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BIO 300
BIOLOGICAL TECHNIQUES AND SKILLS

     SARINI BINTI AHMAD WAKID
     FACULTY OF APPLIED SCIENCE


                                  2
Techniques in field investigation




                                3
Population Ecology
Population—
A group of individuals of the same species
living in a particular geographic area

Population Ecology—
Concentrates mainly on the factors that
affect how many individuals of a particular
species live in an area
                                        4
What is a sample?

“A portion, piece, or segment that is
  representative of a whole”




                                        5
Why do we sample?

Because it is usually impossible to
count all the plants or animals present
in a given area
– e.g. # dragonfly larvae in a pond
– e.g. plant cover on a river terrace
– e.g. species of plants in the estate



                                          6
NON-INVASIVE SAMPLING

Avoid any degradation of the habitat when
sampling
 􀁺 Removal of whole or parts of organisms
should be limited to species that can quickly
  recover




                                                7
REPRESENTATIVE
SAMPLING
Take a number of samples from around the
  sampling site so as to be reasonably sure
  that the samples represent the site in general
Necessities…
 􀁺 the samples represent the whole
– It is necessary to take enough samples so
  that an accurate representation is obtained
– It is necessary to avoid bias when sampling

                                                   8
SAMPLING UNITS

Type determined by the organisms and the
  physical nature of the habitat being sampled
– Area of ground surface
– Volume of air, water or soil
 􀁺 Standard units enable comparison of results




                                                 9
QUADRATS
A standard, area sampling unit consisting of a square frame
 􀁺 Consistent size and shape is essential for comparing samples
   from different places and/or times
Quadrat size
Chosen to suit sampling goals
 􀁺 A balance between what is best and what is practical is always
   necessary
 􀁺 Should suit:
– practical constraints
– habitat
– organism



                                                                    10
Quadrat Method
   A Quadrat is a sampling area of any shape
    randomly deployed. Each individual within
    the quadrat is counted and those numbers
    are used to extrapolate population size.
       Example: a 100 square centimeter metal
        rectangle is randomly thrown four times and all
        of the beetles of a particular species within the
        square are counted each time: 19, 21, 17, and
        19. This translates to 19 beetles per 100 cm2
        or 1900 per m2.


                                                            11
QUADRAT & TRANSECT
ACTIVITIES
How, Where & Why Scientists Do Sampling
Scientists often collect data “in the field” which could
mean underwater, in a forest, in a cave, on a reef, or
even the moon! Two essential methods to gather
ecological information in a standardized way are:
Transect Sampling (using a single line) and
  Quadrat
Sampling (counted within a grid). These sampling
methods provide more accurate data than random
sampling or simply guessing, but they take less time
than counting every specimen in a certain area.
                                                           12
Quadrats
- a   sturdily built
  wooden frame,
   can be folded for
  easy transport and
  storage




                       13
Using a quadrat along
                            a belt transect




Quadrats
- When placed on the ground, the species
   present within the frame are identified and
   their abundance recorded
- Sampling could be random or systematic
                                                    14
Practical Constraints

Small quadrats are quicker to survey but yield a
  smaller individual sample of habitat
– Often require a larger # of samples to
  represent the habitat
 􀁺 Large quadrats require more time and effort
  to survey but provide a larger individual
  sample of habitat
– Often require a smaller # of samples to
  represent the habitat
                                               15
Habitat size
Appropriate sample unit size depends on size scale of
  the habitat
– Small scale habitats require smaller sized samples
 􀁺 Ex. Boulders
– Large scale habitats require larger sized samples
 􀁺 Ex. Forests




                                                        16
Organism size and density

Depends on size and density of organisms
– Small, dense organisms require smaller
  samples
 􀁺 Ex. grass
– Large, scattered organisms require larger
  samples
 􀁺 Ex. Trees


                                              17
TYPES OF SAMPLING

   􀁺 Systematic
   􀁺 Stratified
   􀁺 Random




                    18
SYSTEMATIC SAMPLING

    􀁺 Often used when the area being studied is
    varied, not very large, or
   when time is available
    􀁺 Samples are taken at fixed intervals




                                               19
How to sample systematically
Systematic samples are usually taken along a transect
  line marked by a tape measure
 􀁺 Transect- a line laid across an area




                                                        20
Sampling along gradients

Transects are set
up along a
environmental
gradients
– down a hillside
– across a streambed
– out from a source of
pollution


                           21
Types of transect sampling

   􀁺 Line transect
   􀁺 Belt transect




                             22
Line transect method
A measured line is laid
across the area in the
direction of the
environmental gradient
– The species touching the
line can be recorded
along the whole length of
the line (continuous
sampling) or at specific
points along the line
(systematic sampling)

                             23
Line Transect
- useful where a transition of flora and/or fauna
   occurs
- a string or tape is stretched out along the
   ground in a straight line;
    record the organisms touching or covering
   the line all along its length or at regular
   intervals
- Profile transect: when there is appreciable
   height change along the transect and thus
   affecting the distribution of its species

                                                    24
Belt transect method

Similar to line transect but
widens the sampling area
– Transect line is laid out
– Samples are taken by
determining abundance or %
cover in an area that is a
defined distance from the line
– Samples can be taken all the
way along the line, at specific
intervals or even randomly


                                  25
Using a quadrat
                             along a belt transect,
                             e.g. ladder transect
                             (every 5m)



Belt Transect
It is a strip, usually a metre wide, marked by putting a
   second line parallel to the other. The species
   between the lines are carefully recorded, working a
   metre at a time.
Alternatively, a frame quadrat in conjunction with a
   single line transect could be used.                26
Point Frames
- for grassland field study of dense vegetation

                                                  27
STRATIFIED SAMPLING
  􀁺 Often used when
there are small
areas within a
larger habitat that
are clearly different
  􀁺 Strata- major
differences within
communities
recognized before
sampling begins

                        28
RANDOM SAMPLING

  􀁺 Often used when the area being studied is
fairly uniform, very large, or when there is a
limited amount of time available
  􀁺 Random = chosen by chance rather than
according to a plan; all outcomes are equally
likely
  􀁺 Samples are taken from different positions
within a habitat and those positions are
chosen randomly
                                                 29
How to sample randomly
Choose individuals or Place
“sampling units” haphazardly
– This is rarely completely
   random
 􀁺 OR…
 􀁺 Assign numbers to the
   areas
or individuals to be sampled
– Use a random number
   table to
select which areas or
   individuals
will be sample
                               30
Population Attributes
   Density – size of a population in relation to a definite unit of
    space
   Affected by:
       Natality – the reproductive output (birth rate) of
        a population
       Mortality – the death rate of organisms in a
        population
       Immigration – number of organisms moving
        into the area occupied by the population
       Emigration – number of organisms moving out
        of the area occupied by the population

