk a couple of weekends ago on the topic of feathers. So later in the day, we got together to have a go ourselves. In addition to some feathers we had, we were able to borrow some from my neighbours here in Hall End.

Selection of feathers

That doesn't mean that the flight feathers are uninteresting. A closer look at the rigid part of the pheasant feather reveals the hooking mechanism on the barbules. These are the fine filaments that create the interlocking between the regular rows of  barbs radiating from the main feather stem.

Pheasant feather at 40x, 100x and 400x magnification, showing hooks and notches on barbules

Downy barbs can not only be found on (surprise surprise) down, but also on the bases of other feathers on a bird. Looked at closely, the barbs have fine filamentous barbules with distinctive nodes that can be seen at higher magnification. Below are some of the examples that we discovered.

Pheasant down at 40x, 100x and 400x magnification

Macaw down at 40x, 100x and 400x magnification

Goldfinch down at 40x, 100x and 400x magnification

Possible swan down at 40x, 100x and 400x magnification

Unknown down, possily pigeon at 40x, 100x and 400x magnification

Just to add a bit of colour, I had a go at making a stitched image of a peacock's feather, using the software Image View (which is very like Toupe View). Normally I would take separate pictures and use Hugin to stitch them together. Image View allows you to do the stitching automatically by moving the sample in rows or columns. The second attampt sort of worked, as shown below.

Eye of a peacock feather scanned with Imave View at about 40 magnification, equivalent to about 8 images stitched together.

All in all, it was a very enjoyable exercise and occupied a Sunday afternoon plus a bit extra for picture editing.

Useful identification guides for feathers under the microscope:

Microscopy of Feathers: Carla J. Dove & Sandra L. Koch, 2011. A Practical Guide for Forensic Feather Identification.  THE MICROSCOPE • Vol 59:2, pp 51-71.

PDF from https://www.researchgate.net/publication/289232335_Microscopy_of_feathers_A_practical_guide_for_forensic_feather_identification

Tim G. Brom, 1986. Microscopic identification of feathers and feather fragments of Palearctic birds. Bijdragen tot de Dierkunde, 56 (2): 181-204

PDF from https://brill.com/view/journals/btd/56/2/article-p181_1.xml?language=en

A better gluten free bread loaf crumb using aquafaba foam

 

Wheat free/gluten free bread loaf with a better crumb using aquafaba foam

Once an ardent bread baker, you can imagine my disappointment, nay, despair at having to bake wheat free bread. Crumbly, developing a grainy texture if left for any period of time. I therefore made it my objective to find a better solution. The solution first and my recipe second.

Solution: My elements for palatable gluten free bread are:

1. Creating a stiff peaked aquafaba foam and folding it into the bread dough.
2. Slicing and freezing a freshly baked loaf once it has cooled to room temperature. Take however many slices you need out of the freezer and heat them before using and eating.

Recipe for gluten free bread using aquafaba foam

Adapted from recipe on Doves Farm Freee White bread flour recipe.

Ingredients

Ingredients clockwise from top: Yeast in water with some sugar, olive oil, all other dry ingredients plus most of sugar, lemon juice, aquafaba

• 300g Doves Farm Freee White Bread Flour blend: rice, tapioca, potato, thickener: xanthan gum
• 75g Buckwheat flour
• 75g Chickpea aqua faba at room temperature (you can use egg white from 2 eggs)
• 5ml lemon juice
• 27ml Olive oil
• 36g Sugar
• 6g Dried east: yeast, emulsifier sorbitan monostearate, vitamin C
• 6g Salt
• 0.75 tsp/1.5g Psyllium husk
• 250ml Water

Method

• Mix a teaspoon of sugar, water and yeast and leave at least 5 mins to activate at no more than 33 degC
• In new large bowl, add lemon juice to aqua faba and using hand mixer, mix until stiff peaks achieved

Aquafaba and lemon juice mixed to stiff peaks

• Add flours, salt and Psyllium husk to a bowl
• Add remaining bulk of sugar, yeast and water and mix
• Add oil to flour mix and mix

Right: Dry ingredients mixed with oil and yeast in water

• Add the aquafaba stiff foam to flour mix and fold under with a large spoon until completely even

Aquafaba foam added to mixed dough

Aquafaba foam folded evenly into dough with spoon

• Preheat oven to 200 degC
• Line a 2lb loaf tin with non stick paper
• Pour and spoon the thick-batter like mix into the tin

Pour and spoon the thick batter mix into the lined bread tin

• Cover gently and leave to rise till doubled in size

bread mix in tin at start of rise.

Mix covered with a sheet of paper

bread dough doubled in size

• OPTIONAL: gently spray surface with milk
• Bake at 200 degC till internal temperature over 90 degC, about 45 minutes

Check internal temperature over 90 degrees C

• Remove from tin and allow to cool before slicing and freezing any bread not used immediately.

Loaf cooling on wire rack

A bit of science

Why whip the aquafaba?

We know that with normal wheat bread, it is the gluten that provides the structure that hold the bubbles created as the yeast ferments any natural sugars in the dough. The gluten forms long chains of gluten molecules.

