The Ames test is a bacterial mutagenicity assay used to identify potential carcinogens. It involves exposing histidine-requiring strains of Salmonella typhimurium bacteria to test compounds or chemicals. If a compound is mutagenic, it will induce mutations in the bacteria that allow their growth in the absence of histidine. The Ames test is quick, inexpensive, and has identified many carcinogens and mutagens. However, its sensitivity is sometimes limited as bacteria do not fully replicate human metabolism.
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
A genotoxin is a chemical or agent that can cause DNA or chromosomal damage. Such damage in a germ cell has the potential to cause a heritable altered trait (germline mutation). DNA damage in a somatic cell may result in a somatic mutation, which may lead to malignant transformation (cancer). Many in vitro and in vivo tests for genotoxicity have been developed that, with a range of endpoints, detect DNA damage or its biological consequences in prokaryotic (e.g. bacterial) or eukaryotic (e.g. mammalian, avian or yeast) cells. These assays are used to evaluate the safety of environmental chemicals and consumer products and to explore the mechanism of action of known or suspected carcinogens. Many chemical carcinogens/mutagens undergo metabolic activation to reactive species that bind covalently to DNA, and the DNA adducts thus formed can be detected in cells and in human tissues by a variety of sensitive techniques. The detection and characterisation of DNA adducts in human tissues provides clues to the aetiology of human cancer. Characterisation of gene mutations in human tumours, in common with the known mutagenic profiles of genotoxins in experimental systems, may provide further insight into the role of environmental mutagens in human cancer.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research paper publishing, where to publish research paper, journal publishing, how to publish research paper, Call for research paper, international journal, publishing a paper, call for paper 2012, journal of pharmacy, how to get a research paper published, publishing a paper, publishing of journal, research and review articles, Pharmacy journal, International Journal of Pharmacy, hard copy of journal, hard copy of certificates, online Submission, where to publish research paper, journal publishing, international journal, publishing a paper
Principles of cell viability assays by surendra.pptxSurendra Chowdary
1.DYE EXCLUSION ASSAYS:
Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells.
For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin.
Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension.
Principle:
The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not.
Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain.
A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained.
Trypan blue stain should be stored in dark and filtered after prolonged storage.
As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results.
Assay Protocol:
The cell suspension to be tested is centrifuged at 100 g for 5 min.
The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium.
Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts.
Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope.
Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately.
.
Calculation:
After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue.
Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2.
Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot
2.COLORIMETRIC ASSAYS:
Colorimetric assays
In vitro methods for the assessment of general cellular toxicity,
End-points for the assessment of general cellular toxicity
Specialized cells commonly used in toxicology
Genotoxicity studies can be defined as various in-vitro and in-vivo tests designed to identify any substance or compounds which may induce damage to genetic material either directly or indirectly by various mechanisms. These tests should enable the identification of hazard with respect to DNA damage and fixation.
ABSTRACT- Microbial source of amylase is preferred to other sources because of its plasticity, vast availability, higher yield and
thermostability even at elevated temperatures.Various physical and chemical factors have been known to affect the production of α-
amylase such as temperature, pH, period of incubation, carbon sources acting as inducers, surfactants, nitrogen sources, phosphate,
different metal ions, moisture. Interactions of these parameters are reported to have a significant influence on the production of
the enzyme.Study was mainly aimed to isolate a bacterium capable of hydrolyzing a starch source and to check effect of different physiological
parameters on amylase enzyme activity. To conduct this research, study was mainly focused on three objectives i.e. 1st Screening
and morphological characterization of the isolated bacteria. 2nd Characterization of amylase production by selected isolates. 3rd
Time course of Enzyme production and Partial purification with Ammonium Sulphate saturation.Amylases of isolate-6 and isolate-9
were concentrated by ammonium sulfate precipitation which can be used as partially purified enzyme for further study. Isolate-6 and
Isolate-9 showed the activity 0.34 and 0.28 units/ml/min respectively.Enzyme derived from isolate-6 and isolate-9 was stable at different
physiological conditions. So, it is useful in fermentation industry and in pharmaceuticals.
Key words- Amylase, Starch hydrolyzing bacteria, fermentation and pharmaceutical industries
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
A genotoxin is a chemical or agent that can cause DNA or chromosomal damage. Such damage in a germ cell has the potential to cause a heritable altered trait (germline mutation). DNA damage in a somatic cell may result in a somatic mutation, which may lead to malignant transformation (cancer). Many in vitro and in vivo tests for genotoxicity have been developed that, with a range of endpoints, detect DNA damage or its biological consequences in prokaryotic (e.g. bacterial) or eukaryotic (e.g. mammalian, avian or yeast) cells. These assays are used to evaluate the safety of environmental chemicals and consumer products and to explore the mechanism of action of known or suspected carcinogens. Many chemical carcinogens/mutagens undergo metabolic activation to reactive species that bind covalently to DNA, and the DNA adducts thus formed can be detected in cells and in human tissues by a variety of sensitive techniques. The detection and characterisation of DNA adducts in human tissues provides clues to the aetiology of human cancer. Characterisation of gene mutations in human tumours, in common with the known mutagenic profiles of genotoxins in experimental systems, may provide further insight into the role of environmental mutagens in human cancer.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research paper publishing, where to publish research paper, journal publishing, how to publish research paper, Call for research paper, international journal, publishing a paper, call for paper 2012, journal of pharmacy, how to get a research paper published, publishing a paper, publishing of journal, research and review articles, Pharmacy journal, International Journal of Pharmacy, hard copy of journal, hard copy of certificates, online Submission, where to publish research paper, journal publishing, international journal, publishing a paper
Principles of cell viability assays by surendra.pptxSurendra Chowdary
1.DYE EXCLUSION ASSAYS:
Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells.