                                                                       31
Population Density
Four primary population parameters:




                                      32
Two Types of Density
       Estimates
• Absolute Density – a known density
  such as #/m2


• Relative Density – we know when one
  area has more individuals than
  another


                                        33
Measuring Absolute Density
   Total Count – count the number of
    organisms living in an area
       Human census, number of oak trees in a
        wooded lot, number of singing birds in an area
       Total counts generally are not used very often
   Sampling Methods – use a sample to
    estimate population size
       Either use the quadrat or capture-recapture
        method


                                                         34
Measurement of Environmental Parameters
Abiotic factors are important in determining
   both the distribution of the organisms and
   their physical and physiological adaptations.
Temperature
- diurnal and seasonal temperature variations
   are significant in affecting different species of
   plants and animals
- equipment: mercury thermometer,
 maximum-minimum thermometer,
   miniaturized thermistor


                                                       35
pH meter in use




 pH
 -measure pH of a solution by universal
   indicator, pH paper, pH meter, etc.
 Light
 -measure its duration and intensity; duration
   by predication from Royal Observatory;
   intensity by photographic light meter
                                                 36
Humidity
Relative humidity: the water content of a
   given volume of air relative to the same
   volume of fully saturated air
- equipment: whirling hygrometer

                                              37
Wind and Water Speed
- wind speed:

- anemometer    or wind
  gauges
- water speed:

- time the movement of a
  floating object over a
  measured distance



                           38
Salinity
- using a conductivity meter: greater salinity has
  greater conductivity
Oxygen Level
- using an oxygen meter or chemical method
  (Winkler method)




                                                39
Collecting Methods
Collecting all organisms within a habitat is normally
  impractical and therefore small areas are selected.
Remember to return all material to its original position
  after searching & collecting sufficient specimens.
Some collecting apparatus for general use are listed
  below:




                                                           40
1. specimen tube
2. screwed-topped jars
3. polythene bags
4. forceps
5. paint brush
6. bulb pipette
7. pooter




                         41
8. widger              9. sieve              10. hand lens
11. enamel dish       12. beating tray      13. light traps
14. Tullgren funnel   15. Baermann funnel   16. mammal traps
17. pitfall traps      18. netting




                                                               42
Estimating Population Size
The exact methods used for
  estimation depend not only the
  nature of the habitat but also on
  the organisms involved,
 e.g. animals - population ;
      plants - percentage cover




                                      43
Using Quadrats
- By sampling an area using quadrats and
  counting the number of individuals within
  each quadrat, it is possible to estimate the
  total number of individuals within the area
- confined to plants and sessile, or very slow-

  moving animals;
- fast-moving animals are disturbed and run

  away


                                                  44
Capture-recapture Techniques
- useful for mobile animals which can be marked
- capture, marked, released, randomly recaptured
 and marked individuals recorded

 no. of marked individuals recaptured             total no. of individuals in 1st sample
-------------------------------------------- =   ------------------------------------------
total no. of individuals in 2nd sample             estimated size of population

(the Lincoln Index)



                                                                                              45
Capture-recapture Techniques
Factors affecting the accuracy of the estimation:
deaths, migration, individuals become more liable to
  predation, etc.
Examples:
-   arthropods marked on their backs with non-
    toxic paint,
-   fish have tags attached to opercula,
-   mammals have tags clipped to their ears,
    birds have their legs ringed


                                                       46
Capture-recapture Method
   Important tool for estimating density, birth
    rate, and death rate for mobile animals.
   Method:
       Collect a sample of individuals, mark them,
        and then release them
       After a period, collect more individuals from the
        wild and count the number that have marks
       We assume that a sample, if random, will
        contain the same proportion of marked
        individuals as the population does
       Estimate population density
                                                            47
Assumptions For All Capture-
Recapture Studies
   Marking technique does not increase
    mortality of marked animals
   Marked individuals are allowed to mix with
    population
   Marking technique does not affect catch
    probability
   Marks are not lost or overlooked
   No significant immigration or emigration
   No significant mortality or natality
                                                 48
Peterson Method or Lincoln Index


  Marked animals in       Marked animals in
   second sample            first sample
                      =
   Total caught in         Total population
   second sample                 size




      5 = 16          N = (20)(16)     N = 64
      20 N                   5

                                                49
Some Indices Used
   Traps                       Number of Artifacts
   Number of Fecal             Questionnaires
    Pellets                     Cover
   Vocalization Frequency      Feeding Capacity
   Pelt Records                Roadside Counts
   Catch per Unit Fishing
    Effort




                                                       50
Abundance Scales
The population size may be fairly accurately determined by
  making some form of frequency assessment.
These are subjective and involve an experimenter making some
  estimate of the number of individuals in a given area, or the
  % cover of a particular species.
This is especially useful where individuals are very numerous,
  e.g. barnacles on a rocky shore, or where it is difficult to
  distinguish individuals, e.g. grass plants in a meadow.




                                                            51
The assessments are usually made on an
  abundance scale of 5 categories:
 Abundance,

 Common,

 Frequent,

 Occasional,

 Rare.



Barnacles exposed at
low water
                                         52
   Environmental resistance are the factors
    which limit the growth of a particular
    population,
   e.g. predation,
         disease,
         availability of light, food,
         water,
         oxygen and shelter,
         the accumulation of toxic wastes and
         even the size of the population itself.

                                                   53
Density-dependent Growth
 A population is a density-dependent when its size

  (or density) affects its growth rate because of
  density-dependent factors such as food availability
  and toxic waste accumulation.
Density-independent Growth
 In this type of growth a population increases until

  some factor causes a sudden reduction in its size.
 Its effect is the same regardless of the size of the

  population, e.g. temperature, fires, floods, storms,
  etc.



                                                         54
Regulation of Population Size
 Fecundity is the reproductive capacity of individual
  females of a species.
 Birth rate or natality is used to measure fecundity.

 Death rate or mortality is the number of individuals
  of a species which die per unit time.
 Immigration     occurs when individuals join a
  population from neighbouring ones.
 Emigration occurs when individuals depart from a
  population.
 A cycle occurs when the size of a population
  fluctuates on a regular basis



                                                         55
Ecological Sampling




                      56
Why Do We Sample?
   Determine presence and/or abundance

   Monitor population fluctuations

   Assess ‘ecological damage’

   Assess quality of habitat

   Assess population responses
                                          57
What Do We Sample?
   Physical Environment
       Temperature, DO, pH, salinity, clarity, flow,
        sediment

   Biotic Environment
       All living things




                                                        58
Physical Habitat
   Temperature
       Mercury thermometer
       Electronic thermometer
       Long-term thermometers
   Dissolved Oxygen
       Winkler method (titration)
       DO meter (electrode)
   pH
       Litmus paper
       pH meter (electrode)
   Salinity
       Salinity Meter               YSI 550A DO Meter w/12' cable
                                                             59
Water Clarity
Secchi Disk
    Disk is attached to a calibrated rope. The disk
     is lowered into the water until the white parts
     can no longer be seen. Secchi disk depth is
     then recorded and serves as the waters
     transparency index. The clearer the water, the
     greater the secchi disk depth.




