Red Dye Lab Essay

The consumption of impersonal ruddy dye in a barm cell utilizing different solutions

Abstraction

Every cell transports stuffs in and out throught something called a membrane. There are many different methods of conveyance in the cell Saccharomyces cerevisiae ( Serrano. 1977 ) We want to cognize does adding higher concentrations of azide more efficaciously barricade dye conveyance? We tested the conveyance of dye in barm cells with a metabolic inhibitor. When we did this we showed no difference in the optical density between different azide solutions. and our control. From this we concluded that azide has no consequence on the conveyance through a barm cell membrane.

Introduction

Every cell has a bed of protection called the cell membrane. This cell membrane has many maps. The Saccharomyces cerevisiae cell has a selectively permeabile membrane which means it allows certian stuffs to go through through its membrane more or less than others ( Campbell et al. . 2008 ) . In a certian procedure called active conveyance these cells need energy to travel stuff across the membrane ( Campbell et al. . 2008 ) . This energy that is being used is called adenosine triphosphate or ATP ( Campbell et al. . 2008 ) . The production of ATP. which helps cells maintain a cells pH. helps with the consumption of impersonal ruddy dye because impersonal ruddy dye can non be absorbed if the pH of the cell is reduced ( Repetto. 2008 ) . Saccharomyces cerevisiae has two types of active transporters through the cell membrane: primary and secondary active transporters ( Stambuk. 2000 ) . Sodium azide is a metobolic inhibitor which means it prevents ATP from being produced ( Rowan University. 2009 ) . In a survey of Echerichia coli it was found that ATP was inhibited more than 90 % utilizing sodium azide ( Noumi. 1987 ) .

The survey of how things pass through different cell membranes is really common. So. analyzing how impersonal ruddy base on ballss through a barm cell is something that seems plausable to look at. We want to understand how different things will go through through the the cell membrane of barm. In a old experiment we saw that utilizing a 10 % azide solution. and a control group with impersonal ruddy gave us the same consequences of optical density with the exclusion of two mistakes at 0 % . and 2 % . ( Figure 1 ) . This was concluded by utilizing standard divergence mistake bars to see where the azide and control interventions were similar in optical density.

Since. it was still believed that azide. being a metobolic inhibitor. should barricade dye conveyance we wanted to happen out how much of this azide would do this to go on. In a survey done by Rikhvanov et Al. ( 2001 ) Saccharomyces cerevisiae was compleatly inhibited by Na azide. These consequences helped us to organize a new inquiry for our following experiment. We wanted to cognize does adding higher concentrations of azide more effectivly block dye conveyance? We predicted that higher azide concentrations will suppress the dye conveyance into the barm cell. There is ever a possibility that more azide will hold no consequence on the dye conveyance.

Methods

First. we calculated how much azide we needed to do our concentrations of 10 % . 20 % . and 30 % . We following made a yeast suspension utilizing yeast growing medium ( 56mM Glucose. 20mM HEPES. pH 6. 8 ) and Saccharomyces cerevisiae. After doing our azide concentrations we made four dye concentrations ( 0 % . 0. 5 % . 1. 25 % . 2. 5 % ) from a stock dye solution and barm growing medium ( YGM ) . Following. we took the barm suspension and added it to fresh YGM. to do a more diluted yeast solution. We seperated four 15-mL extractor tubings and added some of this diluted barm solution to each. In tube 1. we added fresh barm growing medium. In tube 2. we added 10 % Na azide. tube 3 we added 20 % Na azide. and tube 4 we added 30 % Na azide. 16 microfuge tubings were obtained and labled in groups of 4 every four tubings contained our 4 different dye concentrations.

We so transfered the control ( diluted yeast suspension ) . and our Azide concentrations into smaller microfuge tubings that contained the different dye concentrations ( 0 % . 0. 5 % . 1. 25 % . 2. 5 % ) respectivly. We so allowed these microfuge tubes to sit for 30 proceedingss. so transport could take topographic point. We following placed these tubings in a microcentrifuge on 5000 rpm’s for 2 proceedingss. Finally. we removed the liquid part from these tubings. doing certain non to upset the pellets on the underside. and so resuspended each tubing with fresh YGM. we did this 3 times. We did one more resuspension before we transfered these solutions into a microtitrate home base. We used a microspectrophotometer at 520 nanometers to read the optical densities of our solutions.

Consequences

Figure 2: Dye per centums versus optical density in a control. 10 % . 20 % . and 30 % azide solutions. In this graph you can see the mistake bars ( utilizing standard dieviation ) are overlaping at every point except at the outliner which was at 2. 5 % dye concentration with 20 % azide. Note how all the solutions ( control. 10 % azide. 20 % azide. and 30 % azide ) show similar aborbance with each concentration of dye.

Disscussion

The consequences that were obtained showed us that higher azide solutions did non suppress the dye conveyance as we thought. Therefore. our alternate hypothesis was non supported. Although. azide is a meabolic inhibitor it did non demo any effects during this experiment. The convergences in the mistake bars suggest that there is no important difference in the optical density with different dye concentrations. The beginning of mistake in this experiment is the outliner that was antecedently mentioned. This could hold been caused from several different things. most likely a pipetting mistake. In other experiments they found that the optical density of impersonal ruddy dye into a barm cell will increase the longer you leave the dye to sit with the cells ( Repetto. 2008 ) .

Since. the dye and the cells were merely incubated for 30 proceedingss it is possible that the optical density was low because of the minimum sum of clip. If azide is known to suppress transport why in this state of affairs did it non? In an experiment conducted by Rikhvanov et Al. ( 2001 ) they found that azide compleatly inhibited the respiration of Saccharomyces cerevisiae. They alternatively grew the Saccharomyces cerevisiae cells at 30*C. Possibly turning these cells utilizing different temperature demands is what allows the Na azide to take consequence.

Citations

2009. Uptake of impersonal ruddy dye by Saccharomyces cerevisiae in the presence of ametabolous inhibitor. Biological Sciences. Rowan University. pp 1-8. Campbell. N. A. . Reece. J. B. . Urry. L. A. . Cain. M. L. . Wasserman. S. A. . Minorsky. P. V. . Jackson. R. B. 2008. Biology. 8th erectile dysfunction. Pearson Benjamin Cummings. San FranciscoCA. pp. 125-138. Repetto. G. . del Peso. A. . LZurita. J. 2008. Impersonal ruddy consumption check for the appraisal ofcell viability/cytotoxicity. Nature Protocols. 3: 1125 – 1131. Rikhvanov. E. G. . Varakina. N. N. . Rusaleva. T. M. . Rachenko. E. I. . Voinkov. V. K. 2002. The effects of Na azide on the thermotolerance of the barms Saccharomycescerevisiae and Candida albicans. Microbiology. 71 ( 6 ) : 662-665

Stambuk. B. U. 2000. A simple research lab exercising exemplifying active transportnext term inprevious termyeast cellsnext term. Biochemistry and Molecular Biology Education. 28 ( 6 ) : 313-317. Serrano. R. 1977. Energy requirments for maltose conveyance in barm. European Journal ofBiochemisrty. 80: 97-102

Noumi. T. . Maeda. M. . Futai. M. 1987. Mode of suppression of Na azide on H+-ATPaseof Escherichia coli. Department of organic chemical science and biochemistry. theinstitute of scientific and industrial research. Osaka university. 213 ( 2 ) : 381-384