For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin.
Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension.
Principle:
The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not.
Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain.
A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained.
Trypan blue stain should be stored in dark and filtered after prolonged storage.
As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results.
Assay Protocol:
The cell suspension to be tested is centrifuged at 100 g for 5 min.
The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium.
Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts.
Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope.
Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately.
.
Calculation:
After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue.
Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2.
Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot
2.COLORIMETRIC ASSAYS:
Colorimetric assays
In vitro methods for the assessment of general cellular toxicity,
End-points for the assessment of general cellular toxicity
Specialized cells commonly used in toxicology
Genotoxicity studies can be defined as various in-vitro and in-vivo tests designed to identify any substance or compounds which may induce damage to genetic material either directly or indirectly by various mechanisms. These tests should enable the identification of hazard with respect to DNA damage and fixation.
ABSTRACT- Microbial source of amylase is preferred to other sources because of its plasticity, vast availability, higher yield and
thermostability even at elevated temperatures.Various physical and chemical factors have been known to affect the production of α-
amylase such as temperature, pH, period of incubation, carbon sources acting as inducers, surfactants, nitrogen sources, phosphate,
different metal ions, moisture. Interactions of these parameters are reported to have a significant influence on the production of
the enzyme.Study was mainly aimed to isolate a bacterium capable of hydrolyzing a starch source and to check effect of different physiological
parameters on amylase enzyme activity. To conduct this research, study was mainly focused on three objectives i.e. 1st Screening
and morphological characterization of the isolated bacteria. 2nd Characterization of amylase production by selected isolates. 3rd
Time course of Enzyme production and Partial purification with Ammonium Sulphate saturation.Amylases of isolate-6 and isolate-9
were concentrated by ammonium sulfate precipitation which can be used as partially purified enzyme for further study. Isolate-6 and
Isolate-9 showed the activity 0.34 and 0.28 units/ml/min respectively.Enzyme derived from isolate-6 and isolate-9 was stable at different
physiological conditions. So, it is useful in fermentation industry and in pharmaceuticals.
Key words- Amylase, Starch hydrolyzing bacteria, fermentation and pharmaceutical industries
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
The Art Pastor's Guide to Sabbath | Steve ThomasonSteve Thomason
What is the purpose of the Sabbath Law in the Torah. It is interesting to compare how the context of the law shifts from Exodus to Deuteronomy. Who gets to rest, and why?
2. Contents
• Introduction
• General procedure
• Strains used by Ames
• Use of liver homogenate
• General procedure
• Importance of Ames test
• Limitations of Ames test
3. Introduction
• Mutagens are agents which causes mutation.
• Many chemical mutagens are found to be carcinogenic
• The Ames test (Salmonella typhimurium reverse mutation assay) is
a bacterial short-term test for identification of carcinogens using
mutagenicity in bacteria as an end point.
• All carcinogens are mutagens but some mutagens are not
carcinogens.
• The Ames test was described by Bruce Ames and his group at the
University of California, Berkeley in a series of papers in the 1970s
4. General procedure
• Bacteria (Salmonella typhimurium) is used as a test organism –
cheap and easy to culture.
• Auxotrophic mutant strain of Salmonella typhimurium is used
which lacks genes for histidine biosynthesis and a mutation for
excision repair system.
• Cells in which a back mutation occurs due to the test compound
grows and produce colony.
• Small amount of histidine is added for initial growth
5. • Rat liver extract is added to the medium which provides activation
by cellular enzymes.
• It permit identification of substances that are not directly
mutagenic (or carcinogenic) but are converted into mutagens by
the enzymatic reactions that take place in the liver of animal
6.
7. Strains used by Ames
The deletion through the uvrB region of the chromosome
eliminates the excision repair system for DNA.
The gal and rfa (deep rough) mutations eliminate, to
different extents, the polysaccharide side chain of the LPS
that coats the bacterial surface, making the bacteria more
permeable and completely nonpathogenic.
The TA1535 set (TA1535, TA1536, TA1537, TA1538), which
is rfa and uvrB, is recommended for general testing for
mutagens and carcinogens in vitro, as it is the most
sensitive to mutagenesis.
The TA1975 set is used for examining the effect of repair
on mutagenesis and killing (in comparison to the TA1535
set).