                                     Secchi Disk       60
Current Velocity (flow)
    Floating-orange method.
        Put an orange (or something else that floats
         just below the water surface) and measure the
         time it takes it to float across a known distance.
    Odometer-type flow meter
        Number of revolutions the propeller makes for
         a given time is calibrated to flow velocity.




                                                          61
Sediment
   Sediment size is important to many
    aquatic organisms.
   Sieve’s are used to separate and grade
    sediment samples.
       Percent of each size grade can be determined




                                                       62
Water Sample
   Water and plankton from various depths
    can be collected.

   A trigger mechanism is used to close the
    sampler.
       Sample is then brought back to the surface




                                                     63
Small Mammals
   Mouse/rat Traps
       Fatal
   Pit Falls
       Bucket is placed in the ground
       Sometimes have ‘leads’ to the buckets
   Live traps
       Havahart
       Sherman
   Spot-light

                        Havahart trap           Sherman trap
                                                        64
Birds
   Stick-under-the-box method
   Bird-trap
       Works like a minnow trap
   Mist net
       Captures birds in flight
   Rocket net
       Uses a propellant to throw a net
        over birds




                                           65
Terrestrial Insects
   Sticky paper
       flies
   Baited Traps
       Fire ants
   Nets
       butterflies
   Foggers
       Collect insects from tree canopies



                                             66
Aquatic Insects
   Drift Net
       Place net in flowing water
   Kick Net
       ‘Kick’ sediment upstream
        from block net and the flow
        will wash them into the net
       Wash bucket
   Serber or Hess Sampler
       Stir up known area of
        sediment
       Animals are collected by a
        catch net
   Multi-plate Sampler
       Become colonized

                                      67
Crawfish and Crab Traps




                          68
Fish Larvae
   Light Traps
       Larvae are attracted to the light
   Ichthyoplankton nets
       Can be towed at various depths
       Fish collect at the ‘cod’end




                                            69
Fish
   Lift net
       Net is placed down, and after
        a set amount of time it is
        quickly lifted
   Pop-net                                            Pop-net
       Similar to a lift net, but floats
        are attached to a framed net.       Lift net
       Operated by a trigger
        mechanism
   Throw net
       A net attached to a heavy
        frame is thrown and every
        thing inside is netted out                 Throw net

                                                                 70
Minnow trap

   Usually use bait to attract small fish
       Light is used sometimes as an attractant




                                                   71
Fish
   Electrofishing
       Electricity is put into the water
       Fish are temporarily stunned and usually swim
        towards the electricity source
       Usually non-fatal but may cause some damage




                                                        72
Fish
   Gill Net
       Gill nets resemble tennis nets
       Fish can not swim completely through the net
        and get caught
       Gill nets are size selective (based on mesh size)
         Square Mesh
                        Stretch mesh




                                        Bar mesh
                                                            73
Fish
   Trammel Net
       Three panels: two large
        mesh on the outside
        and a small mesh on
        the inside
       Fish swim through the
        outer mesh, pushes the
        small mesh through the
        other side and
        becomes entangled


                                  74
Hoop nets (and other
similar nets) can have
bait or not.


Fyke nets have leads
to help guide fish to
the net.




                         75
Seine
Seines are nets that are pulled through
 shallow water to catch fish.




                                           76
Purse Seine
   Used to encircle entire schools of fish
       Usually involves a spotter plane and a second
        boat




                                                        77
Trotline (longline)
    A series of baited drop lines connected to
     a main line.
                           Can be deployed by tying
                           one end to the bank and
                           tying the other end with a
                           heavy weight.




                                                    78
Shrimp (or fish) Trawl
   Net pulled behind a
    boat along the
    bottom
       Either a beam or otter
        boards keep the net
        open




                                 79
Tagging Individuals
   Coded Wire Tags
       Microwire that has a unique
        label
       Magnetic wand detects the tag
       Tag retention should be
        determined
   T-Bar tags
       Can be individually numbered
       External tag

   PIT tags (Passive
    Integrated Transponders)
       Wand induces the tag to
        transmit, individual number is
        displayed
                                         80
Other Tagging Methods

   Toe clip
       Amphibian and reptile
       Clip of one or more toes to
        identify individuals

   Bird Band
       Place a metal band on a
        bird leg
       Generally has
        identification information

                                      81
Preserving plant specimens
   Pressing and drying
   Long-term preservation
    and storage
   Alternative drying
    techniques
   Special preservation
    and processing
    techniques
   Mounting


                             82
Pressing and drying
   Techniques for pressing and drying specimens have been
    established for many years. There are minor variations in
    recommended methods, but they are essentially the same
    worldwide.
   The best specimens are plants that are pressed as soon as
    possible after collection, before wilting and shrivelling. Most
    plants may be kept in sealed containers such as plastic bags for
    up to a day if it is inconvenient to press immediately. However,
    some plants show such rapid wilting, particularly of the flowers,
    that such delays are best avoided. Flowers with a lot of nectar
    may go mouldy very quickly if excess nectar is not shaken off
    before pressing.
   Specimens are pressed flat and dried between sheets of
    absorbent blotters or semi-absorbent paper such as newspaper.
    Papers with a glossy surface should be avoided because they
    are not absorbent enough to aid drying. The plant should be
    carefully laid out between the drying sheets, as their form at this
    stage largely determines their ultimate appearance. The flowers
    should be spread out with the petals carefully arranged, wilted
    leaves should be straightened and unnecessary shoots of
    excessively twiggy shrubs may be cut away.
                                                                      83
Microwave ovens
   Small numbers of specimens can be dried using a microwave oven. The
    technique recommended in the literature is to place the specimens
    between unprinted absorbent paper, for example butcher's paper, not
    newspaper, which is unsuitable because the chemicals present in the
    ink may cause a fire. The specimens should be put in a special press
    which should be of a microwave-safe material (wood, acrylic or
    polycarbonate sheeting e.g. plexiglass or perspex, NO metal
    components). If such a press is not available, sheets of cardboard can
    be placed above and below the specimens and then weighted down.
   Drying time depends on the power of your oven. In most cases drying is
    accomplished by irradiating at maximum power for 1-2 minutes per
    specimen, although it is often a case of trial and error. It is best to
    process no more than 10-12 specimens of average thickness per batch.
    Specimens are usually dried after the moisture that characteristically
    appears on the glass door has disappeared. If the specimen is damp
    when taken out of the oven, allow it to stand before re-radiating as
    moisture continues to evaporate from the specimen for some time. Care
    must be taken not to irradiate the specimens for too long.
   It should be noted that microwave treatment damages seeds and the
    cellular structure of the plants which may reduce the long-term value of
    the specimens.

