1 00:00:13,529 --> 00:00:15,729 Gram staining. So gram staining 2 00:00:15,849 --> 00:00:17,329 is a technique that we use when we're 3 00:00:17,449 --> 00:00:19,849 working with microorganisms, 4 00:00:19,929 --> 00:00:22,769 particularly pathogens, bacteria. 5 00:00:22,849 --> 00:00:25,009 Whenever we're working with any type 6 00:00:25,129 --> 00:00:27,289 of microbiology and pathogens, 7 00:00:27,569 --> 00:00:30,009 they are colourless organisms. 8 00:00:30,329 --> 00:00:31,769 So when we want to view them under 9 00:00:31,889 --> 00:00:34,249 the microscope because it's a light 10 00:00:34,369 --> 00:00:36,849 microscope, we're not able to view those 11 00:00:36,929 --> 00:00:38,889 pathogens effectively. So we need 12 00:00:38,969 --> 00:00:41,249 to stain them in order to see them. 13 00:00:41,769 --> 00:00:43,169 So a gram staining is a technique that 14 00:00:43,289 --> 00:00:47,089 we use to stain our bacteria. The reason 15 00:00:47,209 --> 00:00:48,449 it's called Gram then is because this 16 00:00:48,569 --> 00:00:50,849 was a Danish scientist and he devised 17 00:00:50,929 --> 00:00:53,249 this method of the staining techniques 18 00:00:53,489 --> 00:00:56,369 of bacteria and bacteria is categorized 19 00:00:56,449 --> 00:00:58,969 in two sections. So we have gram 20 00:00:59,049 --> 00:01:02,009 negative bacteria and gram positive 21 00:01:02,129 --> 00:01:04,929 bacteria. Today, we are going to use 22 00:01:05,049 --> 00:01:07,649 E.coli, which is a gram negative 23 00:01:07,729 --> 00:01:11,049 bacteria. So what differentiates 24 00:01:11,169 --> 00:01:12,289 between whether it's positive 25 00:01:12,369 --> 00:01:15,129 or negative is the structure of this 26 00:01:15,249 --> 00:01:17,409 bacteria. So we know generally as 27 00:01:17,529 --> 00:01:20,329 a microorganism that we have the cell 28 00:01:20,409 --> 00:01:23,329 membrane and then we have the outer cell 29 00:01:23,409 --> 00:01:25,649 wall. But it's the chemistry, 30 00:01:25,729 --> 00:01:28,769 the biochemical makeup of this cell wall 31 00:01:28,849 --> 00:01:30,449 to differentiates whether it's a gram 32 00:01:30,529 --> 00:01:33,769 positive or a gram negative. So with 33 00:01:33,849 --> 00:01:38,089 gram positive bacteria, we actually see 34 00:01:38,169 --> 00:01:41,569 this outer peptidoglycan structure 35 00:01:41,689 --> 00:01:43,369 of the cell wall, it's really quite 36 00:01:43,449 --> 00:01:45,129 large in comparison to the gram 37 00:01:45,209 --> 00:01:49,249 negative, the peptidoglycan then reacts 38 00:01:49,369 --> 00:01:52,449 with the stain of Crystal Violet. 39 00:01:52,689 --> 00:01:54,809 So we use this as part of the procedure 40 00:01:55,529 --> 00:01:58,849 so it can stain the outer peptidoglycan 41 00:01:58,969 --> 00:02:02,089 cell wall of gram positive bacteria. 42 00:02:03,809 --> 00:02:07,729 Gram negative bacteria then has a 43 00:02:07,729 --> 00:02:11,289 of lipopolysaccharide outer structure. 44 00:02:12,529 --> 00:02:15,009 Which is actually protecting the inner 45 00:02:15,089 --> 00:02:19,289 peptidoglycan membrane. So Crystal 46 00:02:19,409 --> 00:02:21,889 Violet won't work on gram negative 47 00:02:22,529 --> 00:02:25,809 because of this lipo polysaccharide 48 00:02:25,929 --> 00:02:28,729 structure, and this is what reacts with 49 00:02:28,809 --> 00:02:32,369 safranin. So once we've used the crystal 50 00:02:32,449 --> 00:02:34,969 violet and removed that stain, we then 51 00:02:35,049 --> 00:02:38,169 apply the safranin which will