The TA1950'set and TA1530-set are less sensitive to
mutagens in vitro, but may be required for use in the host
mediated assay in which Salmonella strains are incubated
in the peritoneum of a mouse or rat
8. Use of liver homogenates
The active forms of carcinogens such as aflatoxin, polycyclic hydrocarbons,
dimethyl nitrosamine, and various aromatic amines are formed by
mammalian metabolism, in particular by the TPNH-dependent
microsomal enzymes of liver.
The principal limitation of any bacterial system for detecting carcinogens
as mutagens is that bacteria do not duplicate mammalian metabolism in
activating carcinogens. Mammalian-liver homogenates have been used by
Garner et al., to activate aflatoxin B1 to a compound lethal to our bacterial
tester strain lacking excision repair, by Malling to activate dimethyl
nitrosamine to a compound that reverts one of our bacterial tester strains,
and by Slater et al., to activate dimethyl nitrosamine to a compound lethal
for bacteria lacking polymerase I.
9. Preparation of liver homogenate
• Source of Liver
• Male rats (Sprague-Dawley/Bio-1 strain, Horton Animal Laboratories) were maintained on
Purina laboratory chow. A week before they were killed, their drinking water was made 0.1%
in sodium phenobarbital . The rats (250-500 g) were killed by a blow to the head and
cervical dislocation; the liver was removed and placed in a sterile, ice-cold beaker.
• A portion of human liver was obtained from an autopsy of a 77-year-old man who had died 7
hour earlier of heart failure.
• Preparation of Liver Homogenate Fraction "S-9“
• We have used the procedure of Garner et al.,. All steps were performed at 0-4⁰ with cold
and sterile solutions and glassware. The liver (rat livers were 10-25 g each) was washed in
an equal volume of 0.15 M KCl, minced with sterile scissors in three volumes of 0.15 M KCl
(3 ml/g of wet liver), and homogenized with a Potter-Elvehjem apparatus with a Teflon
pestle. The homogenate was centrifuged (Sorvall RC2-B) for 10 min at 9000 X g, and the
supernatant, which we call the S-9 fraction, was decanted and saved. 1 ml of S-9 fraction
contained microsomes from 250 mg of wet liver; the protein concentrations were fairly
constant from preparation to preparation except for the human S-9 fraction which was
about half, perhaps due to difficulties in homogenization because of its fibrous nature.
• The fresh S-9 fractions (rat and human) were distributed in 2-ml portions in small plastic
tubes (2-ml liquid nitrogen storage tubes/4-Shore-USA, La Jolla, Calif.), quickly frozen in
dry ice, and stored at -80° in a Revco freezer. As required, sufficient S-9 fraction was
thawed (at room temperature) and kept in ice; the unused portion was discarded at the end
of the day.
Preparation of liver homogenate
10. Test procedure
• Addition of S-9 mix to the top agar the S-9 Mix contains per ml: 0.3 ml of S-9
fraction, 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM TPN
(triphosphopyridine nucleotide), and 100 mM sodium phosphate (pH 7.4).
• To 2 ml of molten top agar at 45⁰C are added 0.1 ml of the bacterial tester
strain culture (2 to 3 X 109/ml), up to 0.1 ml of a solution (Me2SO or water) of
the compound to be tested, and 0.5 ml of S-9 Mix
• Then the tube is rotated quickly and the contents are poured on the agar plate.
The additions and pouring should take less than a minute. The colonies on the
plates (his+ revertants) are counted after a 2-day incubation at 37°.
14. Importance of Ames test
• Compared to the carcinogenic assays on mice and rats (takes few years) the Ames test
is quick and inexpensive.
• Used in wide variety of fields such as pharmaceutical, chemical, fertilizer, cosmetics,
food and many more industries for testing chemicals for mutagenicity and
carcinogenicity.
• In 1972-77 a compound known as Tris-BP was used as a flame retardant in children’s
polyester pyjamas which was later found to be a potential mutagen in Salmonella and
Drosophila
• And it also interacts with human DNA and damages mammalian chromosomes.
• Before its use was discontinued, more than 50 million children were exposed to the
chemical.
15. Limitations of Ames test
• Salmonella typhimurium is not a perfect model for humans as it is
a prokaryote.
• The sensitivity for some of the known carcinogen is less in the
Ames test.
16. References
1. Ames, B. N. (1971) in Chemical Mutagens: Principles and Methods for their Detection,
ed. Hollaender, A. (Plenum Press, New York), Vol. 1, pp. 267-282.
2. Ames, Bruce N., Frank D. Lee, and William E. Durston. 1973. “An Improved Bacterial
Test System for the Detection and Classification of Mutagens and Carcinogens.”
Proceedings of the National Academy of Sciences, USA 70: 782
3. Ames, Bruce N., William E. Durston, Edith Yamasaki, and Frank D. Lee. 1973.
“Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for
Activation and Bacteria for Detection.” Proceedings of the National Academy of
Sciences, USA 70: 2281–85.
4. Essential Genetics- a genomics perspective Daniel L. Hartl
5. Genetics Analysis & principles- Robert J. Brooker