                                                                          84
Alternative drying techniques
Silica gel/other desiccants & freeze drying
   Alternative methods of drying plant specimens have been used
    for some time, but are mostly restricted to special purpose
    collections. The main alternatives are freeze-drying and drying in
    a desiccant powder such as desiccant silica gel. In general these
    techniques are used where it is essential to preserve the shape
    of a delicate plant of organ of the plant such as the flower.
    Freeze-drying has also been used to preserve the chemical
    composition of a plant as accurately as possible for later study.
   Disadvantages and special conservation problems of specimens
    dried in these manners are that they are particularly susceptible
    to damage. The dried parts are fragile, lack support and often
    catch on packing materials. They must, therefore, be packed
    especially carefully and stored in small boxes or tubes with some
    appropriate packing material that does not snag and break small
    projections. Acid-free tissue paper is often used. Drying in
    desiccant silica gel crystals or powder can also have the
    disadvantage that it is difficult to remove all traces of the silica
    gel after drying.
                                                                       85
Special preservation and processing
    techniques
    Wet or spirit collections
   Very fleshy or delicate structures, including small
    algae and orchid flowers, are best preserved in an
    air-tight glass or plastic jar with a liquid preservative
    rather than by drying. The type of preservative used
    should be clearly labelled in the jar. Such material is
    often referred to as a spirit collection or wet
    collection.
   Most material can be satisfactorily preserved in 70%
    ethyl alcohol (or 70% methylated spirit or denatured
    alcohol) with 30% water. Colours will fade quickly in
    spirit, however, so it is a good idea to keep
    comprehensive notes and photographs.

                                                                86
Small algae
   Microscopic algae are often collected in a jar and in the water in
    which they were found. If the algae are to be stored for more
    than 2-3 days, a preservative needs to be used. Traditionally this
    has been the extremely toxic formalin - a small amount can be
    added to the water to make a 5% final solution, and the container
    labelled. This must not be sent through the post or by courier.
   There are some other equally toxic options, for example
    propylene phenoxytol, but none should be sent through the post.
    A safer option is to add sufficient concentrated alcohol or
    methylated spirits  to the water containing the algae to make a
    final solution of 70% alcohol. This treatment dilutes the algae
    making them difficult to find, so if they can be concentrated
    somehow first (e.g. by filtering) they can be stored in much less
    liquid. Another option is to fix the algae in formalin (or something
    similar) first, and then prepare a microscope glass slide with a
    permanent water-soluble mounting medium.


                                                                      87
Mounting
   Mounting specimens prevents most fragile material from
    fragmenting and prevents specimens becoming separated from
    their labels. If the plant collection is a long-term project,
    specimens should be mounted on sheets of archival (permanent)
    cardboard or paper with archival-quality fixing media. These
    include stitching with cotton thread, dental floss, nickel-plated
    copper wire (for heavier specimens), narrow strips of archival
    paper, linen tape, or by using an archival adhesive such as
    methyl cellulose adhesive
   One disadvantage of mounting specimens is that it can make
    parts of the specimen inaccessible for examination, so it is
    essential that this be borne in mind during specimen
    arrangement and mounting. For example, easily reversible
    mounting media should be used, specimens should be strapped
    to the sheet, rather than glued all over, and the specimen should
    be carefully arranged before it is attached so that it shows all
    features.

                                                                   88
   Full-size herbarium mounting sheets are
    usually about 43 cm long x 28 cm wide. The
    plant name and accompanying field notes
    should be transcribed on a permanent label
    stuck to one corner of the herbarium sheet
    (the bottom right-hand corner being the most
    common) or, sometimes, annotations may be
    written directly on the sheet or card.
   Small pieces of material which may have
    become separated from the specimen (e.g.
    seeds) can be placed in small plastic bags
    and pinned to the sheet.

                                               89
Long-term preservation and
storage
   The long-term preservation of dry plant
    specimens is largely dependent on protection
    from insect attack. Specimens collected by
    Linnaeus in the eighteenth century, and by
    Banks and Solander on the Endeavour voyage
    in 1788, are still excellently preserved.




                                               90
Pests and their control
   A range of pests attack dried plant material. The
    most common pests are insects and fungi, though
    rodents and other large animals can cause damage
    in poor storage conditions. Insects eat the material,
    the paper surrounding the material, and the
    adhesives and mounting media.
   Such insect pests range from psocids (book lice),
    which attack mainly the softer parts such as flowers
    and soft fruits, to tobacco beetles and carpet
    beetles, which can bore holes through the toughest
    of specimens. Many insects are particularly
    sensitive to relative humidity levels and do not thrive
    at levels below 50%.
                                                          91
The most common and acceptable specimen
treatments for insect control are:
   Freezing
   Microwave
   Poisoning
   Insect deterrents
   Fungal pests




                                          92
Storage

   Dried and pressed plant specimens can be
    stored in cardboard or plastic boxes, or tied in
    bundles in light-weight cardboard folders
    placed in 'pigeon holes'.
   Alternatively, they can be placed in protective
    plastic jackets and displayed in ring folders
    which is recommended if they are to be
    frequently handled, such as for a reference
    collection.

                                                   93
Filing
   Specimens should be filed in a systematic order if a
    relatively permanent collection is being made. The
    major groups, i.e. ferns and fern allies, cycads,
    conifers, dicotyledons and monocotyledons, are
    best kept separately or according to some
    classification scheme, such as that given in a flora
    or handbook.
   Similarly, the genera within each family and the
    species within each genus may be filed
    alphabetically or following some such classification.

                                                            94
Preservation of entire animals

   Types of collection specimens of an entire animal:
    For reference collections, mammals can be prepared as a variety of
    specimens. The condition of the specimen may determine possible
    ways to preserve it; if for instance decomposition of the skin has
    loosened the hair of a carcass so much that it can easily be pulled
    out or removed by rubbing (“slipping” fur), it will be very difficult or
    impossible to produce a study skin or mounted specimen.
    The most usual types of specimens (based on Nagorsen and
    Peterson, 1980) are:
    1) entire fluid-preserved animals (for studying anatomy and
    histology; fluid preservation may change the fur colour)
    2) study skins with accompanying skulls / partial skeletons (some
    bones remain in the skin), for studying pelage colour, hair quality
    and moulting patterns,
    3) mounted skins with accompanying partial or entire skeleton
    (some bones may remain in the skin, dependant on the method of
    preservation) or freeze-dried specimens,
    4) entire skeletons, for instance for studying anatomy, geographic
    variation or for age determination (entire skeletons are poorly
    represented in collections, so Nagorsen and Peterson (1980)
    recommend preparation of at least one male and one female
    skeleton per species.
                                                                               95
Preservation of specimens in the
field
   Formalin preservation
   Preservation in alcohol
   Preservation by cooling or freezing




