Hi

Cytotoxicity

Cytotoxicity Test

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).

Cell physiology

Treating cells with the cytotoxic compound can result in a variety of cell fates. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death (apoptosis).

Cells undergoing necrosis typically exhibit rapid swelling, lose membrane integrity, shut down metabolism and release their contents into the environment. Cells that undergo rapid necrosis in vitro do not have sufficient time or energy to activate apoptotic machinery and will not express apoptotic markers. 

Apoptosis is characterized by well defined cytological and molecular events including a change in the refractive index of the cell, cytoplasmic shrinkage, nuclear condensation and cleavage of DNA into regularly sized fragments. Cells in culture that are undergoing apoptosis eventually undergo secondary necrosis. They will shut down metabolism, lose membrane integrity and lyse.

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 ATP content 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.

Cytotoxicity in cancer

Chemotherapy as a treatment of cancer often relies on the ability of these agents to preferentially kill or damage cancer cells as opposed to healthy cells.

Immune system cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADCC) describes the cell-killing ability of certain lymphocytes, which requires the target cell being marked by an antibody. Lymphocyte-mediated cytotoxicity, on the other hand, does not have to be mediated by antibodies; nor does complement-dependent cytotoxicity (CDC), which is mediated by the complement system.

Three groups of cytotoxic lymphocytes are distinguished:

·         Cytotoxic T cells

·         Natural killer cells

·         Natural killer T cells

Atomic Absorption Spectroscope (AAS)

Atomic Absorption Spectroscopy

>What is Atomic Absorption Spectroscopy?

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.

>Instruments Used in Atomic Absorption Spectroscopy:

• Atomizers- devices that converts liquid into particles.

• Spectrometer- it is an instrument used to measure properties, such as massenergy, wavelength, or index of refraction, and disperse radiant energy.

• Radiation Sources- We have to distinguish between line source AAS (LS AAS) and continuum source AAS (CS AAS). In classical LS AAS, as it has been proposed by Alan Walsh, the high spectral resolution required for AAS measurements is provided by the radiation source itself that emits the spectrum of the analyte in the form of lines that are narrower than the absorption lines.

Colors in Absorption line

>Uses

• 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.

Phytochemicals

Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. They are nonessential nutrients, meaning that they are not required by the human body for sustaining life. It is well-known that plant produce these chemicals to protect themselves but recent research demonstrate that they can also protect humans against diseases. 

Table of Some Phytochemicals

Alkaloids
 In Determinig the presence or absence of alkaloids, Mayer’s reagent test was used. It is a mixture of mercuric chloride solution and potassium iodide solution. Alkaloid is present if there is a formation of green or white precipitations

Carbohydrates 
To determine if carbohydrates were present, Molisch’s reagent is used. It was prepared by mixing naphthol and ethanol. Carbohydrates were present if there is a formation of red or purple when the extract was mixed with Molisch’s reagent.

 Glycosides 
In testing the presence of glycosides, acetic acid and ferric chloride was used. There is a presence of glycosides if the color of the mixture extract and reagent became blue-green. 

 Saponins 
To test of Saponins were present on the extract, ditilled water was added and then chook for fifteen minutes. When there is a formation of foam or frothy bubbles on the mixture, it indicates that saponins were present. Phenols The picture shows that traces of Phenols were present in the extract. The brown extract changes into the green color.The formation of blue or green color indicates the presence of phenols. 

 Tannins 
To determine the presence of tannins, ferric chloride solution was used. When there is a formation of dark blue or greenish black color indicates the presence of tannins.

 Anthocyanin 
The presence or absence of anthocyanin as determined by the use of sodium hydroxide solution., when there is a formation of blue or green precipitates. It indicates that anthocyanin Is present.

 Protein 
The formation of yellow, yellow-green or light green color when the extract was mixed with nitric acid indicates the presence of protein.

Tomatoes contain lycopene

                                              Bacteria

Bacteria (singular: bacterium) constitute a large domain of prokaryotic microorganisms. Typically a fewmicrometres in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs,radioactive waste, and the deep portions of Earth's crust. Bacteria also live in symbiotic and parasitic relationships with plants and animals. They are also known to have flourished in manned spacecraft.

Different Kinds of Bacteria

Cocci- Cocci are round, spherical-shaped bacteria. They can occur as a single bacterium or be arranged in a pair, chain or cluster of bacteria depending on which type of coccus it is.Its Gram stain is Gram-positive.

Bacilli- Bacilli are rod-shaped bacteria that look like cylinders, arranged singly or in chains. Its Gram stain is Gram-positive.

