IN VITRO ASSAY

assays , sterile measure assays drug . , . For eg; . . . in vitro assay . . in vivo test ! assays replenish . i . . , , I . . scientists . . , TEST assay . . .

EMBRYO CRYOPRESERVATION

IN VIVO ASSAY
: TESTING). . . / Difference between in vitro and in vivo In vivo means "within the living" in Latin, and refers to experimentation done in or on the living tissue of a whole, living organism as opposed to a partial or dead one or a controlled environment. Animal testing and clinical trials are forms of in vivo research. In vitro means "within the glass" in Latin, and refers to the technique of performing a given procedure in a controlled environment outside of a living organism. Test tubes and petri dishes are examples of objects used in vitro research. (ANIMAL TESTING) . IN vivo (CLINICAL .

SAMPLE OR DRUG vitro

MICROBIAL ASSAYS anti microbial activity : . ). , incubation 2. SpectrophotometerSpectrophotometer measure . , diameter . i . . absorbance . heat : . . microbes microbial assays growth . Microbial assays .

microbial sterilize .

1. Temperature control- ( inoculum

microbial assay microbial culture ,

3. Plastic test tubes or plastic petri dishes and width

Plastic test tubes or plastic or porcelain ki petri dishes .

4. Media grow . . units . , antibiotics

5. Organisms and inoculum Antibiotics inhibit

Preparation of standard: antibiotic k reference standard k standard . . Preparation of sample: solution . Microbial Assay procedure data potencies calculations treat . microbes . inoculate , Assay test dilution slant cultutre sample . sample

stock

potencies

IMMUNOTECHNIQUES BASED ON ANTIGEN ANTIBODY INTERACTIONS I. Radioimmunoassay

Radioimmunoassay (RIA) , radioactivity antibody-antigen

quantity

. RIA technique . .e.g.,

blood banking , diagnosis of allergies , endocrinology etc. Berson and Yalow concentration 1960 !

! .\

Procedure: 1. Radioactive antigen (iodine protein or

131

125

Antibodies against that antigen. 2. unlabeled ("cold") antigen . . 3. unlabelled antigen radioactive antigen antibody antigen 4. (Unknowns) antigen assays . . supernatant fluid free antigen . . concentration remove . , unlabelled antigens antibodies binding sites SAMPLES

Separating Bound antigens from Free Antigenadd

antiboidy

antigen-antibody complexes .

precipitate

bound antigens

 Radioimmunoassay . Radioimmunoassay

picograms (10-12

. 125I 131I

USES: 1.)

: . 2.) .

abused drugs

(HBsAg)

ENZYME LINKE IMMUNO SORBENT ASSAY (ELISA) ( . ) , ( ) , . DETECT

ANTIBODIES MOLECULE . :

FIXED ANTIBODIES ( )

SUBSTRATE galactosidase)

SAME ANTIBODY ( e.g., -

PROCEDURE: 1. . . ANTIGEN SOLUTION antigen immobilized antibody

2.

reaction mixture

. 3. 4. adding 1 N unbound .

antibody-antigen-antibody "sandwich"

solution

. (e.g., by

NaOH), spectrophotometer .

concentration . (proportional)

intentsity

USES OF ELISA: 1. 2. . 3. HIV, HEPATITS, SEXUAL DISEASES 4. " autoimmune . autoantibodies . , , , THROYID FUNCTION . .

5. "rheumatoid 6. .

. e.g., cocaine , opiates etc.

III.
, ( ) microscope

Fluorescence In Situ Hybridisation (FISH):

. . flouroscent tags . ,

/ fluorescent complementary DNA

short sequences of single-stranded DNA (probes)

Procedures Sample preparation and hybridization

1. denature denature 37oC . incubate . .

2. 3. 70 -C

4. Label probe 5. 6

ADVANTAGES OF FISH
. .

PHOTOMICROGRAPHY
Photomicrography . :
. . . . .

: 1.)

i 2)

. Photomicrography

Photomicrograph

1.

Light micrograph or photomicrograph photomicroscopy

photomicrograph . Vishniac

photomicroscopy

2. Electron micrograph - 3. Digital micrograph

IMAGE ANALYSIS . Computer image analysis processing 1950 Digital Image analysis . : , ,. MRI scan , remote sensing , . , . . (2 ) . (3 ) medical imaging, heavy use of pattern recognition, digital geometry, and signal . . , . .