                                          96
Formalin preservation
   After weighing and measuring the animal and attaching an adequate
    label very small specimens (up to 100 g) can be fixed whole by
    submerging them in 10 % buffered formalin (tissue - formalin
    solution ratio of at least 1 : 12). the body cavity can be filled with
    formalin solution by injection until it is turgid and firm; some formalin
    may also be injected under the skin, into the body cavity, larger
    muscles and organs. If hypodermic needles are not available, the
    body cavity can be opened ventrally by making a slit instead,
    allowing the formalin to enter.
   Keeping the mouth open with a piece of wood or cotton may later
    allow examination of teeth. Then the whole body can be immersed
    in formalin, in the posture in which it is supposed to stay
    permanently because it will harden. The ratio of formalin to carcass
    must be at least 12 to 1 to assure a good fixation. Tissues can be
    left in buffered neutralized formalin for several months, but  formalin
    hardens specimens; therefore, after fixation, longterm storage in
    alcohol may be better. After preservation the carcass should
    therefore be washed in water and transferred into ethanol for
    permanent storage
   Disadvantages; for instance it discolours the fur, after a longish
    immersion, softens the bones and prevents further examination for
    microbiology.
                                                                           97
Preservation in alcohol

   After weighing, a whole animal can be
    preserved in a container of alcohol (70-90%).
    Removal of the intestine prior to storage of
    the animal in alcohol is recommended




                                                98
Preservation by cooling or
freezing
   Removal of the skin with insulating fur before
    cooling or freezing may help to cool the
    carcass down more quickly.
   Freezing is not recommended if histological
    examination is planned




                                                 99
THANK YOU




            100

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Biological Sampling Techniques