react with 52 00:02:38,329 --> 00:02:40,489 the lipopolysaccharide structure 53 00:02:40,569 --> 00:02:44,089 of gram negative bacteria. So once we've 54 00:02:44,169 --> 00:02:46,609 completed our stain, we will be able 55 00:02:46,689 --> 00:02:49,449 to view any gram positive bacteria that 56 00:02:49,569 --> 00:02:52,169 is present as being stained as a purple 57 00:02:52,249 --> 00:02:55,209 colour. And the gram negatives will have 58 00:02:55,329 --> 00:02:58,369 been stained quite a pink, very pale red 59 00:02:58,569 --> 00:03:02,769 in its appearance. So for this procedure 60 00:03:02,889 --> 00:03:03,849 then we are going to be using 61 00:03:03,929 --> 00:03:07,169 the aseptic technique, so my surface 62 00:03:07,249 --> 00:03:09,689 area I'm working with is already being 63 00:03:09,769 --> 00:03:14,689 sterilized. From edge to edge covering 64 00:03:14,809 --> 00:03:17,049 that full surface area, so I am ensuring 65 00:03:17,129 --> 00:03:19,289 and what goes on my slide is only 66 00:03:19,369 --> 00:03:21,489 the microbes that I wish to use, in this 67 00:03:21,569 --> 00:03:27,089 case, E.coli. I've introduced stains 68 00:03:27,209 --> 00:03:30,809 of Crystal Violet safranin. I'm also 69 00:03:30,929 --> 00:03:33,689 going to be using iodine, which enhances 70 00:03:33,769 --> 00:03:35,769 the color of the crystal violet. 71 00:03:37,649 --> 00:03:40,969 I'm also going to be using water 72 00:03:41,049 --> 00:03:45,649 to remove my stains as well as alcohol, 73 00:03:45,969 --> 00:03:47,769 just to dissolve the crystal violets 74 00:03:47,889 --> 00:03:50,249 and safranin making sure that 75 00:03:50,329 --> 00:03:52,169 they are completely removed. So all I've 76 00:03:52,249 --> 00:03:55,049 left behind is the successful stain 77 00:03:55,129 --> 00:03:58,689 of the bacteria. I will be using 78 00:03:58,809 --> 00:04:00,809 an inoculation loop to gather 79 00:04:00,929 --> 00:04:03,329 the bacteria and smear it onto my slide. 80 00:04:06,489 --> 00:04:09,009 And on my glass slide, then, this 81 00:04:09,129 --> 00:04:12,609 is where I will heat fix my bacteria, 82 00:04:12,929 --> 00:04:15,609 so therefore I need the Bunsen burner 83 00:04:15,929 --> 00:04:17,209 and you can see the Bunsen burner 84 00:04:17,329 --> 00:04:19,929 is on an orange flame; safety flame. 85 00:04:20,529 --> 00:04:22,809 My hair is tied back. I have my gloves 86 00:04:22,929 --> 00:04:24,929 on to protect me from the bacteria. 87 00:04:25,369 --> 00:04:27,129 I can see this flame, so I'm not going 88 00:04:27,209 --> 00:04:29,049 to hurt myself and anybody else that's 89 00:04:29,129 --> 00:04:31,089 working in my environment can also see 90 00:04:31,209 --> 00:04:33,209 it and keep themselves safe as well. 91 00:04:34,249 --> 00:04:36,209 I've also got my microscope so I can 92 00:04:36,329 --> 00:04:38,449 view my bacteria when I've completed it. 93 00:04:38,569 --> 00:04:41,729 To get me started, a first need to smear 94 00:04:41,809 --> 00:04:43,969 my bacteria on the glass slide, 95 00:04:44,209 --> 00:04:47,289 so in order to do that, I need to heat 96 00:04:47,409 --> 00:04:50,889 sterilize my inoculation loop. So I'm 97 00:04:50,969 --> 00:04:53,689 going to move my safety flame onto 98 00:04:54,529 --> 00:04:56,849 the blue flame. And in the blue flame, 99 00:04:56,929 --> 00:04:58,329 we can actually see that we've got this 100 00:04:58,409 --> 00:05:00,809 triangular structure and it's the peak 101 00:05:00,889 --> 00:05:02,449 of the triangle that it's