. medicine, security, and remote sensing

HETEROKARYON A heterokaryon is a cell that contains multiple, genetically different nuclei. This can occur naturally, such as in the mycelium of fungi during sexual reproduction, or artificially as formed by the experimental fusion of two genetically different cells. A medical example is a heterokaryon composed of nuclei from Hurler syndrome and Hunter syndrome. Both of these diseases result in problems in mucopolysaccharide metabolism. However, a heterokaryon of nuclei from both of these diseases exhibits normal mucopolysaccharide metabolism, proving that the two syndromes affect different proteins and so can correct each other in the heterokaryon. In an experimental setting, polyethylene glycol may be applied to a culture of cells to induce the individual cells to fuse together, forming a heterokaryon. INTERSPECIFIC COMPETITION Interspecific competition, in ecology, is a form of competition in which individuals

of different species compete for the same resource in an ecosystem (e.g. food or living space). The other form of competition is intraspecific competition, which involves organisms of the same species. If a tree species in a dense forest grows taller than surrounding tree species, it is able to absorb more of the incoming sunlight. However, less sunlight is then available for the trees that are shaded by the taller tree, thus interspecific competition. Cheetahs and lions can also be in interspecific competition, since both species feed on the same prey, and can be negatively impacted by the presence of the other because they will have less food. Competition is only one of many interacting biotic and abiotic factors that affect community structure. resource in competition Moreover, the has competition area. potential If is the to not always a straightforward, support both direct, interaction. then of Interspecific competition may occur when individuals of two separate species share a limiting same the resource cannot populations, the lowered fecundity, growth, or survival may result in at least one species. Interspecific alter populations, communities and evolution interacting species. On an individual organism level, competition can occur as interference or exploitative competition. Direct competition has been observed between individuals, populations and species, but there is little evidence that competition has been the driving force in the evolution of large groups. For example, between amphibians, reptiles and mammals. [1]

Consequences of interspecific competetion 1.Competitive exclusion- The competitive exclusion principle states that two species that use the same resource in the same way in the same space and time cannot coexist and must diverge from each other over time in order for the two species to coexist. One species will often exhibit an advantage in resource use. This superior competitor will outcompete the other with more efficient use of the limiting resource. As a result, the inferior competitor will suffer a decline in population over time. It will be excluded from the area and replaced by the superior competitor. 2. Niche differentiation - Niche differentiation is a process by which competitive exclusion leads to differences in resource use. In the previous example, niche differentiation resulted in spatial displacement. In other cases it may result in other changes that also avoid competition. If competition avoidance is achievable, each species will occupy an edge of the niche and will become more specialized to that area thus minimizing competition. This phenomenon often results in the separation of species over time as they become more specialized to their edge of the niche, called niche differentiation. The species do not have to be in separate habitats however to avoid niche overlap. Some species adapt regionally to utilizing different resources than they ordinarily would in order to avoid competition. 3. Local extinction- although local extinction of one or more competitors has been less documented than niche separation or competitive exclusion, it does occur. In an experiment involving zooplankton in artificial rock pools, local extinction rates were significantly higher in areas of interspecific competition.[5] In these cases, therefore, the negative effects are not only at the population level but also species richness of communities.

Intraspecific competition
Intraspecific competition is a particular form of competition in which members of

the same species vie for the same resource in an ecosystem (e.g. food, light, nutrients, space). This can be contrasted with interspecific competition, in which different species compete. For example, two trees of the same species growing close together will compete for light, water and nutrients in the soil. Getting less resources, they will perform more poorly than if they grew by themselves (for example lowered growth rates and fewer seed output). Trees

have therefore adapted to grow taller or develop larger root systems through natural selection. Grasshoppers provide an animal example. By eating grass, individual grasshoppers deprive their fellow conspecifics of food. This is an example ofexploitation competition, which means that the grasshoppers do not interact directly with each other, but rather have a negative effect on others' growth and reproduction by their effect on a resource (in this case, grass). In other cases, intraspecific competition may be a case of interference competition, in which the animals interact directly. This is the case, most notably, in territorial animals: some individuals actively prevent others from exploiting a given resource, usually food or space. Intraspecific competition is a major factor affecting the carrying capacity of a population (maximum population level supported by the environment). The levelling of population growth at high densities (known as density dependent inhibition) can be seen as an effect of intraspecific competition. Indeed, whereas at low densities organisms do not compete for resources, at higher densities resources become limiting, and the population size can no longer increase. In terms of population growth rate, this produces a sigmoidal curve, which is a familiar sight for ecologists.

Population Fluctuations Density dependence tends to push populations toward carrying capacity, K

Consequently, populations do not grow indefinitely (over long term) density dependence doesnt always lead to a static equilibrium

Instead, at least some variability around K is the rule of Population Fluctuations Fluctuations around K can be dramatic, sometimes leading many ecologists to they occur Population Size K Population fluctuations can be erratic (irruptive) Often due to variation in density-independent environmental immediate impact on population size (e.g., fires, catastrophes) factors that have a large, exceeding 1000 -fold and ask why