  • 1. 1
  • 2. BIO 300 BIOLOGICAL TECHNIQUES AND SKILLS SARINI BINTI AHMAD WAKID FACULTY OF APPLIED SCIENCE 2
  • 3. Techniques in field investigation 3
  • 4. Population Ecology Population— A group of individuals of the same species living in a particular geographic area Population Ecology— Concentrates mainly on the factors that affect how many individuals of a particular species live in an area 4
  • 5. What is a sample? “A portion, piece, or segment that is representative of a whole” 5
  • 6. Why do we sample? Because it is usually impossible to count all the plants or animals present in a given area – e.g. # dragonfly larvae in a pond – e.g. plant cover on a river terrace – e.g. species of plants in the estate 6
  • 7. NON-INVASIVE SAMPLING Avoid any degradation of the habitat when sampling 􀁺 Removal of whole or parts of organisms should be limited to species that can quickly recover 7
  • 8. REPRESENTATIVE SAMPLING Take a number of samples from around the sampling site so as to be reasonably sure that the samples represent the site in general Necessities… 􀁺 the samples represent the whole – It is necessary to take enough samples so that an accurate representation is obtained – It is necessary to avoid bias when sampling 8
  • 9. SAMPLING UNITS Type determined by the organisms and the physical nature of the habitat being sampled – Area of ground surface – Volume of air, water or soil 􀁺 Standard units enable comparison of results 9
  • 10. QUADRATS A standard, area sampling unit consisting of a square frame 􀁺 Consistent size and shape is essential for comparing samples from different places and/or times Quadrat size Chosen to suit sampling goals 􀁺 A balance between what is best and what is practical is always necessary 􀁺 Should suit: – practical constraints – habitat – organism 10
  • 11. Quadrat Method  A Quadrat is a sampling area of any shape randomly deployed. Each individual within the quadrat is counted and those numbers are used to extrapolate population size.  Example: a 100 square centimeter metal rectangle is randomly thrown four times and all of the beetles of a particular species within the square are counted each time: 19, 21, 17, and 19. This translates to 19 beetles per 100 cm2 or 1900 per m2. 11
  • 12. QUADRAT & TRANSECT ACTIVITIES How, Where & Why Scientists Do Sampling Scientists often collect data “in the field” which could mean underwater, in a forest, in a cave, on a reef, or even the moon! Two essential methods to gather ecological information in a standardized way are: Transect Sampling (using a single line) and Quadrat Sampling (counted within a grid). These sampling methods provide more accurate data than random sampling or simply guessing, but they take less time than counting every specimen in a certain area. 12
  • 13. Quadrats - a sturdily built wooden frame, can be folded for easy transport and storage 13
  • 14. Using a quadrat along a belt transect Quadrats - When placed on the ground, the species present within the frame are identified and their abundance recorded - Sampling could be random or systematic 14
  • 15. Practical Constraints Small quadrats are quicker to survey but yield a smaller individual sample of habitat – Often require a larger # of samples to represent the habitat 􀁺 Large quadrats require more time and effort to survey but provide a larger individual sample of habitat – Often require a smaller # of samples to represent the habitat 15
  • 16. Habitat size Appropriate sample unit size depends on size scale of the habitat – Small scale habitats require smaller sized samples 􀁺 Ex. Boulders – Large scale habitats require larger sized samples 􀁺 Ex. Forests 16
  • 17. Organism size and density Depends on size and density of organisms – Small, dense organisms require smaller samples 􀁺 Ex. grass – Large, scattered organisms require larger samples 􀁺 Ex. Trees 17
  • 18. TYPES OF SAMPLING  􀁺 Systematic  􀁺 Stratified  􀁺 Random 18
  • 19. SYSTEMATIC SAMPLING  􀁺 Often used when the area being studied is varied, not very large, or  when time is available  􀁺 Samples are taken at fixed intervals 19
  • 20. How to sample systematically Systematic samples are usually taken along a transect line marked by a tape measure 􀁺 Transect- a line laid across an area 20
  • 21. Sampling along gradients Transects are set up along a environmental gradients – down a hillside – across a streambed – out from a source of pollution 21
  • 22. Types of transect sampling  􀁺 Line transect  􀁺 Belt transect 22
  • 23. Line transect method A measured line is laid across the area in the direction of the environmental gradient – The species touching the line can be recorded along the whole length of the line (continuous sampling) or at specific points along the line (systematic sampling) 23
  • 24. Line Transect - useful where a transition of flora and/or fauna occurs - a string or tape is stretched out along the ground in a straight line; record the organisms touching or covering the line all along its length or at regular intervals - Profile transect: when there is appreciable height change along the transect and thus affecting the distribution of its species 24
  • 25. Belt transect method Similar to line transect but widens the sampling area – Transect line is laid out – Samples are taken by determining abundance or % cover in an area that is a defined distance from the line – Samples can be taken all the way along the line, at specific intervals or even randomly 25
  • 26. Using a quadrat along a belt transect, e.g. ladder transect (every 5m) Belt Transect It is a strip, usually a metre wide, marked by putting a second line parallel to the other. The species between the lines are carefully recorded, working a metre at a time. Alternatively, a frame quadrat in conjunction with a single line transect could be used. 26
  • 27. Point Frames - for grassland field study of dense vegetation 27
  • 28. STRATIFIED SAMPLING 􀁺 Often used when there are small areas within a larger habitat that are clearly different 􀁺 Strata- major differences within communities recognized before sampling begins 28
  • 29. RANDOM SAMPLING 􀁺 Often used when the area being studied is fairly uniform, very large, or when there is a limited amount of time available 􀁺 Random = chosen by chance rather than according to a plan; all outcomes are equally likely 􀁺 Samples are taken from different positions within a habitat and those positions are chosen randomly 29
  • 30. How to sample randomly Choose individuals or Place “sampling units” haphazardly – This is rarely completely random 􀁺 OR… 􀁺 Assign numbers to the areas or individuals to be sampled – Use a random number table to select which areas or individuals will be sample 30
  • 31. Population Attributes  Density – size of a population in relation to a definite unit of space  Affected by:  Natality – the reproductive output (birth rate) of a population  Mortality – the death rate of organisms in a population  Immigration – number of organisms moving into the area occupied by the population  Emigration – number of organisms moving out of the area occupied by the population 31
  • 32. Population Density Four primary population parameters: 32
  • 33. Two Types of Density Estimates • Absolute Density – a known density such as #/m2 • Relative Density – we know when one area has more individuals than another 33
  • 34. Measuring Absolute Density  Total Count – count the number of organisms living in an area  Human census, number of oak trees in a wooded lot, number of singing birds in an area  Total counts generally are not used very often  Sampling Methods – use a sample to estimate population size  Either use the quadrat or capture-recapture method 34
  • 35. Measurement of Environmental Parameters Abiotic factors are important in determining both the distribution of the organisms and their physical and physiological adaptations. Temperature - diurnal and seasonal temperature variations are significant in affecting different species of plants and animals - equipment: mercury thermometer, maximum-minimum thermometer, miniaturized thermistor 35
  • 36. pH meter in use pH -measure pH of a solution by universal indicator, pH paper, pH meter, etc. Light -measure its duration and intensity; duration by predication from Royal Observatory; intensity by photographic light meter 36
  • 37. Humidity Relative humidity: the water content of a given volume of air relative to the same volume of fully saturated air - equipment: whirling hygrometer 37
  • 38. Wind and Water Speed - wind speed: - anemometer or wind gauges - water speed: - time the movement of a floating object over a measured distance 38
  • 39. Salinity - using a conductivity meter: greater salinity has greater conductivity Oxygen Level - using an oxygen meter or chemical method (Winkler method) 39
  • 40. Collecting Methods Collecting all organisms within a habitat is normally impractical and therefore small areas are selected. Remember to return all material to its original position after searching & collecting sufficient specimens. Some collecting apparatus for general use are listed below: 40
  • 41. 1. specimen tube 2. screwed-topped jars 3. polythene bags 4. forceps 5. paint brush 6. bulb pipette 7. pooter 41
  • 42. 8. widger 9. sieve 10. hand lens 11. enamel dish 12. beating tray 13. light traps 14. Tullgren funnel 15. Baermann funnel 16. mammal traps 17. pitfall traps 18. netting 42
  • 43. Estimating Population Size The exact methods used for estimation depend not only the nature of the habitat but also on the organisms involved, e.g. animals - population ; plants - percentage cover 43
  • 44. Using Quadrats - By sampling an area using quadrats and counting the number of individuals within each quadrat, it is possible to estimate the total number of individuals within the area - confined to plants and sessile, or very slow- moving animals; - fast-moving animals are disturbed and run away 44
  • 45. Capture-recapture Techniques - useful for mobile animals which can be marked - capture, marked, released, randomly recaptured and marked individuals recorded no. of marked individuals recaptured total no. of individuals in 1st sample -------------------------------------------- = ------------------------------------------ total no. of individuals in 2nd sample estimated size of population (the Lincoln Index) 45