at its 102 00:05:02,569 --> 00:05:05,409 hottest. So putting in the innoculation 103 00:05:05,489 --> 00:05:09,849 loop, we can see it's glowing with 104 00:05:09,929 --> 00:05:12,049 the heat. So I am completely sterilized 105 00:05:12,169 --> 00:05:16,489 in that area of anything that might 106 00:05:16,609 --> 00:05:20,289 already be existing, remove 107 00:05:20,369 --> 00:05:23,889 it from the heat back to safety flame 108 00:05:24,009 --> 00:05:25,369 so myself and anyone else 109 00:05:25,489 --> 00:05:27,129 in the environment, are nice and safe. 110 00:05:27,249 --> 00:05:37,649 Then going to open my E.coli. And again, 111 00:05:37,729 --> 00:05:42,329 using my blue flame, I'm going to pass 112 00:05:42,449 --> 00:05:46,089 the rim of the bottle through. So if 113 00:05:46,209 --> 00:05:47,969 there is any preexisting bacteria other 114 00:05:48,049 --> 00:05:51,249 than E.coli I'm sterilizing that way. 115 00:05:58,089 --> 00:06:01,849 So taking my innoculation loop. I want 116 00:06:01,929 --> 00:06:03,449 to make sure I'm not touching any 117 00:06:03,569 --> 00:06:05,249 of the glass edges, so it's only having 118 00:06:05,369 --> 00:06:10,049 contact with my E.coli. Placing that in, 119 00:06:10,169 --> 00:06:13,209 dip it into the E.coli and very gently 120 00:06:13,289 --> 00:06:15,009 just swirling around, make sure there's 121 00:06:15,089 --> 00:06:17,889 full contact on its surface area without 122 00:06:18,009 --> 00:06:21,969 touching the glass vial. We're going 123 00:06:22,089 --> 00:06:26,849 to remove that and place my lid back 124 00:06:26,969 --> 00:06:31,649 on my E.coli. Again, insuring safety 125 00:06:31,769 --> 00:06:33,769 so when I place it down, it's not going 126 00:06:33,849 --> 00:06:35,329 to get knocked over and if it did, 127 00:06:35,409 --> 00:06:37,409 it's not going to spill anywhere. 128 00:06:38,489 --> 00:06:41,969 With my glass slide, I'm going to place 129 00:06:42,089 --> 00:06:44,289 my innoculation loop on the surface, 130 00:06:45,089 --> 00:06:49,609 smear in that thinly over the surface 131 00:06:49,689 --> 00:06:52,369 area. You want to do it nice and thin, 132 00:06:52,449 --> 00:06:54,529 because if it's just this blob 133 00:06:54,649 --> 00:06:56,209 in the middle, they're all going to be 134 00:06:56,289 --> 00:06:57,529 sat on top of each other and you're not 135 00:06:57,649 --> 00:06:59,089 going to see it properly. 136 00:06:59,169 --> 00:07:01,169 So when you spread it out thin, 137 00:07:01,409 --> 00:07:03,409 you've got it nicely distributed 138 00:07:03,529 --> 00:07:06,849 and you'll get a better image. So, 139 00:07:06,969 --> 00:07:08,409 again, because I've got E.coli 140 00:07:08,529 --> 00:07:10,689 on my occulation loop; back to my blue 141 00:07:10,769 --> 00:07:14,649 flame and I'm going to heat, 142 00:07:14,769 --> 00:07:18,009 sterilize and make sure that 143 00:07:18,089 --> 00:07:22,169 I and my colleagues are safe. I'm going 144 00:07:22,289 --> 00:07:24,369 to keep a blue flame now, because I'm 145 00:07:24,489 --> 00:07:26,009 going to continue using it, my heat 146 00:07:26,129 --> 00:07:29,769 fixing my glass slide, so I want to feel 147 00:07:30,049 --> 00:07:31,689 where it's nice and warm, but not too 148 00:07:31,769 --> 00:07:32,809 hot where I'm burning myself 149 00:07:32,889 --> 00:07:34,409 because I don't want to cook 150 00:07:34,529 --> 00:07:37,969 the bacteria. All I'm doing is holding 151 00:07:38,049 --> 00:07:43,529 it over the top so I can use that heat 