Vibrio- Vibrios are one of three types of bacteria with a spiral-like shape. Vibrios are comma-shaped bacteria that look like a curved rod. They typically live in aquatic environments. Vibrio cholera moves in a darting motion by a single flagellum, a whiplike structure, and is the bacteria that causes cholera. Its Gram stain is Gram-negative.

Spirilla- Spirilla are another subgroup of bacteria with a more rigid spiral shape. One such bacteria is Campylobacter jejuni, which causes diarrhea. Campylobacter jejuni is typically acquired in places where sanitation is poor or by eating raw or undercooked poultry. Its Gram stain is Gram-negative.

Spirochete- Spirochetes are long, thin and flexible corkscrew-shaped bacteria. They typically move in a distinctive rotating manner that allows them to be mobile in mucus-lined tissue or where it is viscous. It is not classified as Gram-positive and Gram-negative.

Diseases That Each Type of Bacteria Cause

Coccus- These bacteria cause many different types of common diseases. Among the more common cocci are Staphylococcus aureus, which appears as a cluster of cocci. Staph aureus can often be found in the nose and on the skin without causing disease, but it is also responsible for causing conditions such as boils, pneumonia, meningitis and toxic shock syndrome. Streptococcus pyogenes occurs as a chain of cocci and causes diseases that originate in the throat or skin, including strep throat and scarlet fever. Neisseria meningitidis occurs in pairs of cocci. This bacteria causes bacterial meningitis, an inflammation of the brain and spinal cord, after entering the body through the nose or throat.


Bacilli- Escherichia coli is a rod-shaped bacteria that normally lives in your intestinal tract without causing disease. However, a few strains of E. coli do cause disease that's spread typically by eating or drinking contaminated food or water; a typical symptom is diarrhea. Corynebacterium diphtheriae, another rod-shaped bacterium, infects the respiratory tract and causes diphtheria. Diphtheria causes a thick coating on the back of the nose and throat, making it difficult to swallow or breathe, followed by swelling of the neck and potentially death. Bacillus anthracis is the bacteria that cause anthrax. This rod-shaped bacterium grows in long chains and can infect you through broken skin, ingestion or inhalation.


Vibrio- Vibrio cholera moves in a darting motion by a single flagellum, a whiplike structure, and is the bacteria that causes cholera. Cholera is an intestinal infection that causes severe diarrhea and dehydration, typically transmitted by drinking contaminated water. It is a very serious disease that can lead to death if not treated promptly.


Spirilla- One such bacteria is Campylobacter jejuni, which causes diarrhea. Campylobacter jejuni is typically acquired in places where sanitation is poor or by eating raw or undercooked poultry. Helicobacter pylori is a similar-shaped bacteria found in your stomach. While some people infected with Helicobacter pylori have no symptoms, it is a common cause of stomach inflammation and ulcers.


Spirochete- Two well-known spirochetes that cause disease in humans are Treponema pallidum and Borrelia burgdorferi. Treponema pallidum causes the sexually transmitted disease syphilis. Infection typically begins as a single sore at the site of infection. Additional lesions or rashes can develop elsewhere on the body if left untreated. Borrelia burgdorferi is transmitted through the bite of an infected tick and causes Lyme disease. Infection with Borrelia burgdorferi causes a typical "bull's-eye" rash. If left untreated, it can affect your heart and nervous system and cause arthritis.

Difference Between Gram-positive and Gram-negative Bacteria

Gram-positive Bacteria- Gram-positive bacteria are a class of bacteria that take up the crystal violet stain used in the Gram staining method of bacterial differentiation. The thick peptidoglycan layer in the cell wall that encases their cell membrane retains the stain, making definitive identification possible.

Gram-negative Bacteria- Gram-negative bacteria are a class of bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation,making positive identification possible. The thin peptidoglycan layer of their cell wall is sandwiched between an inner cell membrane and a bacterial outer membrane. In Gram staining, the outer lipid-based membrane of gram-negative bacteria is removed by an alcohol solution. The alcohol also decolorizes the then exposed peptidoglycan layer by dissolving away the previously applied crystal violet. A counterstain (safranin or fuchsine) is then added which recolorizes the bacteria red or pink.

What I Learned

• I learned that there are 5 different kinds of bacteria. 
• I learned the diseases each type of bacteria can cause.
• I learned the differences between a Gram-positive and Gram-negative bacteria.

What I Did

      I search through the internet for information about bacteria and learned much more about it.

I Can Apply My Learning To

• I can apply my learning to my course that I will take in college.
• I can apply my learning to research proposals that are needed to be done.

Posted by 

Mark Oniel M. Binuya