  • 46. Capture-recapture Techniques Factors affecting the accuracy of the estimation: deaths, migration, individuals become more liable to predation, etc. Examples: - arthropods marked on their backs with non- toxic paint, - fish have tags attached to opercula, - mammals have tags clipped to their ears, birds have their legs ringed 46
  • 47. Capture-recapture Method  Important tool for estimating density, birth rate, and death rate for mobile animals.  Method:  Collect a sample of individuals, mark them, and then release them  After a period, collect more individuals from the wild and count the number that have marks  We assume that a sample, if random, will contain the same proportion of marked individuals as the population does  Estimate population density 47
  • 48. Assumptions For All Capture- Recapture Studies  Marking technique does not increase mortality of marked animals  Marked individuals are allowed to mix with population  Marking technique does not affect catch probability  Marks are not lost or overlooked  No significant immigration or emigration  No significant mortality or natality 48
  • 49. Peterson Method or Lincoln Index Marked animals in Marked animals in second sample first sample = Total caught in Total population second sample size 5 = 16 N = (20)(16) N = 64 20 N 5 49
  • 50. Some Indices Used  Traps  Number of Artifacts  Number of Fecal  Questionnaires Pellets  Cover  Vocalization Frequency  Feeding Capacity  Pelt Records  Roadside Counts  Catch per Unit Fishing Effort 50
  • 51. Abundance Scales The population size may be fairly accurately determined by making some form of frequency assessment. These are subjective and involve an experimenter making some estimate of the number of individuals in a given area, or the % cover of a particular species. This is especially useful where individuals are very numerous, e.g. barnacles on a rocky shore, or where it is difficult to distinguish individuals, e.g. grass plants in a meadow. 51
  • 52. The assessments are usually made on an abundance scale of 5 categories:  Abundance,  Common,  Frequent,  Occasional,  Rare. Barnacles exposed at low water 52
  • 53. Environmental resistance are the factors which limit the growth of a particular population,  e.g. predation, disease, availability of light, food, water, oxygen and shelter, the accumulation of toxic wastes and even the size of the population itself. 53
  • 54. Density-dependent Growth  A population is a density-dependent when its size (or density) affects its growth rate because of density-dependent factors such as food availability and toxic waste accumulation. Density-independent Growth  In this type of growth a population increases until some factor causes a sudden reduction in its size.  Its effect is the same regardless of the size of the population, e.g. temperature, fires, floods, storms, etc. 54
  • 55. Regulation of Population Size  Fecundity is the reproductive capacity of individual females of a species.  Birth rate or natality is used to measure fecundity.  Death rate or mortality is the number of individuals of a species which die per unit time.  Immigration occurs when individuals join a population from neighbouring ones.  Emigration occurs when individuals depart from a population.  A cycle occurs when the size of a population fluctuates on a regular basis 55
  • 57. Why Do We Sample?  Determine presence and/or abundance  Monitor population fluctuations  Assess ‘ecological damage’  Assess quality of habitat  Assess population responses 57
  • 58. What Do We Sample?  Physical Environment  Temperature, DO, pH, salinity, clarity, flow, sediment  Biotic Environment  All living things 58
  • 59. Physical Habitat  Temperature  Mercury thermometer  Electronic thermometer  Long-term thermometers  Dissolved Oxygen  Winkler method (titration)  DO meter (electrode)  pH  Litmus paper  pH meter (electrode)  Salinity  Salinity Meter YSI 550A DO Meter w/12' cable 59
  • 60. Water Clarity Secchi Disk  Disk is attached to a calibrated rope. The disk is lowered into the water until the white parts can no longer be seen. Secchi disk depth is then recorded and serves as the waters transparency index. The clearer the water, the greater the secchi disk depth. Secchi Disk 60
  • 61. Current Velocity (flow)  Floating-orange method.  Put an orange (or something else that floats just below the water surface) and measure the time it takes it to float across a known distance.  Odometer-type flow meter  Number of revolutions the propeller makes for a given time is calibrated to flow velocity. 61
  • 62. Sediment  Sediment size is important to many aquatic organisms.  Sieve’s are used to separate and grade sediment samples.  Percent of each size grade can be determined 62
  • 63. Water Sample  Water and plankton from various depths can be collected.  A trigger mechanism is used to close the sampler.  Sample is then brought back to the surface 63
  • 64. Small Mammals  Mouse/rat Traps  Fatal  Pit Falls  Bucket is placed in the ground  Sometimes have ‘leads’ to the buckets  Live traps  Havahart  Sherman  Spot-light Havahart trap Sherman trap 64
  • 65. Birds  Stick-under-the-box method  Bird-trap  Works like a minnow trap  Mist net  Captures birds in flight  Rocket net  Uses a propellant to throw a net over birds 65
  • 66. Terrestrial Insects  Sticky paper  flies  Baited Traps  Fire ants  Nets  butterflies  Foggers  Collect insects from tree canopies 66
  • 67. Aquatic Insects  Drift Net  Place net in flowing water  Kick Net  ‘Kick’ sediment upstream from block net and the flow will wash them into the net  Wash bucket  Serber or Hess Sampler  Stir up known area of sediment  Animals are collected by a catch net  Multi-plate Sampler  Become colonized 67
  • 68. Crawfish and Crab Traps 68
  • 69. Fish Larvae  Light Traps  Larvae are attracted to the light  Ichthyoplankton nets  Can be towed at various depths  Fish collect at the ‘cod’end 69
  • 70. Fish  Lift net  Net is placed down, and after a set amount of time it is quickly lifted  Pop-net Pop-net  Similar to a lift net, but floats are attached to a framed net. Lift net  Operated by a trigger mechanism  Throw net  A net attached to a heavy frame is thrown and every thing inside is netted out Throw net 70
  • 71. Minnow trap  Usually use bait to attract small fish  Light is used sometimes as an attractant 71
  • 72. Fish  Electrofishing  Electricity is put into the water  Fish are temporarily stunned and usually swim towards the electricity source  Usually non-fatal but may cause some damage 72
  • 73. Fish  Gill Net  Gill nets resemble tennis nets  Fish can not swim completely through the net and get caught  Gill nets are size selective (based on mesh size) Square Mesh Stretch mesh Bar mesh 73
  • 74. Fish  Trammel Net  Three panels: two large mesh on the outside and a small mesh on the inside  Fish swim through the outer mesh, pushes the small mesh through the other side and becomes entangled 74
  • 75. Hoop nets (and other similar nets) can have bait or not. Fyke nets have leads to help guide fish to the net. 75
  • 76. Seine Seines are nets that are pulled through shallow water to catch fish. 76
  • 77. Purse Seine  Used to encircle entire schools of fish  Usually involves a spotter plane and a second boat 77
  • 78. Trotline (longline)  A series of baited drop lines connected to a main line. Can be deployed by tying one end to the bank and tying the other end with a heavy weight. 78
  • 79. Shrimp (or fish) Trawl  Net pulled behind a boat along the bottom  Either a beam or otter boards keep the net open 79
  • 80. Tagging Individuals  Coded Wire Tags  Microwire that has a unique label  Magnetic wand detects the tag  Tag retention should be determined  T-Bar tags  Can be individually numbered  External tag  PIT tags (Passive Integrated Transponders)  Wand induces the tag to transmit, individual number is displayed 80
  • 81. Other Tagging Methods  Toe clip  Amphibian and reptile  Clip of one or more toes to identify individuals  Bird Band  Place a metal band on a bird leg  Generally has identification information 81
  • 82. Preserving plant specimens  Pressing and drying  Long-term preservation and storage  Alternative drying techniques  Special preservation and processing techniques  Mounting 82
  • 83. Pressing and drying  Techniques for pressing and drying specimens have been established for many years. There are minor variations in recommended methods, but they are essentially the same worldwide.  The best specimens are plants that are pressed as soon as possible after collection, before wilting and shrivelling. Most plants may be kept in sealed containers such as plastic bags for up to a day if it is inconvenient to press immediately. However, some plants show such rapid wilting, particularly of the flowers, that such delays are best avoided. Flowers with a lot of nectar may go mouldy very quickly if excess nectar is not shaken off before pressing.  Specimens are pressed flat and dried between sheets of absorbent blotters or semi-absorbent paper such as newspaper. Papers with a glossy surface should be avoided because they are not absorbent enough to aid drying. The plant should be carefully laid out between the drying sheets, as their form at this stage largely determines their ultimate appearance. The flowers should be spread out with the petals carefully arranged, wilted leaves should be straightened and unnecessary shoots of excessively twiggy shrubs may be cut away. 83
