Cytotoxicity

1. What is Cytotoxicity?

             Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are an immune cell or some types of venom, e.g. from the puff adder (Bitis arietans) or brown recluse spider (Loxosceles reclusa).

             Cytotoxicity is the quality of being toxic to cells. Cells exposed to a cytotoxic compound can respond in a number of ways. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis; they can stop growing and dividing; or they can activate a genetic program of controlled cell death, termed apoptosis.

MEASURING CYTOTOXICITY 

 

               Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical.

              Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components.Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. One molecule, lactate dehydrogenase (LDH), is commonly measured using LDH assay. Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity.

                Cytotoxicity can also be monitored using the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) or MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATPcontent as a marker of viability.Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction.

             Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

             A label-free approach to follow the cytotoxic response of adherent animal cells in real-time is based on electric impedance measurements when the cells are grown on gold-film electrodes. This technology is referred to as electric cell-substrate impedance sensing (ECIS). Label-free real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like many colorimetric endpoint assays.

  

1. What is Atomic absorption spectroscopy (AAS) ?
               
                Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state.
                In analytical chemistry the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different elements in solution or directly in solid samples used in pharmacology, biophysics and toxicology research.
Atomic absorption spectroscopy was first used as an analytical technique, and the underlying principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany.
              Atomic absorption spectrometry has many uses in different areas of chemistry such as:

• Clinical analysis: Analyzing metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, muscle tissue, semen
• Pharmaceuticals: In some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product
• Water analysis: Analyzing water for its metal content.                                                                          How it works
  Atoms of different elements absorb characteristic wavelengths of light. Analysing a sample to see if it contains a particular element means using light from that element. For example with lead, a lamp
  containing lead emits light from excited lead atoms that produce the right mix of wavelengths to be
  absorbed by any lead atoms from the sample. In AAS, the sample is atomised – ie converted into
  ground state free atoms in the vapour state – and a beam of electromagnetic radiation emitted from
  excited lead atoms is passed through the vaporised sample. Some of the radiation is absorbed by the lead atoms in the sample. The greater the number of between 1 Nm–2 and 5 Nm–2. The ionisation of some gas atoms occurs by applying a potential difference of about 300–400 V between the anode and the cathode. These gaseous ions bombard the cathode and eject metal atoms from the cathode in a process called sputtering. Some sputtered atoms are in excited states and emit radiation characteristic of the metal as they fall back to the ground state – eg Pb* → Pb + h (Fig. 2). The shape of the cathode concentrates the radiation into a beam which passes through a quartz window, and the shape of the lamp is such that most of the sputtered atoms are redeposited on the cathode.
   

  Atomic Absorption Theory

  Atomic absorption spectroscopy relies on the Beer-Lambert law to determine the concentration of a particular analyte in a sample. The absorption spectrum and molar absorbance of the desired sample element are known, a known amount of energy is passed through the atomized sample, and by then measuring the quantity of light it is possible to determine the concentration of the element being measured. Aurora’s TRACE AI1200 and TRACE 1300 Atomic Absorption Spectrometers are available with flame, graphite furnace and vapor hydride generation atomizers. These atomizers aspirate the sample into the light path where it is illuminated by a hollow-cathode lamp (HCL), which emits light at the wavelength characteristic of the desired elements. A built-in detector measures the light emissions both in presence and absence of sample, and the ratio of the absorbances are used to determine the analyte concentration.
   
  

PHYTOCHEMICAL

1. What is phytochemical?
                 Phytochemicals are chemical compounds that occur naturally in plants (phyto means "plant" in Greek). Some are responsible for color and other organoleptic properties, such as the deep purple of blueberries and the smell of garlic. The term is generally used to refer to those chemicals that may have biological significance, for example carotenoids orflavonoids, but are not established as essential nutrients.
2. What is Phytochemical Screening?
              Phytochemical screening is a process of tracing plant constituents. For example you want to found out if a certain plant contains alkaloids (a plant constituent) then, you will be performing a phytochemical screening procedures for alkaloids (in this case mayer's and Wagner's test). There are general plant constituents that can be performed with a standard test. And these are screenig for:  Alkaloids  Saponin glycosides  Cardenolides and Bufadionolides  Flavonoids  Tannins and Polyphenolic compounds  Anthraquinones  Cyanogenic glycosides  Carbohydrates  Fixed oils, Fats, and Volatile oils.-Marian m. elevera.
3. What are the different reagents used in Phytochemical Screening?