152 00:07:44,329 --> 00:07:48,129 to dry out the nutrient broth. What 153 00:07:48,249 --> 00:07:51,249 the E.coli is being suspended. So all 154 00:07:51,369 --> 00:07:54,089 I'm left behind way is the E.coli 155 00:07:54,209 --> 00:07:57,409 on the surface of that slide. Just using 156 00:07:57,489 --> 00:08:00,249 the light in the room, you can see, 157 00:08:00,329 --> 00:08:03,129 obviously, if it is still quite damp 158 00:08:03,249 --> 00:08:05,849 or if it's completely dried. So once 159 00:08:05,929 --> 00:08:07,369 you're happy that your bacteria 160 00:08:07,449 --> 00:08:09,769 is completely dry and just checking all 161 00:08:09,889 --> 00:08:12,889 the moisture is being removed, you can 162 00:08:13,009 --> 00:08:17,009 remove your slide and back to safety 163 00:08:17,089 --> 00:08:19,289 flame; then going to move over 164 00:08:19,369 --> 00:08:21,809 to the sink and we can see the glass 165 00:08:21,889 --> 00:08:23,649 rods which we're going to place 166 00:08:23,769 --> 00:08:27,369 the slide over the top. So while I'm 167 00:08:27,489 --> 00:08:31,049 conducting my staining. All 168 00:08:31,129 --> 00:08:33,409 of the excess of the stain is going 169 00:08:33,529 --> 00:08:35,409 to go directly into my sink, 170 00:08:35,529 --> 00:08:37,449 which is an easy to wash away, and we're 171 00:08:37,569 --> 00:08:41,529 not ruining our surfaces. So firstly, 172 00:08:41,609 --> 00:08:45,609 I'm going to take the crystal violet. 173 00:08:47,089 --> 00:08:49,289 So as we said the crystal violet 174 00:08:49,501 --> 00:08:52,929 is going to stain the outer peptidoglycan 175 00:08:52,929 --> 00:08:55,329 structure of the cell wall 176 00:08:55,409 --> 00:08:58,609 of any gram positive bacteria that may 177 00:08:58,689 --> 00:09:01,569 be present on that slide. So taking 178 00:09:01,649 --> 00:09:09,809 a few drops. I'm going to cover that 179 00:09:09,889 --> 00:09:16,449 surface area. Feel free to be generous 180 00:09:16,569 --> 00:09:18,209 with it, because you want to get as many 181 00:09:18,329 --> 00:09:20,329 of them as covered as possible. 182 00:09:22,889 --> 00:09:24,729 So you can see them under the microscope 183 00:09:24,809 --> 00:09:26,889 point. So you want to leave that now 184 00:09:26,969 --> 00:09:37,329 for 30 seconds. So once your 30 seconds 185 00:09:37,409 --> 00:09:39,369 have been completed, we're going to use 186 00:09:39,449 --> 00:09:43,129 water in order to remove the crystal 187 00:09:43,249 --> 00:09:45,009 violet that is staining our gram 188 00:09:45,089 --> 00:09:47,569 positive bacteria, which may be present 189 00:09:47,649 --> 00:09:50,369 on our slide. So, again, being very 190 00:09:50,489 --> 00:09:54,049 generous with this, we're removing with 191 00:09:54,129 --> 00:09:57,529 water and we can see it's going to go 192 00:09:57,609 --> 00:10:01,929 over the slide. May want to tip it, 193 00:10:02,049 --> 00:10:08,009 absolutely fine. Rinsing down the slide 194 00:10:08,089 --> 00:10:12,209 any of the excess crystal Violet. 