Chapter 1 bio 300

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  • 1. 1
  • 2. BIO 300BIOLOGICAL TECHNIQUES AND SKILLS SARINI BINTI AHMAD WAKID FACULTY OF APPLIED SCIENCE 2
  • 3. Techniques in field investigation 3
  • 4. Population EcologyPopulation—A group of individuals of the same speciesliving in a particular geographic areaPopulation Ecology—Concentrates mainly on the factors thataffect how many individuals of a particularspecies 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 tocount all the plants or animals presentin 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 SAMPLINGAvoid any degradation of the habitat whensampling 􀁺 Removal of whole or parts of organismsshould be limited to species that can quickly recover 7
  • 8. REPRESENTATIVESAMPLINGTake a number of samples from around the sampling site so as to be reasonably sure that the samples represent the site in generalNecessities… 􀁺 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 UNITSType 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. QUADRATSA standard, area sampling unit consisting of a square frame 􀁺 Consistent size and shape is essential for comparing samples from different places and/or timesQuadrat sizeChosen 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 & TRANSECTACTIVITIESHow, Where & Why Scientists Do SamplingScientists often collect data “in the field” which couldmean underwater, in a forest, in a cave, on a reef, oreven the moon! Two essential methods to gatherecological information in a standardized way are:Transect Sampling (using a single line) and QuadratSampling (counted within a grid). These samplingmethods provide more accurate data than randomsampling or simply guessing, but they take less timethan 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 transectQuadrats- 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 ConstraintsSmall 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 sizeAppropriate 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 densityDepends 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 systematicallySystematic samples are usually taken along a transect line marked by a tape measure 􀁺 Transect- a line laid across an area 20
  • 21. Sampling along gradientsTransects are setup along aenvironmentalgradients– down a hillside– across a streambed– out from a source ofpollution 21
  • 22. Types of transect sampling 􀁺 Line transect 􀁺 Belt transect 22
  • 23. Line transect methodA measured line is laidacross the area in thedirection of theenvironmental gradient– The species touching theline can be recordedalong the whole length ofthe line (continuoussampling) or at specificpoints 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 methodSimilar to line transect butwidens the sampling area– Transect line is laid out– Samples are taken bydetermining abundance or %cover in an area that is adefined distance from the line– Samples can be taken all theway along the line, at specificintervals or even randomly 25
  • 26. Using a quadrat along a belt transect, e.g. ladder transect (every 5m)Belt TransectIt 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 whenthere are smallareas within alarger habitat thatare clearly different 􀁺 Strata- majordifferences withincommunitiesrecognized beforesampling begins 28
  • 29. RANDOM SAMPLING 􀁺 Often used when the area being studied isfairly uniform, very large, or when there is alimited amount of time available 􀁺 Random = chosen by chance rather thanaccording to a plan; all outcomes are equallylikely 􀁺 Samples are taken from different positionswithin a habitat and those positions arechosen randomly 29
  • 30. How to sample randomlyChoose individuals or Place“sampling units” haphazardly– This is rarely completely random 􀁺 OR… 􀁺 Assign numbers to the areasor individuals to be sampled– Use a random number table toselect which areas or individualswill 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 DensityFour 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 ParametersAbiotic 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. HumidityRelative 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 conductivityOxygen Level- using an oxygen meter or chemical method (Winkler method) 39
  • 40. Collecting MethodsCollecting 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 tube2. screwed-topped jars3. polythene bags4. forceps5. paint brush6. bulb pipette7. pooter 41
  • 42. 8. widger 9. sieve 10. hand lens11. enamel dish 12. beating tray 13. light traps14. Tullgren funnel 15. Baermann funnel 16. mammal traps17. pitfall traps 18. netting 42
  • 43. Estimating Population SizeThe 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 TechniquesFactors 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 ScalesThe 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 atlow 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
  • 56. Ecological Sampling 56
  • 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 othersimilar nets) can havebait or not.Fyke nets have leadsto help guide fish tothe 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 butchers 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 techniquesSilica 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 andstorage 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 specimentreatments 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 thefield 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 orfreezing 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