- explore their occurrence in different species

Or they can be periodic (cyclic) Populations exhibiting peri odic cycles hit peaks and valleys in abundance at regular intervals Irregular vs. Cyclic Population Fluctuations Records of gyrfalcons (Falco rusticolus) exported from Iceland in the mideighteenth century indicate dramatic and regular population fluctuations Evidence for Cycles Popularity of falconry waned One answer: Cycles are the result of time lags in responses of populations to their own density i.e., delayed density dependence Why Do Cycles Occur? That is, cycles derive from intrinsic properties of populations

 No environmental (extrinsic) change needed Populations acquire momentum when high birth rates at low densities cause the populations to overshoot K Overshoot of K causes very low survival and birth rates, population falls well below K Recovery occurs when birth rates again reflect current, lowdensity, conditions Key point: Population cycles derive from time delays in the responses of birth and death rates to current environmental conditions nature of cycle depends on nature of time delay Instant updating, no cycle Intrinsic Mechanism For Population Cycles Laboratory populations of Daphnia at higher temperature (25o C), Daphnia magna exhibits oscillations: period of oscillation is 60 days (delay 12-15 days) At high density, reproduction stops; doesnt begin again until senescent individuals die off (takes at least 10 days; a slow decline) Next increase phase needs recruitment of young, fecund individuals at lower temperature (18o

C), the population fails to cycle, because of little or no time delay of responses Minimal senescent period (rapid die off) The Importance of a Time Delay: An Empirical Example Pratt (1944) Biol Bull Daphnia example neat because is implicates demographic process as source of time delay High survival of senescent, non-reproductive adults under crowded conditions in warm environment Other sources of time delays might incl ude Slow development (current generation a product of past environment) Food (nutrient) storage: holdover from period of plenty buffers population from current conditions, for a while Sources of Intrinsic Time Delays Another mechanism: Cycles a re the result of time lagged influence of density-dependent extrinsic factors e.g., delayed density-dependent predation, parasitism, disease Why Do Cycles Occur? An Extrinsic Mechanism For Population Cycles Time Prey Population Size

Time Lag Predation Rate Because of time lag, prey population overshoots K before predation (parasitism) drives it back down; cant grow toward K again until predation (parasitism) rate drops off Across boreal forests of North America Trapping records of the Hudsons Bay Company show cycles in populations of snowshoe hares (Lepus americanus) and Canada lynx (Lynx canadensis) A Cycle Caused by Extrinsic Factors Fluctuations are dramatic, characterized by a roughly 10-year period Lynx cycle lags a year or two behind that of hare cycle The Snowshoe Hare Cycle # of Pelts Mechanics of the 10-Year Hare Cycle The 10-year snowshoe hare cycle consists of three phases Increase Phase Decline Phase Low Phase Krebs et al. (1995) Science Mechanics of the 10-Year Hare Cycle Increase phase Time of plenty (lots of winter browse)

 High productivity, rapid (exponential) population growth Before long (2-3 years), K is exceeded and, all the while, specialized predator (lynx) populations growing as well Krebs et al. (1995) Science Mechanics of the 10 -Year Hare Cycle Decline phase Huge momentum shoots hare populations well beyond K Food becomes limiting (see picture), predation heavy So, productivity and survival plummet, triggering decline Decline is swift (1-2 years), driven by time-lagged predation Krebs et al. (1995) Science Mechanics of the 10 -Year Hare Cycle Low phase Following decline, hares are scarce, allowing food to recover Predators die off, disperse so survival is high Yet, low phase can persist for 4-5 years Why? Answer seems to be stress (time lag); i.e., the ghost of predation risk Boonstra et al. (1998) Ecology Southern Snowshoe Hare Populations Along the southern periphery of the species range, cycle doesnt seem to exist. Why? Still debated, but

 Apparently, increase phase never occurs Instead, hares perpetually suppressed by suite of generalist carnivores (never die off, disperse) No predation time lag also, fragmentation allegedly intensifies predation

Mathematical modelling is the use of mathematics to describe real-world phenomena investigate important questions about the observed world explain real-world phenomena test ideas make predictions about the real world The real world refers to engineering physics physiology ecology wildlife management
A mathematical model is a description of a system using mathematical concepts and language. The process of developing a mathematical model is termed mathematical modelling. Mathematical models are used not only in the natural sciences (such as physics, biology, earth science, meteorology) and engineering disciplines (e.g. computer science, artificial intelligence), but also in the social sciences (such as economics, psychology, sociology and political science); physicists, engineers, statisticians, operations research analysts and economists use mathematical models most extensively. A model may help to explain a system and to study the effects of different components, and to make predictions about behaviour. Mathematical models can take many forms, including but not limited to dynamical systems, statistical models, differential equations, or game theoretic models. These and other types of models can overlap, with a given model involving a variety of abstract structures. In general, mathematical models may include logical models, as far as logic is taken as a part of mathematics. In many cases, the quality of a scientific field depends on how well the mathematical models developed on the theoretical side agree with results of repeatable experiments. Lack of agreement between theoretical mathematical models and experimental measurements often leads to important advances as better theories are developed.