Sunday 19 March 2023

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


Posted by Miltoncontact at 00:11

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