Cytotoxicity

1. What is Cytotoxicity?

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.

2. How is Cytotoxicity being measured?

There are many ways to measure cytotoxicity, but most involve assessment of cell membrane integrity. Membrane integrity can be evaluated by using vital dyes (such as trypan blue or propidium iodide), by protease biomarkers, with MTT or MTS redox potential assays, or by measuring ATP content. Many of these assays involve colorimetric, fluorescence, or luminescencedetection.

With one of the widest ranges of detection technologies on the market today, Molecular Devices can provide the bioanalytical and analytical products that you need to conduct cytotoxicity measurements, from cytometry and high-content screening systems to multi-mode microplate analysis systems to high-throughput cellular screening systems.

Cell Cytotoxicity

Atomic Absorption Spectroscopy (AAS)

1. What is 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.

The modern form of AAS was largely developed during the 1950's by a team of Australian chemists. They were led by Sir Alan Walsh at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Division of Chemical Physics, in Melbourne, Australia.

An Example of AAS Machine

2. What is the use of AAS?

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.

3. How does the AAS Machine works?

In order to analyze a sample for its atomic constituents, it has to be atomized. The atomizers most commonly used nowadays are flames and electrothermal (graphite tube) atomizers. The atoms should then be irradiated by optical radiation, and the radiation source could be an element-specific line radiation source or a continuum radiation source. The radiation then passes through amonochromator in order to separate the element-specific radiation from any other radiation emitted by the radiation source, which is finally measured by a detector.

Atomic absorption spectrometer block diagram

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.

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).

3. What are the different reagents used in phytochemical screening? How are these reagents are prepared?

A. For Alkaloids: 

 Mayer’s reagent - is freshly prepared by dissolving a mixture of mercuric chloride (1.36 g) and of potassium iodide (5.00 g) in water (100.0 ml).

B. For Carbohydrates:

Molisch's reagent- dissolve 25 g of α-naphthol in 95% ethanol and dilute it to 500 mL with ethanol.

C. For Glycosides:

Modified Borntrager’s Test- Extracts were treated with Ferric Chloride solution and immersed in boiling water for about 5 minutes. The mixture was cooled and extracted with equal volumes of benzene.

D. For Saponins:

Froth Test-  Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the 

presence of saponins.

E. For Phenols:

Ferric Chloride Test- Extracts were treated with 3-4 drops of ferric chloride solution.

F. For Tannins:

Ferric Chloride Test- Extracts were treated with 3-4 drops of ferric chloride solution.

G. For Anthocyanins:

Sodium Hydroxide Test- Extracts are added with colorless sodium hydroxide.

H. For Proteins:

Xanthoproteic Test- The extracts were treated with few drops of concentrated nitric acid. 

I. For Flavonoids:

Alkaline Reagent Test- Extracts were treated with few drops of sodium hydroxide solution. 

4. What are the indicators of the presence of each phytochemical?

A. Mayer's Reagent-  to give a cream colored precipitate

B. Molisch's Reagent- a positive reaction is indicated by appearance of a purple ring at the interface between the acid and test layers.

C. Modified Borntrager’s Test- formation of rose-pink color in the ammonical layer indicates the presence of anthranol glycosides. 

D. Froth Test- formation of 1 cm layer of foam indicates the presence of saponins.

E. Ferric Chloride Test- formation of bluish black color indicates the presence of phenols.

F. Ferric Chloride Test- formation of bluish black color indicates the presence of tannins.

G. Sodium Hydroxide Test- when there is a formation of blue or green precipitates. It indicates that anthocyanin Is present.

H. Xanthoproteic Test- formation of yellow color indicates the presence of proteins. 

I. Alkaline Reagent Test- formation of intense yellow color, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids.

Bacteria

A. Definition of Bacteria

Bacteria are microscopic living organisms, usually one-celled, that can be found everywhere. They can be dangerous, such as when they cause infection, or beneficial, as in the process of fermentation (such as in wine) and that of decomposition.

Picture of Bacteria

B. Different kinds of bacteria and their gram stain

Gram-positive bacterias : 

• Cocci
• Bacillus

Gram-negative bacterias : 

• Spirillum
• Spirochaete
• Vibrios

C. Diseases that each bacteria cause

Cocci- it causes strep throat, some skin diseases and pneumonia, among many others. They can also cause gonorrhea, meningitis, and skin lesions.

Bacillus-  Escherichia coli do cause disease that's spread typically by eating or drinking contaminated food or water; a typical symptom is diarrhea. Corynebacterium diphtheriae, 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.

Spirillum- Rat-Bite fever is one disease caused by spirilla minus

Spirochaete- 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.

 
Vibrios-  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.

D. Difference between gram positive and gram negative

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 cannot retain the violet stain after the decolorization step; alcohol used in the decolorization process degrades the outer membrane of gram-negative cells making the cell wall more porous and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take up the counterstain (safranin or fuchsine) and appear red or pink.

Gram-positive and -negative cell wall structure
Photo From: Wikipedia