195 00:10:12,289 --> 00:10:14,729 You can see the mess it's making 196 00:10:14,849 --> 00:10:16,849 which is exactly we're over the sink. 197 00:10:22,609 --> 00:10:24,489 We can actually start to see as well 198 00:10:24,609 --> 00:10:28,049 that it has worked. So what this 199 00:10:28,129 --> 00:10:30,009 is telling is we know we use E.coli, 200 00:10:30,129 --> 00:10:32,009 which is gram negative, we've actually 201 00:10:32,089 --> 00:10:33,609 stained some gram positives there 202 00:10:33,729 --> 00:10:35,289 so my slide wasn't sterile 203 00:10:35,409 --> 00:10:40,569 when we actually started using it. Now 204 00:10:40,649 --> 00:10:42,889 I want to enhance that colour, 205 00:10:43,009 --> 00:10:44,489 so in order to do that, I'm going to add 206 00:10:44,609 --> 00:10:49,289 some iodine to my slide so again, 207 00:10:49,409 --> 00:10:51,209 I'm being extremely generous all over 208 00:10:51,289 --> 00:10:55,649 the surface area. We're going to add 209 00:10:55,769 --> 00:10:58,209 iodine, sometimes referred to as gram 210 00:10:58,329 --> 00:11:04,609 iodine with this procedure, it's iodine; 211 00:11:04,689 --> 00:11:07,649 And once again, I'm going to leave that 212 00:11:07,769 --> 00:11:17,569 for 30 seconds. So when 30 seconds have 213 00:11:17,689 --> 00:11:22,089 passed, once again, you can tilt that 214 00:11:22,209 --> 00:11:25,209 way. I'm just going to give it another 215 00:11:25,289 --> 00:11:28,489 rinse without water, removing any excess 216 00:11:28,569 --> 00:11:34,689 iodine. So any crystal violet that has 217 00:11:34,809 --> 00:11:36,089 successfully stayed with any gram 218 00:11:36,209 --> 00:11:41,209 positive bacteria we've now stained, 219 00:11:41,289 --> 00:11:43,889 enhanced the color and we just want 220 00:11:44,009 --> 00:11:46,689 to fix it. So we're going to cover 221 00:11:46,809 --> 00:11:50,849 it now with alcohol. So it's ninety five 222 00:11:50,969 --> 00:11:54,969 percent alcohol. And again, 223 00:11:55,089 --> 00:12:00,649 covering that surface area. And what 224 00:12:00,769 --> 00:12:04,769 it's going to actually do now, it's going 225 00:12:04,889 --> 00:12:07,209 to break down any of the excess Crystal 226 00:12:07,329 --> 00:12:11,729 Violet that still may be present. 227 00:12:11,809 --> 00:12:14,649 Just giving it a really good rinse, so I'm 228 00:12:14,729 --> 00:12:16,729 going to leave it on for 20 seconds. 229 00:12:21,849 --> 00:12:24,089 So when 20 seconds have passed once 230 00:12:24,209 --> 00:12:29,209 again, if you want to tilt your slide, 231 00:12:29,289 --> 00:12:48,329 you can clean it with water. So now 232 00:12:48,449 --> 00:12:51,929 I want to see if I've got any successful 233 00:12:52,049 --> 00:12:54,529 gram negative bacteria, which hopefully 234 00:12:54,649 --> 00:12:57,889 I do because of my E.coli, so I'm going 235 00:12:57,969 --> 00:13:02,329 to add my safranin. Which is going to be 236 00:13:02,449 --> 00:13:05,369 staining the Lipo Polysaccharides 237 00:13:05,489 --> 00:13:08,529 structure of gram negative cell wall. 238 00:13:10,689 --> 00:13:12,249 So once again, I'm going to take 239 00:13:12,329 --> 00:13:14,889 the safranin and cover that surface 240 00:13:14,969 --> 00:13:24,969 area. And this will be left once again 241 00:13:25,289 --> 00:13:34,009 for 30 seconds. When the 30 seconds have 242 00:13:34,129 --> 00:13:37,609 passed, I'm going to do my final rinse, 243 00:13:38,569 --> 00:13:47,369 remove my safranin and clean it with water. 244 00:13:55,609 --> 00:13:57,409 So hopefully all I'm going 245 00:13:57,409 --> 00:13:59,209 to have left behind is the E.coli 246 00:13:59,329 --> 00:14:02,809 that I've used, maybe any gram positive 247 00:14:02,889 --> 00:14:04,489 because my slide wasn't sterile 248 00:14:04,609 --> 00:14:07,729 when I used it. And when I view it down 249 00:14:07,809 --> 00:14:09,689 the microscope, I should now see 250 00:14:09,809 --> 00:14:13,649 some nice pink gram negative bacteria, 251 00:14:14,009 --> 00:14:16,889 possibly some purple gram positive 252 00:14:16,969 --> 00:14:20,009 bacteria. So I'm going to take my slide 253 00:14:20,089 --> 00:14:23,569 back over to my blotting paper. 