Lugol’s Iodine Solution

Lugol’s solution is the preservative preferred by Dalcon Environmental for algal samples.

Lugol’s solution is an iodine-based preservative with several advantages compared to other preservatives. First and foremost, Lugol’s solution is relatively harmless (not very toxic) compared to aldehyde-based or other more toxic fixatives.  It is also better for accurately quantifying cells than many aldehyde-based fixatives as it stains cells a dark brown colour and stains starch bodies black.  This facilitates enumeration and aids with identification; it also adds weight to cells and helps phytoplankton cells sink to bottom of settling chambers.

Lugol’s solution (neutral Lugol’s solution) is prepared as follows:

  1. Measure 1,000 ml distilled H20 into a brown glass bottle
  2. Dissolve 100 g of potassium iodide (KI) in the 1,000 ml distilled H20
    This may take from 30-45 minutes, and a small amount of residue may remain in the bottom of the flask.
  3. Add 50 g of crystalline iodine (I2) and mix thoroughly ensuring it is fully dissolved
  4. Securely stopper the bottle and store in a dark place
  5. Transfer some of the prepared solution to a small (100 ml) amber glass bottle for routine use and store the rest.
    Before transferring solution to the small amber glass bottle, swirl to mix any material that has settled out during storage.
Neutral Lugol’s solution can also be purchased from most scientific chemical suppliers (but be sure to confirm it contains the same proportion of chemicals as above before purchase).

Typically an acidified version of Lugols solution is used to preserve most algal samples.  Acid Lugols solution is prepared as above but with the addition of 100 ml of glacial acetic acid (CH3COOH).

Acid Lugol’s solution is  good  for  all  phytoplankton  but  not  for  coccolithophorids  as  the  acid may dissolve the coccoliths. If coccolithophorids need to be preserved with the coccoliths  intact,  a  parallel  sub-sample  should  be  fixed  with  alkaline  Lugol’s solution.

Alkaline Lugols solution is prepared as above substituting the 100 ml of glacial acetic acid for 50 g of sodium acetate  (CH3COONa).  It is best prepared by first dissolving the sodium acetate in 50 ml of the water first.

The amount of acid Lugol’s solution which should be added to an algae sample depends on the concentration of algae in the sample, but typically 0.3 ml to 1.0 ml of  acid Lugol’s solution per 100 ml of sample is sufficient with the larger volume of Lugol’s being used for particularly dense algal samples.

A good rule of thumb is to add the Lugol’s to the sample a few drops at a time until the colour of the water in the sample is that of weak tea or light beer (as illustrated below) allowing a little time between each addition for the Lugol’s to be absorbed into the algal cells.

If too much Lugol’s is added, or if samples are received already preserved with too much Lugol’s, sodium thiosulphate can be used to neutralise it.  It is best to neutralise a sub-sample of the original sample before to analysis rather than the whole sample.

To neutralise the Lugol’s in a sample, add 100 ml of a 3% sodium thiosulphate stock solution (3g of Na2S2O3 in dissolved 100ml of deionised water) to 5 ml of sample.

Although this is our preferred preservative, it is important to note that there are some disadvantages to using Lugol’s solution as follows:

  • Lugol’s solution breaks down in sunlight – samples, in-use bottles of Lugol’s and stock solutions of Lugol’s must all be stored somewhere dark and cool.
    • Samples preserved in Lugol’s solution do not have a very long shelf life. Samples stored for more than a few years are of little use.
    • Lugol’s solution loses its preservative power over time, and stored samples must be checked routinely (every three months) with more Lugol’s added if necessary.
      • Plastic sample bottles will absorb some of the Lugol’s colour; this needs to be taken into account when assessing the amount of preservative to add.
    • Lugol’s solution masks  chlorophyll  fluorescence,  which  may  be  needed  to  recognise  mixotrophic and very small species.
    • Lugol’s solution may dissolve hard  structures  such  as  coccoliths  and  diatom  frustules  and, therefore, is not ideal for long-term storage of many plankton taxa
    • Lugol’s solution stains   cells   a   dark   brown   colour,   which   may obscure   some   of   the    characteristic features of used to identify certain taxa.
    • Lugol’s does not necessarily preserve the cell shape and size of live specimens, and it can cause a 30% to 40% shrinkage from live biovolume.
    • Plastic sample bottles are permeable to Lugol’s solution. Over time, the sample bottle (and possibly surrounding items) will become discoloured due to the iodine.

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