After Alamar Blue dye is added, the plate is incubated for 4 hours and then is observed under a spectrophotometer. The spectrophotometer can measure fluorescence of the dye at 570 nm, which is the wavelength of the fluorescence of Alamar Blue dye. The spectrophotometer will return values for each well that correlates with the measured absorbance. These absorbance values can be converted to survivability percentage and can be graphed with a GraphPad program as shown in Graph 1A and 1B. The software can calculate survivability of cells by normalizing the absorbance value of the experimental group with absorbance value of the positive control group (Larson et al., 1997).Alamar Blue Assay detects cell viability through the compound resazurin in the Alamar Blue dye. Resazurin in its natural state is a blue non-fluorescing compound, but when reduced, resazurin fluoresces. This …show more content…
The dye is simple to use, as it detects cell viability by just the application of the dye onto the cells. Furthermore, the dye is non-toxic, meaning the assay is not an end point assay. Therefore, the cells plated onto the plate would still be viable for use in further experiments. The assay is also very sensitive, and can detect down to 50 mammalian cells. The downside to this technique is that the fluorescence is dependent on temperature and pH of the solution, so in order to obtain an accurate reading of the plate, the plate would have to be at a consistent condition. To prevent this issue, the plate would be incubated throughout the majority of the experiment. (Obrien et al., 2000) Another weakness is the possibility of false positives or negatives that would result from the drug inhibiting or inducing enzymatic activity that fluoresces the Alamar Blue dye without actually altering viability of the cells (Quent et al.,
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Using two separate aseptic pipettes, 250 µl of LB broth were added to each micro test tube and mixed gently. Likewise, using two separate, aseptic pipettes for each tube, 100 µl of solution was added to the appropriate agar plate. After, using a new loop for each plate, the solution was spread gently across their surfaces. Lastly, the plates were stacked, taped together, and labelled before placing them upside down in an incubator set at 37°C
It would help out rule compounds that won’t work in humans from animal tests. According to PETA with this technique it ‘allows the human brain to be safely studied down to the level of a single neuron, and researchers can even temporarily and reversibly induce brain disorders.’ Human volunteers can help replace the animals used for animal testing in which they have their brains damaged. Therefore, the alternatives will potentially start us in reaching the goal of reducing and possibly eliminating the need for any type of research on animals without compromising our ability to work toward discoveries that may ease suffering in humans as well as
Starch solution is then placed into the test tube at a quantity of 5 mL. 5 drops of Lugol’s Iodine solution is added to the test tube. If the color changes, then it is known that starches are present in the solution. Proteins are next tested. In order to do this, 5 mL of gelatin solution is added to the test tube. 10 drops of Biuret’s reagent are added to test for protein.
An animal, specially mouses and rabbits, are exposed to the test substance, either being forced to eat it, breathe it, have it rubbed on their skin, or have it injected. The test substance is often a household chemical, an industrial chemical, such as: pesticide, a food addictive, a cosmetic ingredient, or a pharmaceutical product. After that step is done, animals are observed for toxic reactions such as: vomiting, diarrhea, convulsions, respiratory distress, appetite or weight loss, rashes and allergic reactions, skin and eye irritation, and many other harmful effects. After the scientists collect all the needed information the animal is then killed to end the experiment, worse still, their internal organs are often examined for harmful effects- depending on the type of tests, the experiment may last for a few hours or may last up to several days or even months. Frankly, animal testing is unethical on many levels.
For the remaining 6 tubes, 2mL Buffer A and 500 µL 5 mM ONPG was added and mixed, keeping the ratio of Buffer A to ONPG constant. Immediately following the preparation of each tube, as an independent variable, they were all placed into temperature baths of 4˚C, 22˚C, 37˚C, 45˚C, 65˚C, and 95˚C for a total of ten minutes. The 22˚C tube was left to incubate at room temperature. After incubation, 750µL of Buffer B was added and mixed in, in order to halt the reaction in each tube. The absorbance level of each was read with a photospectrometer at 421nm, with the blank used as a zero.
In the lab, starch was mixed with ten drops of Benedict's and tested negative for sugar and the color changed to blue.
Eosine and Methylene Blue EMB The test for Unknown 361 showed a pink color, which signified that the fermentation occurred very slowly and poorly. Procedure: 1. Obtain an EMB agar plate 2. Streak it with the appropriate bacterial culture using the quadrant streak plate method 3.
Next, I dye the Unknown with Gram’s iodine to create a complex only have on gram positive. The slide is rinsed by water after 30 seconds. Decolorization is the next step of the whole process. I let the alcohol flow on 45-degree angle slide within 15 seconds and wash it with water to remove colors on the surface. Lastly, the unknown is once again dyed with safranin for 1 minute then wash it off with water for the last time and dry it using bibulous paper.
(2004) as follows: The reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 µL. Sample (20µL) and fluorescein (120 µL; 70 nM, final concentration) were placed in the well of the microplate. The mixture was pre-incubated for 15 min at 37 °C. AAPH solution (60 µL; 12 mM, final concentration) was added rapidly using a multichannel pipet. The microplate was immediately placed in the reader and the fluorescence recorded every minute for 240 min.
This reflects the variability in toxicity of naturally occurring LPS (rapidmicrobiology n.d.). The chromogenic method uses a synthetic substrate that brings about a colour change when it is cleaved by endotoxin-activated protease. The turbidimetric method on the other hand, relies on the a coagulin gel cot forming which will alter the turbidity of the sample. Both the turbidimetric and the chromogenic methods can be used as quantitative kinetic methods simply by plotting standard curves of time vs endotoxin concentration. Spectrophotometric instruments can be used to detect changes in colour and turbidity at much lower concentration than that need to form a visible gel-clot.
Biochemical tests are the tests used for the identification of bacterial species based on the differences in the biochemical activities of different bacteria. Bacterial physiology differs from one species to the other. These differences in carbohydrate metabolism, protein metabolism, fat metabolism, production of certain enzymes and ability to utilize a particular compound help them to be identified by the biochemical tests. Gram’s stain was originally devised by histologist Hans Christian Gram in 1884. Gram-positive bacteria stain purple, while Gram-negative bacteria stain pink when subjected to Gram staining.
Conclusion and Recommendations Ultimately, testing cell viability and knowing the count of viable and non-viable cells in a cell line is an important key in any research using cells. Having enough number of viable cells in a suspension would give accurate results in an experiment. This also shows how trypan blue is an important dye in cell viability and in the study of cells. This dye makes researcher’s life convenient in identifying and differentiating viable versus non-viable cells or live versus dead cells. It is really needed that researchers should ensure new and fresh cell cultures to ensure more viable cells in a culture.