254 00:14:28,409 --> 00:14:30,929 Very carefully just going to blot 255 00:14:31,049 --> 00:14:36,689 the slide of any excess stains on water 256 00:14:36,809 --> 00:14:40,769 that could still be present on there. 257 00:14:40,889 --> 00:14:45,449 Then I can view it down the microscope, 258 00:14:45,849 --> 00:14:47,849 so when we're using the microscope. 259 00:14:50,369 --> 00:14:52,089 Place you slide onto the stage. 260 00:14:52,169 --> 00:14:57,129 The stage clips in place to hold 261 00:14:57,249 --> 00:14:59,889 the slide, always using the lowest 262 00:15:00,009 --> 00:15:03,449 magnification first. We're going to view 263 00:15:03,529 --> 00:15:07,689 down the eyepiece and use the coarse 264 00:15:07,769 --> 00:15:12,969 adjustment to be able to change 265 00:15:13,089 --> 00:15:19,649 our view. I'm confident now I am looking 266 00:15:19,729 --> 00:15:23,849 at my gram negative bacteria of E.coli, 267 00:15:24,249 --> 00:15:26,129 lovely and pink in their appearance, 268 00:15:26,209 --> 00:15:28,089 a couple of purple's in there, so as 269 00:15:28,169 --> 00:15:29,729 I said my slide wasn't sterile 270 00:15:29,809 --> 00:15:32,289 when I used it, but I can use my fine 271 00:15:32,409 --> 00:15:36,249 adjustment just to enhance the clarity. 272 00:15:37,609 --> 00:15:40,689 So now I want to see my bacteria 273 00:15:40,809 --> 00:15:43,689 at a larger magnification, so just 274 00:15:43,849 --> 00:15:48,449 rotate, to times 10, use the coarse 275 00:15:48,529 --> 00:15:51,729 adjustment to find my bacteria once 276 00:15:51,849 --> 00:15:55,969 again and then my fine adjustment to get 277 00:15:56,089 --> 00:15:59,649 a clear enhanced image of my bacteria. 278 00:15:59,729 --> 00:16:05,209 Lovely. And after that, you may want 279 00:16:05,289 --> 00:16:07,049 to conduct some biological drawings 280 00:16:07,129 --> 00:16:09,249 or even use your mobile phone to take 281 00:16:09,369 --> 00:16:12,009 photographic images of your work, 282 00:16:12,209 --> 00:16:14,249 and you can even which is great with 283 00:16:14,329 --> 00:16:15,329 phones, once you've taken that 284 00:16:15,449 --> 00:16:17,369 photograph, you can then even enhance 285 00:16:17,449 --> 00:16:21,489 that image even more. So with gram 286 00:16:21,609 --> 00:16:23,769 staining, as we said, this would be 287 00:16:23,889 --> 00:16:26,929 to identify a gram positive and our gram 288 00:16:27,049 --> 00:16:29,729 negative bacteria, gram positive, 289 00:16:29,809 --> 00:16:31,689 we're using our crystal violet, 290 00:16:31,809 --> 00:16:34,409 which is staining in that outer peptidoglycan 291 00:16:34,409 --> 00:16:36,129 structure of the cell wall 292 00:16:36,249 --> 00:16:39,889 of gram positive bacteria. Safranin 293 00:16:40,009 --> 00:16:42,089 is going to be staining our 294 00:16:42,089 --> 00:16:44,889 Lipopolysaccharide, outer structure 295 00:16:45,009 --> 00:16:47,009 of the cell wall, of gram negative 296 00:16:47,089 --> 00:16:49,609 bacteria. Examples of gram negative 297 00:16:49,689 --> 00:16:52,249 bacteria would be our E.coli and one 298 00:16:52,329 --> 00:16:54,329 example of a gram positive bacteria 299 00:16:54,489 --> 00:16:55,929 maybe such as m luteus, 300 00:16:56,049 --> 00:16:58,169 which is commonly used within research 301 00:16:58,169 --> 00:16:59,489 schools and colleges.