TEST METHOD: DIATOM SAMPLING AND IDENTIFICATION

Revised: 30 May 2007 (peroxide method added)

RISK ASSESSMENT

Assess the Quality, Safety and Environmental risks of each step.

OVERVIEW

This test method is used to determine the diatom assemblages occurring in waterbodies such as rivers, streams, wetlands, estuaries and salinas. Assemblages of pollution and/or salinity tolerant diatoms may be used as an early indicator of a deterioration in water quality.

TASK SAFETY REQUIREMENTS

Safety boots are mandatory

Observe the requirements of the Chemical Hygiene Plan

Use safety glasses and wear gloves to protect hands from heat in the laboratory.

SPECIFIC JOB STEPS RISK ASSESSMENT RISK CONTROL
Collecting periphytic diatoms: Place 10 glass slides in a JJ Periphtyometer. Secure with fishing line. Suspend the periphytometer in the water at the sampling location for two weeks. When collecting, keep the samples cool and damp in an esky. It is hard to remove dried diatoms from the slides. Wrap the colonised periphytometers in wet paper towels in the esky during transport.

Counting diatoms (abundance):

Method 1: Remove 5 slides from the periphytometer. Use a single sided razor to scrape the diatoms from each side of the slides into a test tube. Add 4-5 drops of Lugols solution and distilled water to make a storage volume of 30mL.

Shake to break up clumps and suspend diatoms.

Place 1mL in a Sedgewick-Rafter counting chamber. A chamber has 1000 cells arranged in 50 columns of 20. Count the diatoms in as many cells as it takes to obtain a count of 200 to 400 organisms. Record the tally and the number of cells counted.

Lightly colonised slides may not need 30mL volume.

Single sided razors are sharp.

Use a smaller volume, but remember to account for the volume in the calculation.

Use razors with a safety side, NOT snapped double sided blades.

Calculations:

Different storage volumes or different sized slides may have been used. Modify the calculations to reflect the non-standard volume or slide size.

Method 2: Remove several slides from the periphtometer and scrape away the diatoms from one side of the slide while leaving the coating of slides on the other. Add a drop of water (sea or fresh, as appropriate) to the diatom coating and place a large coverslip over the diatoms. Count diatoms in several fields of view (at least 200 diatoms) and record the number of FOV, number of diatoms counted and magnification used to view the slide. Record the most dominant species and the presence of any delicate species.

You do not know the names of the species. Use literature in the Delta library and photographs in the Delta electronic herbarium to identify diatoms.

Calculations:

Determine the area of the FOV for the magnification you used to view the diatoms.

 

You do not know how large the FOV is.

 

Use the slide micrometer to determine the diameter of the FOV and then calculate the area of the circle in mm2.

Determining species assemblage/composition using permanent mounts: The remaining 5 slides are removed from the periphytometer and are scraped into a test tube or small beaker with a single-sided razor. A few drops of Lugol's solution is used to preserve the diatoms. The frustules need 'clearing' of organic material to display their ornamentation, for identification. The organic matter is removed from the frustures by either boiling in nitric acid or in peroxide solution.

Single sided razors are sharp.

Delicate frustules may not withstand the cleaning process.

Use razors with a safety side, NOT snapped double sided blades.

Note any delicate species (eg Nitszchia closterium, Nitszchia flexa) seen in the abundance counts and if they do not appear in the cleaned sample, record this in a note.

Nitric acid cleaning: The volume of diatom scrapings is made up to 30mL with distilled water. The sample was shaken to break up clumps and suspend the diatoms.10mL of sample is transferred to a 100mL Pyrex beaker, and 10mL of strong nitric acid (60-90%) added also. The beaker is placed on a hot plate in a fume cupboard or in the open air, and allowed to come to the boil, then simmered until the volume is reduced by half.

