A Methods Manual for the Collection, Preparation and Analysis of ...

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2.3.2 Examination of fresh material and extraction of diatoms for acidtreatmentThe living, motile component of the sampled diatom population may be extracted andseparated in the following manner: The fresh sediment/diatom mix is spread over the bottom of a petri dish or flat-bottomedplastic tray and the heavier sediment is allowed to settle for a few hours (e.g. overnight). The following day the excess supernatant is drained from the petri dish until the moistsediment is exposed. Several coverslips are allowed to gently ‘float’ and rest on the damp sediments for a 4hour period of exposure to natural light. The coverslips are then carefully removed and gently rinsed to remove unwanted sandparticles. The coverslips are placed on a clean slide for examination of diatom cells.2.3.2.1 Alternative separation techniques Tissue paperIf the original sample contains large sand grains, it is advisable to place tissue paper betweenthe coverslip and the sediment. This allows the passage of the motile diatoms on to thecoverslip but prevents the transfer of unwanted sediment grains to the slide. Submersed coverslipsIt is not necessary to remove all the supernatant from the fresh material if living diatoms arenot required for initial examination. Coverslips are submerged and ‘floated’ on to thesediment/diatom mix after the material has settled in a tray / petri dish. Living diatomsactively adhere to the surface of the coverslip under the water. This technique ensures thatsand grains are washed off the coverslips as they are carefully withdrawn and placed in asample bottle containing ethanol for preservation or allowed to air dry for acid treatment.2.4 PhytoplanktonPhytoplankton sampling can be achieved in one of two ways. The most simple method is tocollect water in a two litre container, add preservative (Lugol’s iodine), and allow the deadplanktonic organisms to settle out. The sedimentation rate of most phytoplankton allows forcomplete settling within 16-24 hours from a two litre measuring cylinder.Alternatively a plankton net may be used with a mesh size of not more than 25 µm. Theplankton net should be dragged back and forth just below the surface of standing waters orheld in the stream of moving waters for a few minutes. This should allow for the collection of15
ample cells. The contents of the net should then be emptied into a wide-mouthed plasticstorage bottle and preservative added if required (see Section 11 for further details).In standing waters (such as dams, lakes, estuaries) one should be aware of verticalstratification of phytoplankton during certain times of the day. Where the system is deepenough (i.e. >10m) take a vertical haul with the plankton net over a 5 metre depth to cover thezone where light penetration is sufficient (euphotic zone) to encourage algal growth.2.5 Terrestrial or soil diatomsSoil diatoms and aerophilic diatoms have seldom been investigated in South Africa. Thesediatoms are an interesting group with many adaptations for the arid climate in which they live.Soil diatoms can be collected from moist sub-aerial habitats, as well as from aerial and aridaerial habitats. Six sub-samples (± 5 cm 2 ) should be collected within a 10 m radius. Once thesite has been selected detritus or other material covering the surface of the soil should becarefully moved aside. The soil should be collected to a depth of about 1 cm using a knife,spoon, perspex corer or other similar implement (a slightly concave butter knife is ideal!). Thesix sub-samples should, in total, amount to about 200 grams of soil. This soil should be storedin a paper envelope to prevent the build up of moisture which promotes the growth ofundesirable fungi.In order to separate the diatoms from the soil, a portion of the soil sample is placed in a sterilepetri dish and wetted with distilled water until the soil is saturated. One or two wettings maybe required before the soil becomes saturated depending on the amount of organic materialpresent. Once the soil is saturated it should be left for several days exposed to light (but not indirect sunlight), where after pre-cleaned coverslips can be placed gently on the surface of thesoil. After two weeks the coverslips can be removed and the living cells examined under themicroscope. If cleaned material is required the coverslips may be treated using any of themethods detailed below under Laboratory Procedures. A simple method to check for thepresence of diatoms is to invert the coverslips and heat them until the organic content isburned away (incineration). Permanent mounts can then be made in the standard fashion seeLaboratory Procedures, section 2. Many soil diatoms are very small and are best examinedunder the scanning electron microscope (SEM) - see details of preparation techniques below.16
Page 1 and 2: A Methods Manual for theCollection,
Page 3 and 4: This report is part of a set on Dia
Page 5 and 6: FOREWORDThis method manual is inten
Page 7 and 8: TABLE OF CONTENTSNOTE TO THE USER .
Page 9 and 10: GLOSSARYAerophilic - Depending on f
Page 11 and 12: Phytoplankton - The phytoplankton c
Page 13 and 14: photomicrographs earned high praise
Page 15 and 16: 2. How do you recognise diatoms in
Page 17 and 18: 4. Diatoms - Colony formation and a
Page 19 and 20: 5.1 Pennate and Centric diatomsPenn
Page 22 and 23: SECTION 2: FIELD PROCEDURESTo facil
Page 24 and 25: depending on the period of exposure
Page 28 and 29: 3. Toolkit for Fieldwork (field app
Page 30 and 31: 5. Site selection for water quality
Page 32 and 33: 6. Sampling locality details and fi
Page 34 and 35: 8. Choice of substrata (detail)Samp
Page 36 and 37: 10 Preservation of diatom material
Page 38 and 39: 1: Micro-pipette 1 ml with disposab
Page 40 and 41: 1.2 Pre-preparation examination of
Page 42 and 43: 1.3.2.3 Shake the sample well and p
Page 44 and 45: one is available the peroxide can b
Page 46 and 47: 2 Preparation of diatom slidesHealt
Page 48 and 49: 2.1.9 After the diatom-coated cover
Page 50 and 51: 4. ArchivingIt is important to reta
Page 52 and 53: affected at counts of 300 and above
Page 54 and 55: 6. Counting records (electronic and
Page 56 and 57: The main source of variability is,
Page 58 and 59: 11. Key reference worksThere are se
Page 60: HARDING WR, ARCHIBALD CGM, TAYLOR J

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