Field Collection and Sampling 1courtesy of Carol Ritland

Starting your collection What to sample: Tissue What to consider: Age, Season What methodology to used for your biological questions What is the natural history of your organism eg. Life history etc. nd2.html&h=375&w=500&sz=70&hl=en&start=5&usg=__d6sSz-0SC0vP_NuogTY5sRsvsxY=&tbnid=a9- 2ftNtkcqeBM:&tbnh=98&tbnw=130&prev=/images%3Fq%3Danimals%26gbv%3D2%26hl%3Den%26client%3Dfirefox- a%26rls%3Dorg.mozilla:en-US:official%26sa%3DG 2

Starting your collection….cont’d Check for permission and necessary permits for sampling Check for location of field sites Check on logistics of field collection 3

4 Conifer abundance: affect on soil richness Photos = L. Ritland courtesy of Carol Ritland

5 Starfish abundance: affect on barnacles Photos = L. Ritland

6 1)What should you collect? 2)What could you collect? 3)Where would you collect? 4)When would you collect? 5) How would you collect? 6) How much would you collect? 7) Why did you collect them?

Tissue (Always go fresh if possible): –Plant: Seed, Leaves, Flower, Pollen, Bark, Xylem, Roots –Animal: Reproductive tissue, muscle, skin, hair, scat, blood, ear/toe/tail clip, fin, tooth, sloughed skin, saliva –Fungal: Hyphae, spores, fruiting bodies –Bacterial: single isolate culture –Destructive vs Non Destructive methods 7

Factors to consider when collecting: Age: –Plant = actively growing material such as apical points, seedlings –Animal = actively dividing tissue (buccal and blood cells) –Fungus = young fruiting bodies (pure culture) –Bacteria = liquid culture Season: –For conifer = use early spring bud burst especially for DNA markers 8

How to sample: Sampling schemes: –Linear –Quadratic squares –Distance between samples –For animals: migration, reproductive strategies, life cycle –For plants: clonality, roots, reproductive strategies, life cycle –Consult a statistician? (Gene expression studies) 9courtesy of Carol Ritland

Sampling cont’d Tools for sampling: –Ideally flash freezing samples with liquid nitrogen and transportation under ultra low temperature –Clean and if possible sterilize collection tools between samples –Pack samples with foil or proper containers eg. cryovials –Label all samples with non water based ink and protect with clear tape/paper and pencil 10courtesy of Carol Ritland

Liquid nitrogen vapour tanks 11courtesy of Carol Ritland

Ultralow temp. Liq N2 tanks Pending on size Ultralow temp can last from 10 days to 3 weeks Can be used on aircrafts 12courtesy of Carol Ritland

Use sterile technique when sampling Keep meticulous records Identify any problems in the field for given sample Do not depend on your memory alone 13courtesy of Carol Ritland

Long Term Storage: –Ideally all tissues should be kept at ultra low temperature (minus 70 to 80°C) –Certain tissues (blood, animal tissues cut into small pieces <1mm 2 ) could be stored in 100% ethanol and saturated EDTA –Plants (small amounts) could be desiccated with lots of silica beads with lots of changes of beads 14courtesy of Carol Ritland

No No..s Items to avoid: –Unnecessary chemicals eg. Formalin –historical samples that has been treated with fixatives –degraded samples –freezing and thawing of tissue –freezer burn (improper storage conditions) –improper inventory of samples 15courtesy of Carol Ritland

More to ponder….. Items to consider: –For certain molecular marker (eg. AFLP, microarray) use the same tissue type (developmental differences could cause error when genotyping) –If possible collect all samples within a season over a same span of time (eg. For microarray analysis) –Collect more samples than required for pilot study and lost of samples 16courtesy of Carol Ritland