The samples are poured into test tubes and allowed to cool and settle overnight. OR

The cooled samples are placed into plastic centrifuge tubes and topped up with distilled water then centrifuged at 3500 rpm for 5 minutes

Nitric acid is a strong acid

Handling hot glassware roughly may cause cracking of the glass or injury to the operator.

Settling too long allows lightly silicified diatoms to dissolve

Use safety glasses, a fume cupboard or external workbench and heat proof gloves.

Handle gently.

Only settle overnight. Preferentially use the centrifuge.

 

The supernatent is pipetted off and replaced with distilled water. The ‘pellet’ of diatoms is resuspended and allowed to settle overnight. One wash may not remove all the acid. Repeat the wash cycle 2-3 times.
Peroxide cleaning: Hydrogen peroxide is added to the diatom scrapings in the test tube or small beaker, and the tubes are placed in a water bath at approximately 60-80° C, or onto a temperature-controlled hot plate. Two hours is sufficent for the removal of small amounts of organic matter from the frustules, however in areas with heavy sediment of overgrowth of other organisms it may take a lot longer.

Clearing of the frustules is complete when effervescence ceases.

The samples are poured into test tubes and allowed to cool and settle overnight. OR

The cooled samples are placed into plastic centrifuge tubes and topped up with distilled water then centrifuged at 3500 rpm for 5 minutes

The pellet of diatoms is resuspended in ethanol. This process should be repeated twice, and after the final rinse in ethanol, the sediment can be stored in a vial, ready for mounting.

Hydrogen peroxide reacts very vigorously with organic matter

 

Handling hot glassware roughly may cause cracking of the glass or injury to the operator.

Settling too long allows lightly silicified diatoms to dissolve

Make sure the test tubes or beakers are large enough to prevent any overflowing. Reduce the temperature if the vessels continue to effervesce to the point of overflow.

Handle gently.Use safety glasses and heat proof gloves.

Only settle overnight. Preferentially use the centrifuge.

Mounting: Resuspend the diatoms and observe a wet mount under the microscope. The frustules should not overlap. Adjust the dilution until the required density is reached. Frustules overlap

There is lots of space between individual frustules.

Dilute the sample.

Let the sample settle overnight and remove some supernatent.

Pipette a drop of sample onto several coverslips. Allow to settle and evaporate for several hours. If speed is of the essence, finish the drying by warming the coverslips at a low temperture (60oC) on a tile. Otherwise leave to dry naturally, to maximise the number of valves that will settle in valve view, rather than girdle view. Not all diatom species may be in a pipette. Several replicates ensures maximum representation. It also allows for slides to be sent away to the diatom herbarium for identification.
Place a drop of Napthrax mounting medium on several glass slides and "touch" the slides onto the coverslips. Flip the slides so the coverslips are uppermost and keep them warm until the toluene solvent in the Napthrax stops bubbling. Allow the slides to cool and harden at room temperature.Check they have set properly by trying to shift the coverslip gently with your fingernail.

Napthrax contains a flammable solvent – toluene.

The covers move on the cooled slides when you test them

NEVER use an open flame near toluene, use a sealed warming element.

Reheat.

Identification: Diatoms are identified to species level or genus level, depending on the project. Identification uses literature from the Delta library and photographs in the Delta electronic herbarium. One approach is to photograph each species seen on a slide, for later identification. The species are given a numeric code and the counting may proceed on that basis, with names being applied later. Some diatoms resist identification. Assistance is available at the:

International Diatom Herbarium, Curtin University of Technology, GPO Box U 1987, Perth 6845 Western Australia

Provide the slide and a digital photograph of the recalcitrant diatom to them.

Once the diatoms on a slide are identified or photographed, the species are tallied, by counting the diatoms in “fields of view” until 400 frustules have been tallied.

The permanent mount slides shall be labelled and stored as voucher specimens.

   
Calculation: The relative frequency of each species is calculated from the tally sheet.    
The information collected from the counts may be used to assess sites using the Generic Diatom Indices or may be used in other forms of analysis.    

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