Embryo in embryogenesis of Kuhol

The embryo’s of the P.canaliculata contains 90% of perivitellin fluid proteins. This fluid proteins sharply decreases as the embryo’s development proceed to stage 4 and 5. Most proteins are incorporated and used at these stages must have been consumed as energy source, nutrients and converted into other components. Basically, there is no net increase in embryo protein content until hatchlings stages 3 and 4. these observed stages has the trend to increase protein level. Temporarily stored protein ( egg perivitellins) remain unaltered by the embryos. After hatchlings (stage 6) these proteins become depleted in juveniles. Therefore, protein conversion efficiency ( calculated as % of perivitellins energy transformed into embryonic tissues) was calculated between stages 1 and 4 and the 29% low value observed are not only used as embryo structure but also as source of energy.

source:           Heras, H., & Pollero, R. J. (2002). Lipoproteins from plasma and perivellin fluid of the apple snail Pomacea canaliculata. Retrieved September 11, 2011, from http://www.cricyt.edu.ar/biocell/vol/pdf/26/12.pdf

over view on culture media

An appropriate culture media is needed for the proper growth and maintenance of a microorganism that is done in the laboratory. These specialized culture media are important in the identification and isolation of microorganism. Each microorganism varies in terms of their needs, such as energy, carbon, nitrogen, phosphorus, sulfur, and various minerals that they acquired in their specific culture media. It is necessary to have knowledge in identifying the normal habitat of microorganism so that it would be easier in selecting specific culture media that is suitable for them.  

Complex media is composed of peptones, meat extract and yeast extract. A partial proteolytic digestion of meat, casein, soya meal, gelatin, and other protein sources produces protein hydrolysates called peptones. Meat extract contains amino acids, peptides, nucleotides, organic acids, vitamins, and minerals. Both meat and yeast extract are aqueous extracts of élan beef and brewer’s yeast. They serve as sources of carbon, energy, and nitrogen.

 Selective media favor the growth of particular microorganisms. Bile salts or dyes like basic fuschin and crystal violet favor the growth of gram negative bacteria by inhibiting the growth of gram positive bacteria without affecting the gram negative organisms. Endo agar, eosin methylene blue agar, and Mac Conkey agar are wisely used for the detection of E. coli.  

Morpholigical Description of E.coli

Escherichia coli is quickly identified in urine by the process of hemolysis that occurs in blood agar.  It is determined in a differential media such as EMB having a morphological feature of an iridescent “sheen”. It gives positive test for indole, lysine decarboxylase, and manitol fermentation and produces gas from glucose.

Culture Media Contains Nutrients for Culture Growth

Since Pasteur and Koch, microbiologists have tried to mimic the natural environment of bacteria in order to grow them in a laboratory. Beef broth is one medium used for cultivation of bacteria, or nutrient broth which contains water, beef extract and peptine, a nitrogen preparation from plant or animal sources. Agar is used to solidify the medium, upon the addition of this, the product is called nutrient agar.

"Agar is a polysaccharide derived from marine red algae. Introduced by the school of bacteriology of Robert Koch, agar is a unique colloid that remains liquid until cooled top below approximately 36˚ C. This allows for mixing blood with culture media for determination of haemolytic reactions. The solidified medium can be used to cultivate bacteria, isolate pure cultures, or accomplish other tasks, such as a medium for measuring bacterial growth. Once solidified, agar will remain solid at room temperature. It will not melt until it reaches a temperature of 85˚C, making the material excellent for growing thermophilic bacteria on nutrient agar. Agar adds no macronutrients to the nutrient medium. Sometimes it is valuable to use semi-solid medium, such as when testing bacterial motility. In this case, a lower concentration of agar is added to the medium to make it stiff but not as solid as nutrient agar"

  Nutrient broth and nutrient agar are examples of chemically undefined medium or complex medium.  We cannot be certain of the exact components or the quantity thus it is called complex. We do not precisely know what carbon and energy sources are present nor what other factors that facilitates growth are present. Complex media are commonly used in the teaching laboratory. Because you simply want to grow bacteria and are not concerned what specific nutrients are needed to accomplish this.

Another type of medium is a chemically defined medium or synthetic medium. In this type of medium, the components are known. This is commonly used when understanding the growth conditions necessary for bacterial growth.

A Bacterial Growth Curve Illustrates the Dynamics of Growth in Culture Medium

In growing bacteria, a standard growth curve can be observed; it illustrates the events that occur over time within the population of bacteria. There are four distinct phases that take place: the lag phase, the logarithmic phase, the stationary phase and the decline phase.

In the lag phase cover the first few hours of the curve, no cell divisions occur, because bacteria are adapting to the new environment. In cases of infection in the respiratory tract, white blood cells may swallow up the bacteria, in culture medium; they may die from the inability to adapt to the new environment. The actual length of the lag phase depends on the metabolic activity of the remaining bacteria which were able to adapt. Then the preparation for binary fission begins, they grow in size, store nutrients, and synthesize enzyme. Then the population now enters the active stage called the logarithmic phase or log phase. This is the exponential growth for E.coli. During the log phase, all cells undergo binary fission; the length of the lag phase depends on the species and the environmental conditions. As each generation time passes, the number of bacteria doubles and the graph rises in a straight line on a logarithmic scale.

In human infection, symptoms develop during the lag phase. Bacteria and their metabolic wastes cause tissue damage. Coughing or fever may occur, and fluid may enter the lungs if air sacs are damaged. However, this is also the stage where bacteria are very susceptible antibiotics. In liquid medium preparations, bacterial growth is implies bacterial growth. If prepared in solid medium, growth seen through the emergence of colonies, a single colony may consist of millions of organisms.