Aquafaba (or egg white) contain proteins that can also be usedd to create a bubble structure by whipping them. 

During whipping, the natural structure of these proteins is broken open (denatured) and the protein molecules form chains that can coat air bubbles.

Room temperature and a bit of acidity from lemon juice or tatrate make the proteins easier to denature and foam during whipping.

Folding the foam into the dough mimics the effect of the gluten in wheat bread sufficiantly to hold the dough and the bubbles created during yeast fermentation.

Why check if the internal temperature is 90 degC or above after baking?

The bread dough contains a significant amount of water and during baking, only the crust loses sufficient water to be dry enough to brown. The interior of the loaf will only heat up to the boiling point of water at 100 degC unless you bake it to a charred crisp.

If the dough reaches a temperature of 90 degC or above, all the starch grains that make flour gritty will have been dissolved into the dough, making it smooth and all the proteins will have been completely denatured, fixing the bubbles in the bread permanently. 

Whilst you can check if the bread has baked by tapping it to see if it sounds hollow, I simply prefer the certaintly of knowing absolutely that the bread has been baked all the way through.

As the bread cools to room temperature, the dissolved starch will also create a jelly, just like a thick gravy that has been allowed to cool and set. The bread is easier to slice and ready to eat.

Why freeze gluten free bread and reheat slices to eat?

If you leave your gluten free bread out over time, it can acquire that irritating gritty texture. As the bread stands, water begins to migrate towards the crust and small starch crystals begin to form (see https://onlinelibrary.wiley.com/doi/abs/10.1002/star.200400330). Many gluten free recipes have a higher water content than the equivalent gluten wheat based ones to combat this.

By slicing and freezing the bread you slow down this crystallisation dramatically.

By heating the slices, you then melt any gritty crystals that are present and recreate a better bread texture.

Blog Reset - from Business to Personal

 

I finally closed Milton Contact Ltd in March, leaving just a legacy website www.miltoncontact.com. 

I have retired and this blog will reflect my personal interest and activities, which range from microscopy to museum volunteering to getting to grips with gluten free cooking!

GB-DE Rail sectors on track in London this week

Trains ready at Kings Cross (2020)

This time, a trip to London was quite an adventure, in this Covid era! I was off to Belgrave Square to spend two days chairing an event between a delegation of German companies in the rail sector, with a combined annual turnover of over a billion pounds, and GB representatives of key organisations, companies, and government departments. 

The event had been planned and implemented by Europartnerships, on behalf of the BMWi, the German Federal Ministry for Economic Affairs and Energy and the ministry's program "Mittelstand Global", which supports exporting German SMEs.

Day one, Hendrik, Stefan, Johann, Stuart, Robert, Yannik and Thomas, representing the seven participation companies (see links at bottom of article), were the audience to a range of presentations. Although their companies already had global business presences, including the GB, Covid and Brexit meant that it was important to get up to speed with the current situation vis a vis  GB-DE trade.

Some of the points that struck me personally follow.

Topics covered were:

• The excellent support available by their own BMWi through export development initiatives and portal IXPOS. One of the reasons for the success of German businesses abroad is the integrated assistance provided to SMEs, from theri local regions upwards to the federal level.
• Different distrubution channels and important cultural factors (how to understand us Brits), presented by the German Chamber of Commerce in the United Kingdom - AHK. (Do accept the invitation to go to the pub after meetings!). Perhaps the most worrying part of the presentation is that our (UK) importance for the German market (the 4th largest gloal economy), in terms of imports has rapidly declined down from 5th to 11th place and still sinking.
• The UKs decline in trade with Germany was also repeated in figures shown by the GTAI (Germany Trade and Invest) and was coupled with uncertainty about the GB reorientation post brexit. That said, the UK was still the 5th largest economy, expected to have strong growth in the coming years, and therefore a major partner to seek out.
• The Department for Transport gave us an update on Rail in the UK, showing that in terms of passenger miles, the UK was outperforming our neighbours - and that this was coupled with an exemplary safety record, including only 10 fatalities on the whole network in 2020-21. HS2 and the planned investment in the eastern parts of the North were in progress and a major organisatory change, in the formation of Great British Railways, was imminent.
• The Department for International Trade gave a very positive presentation on the opportunities and support for companies based in the UK. This was complemented by a useful list of project opportunities and places to look out for forthcoming contracts, as well as contacts for our German delegates to get in touch with at the DIT.
• With the UK being the birthplace of the rail sector, it is no surprise that one of the key membership organisations, the RIA (Rail Industry Association) has a 145 year history, a substantive membership of relevant companies in the sector, of which more than 60% are SMEs. As influencers of policy and public affairs, they also provide a comprehensive program of events and support, as well as encouraging innovation and providing trade missions overseas themselves.
• I suppose we all anticipated an opportunity for a nap during the presentation on Tax law peculiarities in the UK, by the speaker from Blick Rothenberg. Instead, the consequences of Brexit had us gripped by their impact on the import and export of goods across the new border with Great Britain. Theoretically The EU–UK Trade and Cooperation Agreement (TCA) allows for more than 99% of trade between us to go on without tariffs, if the goods are made more than 70% in the EU. Unfortunately, the sticking point for complex items is that the supplier has to do considerable checks to assess the percentage of non-EU items in multi component items or complex equipment and confirm it is below the 30% level to comply. More fundamentally, all your accompanying paperwork has to be in order, otherwise your goods get stuck in the Customs quagmire. Audience tip: if importing goods from EU to UK, get your UK recipient to sort out the paperwork and carriage. I was already aware of the risk of double taxation, by both the sender and recipient country from another as goods transferred across borders. This was another detailed presentation and emphasised the importance of getting the right advice to avoid pitfalls in trade.
• The talk by Entreprenör covered the assistance in setting up a copany in the UK (much simpler than in Germany but with public transparancy re accounts and personell information held at Companies House. One of the new hazards to look out for was that an audit may be required based on the size of the worldwide group.
• Day 1 ended with a very informative talk on Transport for London (TFL) and Cross Rail, which shone a light on the recovery after the initial covid Epidemic.
• 
  

