Active Transport of Vital Dyes
Active Transport of the Sugar Lactose
..... INTRODUCTION: Diffusion is a purely physical and non-biological phenomenon which, taken alone, plays an extremely fundamental role in living cells. ..Current understanding leads us to believe that most small molecules inside of cells move about mostly by diffusion. ..Just how important diffusion is might be seen if one looks at a lowly bacterial cell. ..Were the cytoplasm of that cell pH 7, that very small volume would contain - at any given instant - only about ten H+ ions! ..Biochemists have identified hundreds of reactions that entail the use of such free protons - each millisecond. ..Wow, those protons must diffuse around rapidly! ..Well, of course they can move around rapidly as they are only a type of electricity! But glucose molecules with 180 times the mass of a proton, can bounce back and forth across an E. coli cell maybe about 500 times a second. ..WOW, again! ..So this can fully explain how the small biochemical nutrient and waste molecules can move around inside the cell to be in the right places at just the right instant. ..Of course the same would hold true for toxins that could seep in from the outside environment - heavy metals, and even antibiotics from the myriad of neighboring bacteria and fungi in, for example, the soils.
Diffusion: the Use of Antibiotic Sensitivity Disks and the Making of Antibiotic Resistant Bacteria
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Let us now take a look at how we can make a simple device that will produce an antibiotic that inhibits the growth of surrounding bacteria: ..it is only a thick disk of blotting paper! ..Hospitals use this method to determine what sorts of antibiotics with which to treat you when you have a bacterial infection. ..The small circle of blotting paper contains a measured quantity of a selected antibiotic. ..This disk is placed onto the surface of the agar in a petri plate which has just been thoroughly coated with an invisible layer of bacteria. ..Then the race is on: the antibiotic begins diffusing out of the disk, while the bacteria try to grow as fast as they can. ..By the next day, you should see a clear halo around disks containing potent antibiotic. ..The diameter of the halo is determined by how fast a lethal concentration of antibiotic can diffuse out into the surrounding agar. (Reference manual: For specific techniques, see pages 94-95 in Pierce and Leboffe: Exercises for the Microbiology Laboratory (1999) (ISBN 0-89582-462-0); and pages 38-39 in Leboffe and Pierce: A Photographic Atlas for the Microbiology Laboratory (1999) (ISBN 0-89582-461-2) Morton Publishing Co. ) .....There is another experiment built right into that of the previous paragraph. ..It is extremely important as a followup to both this lab extension and to the one on genetics. ..If the plates showing antibiotic halos are incubated for several more days, often very small new colonies begin to grow up within the halos. ..These are mutants of the originial bacteria that are now resistant to the antibiotic. ..Obviously the best antibiotics to use are those that are hardest for resistance to develop.
Active Transport of "Vital Dyes"
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Another experiment involving active transport concerns the use of vital dyes. ..There are two types of vital dyes: those which living cells accumulate from the medium such that living cells rapidly become colored, while dead cells remain colorless. ..The other type of vital dye works oppositely: ..living cells exclude the dye, while dead ones take it in my simple diffusion.
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Some of the simplest classroom model systems can make use of amoeba or ciliates. ..Living cells will actively accumulate colored particles from the surrounding water. ..Because this section is still being investigated, no recommendations are given at this moment.
Active Transport of the Sugar Lactose (Reference manual: For specific techniques, reference will be made to pages in Pierce and Leboffe: ..Exercises for the Microbiology Laboratory (1999); Morton Publishing Co. (ISBN 0-89582-462-0)) .....
But the very first living things - primordial life - made use of what we might call "differential diffusion" - allowing somethings to diffuse and not others. ..Thus evolved the semi-permeable membrane, which gave small molecules no resistance to diffusion, but prohibited large molecules from doing so. ..A rather neat way to keep the big molecules - the primordial enzymes and genes - inside the cell, yet allow free influx of nutrients and efflux of wastes.
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We ourselves can invent such a replica of such a primordial cell by obtaining a mutant culture of E. coli in which its genes for degrading lactose sugar are all in good working order EXCEPT one. ..LacY is a cistron* that codes for a protein that is used to "pump" lactose from outside the cell into the interior. ..When such a lacY- cell is placed in a medium containing only water, necessary minerals and lactose, that cell must depend solely on diffusion/osmosis to survive. ..If you wish to see just how important active transport is (the lactose pump), you can add some lactose to greenish Simmons Citrate Agar (page 76) before sterilization and pour a petri plate of it. ..On that plate make two stripes with swabs - one of normal lac+ E.coli, and the other of the mutant lacY- strain. ..Then watch how fast they grow. When acid waste products are made, the bacteria grow into blue colonies surrounded by bluish agar.
* An "operon" is a gene that is made up of several parts: an on/off switch and several "cistrons", which are all linked together right after the "switch". ..When the switch is "off", all the cistrons are "off", and vice versa. ..Each cistron makes a polypeptide. ..Many times the polypeptide is an enzyme (lactase). ..Sometimes it takes several polypeptides to join together to make an enzyme (catalase). ..The lactose pump is a single polypeptide that is embedded in the cell's membrane where it grabs passing lactose molecules and transfers them to inside the cell.
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