After some time: days in cases of infection and hours in culture preparations, the dynamism of the population changes, as the reproductive and death rate equalizes, the population enters a plateau. This is called the stationary phase. In an infection, antibodies, and possibly antibiotics too are now attacking the bacteria. In an in vitro environment, available nutrients become scarce and waste products accumulate.  If the bacterium is a species that secretes antibiotics, they are produced during the stationary phase, if the organism is a species of Bacillus or Clostridium, all vegetative cells will produce endospores.

If such conditions persist, the environment reaches its limit and the population declines thus entering the exponential death phase or decline phase. At this point, the number of dying cells exceeds the number of new cells formed. A bacterial glycocalyx may hinder death by acting as buffer to the environment. For many species, though, the history of the population ends with death of the last cell.

source: please ask joymyrn

Methods of Protein Analysis / Determination

Protein Determination Methods:

1. Kjeldahl Method

The first step in the Kjeldahl method is the digestion of the sample in sulfuric acid with a catalyst which converts nitrogen to ammonia. The next step is the distillation of the ammonia from the previous step into a trapping solution. Lastly, the ammonia is titrated with a standard solution to determine the amount (3.1 Nitrogen Determination by Kjeldahl (rack), 2007). The amount of nitrogen is calculated using a formula. This amount is then applied to another formula to determine protein content. (Blamire, 2003).

2. Dye Binding Method

                At low pH, basic groups of protein are positively charged. These will quantitatively bind a negatively charged dye. In doing this method, you must first mix the protein, dye and buffer which has a pH of two. Then you filter or centrifuge the mixture. You then measure the optical density of the filtrate. The optical density of the dye bound protein is equal to the optical density of the dye initially and the optical density of the filtrate. The factors influencing Dye Binding Determination are temperature, non-proteins, buffer systems, and protein quality.

3. Biuret Method

                This method is a good general protein assay for batches of material for which yield is not a problem. Under alkaline conditions, substances containing two or more peptide bonds form a purple complex with copper salts in the reagent. It uses a spectrophotometer to measure the absorbance and then calculate for the amount of protein. (Caprette, 1997)

4. Lowry Method

                The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogens with the copper ions under alkaline conditions. It is sensitive to low concentrations of protein. A variety of compounds will interfere with the procedure like amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents. (Williamson, 2000)

5. Ultra-violet Absorption (UV) at 280 nm

                Most proteins absorb ultraviolet light at a maximum of 280 nm wavelength because of the presence of Tyrosine and Tryptophan. This has been used as a rapid and fairly sensitive measure of protein concentration. However, nucleic acids absorb light at a wavelength of 280 nm, but they absorb much more strongly at 260 nm UV light. For protein it is the reverse situation. This assay uses this reverse relationship to calculate the interference of nucleic acids in the estimation of protein concentration.

                The concentration of the protein in solution is calculated according to the treatment of Warburg and Christian. With this protocol, it is not necessary to know the nucleic acid concentration of the sample to find the protein concentration of an unknown sample. This method gives considerable error with mixture containing more than 20% nucleic acids or with very turbid solutions. (determination of protein concentratoin by UV absorption)

6. Fluorescence Method

                Tyrosine and Tryptophane is a fluorescent compound. This method excites amino acids at 280 nm and measures the emission at 348 nm. It is more sensitive than UV absorption. (Protein Determinatin Methods)

7.  Combustion method

                Total protein is determined using nitrogen analysis. The sample is combusted with oxygen and the gases containing nitrogen oxides are collected in a ballast tank until a specified pressure is reached. Helium is used as a carrier and an aliquot of combustion gas containing nitrogen oxides is reduced to nitrogen. It is then passed through a tube containing magnesium perchlorate and sodium

hydroxide on a silicate carrier to remove water and carbon dioxide. The nitrogen is measured with a thermal conductivity detector using helium as a reference. Nitrogen is then converted to protein using a conversion factor. (Protein Determination by Combustion, 2009)

 Bibliography

3.1 Nitrogen Determination by Kjeldahl (rack). (2007). Retrieved September 5, 2011, from Foragetesting.org: http://www.foragetesting.org/lab_procedure/sectionB/3/part3.1.htm

Blamire, J. (2003). e-learning for Quantitative Analysis-kjeldahl method. Retrieved September 5, 2011, from science@a distance: http://www.brooklyn.cuny.edu/bc/ahp/SDKC/Chem/SD_KjeldahlMethod.html

Caprette, D. R. (1997). Biuret Protein Assay. Retrieved September 2011, from Experimental Biosciences: Introductory Laboratory - Bioc 211: http://www.ruf.rice.edu/~bioslabs/methods/protein/biuret.html

determination of protein concentratoin by UV absorption. (n.d.). Retrieved 2011, from Center for cocoa biotechnology research and development: http://www.koko.gov.my/CocoaBioTech/Protein%20Quantitation1.htm

Protein Determinatin Methods. (n.d.). Retrieved September 2011, from Ohio State University: class.fst.ohio-state.edu/fst601/food%20chemistry%20601-8.pdf

Protein Determination by Combustion. (2009). Retrieved September 2011, from United States Department of Agriculture: Food Safety and Inspection Service, Office of Public Health Science: www.fsis.usda.gov/PDF/CLG_PRO_4_03.pdf

Williamson, J. (2000). Biology 371 Independent Research week 5: Protein Determination-Lowry Procedure. Retrieved September 2011, from Davidson College: http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html