Diatom Isolation process

Diatoms are a major group of algae, and are among the most common types of phytoplankton. They are photosynthetic and are orange-ish green in colour because of the presence of caroteniods along with chlorophyll. Most diatoms are unicellular, although they can exist as colonies in the shape of filaments, ribbons, zigzags, fans etc. They are generally microscopic. A unique feature of diatom cells is that they are enclosed within a cell wall made of silica (hydrated silicon dioxide) called a frustule. These frustules show a wide diversity in form, but are usually almost bilaterally symmetrical, hence the group name. The symmetry is not perfect since one of the valves is slightly larger than the other allowing one valve to fit inside the edge of the other. Diatom communities are a popular tool for monitoring environmental conditions, past and present, and are commonly used in studies of water quality, by studying their colonies and diversity of species.
Diatoms are a widespread group and can be found in the oceans, in freshwater, in soils and on damp surfaces.

This process of extraction of the Diatoms takes around 1 to 1.5 hours.

In Indonesia, during HackteriaLab 14, we tried to study the diversity of diatoms from the Kali-Code and see if diatoms can be used as a feasible bio-indicator of water pollution.

The first bottleneck was that the diatoms needed a slightly higher magnification than what the webcam microscope has in order to be observed clearly. We modified the webcam microscope by increasing the distance between the lens and the sensor to increase magnification. Later, back in the (Art)ScienceBLR lab, we rigged the webcam lens onto a DSLR to obtain clearer, magnified images of the diatoms.

The process we followed in Indonesia was also dependent on the use of a centrifuge. This made it a bit difficult for the process to be carried out in the field. If access to a centrifuge is difficult, samples can be swirled and left to settle in a test tube on the stand, for a crude separation during sedimentation. This method takes significantly longer, so it is recommended to use a centrifuge.

Using gaudilabs centrifuge to separate the diatoms. The diatoms form a white layer on top of the other algae.

Centrifuging using the dremel at the workshop in Jogja

During the workshop we did with the interns of X-Code in Jogja, we also realized that because of the number of steps and complexity, this process would be better for a workshop which continues over a day or two, so that the information and process can be understood with clarity.

Materials

Plastic tray
Toothbrush with hard bristle preferably
Drain cleaner (Anything containing sodium hypochlorite, Domex in India, Domestos Indonesia )
Bigger eppendorf tubes, which can be used in a suitable centrifuge
Test tubes and stand
Pipet
Plastic pipet/dropper
A device which shows gps coordinates – smart phone in our case ( GPS test app)
Microscpe ( A slighly modified webcam microscope is fine. We need a minimum magnification of around 300-400X.)

Method

1. Identify the water body and location from where the diatoms are to be sampled. Mark the coordinates on the gps.

2. Choose a rock/stone which is flat, covered by the water and is at a shallow part of the water, which receives enough sunlight. We chose the ones which were close to the shore/bank.

3. Pick up the rock. It should feel slimy and have an orange-ish green tinge (maybe just in parts, but that is fine). If it is slimy, place it into the tray or else choose another rock.

4. Scrub the rock using the toothbrush to remove all the diatoms. Using a dropper, pour some clean water on the rock. The orange-ish water collected in the tray contains the diatoms.

5. For a simple observation, just take a drop of this water and observe under the microscope. ( A good reference of the variety of shapes possible is Here. )

6. To isolate the diatoms from other micro algae, samples of the water from the tray can be centrifuged and observed. The top layer would have the highest concentration of diatoms. In the absence of a centrifuge, the water can be placed in a test tube, swirled once and then left on a stand till it settles. The top layer here too has the highest concentration of the diatoms.
The diatoms are live here and some species are moving, which can make them hard to observe. If the concentration of the diatoms is high, this sample on the slide can be dried. After it has dried, The slide can be preserved using several methods. We used Akbars PVLG recipe and after mounting, sealed the slide with transparent nail polish.

7. To observe just the silica casing of the diatoms, take the water from the tray containing the diatoms and put it into eppendorf tubes and centrifuge. Once all the algae is in the bottom, using a dropper, remove all the water without disturbing the diatom and algae layer.

8. Fill the tube with drain cleaner, and mix it through all the layers. Let this solution stand for half an hour, and agitate it 3 to 4 times while it sits.

9. After half an hour centrifuge the tubes again. This time, there should be a white layer which forms between the liquid and muddy layer. This White layer consists of the diatoms. Before observing this, they need to be cleaned of the drain cleaner though.

10. To clean the diatoms, remove the drain cleaner without disturbing the other layers and add clean water to the tubes. Mix it well and centrifuge. Once the layers are separate, again replace the water with some fresh water and repeat the process. This needs to be done around 3 times.

11. After the final wash, the water can be thrown. The diatoms can be carefully removed from the white layer using a pipette and observed under the microscope. They can be mounted using Akbars PVLG recipe.

PVLG Receipe

Material:

100 mL distilled water
100 mL lactic acid
10 mL glycerol
16,6 g polyvinyl alcohol (PVA)

Preparation:

To prepare PVLG, add the polyvinyl alcohol (a dry powder) to the water and put in
60 degree oven until dissolves. Add lactic acid and glycerine and allow the solution
to set for 24 hours before first using. Specimens can be mounted directly in the
PVA solution, or the solution can be added to the sides of the cover slips that
were made with water, lactophenol, or melzer’s reagent. The PVA solution will
infiltrate the material in a day or two. Slides made with PVLG can be hardened by
heating at 40-75oC overnight. Immersion oil can be wiped from these hardened slides
without disturbing the specimen.