Transmembrane Transport - Neutral Red vs. Crystal Violet

Introduction 

The neutral red (NR) and the crystal violet staining (CVS) assay techniques are two commonly practiced test trans-membrane transport system. Although both are used extensively conveniently as popular assay methods, they must be used with a lot of caution. A cellular membrane comprises of lipid layers. It has both polar and nonpolar sections. The neutral red (NR) changes with changes in pH value from yellow in neutral colors to red in acidic environments. On the other hand, the violet color in the crystal violet staining (CVS) absorbed is due to its ability to absorb visible light. Its maximum absorbance occurs at a wavelength of approximately 510 nm. 

Small polar molecules can passively diffuse over the cell membrane, but transporters are integral for big molecules that encompass but not limited to peptides and sugars. Microorganisms and infections have created complex mechanisms to move protein poisons and entire life forms into the cytoplasm. Passive diffusion depends on the existence of an electric slope to occur ( Gautier & Hinner, 2015; Sherwood, 2015) . In the least complicated terms, the process involves three steps where the material saturate first and segments into the membrane, diffuses over, and are discharged into the cytosol. 

In correlation, even marginally, polar metabolites such as glycerol and urea have a lower ability to cross fake membranes. The plasma membrane is for all intents and purposes impermeable against bigger, uncharged polar molecules and all charged molecules, including particles. For sure, regardless of their diminutive size, K+ and Na+ have very low porousness coefficients ( Yang & Hinner, 2015) . Aside from little solutes of moderate polarity, the quantity of characteristic molecules known to diffuse over the cell membrane passively is highly restricted. Steroid hormones have been accepted to do as such, albeit direct experimental proof is rare. Penetrability factors on the request for 1about 10 cm/s have been accounted for various steroids across cell monolayers. It can be hypothesized that actual dyes follow a particular trend or pattern when experiments are repeated severally ( Hardin & Bertoni, 2016) . It can also be hypothesized that external factors, such as extreme environmental factors, are ignored. It is also important to consider the physical properties of the yeast membrane so that that can be put into consideration. 

Materials and Methods 

Materials 

Part 1.A: Reagent preparation Yeast Growth Medium (YGM) 56 mM glucose (Fisher Chemical Company, Waltham, MA) 20 mM HEPES (Fisher Chemical Company, Waltham, MA) pH 6.8 

Solutions made in part 1A. 

Part B: Dye solutions 

Dye NR-2010 solutions: 

1% 

0.5% 

0.25% 

0.125% 

0.0625% 

0.03125% 

Procedure 

After the preparation of solution, it was stored at 4 degrees to the time the experiment was started. Prior to the commencement of the study, it was predicted that H2O and Na+ molecules can get into or out of a cell and sum of OH and NH bonds and the sum of Ns and Os are permeable. This is because most of these Ns and Os are highly porous. During the experiment, it was important to consider the three ways that molecules could gain access into the cytosol or onto the extracellular matrix include passive and active transport. Three approaches enshrined under passive transport comprise of osmosis, facilitated diffusion as well as simple diffusion. The total amount of YGM solution needed for Part 1.B was obtained by adding 56 mM glucose and 20 mM HEPES that totaled to 76 mM HEPES that was added to a good amount extra just in case of spills and disasters. After that, we need to prepare Dye NR-2010 (USB Biologicals, Santa Clara, CA) solutions in YGM at the following concentrations: 

1% - 32 test tubes 

0.5% - 16 test tubes 

0.25% - 8 test tubes 

0.125%- 4 test tubes 

0.0625% - 2 test tubes 

0.03125% - 1 test tube 

It all totaled to the number of test tubes add up to 63. The easiest way to make this set of Dye NR-2010 solutions is to measure two μl and then divide them into two halves that should be divided into 32 test tubes. In that regard, two different ways to make a solution, like a 0.03125% solution were used to minimize the number of divisions such as the first test tube to test the others in terms of accuracy. 

Data Manipulation 

Actual %	Replicate	Replicate	Replicate	Mean	SD A520
dye	1	2	3	A520
A	0.048	0.048	0.046	0.04733333	0.00547
B	0.047	0.046	0.046	0.04633333	0.00057735
C	0.046	0.046	0.047	0.04633333	0.00057735
D	0.046	0.047	0.046	0.04633333	0.00057735
E	0.046	0.054	0.046	0.04866667	0.0046188
F	0.047	0.046	0.046	0.04633333	0.00057735
G	0.046	0.046	0.046	0.046	8.4984E-18
Actual %	Replicate	Replicate	Replicate	Mean	SD A520
dye	1	2	3	A520
A	0.051	0.072	0.049	0.05733333	0.01274101
B	0.048	0.053	0.049	0.05	0.00264575
C	0.050	0.052	0.045	0.049	0.00360555
D	0.052	0.052	0.049	0.051	0.00173205
E	0.049	0.052	0.047	0.04933333	0.00251661
F	0.047	0.070	0.049	0.05533333	0.01274101
G	0.049	0.056	0.055	0.05333333	0.00378594
H	0.047	0.055	0.048	0.05	0.0043589

Data Interpretation 

The choice of multiple measurements was to ensure accuracy and reduce the margin of errors. Creating numerous data charts enables the researcher to analyze multiple data across a broad spectrum. In the chart, the independent variable is represented by actual percentages of dye, while dependent variables are represented by replicate 1, 2, and 3. While comparing the control and +Azide, it is evident that a large pool of data portrays more precise trend lines. In that regard, the treatment data to the three graphs made as predictions above portrayed clear trends that the actual percentages of the dye are declining towards the end. 

To conduct data interpretation, different charts and include trend lines can be created. Trend lines in the line graphs denote the best experimental predictions. The experimental prediction follows the form of if/then statements ( Gautier & Hinner, 2015) . Theoretical prediction developed the hypothetical part of the experimental prediction. The data can be used to indicate how Dye NR-2010 gets into or out of the yeast cells. From that perspective, the experiment is in concurrence with theoretical predictions, and reject the others ( Chiba, Kawakami & Tohyama, 1998) . Here, it can be assumed that external variables will not profoundly impact the experiment. In that regard, one can also conclude that the processes did not consider extreme environmental factors such as extreme weather conditions. When thinking about the movements of solutes across cellular membranes, it was vital to consider that the membranes will resemble ideal membranes. 

In the experiment, I revisited theoretical predictions that are based on the idea that the membranes follow the characteristics of an ideal membrane. It seems suitable to remember the fact that experimental predictions seem better combined with theoretical predictions. For example, we can recall hypothetical aspects of transportation ( Yang & Hinner, 2015) . Experimental predictions can be used to confirm theoretical predictions. The data of the experiments can be used to verify the results of Dye NR-2010 as it moves across the yeast cell membrane. In that regard, it is evident that both theoretical and experimental predictions can be crucial in predicting the permeability. 

References 

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Chiba, K., Kawakami, K., & Tohyama, K. (January 01, 1998). Simultaneous Evaluation of Cell Viability by Neutral Red, MTT and Crystal Violet Staining Assays of the Same Cells.  Toxicology in Vitro: an International Journal Published in Association with Bibra, 12,  3, 251. 

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Gautier, A., & Hinner, M. J. (2015).  Site-Specific Protein Labeling: Methods and Protocols . New York, NY: Springer. 

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Hardin, J., & Bertoni, G. (2016).  Becker's world of the cell . Boston: Pearson. 

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Sherwood, L. (2015).  Human physiology: From cells to systems . Australia: Brooks/Cole. 

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Yang, N. J., & Hinner, M. J. (January 01, 2015). Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins. 29-53. 

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