4.What are the indicators of the presence of each phytochemical?
SteroidsLibermann – Burchard Reaction: 2 ml extract was mixed with chloroform. To this 1-2 ml acetic anhydride and 2 drops concentrated sulphuric acid were added from the side of test tube. First red, then blue and finally green colour appears.
Lignin-
With alcoholic solution of phloroglucinol and concentrated hydrochloric acid appearance of                  red colour shows the presence of lignin.
Glycosides- Alcoholic extract when made alkaline, shows blue or greenfluorescence.

Terpenoids -    Noller’s test: The substance was warmed with tin and thionyl chloride. Pink                                               coloration indicates the presence of triterpenoids.
Proteins- Appearance of red colour shows the presence of proteins and free amino acids.
Biuret test: Equal volume of 5% solution of sodium hydroxide and 1% copper sulphate were added. Appearance of pink or purple colour indicates the presence of proteins and free amino acids.
Carbohydrates - Appearance of reddish orange precipitate shows the presence of carbohydrates.
Gums and mucilages-About 10ml of various extracts were added separately to 25ml of absolute alcohol with constant stirring and filtered. The precipitate was dried in air and examined for its swelling properties and for the presence of carbohydrates.
Phytoserol- Appearance of bluish green colour shows the presence of phytosterol.
Oils and Fats- Appearance of oil stain on the paper indicates the presence of fixed oil.
Tannins and Phenols- Dilute ferric chloride solution (5%) - violet colour
1% solution of gelatin with 10%NaCl - white precipitate
10% lead acetate solution - white precipitate
Flavanoids-With aqueous solution of sodium hydroxide blue to violet colour (Anthrocyanins), yellow colour (Flavones), yellow to orange (Flavonones).
With concentrated sulphuric acid yellowish orange colour (Anthrocyanins), orange to crimson colour (Flavonones).
Shinoda’s test – the extracts were dissolved in alcohol, to that a piece of magnesium and followed by concentrated hydrochloric acid was added drop wise and heated. Appearance of magenta colour shows the presence of flavonoids.

BACTERIA

What comes into your mind when you heard the the word "bacteria"? When we heard bacteria, the frirst thing that we think is all about dirts and diseases. But what is the true meaning of BACTERIA? The word bacteria is a plural form of bacterium comes from Greek word "bakteria" which means "little stick". Bacteria are microscopic living organisms, usually one-celled that can be found everywhere. They can be dangerous, such as when they cause infections. They can also beneficial to our life, because of their usefulness that can help our daily life. They can be Independent (free living organisms) or Parasites (depend on other organisms for life). They can reproduce by fission.

Bacteria can be classified using GRAM STAIN TEST, who developed by Hans Christian Gram in the 1800s. It is a method for classifying different types of bacteria using a chemical stain and viewing through a microscope. Most bacteria are classified into 2 groups. The Gram-positive and Gram-negative. We classified the bacteria as Gram-positive if it has thick layer of peptidoglycans that can stained purple by the crystal violet dye. so that the bacteria apper purple or blue. While the Gram-negative has thin layer of peptidoglycans, so they cannot retain the crystal viole dye and thus they appear red or pink due to the retention of the counter stain.

Gram-positive bacteria can also cause different diseases. Some of these diseases are Anthrax, Rheumatic fever, Diphtheria, Botulism, Septic sone throat, Boils, Tetanus, Gas gangrene, and Toxic shock syndrome.
Gram-negative can cause many types of infections and are spread to humans in a variety of ways. Several species, including Escherichia coli, are commoncausesof food–borne disease. Vibrio cholerae—thebacteria responsible for cholera—is a waterborne pathogen.Gram-negativebacteria can also cause respiratory infections, such as certain typesof pneumonia,and sexually transmitted diseases, including gonorrhea. Yersinia pestis, theGram-negative bacterium responsible for plague, is transmitted to people through the biteof an infected insect or handling an infected animal.