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Biol 140L Final Exam Review

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BIOL 240
Cheryl Duxbury

Experiment 1 Simple Stainingthough all organisms are the same colour a simple stain allows one to distinguish the morphologies of cells such as cocci rods spirals and vibriosmost common dyes are methylene blue basic fuchsin and crystal violetpositive staining procedurestain is basic and has a positive chromophore that is attracted to the negative charge on the outer surface of organisms used Loefflers methylene blue stain for 12 minutes on Escherichia coli Bacillus subtilis and Staphylococcus epidermidis and rinse with water Experiment 2 Operation of the Microscopethe light microscope consists of a lens system a controllable light source and a geared mechanism to adjust the distancethe objective is nearest the specimen and magnifies the specimen and the ocular eyepiece lens is at the top of the microscopecondensor contains lenses that collect and concentrate light the amount of light can be adjusted by opening and closing the iris diaphragm oil is used at 100x magnification only and improves the resolution of the magnified image providing a sharpness of detailwhen using the 100x oil immersion position make sure the condensor is in the uppermost positiion just under stage to allow for maximum light capture start by having the diaphragm in the half open positionresolving power ability to distinguish two separate images the max resolving power is 02 micro metersthe total magnification of the image achieved with the microscope is the product of the ocular 10x and the highdry lens 4x 10x 40x or 100x4xscanning 10xlow power 40xhigh power 100xoil immersionExperiment 3 Basic Bacterial Shapesmorphological variations may be observed due to agecomposition of the culture mediaother types include stalked Caulobacter clubshaped Corynebacterium and commashaped VibrioCharacteristicCoccusBacillusSpirillumShape of CellRoundRodSpiralMotilityMost nonmotileMost motileMost motileArrangementSingle SingleSinglemicrococcusPairs diplobacillusSpirochetesPairs diplococcusChains Chains streptobacillusstreptococcusGroups of four GaffykaCubes of 8 sarcinaClusters staphylococcusSporesNoneProduced by Noneseveral speciesE colismall rod bacillusBacillus subtilislarge rod bacillusS epidermidischained cocciRhodospirillum rubrumspirillum Magnification of specimensize of specimen in drawingestimated size of image Experiment 4 The Gram Staindevised by Danish scholar Christian Gram in 1884used to differentiate types of bacteria depending on their ability to retain a particular stain1primary stain of Huckers crystal violet for 1 minute 2Grams iodine stain the mordant is added to fix the primary stain into cells for 1 minute 3wash the stained smear with a decolourizing agent like 95 ethanol 45 drops 4counterstain safranin is added for minute 5grampositive bacteria will be purple while gramnegative bacteria will be decolorized and will be red from the counterstainthe age of the cultures and the pH of the medium in which the bacteria are grown in will affect their reaction to the Grams stainolder cultures of gram frequently appear gram or gramvariable and gramnegative may sometimes appear gramvariableEscherichia coligram Staphylococcus epidermidisgram Experiment 5 The AcidFast Stainidentifies mycobacterium which contains mycolic acids in their cell wall are very lipid rich making their cell wall waxy and thus are impermeable to most stainsZiehlsNeelson proceduresmear is flooded with carbolfuchsin red dye with 5 phenol which has a high affinity for the lipidrich cell walls and the smear is then heated to facilitate penetration and even when decolorized with alcohol they will retain the dye methylene blue is used as a counterstainKinyoun modificationthe conc of phenol and carbolfuchsin are increased and a detergent is added so heating isnt necessaryall acidfast organisms are grambut not all gramorganisms are acidfastacid fast organisms will be red and non acid fast will be blue1prepare a heatfixed smear of Ecoli2add 45 drops of carbolfuschin for 510 mins3gently rinse with water 4decolorize with alcohol until colourless5counterstain with methylene blue for 2 minutesS epidermidisnonacid fastMycobacterium tuberculosisacid fast
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