[Script Info]
Title: 
[Events]
Format: Layer, Start, End, Style, Name, MarginL, MarginR, MarginV, Effect, Text
Dialogue: 0,0:00:00.00,0:00:03.41,Default,,0000,0000,0000,,The Gram Stain is the most \Nwidely employed staining method
Dialogue: 0,0:00:03.41,0:00:05.28,Default,,0000,0000,0000,,in microbiology.
Dialogue: 0,0:00:05.28,0:00:10.01,Default,,0000,0000,0000,,It is a differential stain because \Nit divides bacteria into 2 classes:
Dialogue: 0,0:00:10.01,0:00:13.19,Default,,0000,0000,0000,,gram positive and gram negative.
Dialogue: 0,0:00:13.19,0:00:16.28,Default,,0000,0000,0000,,In the first step of the gram stain \Nprocedure,
Dialogue: 0,0:00:16.28,0:00:20.09,Default,,0000,0000,0000,,cells from a fresh culture \Nare transferred to a clean slide
Dialogue: 0,0:00:20.09,0:00:21.81,Default,,0000,0000,0000,,and allowed to dry.
Dialogue: 0,0:00:21.81,0:00:25.63,Default,,0000,0000,0000,,If the cells are on an augur plate, \Nthey should first be transferred
Dialogue: 0,0:00:25.63,0:00:28.48,Default,,0000,0000,0000,,to a liquid medium for dilution.
Dialogue: 0,0:00:28.48,0:00:32.43,Default,,0000,0000,0000,,The cells should form a thin,\Nbarely visible film.
Dialogue: 0,0:00:32.43,0:00:34.52,Default,,0000,0000,0000,,This can be achieved\Nby smearing cells
Dialogue: 0,0:00:34.52,0:00:37.23,Default,,0000,0000,0000,,obtained from the surface \Nof an augur medium
Dialogue: 0,0:00:37.23,0:00:39.56,Default,,0000,0000,0000,,or from a liquid culture.
Dialogue: 0,0:00:39.56,0:00:43.31,Default,,0000,0000,0000,,Fresh cultures must be used \Nbecause as cells age,
Dialogue: 0,0:00:43.31,0:00:46.36,Default,,0000,0000,0000,,they lose their ability \Nto retain the stain.
Dialogue: 0,0:00:46.36,0:00:50.11,Default,,0000,0000,0000,,The cells are then fixed to the slide \Nby passing slightly
Dialogue: 0,0:00:50.11,0:00:52.78,Default,,0000,0000,0000,,above the flame of a Bunsen burner.
Dialogue: 0,0:00:52.78,0:00:56.16,Default,,0000,0000,0000,,After passing above the flame, \Nthe slide should feel warm
Dialogue: 0,0:00:56.16,0:01:00.14,Default,,0000,0000,0000,,when touched to the back of the hand; \Nbut should not be too hot.
Dialogue: 0,0:01:00.14,0:01:04.82,Default,,0000,0000,0000,,The fixed cells are then stained \Nwith the basic dye, crystal violet,
Dialogue: 0,0:01:04.82,0:01:07.16,Default,,0000,0000,0000,,for 30 to 40 seconds.
Dialogue: 0,0:01:07.16,0:01:11.24,Default,,0000,0000,0000,,The slide is then rinsed with water \Nto remove excess stain.
Dialogue: 0,0:01:11.24,0:01:15.26,Default,,0000,0000,0000,,At this point, all cells appear purple \Nunder the microscope.
Dialogue: 0,0:01:15.26,0:01:19.74,Default,,0000,0000,0000,,Next, a solution of Grams iodine \Nis added and retained on the slide
Dialogue: 0,0:01:19.74,0:01:21.68,Default,,0000,0000,0000,,for about 1 minute.
Dialogue: 0,0:01:21.68,0:01:24.36,Default,,0000,0000,0000,,The iodine combines \Nwith the crystal violet
Dialogue: 0,0:01:24.36,0:01:29.11,Default,,0000,0000,0000,,to form a dye-iodine complex, \Nthereby decreasing its solubility
Dialogue: 0,0:01:29.11,0:01:30.54,Default,,0000,0000,0000,,within the cell.
Dialogue: 0,0:01:30.54,0:01:34.24,Default,,0000,0000,0000,,At this point, \Nthe cells still appear purple.