  • 84. Microwave ovens  Small numbers of specimens can be dried using a microwave oven. The technique recommended in the literature is to place the specimens between unprinted absorbent paper, for example butcher's paper, not newspaper, which is unsuitable because the chemicals present in the ink may cause a fire. The specimens should be put in a special press which should be of a microwave-safe material (wood, acrylic or polycarbonate sheeting e.g. plexiglass or perspex, NO metal components). If such a press is not available, sheets of cardboard can be placed above and below the specimens and then weighted down.  Drying time depends on the power of your oven. In most cases drying is accomplished by irradiating at maximum power for 1-2 minutes per specimen, although it is often a case of trial and error. It is best to process no more than 10-12 specimens of average thickness per batch. Specimens are usually dried after the moisture that characteristically appears on the glass door has disappeared. If the specimen is damp when taken out of the oven, allow it to stand before re-radiating as moisture continues to evaporate from the specimen for some time. Care must be taken not to irradiate the specimens for too long.  It should be noted that microwave treatment damages seeds and the cellular structure of the plants which may reduce the long-term value of the specimens. 84
  • 85. Alternative drying techniques Silica gel/other desiccants & freeze drying  Alternative methods of drying plant specimens have been used for some time, but are mostly restricted to special purpose collections. The main alternatives are freeze-drying and drying in a desiccant powder such as desiccant silica gel. In general these techniques are used where it is essential to preserve the shape of a delicate plant of organ of the plant such as the flower. Freeze-drying has also been used to preserve the chemical composition of a plant as accurately as possible for later study.  Disadvantages and special conservation problems of specimens dried in these manners are that they are particularly susceptible to damage. The dried parts are fragile, lack support and often catch on packing materials. They must, therefore, be packed especially carefully and stored in small boxes or tubes with some appropriate packing material that does not snag and break small projections. Acid-free tissue paper is often used. Drying in desiccant silica gel crystals or powder can also have the disadvantage that it is difficult to remove all traces of the silica gel after drying. 85
  • 86. Special preservation and processing techniques Wet or spirit collections  Very fleshy or delicate structures, including small algae and orchid flowers, are best preserved in an air-tight glass or plastic jar with a liquid preservative rather than by drying. The type of preservative used should be clearly labelled in the jar. Such material is often referred to as a spirit collection or wet collection.  Most material can be satisfactorily preserved in 70% ethyl alcohol (or 70% methylated spirit or denatured alcohol) with 30% water. Colours will fade quickly in spirit, however, so it is a good idea to keep comprehensive notes and photographs. 86
  • 87. Small algae  Microscopic algae are often collected in a jar and in the water in which they were found. If the algae are to be stored for more than 2-3 days, a preservative needs to be used. Traditionally this has been the extremely toxic formalin - a small amount can be added to the water to make a 5% final solution, and the container labelled. This must not be sent through the post or by courier.  There are some other equally toxic options, for example propylene phenoxytol, but none should be sent through the post. A safer option is to add sufficient concentrated alcohol or methylated spirits  to the water containing the algae to make a final solution of 70% alcohol. This treatment dilutes the algae making them difficult to find, so if they can be concentrated somehow first (e.g. by filtering) they can be stored in much less liquid. Another option is to fix the algae in formalin (or something similar) first, and then prepare a microscope glass slide with a permanent water-soluble mounting medium. 87
  • 88. Mounting  Mounting specimens prevents most fragile material from fragmenting and prevents specimens becoming separated from their labels. If the plant collection is a long-term project, specimens should be mounted on sheets of archival (permanent) cardboard or paper with archival-quality fixing media. These include stitching with cotton thread, dental floss, nickel-plated copper wire (for heavier specimens), narrow strips of archival paper, linen tape, or by using an archival adhesive such as methyl cellulose adhesive  One disadvantage of mounting specimens is that it can make parts of the specimen inaccessible for examination, so it is essential that this be borne in mind during specimen arrangement and mounting. For example, easily reversible mounting media should be used, specimens should be strapped to the sheet, rather than glued all over, and the specimen should be carefully arranged before it is attached so that it shows all features. 88
  • 89. Full-size herbarium mounting sheets are usually about 43 cm long x 28 cm wide. The plant name and accompanying field notes should be transcribed on a permanent label stuck to one corner of the herbarium sheet (the bottom right-hand corner being the most common) or, sometimes, annotations may be written directly on the sheet or card.  Small pieces of material which may have become separated from the specimen (e.g. seeds) can be placed in small plastic bags and pinned to the sheet. 89
  • 90. Long-term preservation and storage  The long-term preservation of dry plant specimens is largely dependent on protection from insect attack. Specimens collected by Linnaeus in the eighteenth century, and by Banks and Solander on the Endeavour voyage in 1788, are still excellently preserved. 90
  • 91. Pests and their control  A range of pests attack dried plant material. The most common pests are insects and fungi, though rodents and other large animals can cause damage in poor storage conditions. Insects eat the material, the paper surrounding the material, and the adhesives and mounting media.  Such insect pests range from psocids (book lice), which attack mainly the softer parts such as flowers and soft fruits, to tobacco beetles and carpet beetles, which can bore holes through the toughest of specimens. Many insects are particularly sensitive to relative humidity levels and do not thrive at levels below 50%. 91
  • 92. The most common and acceptable specimen treatments for insect control are:  Freezing  Microwave  Poisoning  Insect deterrents  Fungal pests 92
  • 93. Storage  Dried and pressed plant specimens can be stored in cardboard or plastic boxes, or tied in bundles in light-weight cardboard folders placed in 'pigeon holes'.  Alternatively, they can be placed in protective plastic jackets and displayed in ring folders which is recommended if they are to be frequently handled, such as for a reference collection. 93
  • 94. Filing  Specimens should be filed in a systematic order if a relatively permanent collection is being made. The major groups, i.e. ferns and fern allies, cycads, conifers, dicotyledons and monocotyledons, are best kept separately or according to some classification scheme, such as that given in a flora or handbook.  Similarly, the genera within each family and the species within each genus may be filed alphabetically or following some such classification. 94
  • 95. Preservation of entire animals  Types of collection specimens of an entire animal: For reference collections, mammals can be prepared as a variety of specimens. The condition of the specimen may determine possible ways to preserve it; if for instance decomposition of the skin has loosened the hair of a carcass so much that it can easily be pulled out or removed by rubbing (“slipping” fur), it will be very difficult or impossible to produce a study skin or mounted specimen. The most usual types of specimens (based on Nagorsen and Peterson, 1980) are: 1) entire fluid-preserved animals (for studying anatomy and histology; fluid preservation may change the fur colour) 2) study skins with accompanying skulls / partial skeletons (some bones remain in the skin), for studying pelage colour, hair quality and moulting patterns, 3) mounted skins with accompanying partial or entire skeleton (some bones may remain in the skin, dependant on the method of preservation) or freeze-dried specimens, 4) entire skeletons, for instance for studying anatomy, geographic variation or for age determination (entire skeletons are poorly represented in collections, so Nagorsen and Peterson (1980) recommend preparation of at least one male and one female skeleton per species. 95
  • 96. Preservation of specimens in the field  Formalin preservation  Preservation in alcohol  Preservation by cooling or freezing 96
  • 97. Formalin preservation  After weighing and measuring the animal and attaching an adequate label very small specimens (up to 100 g) can be fixed whole by submerging them in 10 % buffered formalin (tissue - formalin solution ratio of at least 1 : 12). the body cavity can be filled with formalin solution by injection until it is turgid and firm; some formalin may also be injected under the skin, into the body cavity, larger muscles and organs. If hypodermic needles are not available, the body cavity can be opened ventrally by making a slit instead, allowing the formalin to enter.  Keeping the mouth open with a piece of wood or cotton may later allow examination of teeth. Then the whole body can be immersed in formalin, in the posture in which it is supposed to stay permanently because it will harden. The ratio of formalin to carcass must be at least 12 to 1 to assure a good fixation. Tissues can be left in buffered neutralized formalin for several months, but  formalin hardens specimens; therefore, after fixation, longterm storage in alcohol may be better. After preservation the carcass should therefore be washed in water and transferred into ethanol for permanent storage  Disadvantages; for instance it discolours the fur, after a longish immersion, softens the bones and prevents further examination for microbiology. 97
  • 98. Preservation in alcohol  After weighing, a whole animal can be preserved in a container of alcohol (70-90%). Removal of the intestine prior to storage of the animal in alcohol is recommended 98
  • 99. Preservation by cooling or freezing  Removal of the skin with insulating fur before cooling or freezing may help to cool the carcass down more quickly.  Freezing is not recommended if histological examination is planned 99
  • 100. THANK YOU 100