Millstone phallus comes to Godmanchester Museum

 

Godmanchester Millstone with Phallus held by Curator Kate Hadley (courtesy of Godmanchester Museum)

I left the preparations for the reinstated annual Hall End BBQ for a quick dash up the A14 for a very important date. Godmanchester Museum was celebrating its re-opening with a major event, the display of the new exhibit, part of a Roman millstone emblazoned with a phallus (Saturday 31st July 2021). This is a very rare item indeed - only four are known of amongst the tens of thousands of Roman millstone fragments found to date in Britain. 

My small involvement with this stone began with a phone call taken by my wife Jane, from the Godmanchester Museum's curator Kate Hadley, who wanted the stone photographed. 

"Kate called, saying that she was holding a p....s you might be interested in!" was the message.  How could I resist! Kate and I spent an afternoon in June trying to get the best lighting and positioning of the stone to reveal its true magnificence.

By the time I arrived, a crowd had already gathered for the Godmanchester Museum opening and  David Stokes, Chairman of the museum, began the proceedings at 2:30 pm.

David Stokes, Chairman of the Godmanchester Museum, opening proceedings

Interested crowd at the opening of the Godmanchester Museum

He was followed by Claire Hardy, Director of the Norris Museum, who had generously brought along their rare example of another Roman millstone fragment with a phallus, from the Norris collection, (partner to another fragment held at the Norris showing engraved curves).

Claire Hardy, Director of the Norris Museum, and their phallus millstone

Godmanchester Mayor Councillor Clifford Thomas and Philip Saunders, Chairman of the Huntingdonshire Local History Society

The Godmanchester millstone fragment had been found in a posthole at Offord Hill house, during the excavations prior to the new A14 build. Archaeologist Ruth Shaffrey, realised its significance when when was conducting a routine catalogueing of the finds. She had been researching Roman millstones and was a specialist in ancient worked stone. It was thanks to Quentin Carrol, Historic Environment Assistant Director and Archaeologist, Cambridgeshire County Council, that the remarkable millstone was made available to its nearest hometown museum.

Ruth Shaffrey, Archaeological Worked Stone Specialist, with the Godmanchester millstone phallus (courtesy of Godmanchester Museum)

It was great to hear from Ruth about the origins and possible significance of the millstone. This is what I recall from her talk.

During Roman times, millstones were made using stone excavated from the Millstone grit from the Peak District/Yorkshire. The Godmanchester and Norris millstones were of a medium size and probably animal driven, whilst larger ones could be water powered.

In a typical Roman bakery, the counter would be in the central part of the room, the millstone at one end of the room and the ovens at the other. Customers would therefore have quite likely seen the magnificent carved stones displayed in action. 

The millstone had obviously broken at some time and was later used as a quernstone for hand grinding of grain in its own right.  It was also used as a sharpening tool for blades. It finally ended up in a filled-in post hole, where it was finally discovered in the A14 dig.

Millstones with carvings upon them are rare and would most likely have been commissioned by a wealthy baker or merchant. Whilst the use of a phallus might seem unusual to the modern eye, it was a familiar emblem in Roman times and used both in domestic and other settings, including as jewellery and even being worn by children (https://imperiumromanum.pl/en/curiosities/phallus-symbol-in-roman-and-greek-world/). It is thought that it was a lucky symbol as well as the usual possible links to fertility that we would associate it with.

Much like anvils, millstones were themselves seen as important objects, imbued with symbolism, as they were closely linked to grain, harvests and the production of food. To have an object like a millstone, that is already highly symbolic, and then to have it engraved with another important symbol was therefore unusual, hence their rarity.

Ruth also revealed that our region was regarded as one of the granaries of Britain and that more millstones and fragments had been discovered here than in the rest of the UK.

After a walk around the exhibition afterwards and purchase of one of the magnificent postcards of the millstone, I left Ruth to enjoy her specially made Roman vegetarian meal. It had been prepared by local Godmanchester Roman cookery expert, Sosia Juncina. It was back to Milton for our own annual BBQ and street party!