Dialogue: 0,0:01:34.24,0:01:39.24,Default,,0000,0000,0000,,The cells are then de-colorized \Nby washing with ethanol or acetone.
Dialogue: 0,0:01:39.24,0:01:41.80,Default,,0000,0000,0000,,This is the differential step.
Dialogue: 0,0:01:41.80,0:01:45.42,Default,,0000,0000,0000,,Gram positive bacteria retain \Nthe crystal violet,
Dialogue: 0,0:01:45.42,0:01:48.87,Default,,0000,0000,0000,,whereas gram negative bacteria do not.
Dialogue: 0,0:01:48.87,0:01:52.52,Default,,0000,0000,0000,,The ethanol or acetone \Nshould be added dropwise
Dialogue: 0,0:01:52.52,0:01:56.31,Default,,0000,0000,0000,,with the slide tilted at an angle \Nuntil the drop coming off
Dialogue: 0,0:01:56.31,0:01:59.92,Default,,0000,0000,0000,,the edge of the slide \Njust starts to become colorless.
Dialogue: 0,0:01:59.92,0:02:05.00,Default,,0000,0000,0000,,Even gram positive cells can lose \Nthe crystal violet iodine complex
Dialogue: 0,0:02:05.00,0:02:08.66,Default,,0000,0000,0000,,during prolonged excessive\Nde-coloration.
Dialogue: 0,0:02:08.66,0:02:12.26,Default,,0000,0000,0000,,Excess ethanol is then\Nwashed off with water.
Dialogue: 0,0:02:12.26,0:02:16.54,Default,,0000,0000,0000,,When viewed under the microscope, \Ngram positive cells appear purple
Dialogue: 0,0:02:16.54,0:02:19.92,Default,,0000,0000,0000,,and gram negative cells are colorless.
Dialogue: 0,0:02:19.92,0:02:24.31,Default,,0000,0000,0000,,Finally, the rinsed cells are covered \Nwith the counter stain safranin
Dialogue: 0,0:02:24.31,0:02:26.54,Default,,0000,0000,0000,,for 20 to 30 seconds.
Dialogue: 0,0:02:26.54,0:02:29.82,Default,,0000,0000,0000,,This stains the gram negative bacteria \Npink.
Dialogue: 0,0:02:29.82,0:02:34.49,Default,,0000,0000,0000,,After rinsing with water, \Nthe slide is dried with filter paper.
Dialogue: 0,0:02:34.49,0:02:38.84,Default,,0000,0000,0000,,When viewed microscopically, \Nthe gram positive bacteria are purple
Dialogue: 0,0:02:38.84,0:02:41.91,Default,,0000,0000,0000,,and the gram negative bacteria are pink.
Dialogue: 0,0:02:41.91,0:02:46.22,Default,,0000,0000,0000,,Generally, the gram stain correlates \Nwith the cell wall structure
Dialogue: 0,0:02:46.22,0:02:47.92,Default,,0000,0000,0000,,among the bacteria.
Dialogue: 0,0:02:47.92,0:02:51.67,Default,,0000,0000,0000,,The ethanol is thought to shrink \Nthe thick peptidoglycan
Dialogue: 0,0:02:51.67,0:02:55.77,Default,,0000,0000,0000,,in gram positive cells, \Nthus retaining the dye.
Dialogue: 0,0:02:55.77,0:02:58.65,Default,,0000,0000,0000,,The thick dehydrated \Npeptidoglycan layer
Dialogue: 0,0:02:58.65,0:03:03.18,Default,,0000,0000,0000,,of gram positive bacteria \Nappears to be a permeability barrier
Dialogue: 0,0:03:03.18,0:03:07.16,Default,,0000,0000,0000,,preventing the loss \Nof the crystal violet iodine complex.
Dialogue: 0,0:03:07.16,0:03:12.58,Default,,0000,0000,0000,,In contrast, the peptidoglycan \Nin gram negative bacteria is very thin,
Dialogue: 0,0:03:12.58,0:03:14.66,Default,,0000,0000,0000,,and has large pores.
Dialogue: 0,0:03:14.66,0:03:18.44,Default,,0000,0000,0000,,Ethanol may extract lipids\Nand increase the porosity
Dialogue: 0,0:03:18.44,0:03:21.71,Default,,0000,0000,0000,,thus removing the crystal violet \Niodine complex.