1 00:00:12,390 --> 00:00:15,030 Aseptic technique. So the aseptic 2 00:00:15,110 --> 00:00:17,510 technique is something that we practice 3 00:00:17,630 --> 00:00:20,270 when we're conducting microbiology. 4 00:00:20,390 --> 00:00:22,310 It's used in any of the practicals that 5 00:00:22,430 --> 00:00:25,030 use in microbiology. It basically means 6 00:00:25,110 --> 00:00:28,270 no germ. So you are ensuring 7 00:00:28,390 --> 00:00:30,350 when you are working with your chosen 8 00:00:30,470 --> 00:00:33,230 microbes that you are only working with 9 00:00:33,310 --> 00:00:35,510 those chosen microbes and not something 10 00:00:35,630 --> 00:00:36,670 that's been introduced 11 00:00:36,750 --> 00:00:38,990 from the environment or on the surface 12 00:00:39,110 --> 00:00:41,110 that you are working on. 13 00:00:41,310 --> 00:00:44,390 So before filming, this surface has been 14 00:00:44,510 --> 00:00:45,710 completely sterilized, 15 00:00:45,790 --> 00:00:47,870 using disinfectant and when I've 16 00:00:47,950 --> 00:00:50,110 sterilized, I've ensured there it's gone 17 00:00:50,310 --> 00:00:52,550 from complete edge to edge 18 00:00:52,710 --> 00:00:55,030 of the surface area that we're working 19 00:00:55,150 --> 00:00:59,030 in. For this particular practical then 20 00:00:59,150 --> 00:01:02,870 I'm going to do a lawn spread on an agar 21 00:01:02,990 --> 00:01:07,150 plate using E.coli and mask ring, 22 00:01:07,230 --> 00:01:09,070 and I'll talk about that more as we go 23 00:01:09,190 --> 00:01:12,710 through. During this aseptic technique, 24 00:01:12,830 --> 00:01:13,870 we're going to use two different 25 00:01:13,950 --> 00:01:16,150 methods. We are going to use chemical 26 00:01:16,270 --> 00:01:19,310 aseptic techniques using alcohol 27 00:01:19,430 --> 00:01:22,750 and also heat sterilization with 28 00:01:22,870 --> 00:01:26,590 the Bunsen burner as well. So with 29 00:01:26,670 --> 00:01:28,350 microbiology, there's a few instruments 30 00:01:28,470 --> 00:01:30,470 that you may need to be introduced to, 31 00:01:30,910 --> 00:01:34,630 so firstly our microbes. So for this 32 00:01:34,750 --> 00:01:37,070 one, I'm going to be using E.coli, 33 00:01:37,150 --> 00:01:40,230 so this is an E.coli broth, which we can 34 00:01:40,310 --> 00:01:42,830 see it's quite murky and that's 35 00:01:42,910 --> 00:01:45,030 the growth of those little pathogens 36 00:01:45,150 --> 00:01:49,230 inside. E.coli to a young, 37 00:01:49,350 --> 00:01:52,310 healthy person isn't going to cause too 38 00:01:52,390 --> 00:01:54,350 many problems, just a few more visits 39 00:01:54,470 --> 00:01:56,470 to the bathroom than you expected. 40 00:01:56,990 --> 00:01:59,550 But to someone more vulnerable, it could 41 00:01:59,630 --> 00:02:02,350 cause serious illness, if not death. 42 00:02:03,590 --> 00:02:05,910 So with all forms of microbiology 43 00:02:05,990 --> 00:02:07,590 and this one in particular, we have 44 00:02:07,670 --> 00:02:09,230 to be extremely careful with our health 45 00:02:09,350 --> 00:02:11,550 and safety. So, as you can see, 46 00:02:11,630 --> 00:02:14,750 appropriate PPE and gloves protecting 47 00:02:14,830 --> 00:02:21,190 myself. With our microbes then depending 48 00:02:21,310 --> 00:02:24,910 what they may be, we grow them within 49 00:02:24,990 --> 00:02:27,550 a petri dish and inside my petri dish, 50 00:02:27,990 --> 00:02:30,990 you may be able to see this jelly 51 00:02:31,110 --> 00:02:35,390 substance known as nutrient agar. 52 00:02:35,510 --> 00:02:36,590 So this is a medium that I'm going 53 00:02:36,710 --> 00:02:39,190 to grow the microbes on, this 54 00:02:39,310 --> 00:02:41,910 is providing water and nutrients 55 00:02:42,710 --> 00:02:43,350 and we're going to pop 56 00:02:43,470 --> 00:02:46,750 it into an incubator, 36, 37 degrees C 57 00:02:46,870 --> 00:02:49,350 replicating in our internal body 58 00:02:49,710 --> 00:02:52,390 temperature. So it's creating a perfect 59 00:02:52,510 --> 00:02:59,870 environment for them to grow in. We have 60 00:02:59,990 --> 00:03:02,550 our pipettes, which we are going to use 61 00:03:02,630 --> 00:03:10,710 for our Microbe's. Forceps, this glass 62 00:03:10,830 --> 00:03:14,670 rod known as a lawn spreader 63 00:03:14,790 --> 00:03:17,470 and a common tool within microbiology, 64 00:03:17,590 --> 00:03:19,430 but we won't use it in this particular 65 00:03:19,550 --> 00:03:22,150 practical is the inoculation loop. 66 00:03:22,710 --> 00:03:25,950 The inoculation loop is if we have what 67 00:03:26,070 --> 00:03:30,670 we call a slope where our broth is solid 68 00:03:31,230 --> 00:03:34,910 at an angle, at a slope. All this 69 00:03:34,990 --> 00:03:38,390 is doing is increasing the surface area 70 00:03:38,470 --> 00:03:40,390 in which our microbes can grow 71 00:03:40,510 --> 00:03:43,070 on and we use the innoculation loop 72 00:03:43,750 --> 00:03:46,990 to collect the microbes and streak plat 73 00:03:47,110 --> 00:03:50,030 it onto our agar. But today we're doing 74 00:03:50,150 --> 00:03:52,230 lawn spread so this is not needed. 75 00:03:54,310 --> 00:03:57,470 And then we have our marker pen 76 00:03:57,550 --> 00:04:01,390 for labeling our agar plate. 77 00:04:01,510 --> 00:04:04,510 Bunsen burner. Health and safety again, 78 00:04:04,630 --> 00:04:06,030 then you'll notice with a Bunsen burner, 79 00:04:06,150 --> 00:04:08,150 we've got this lovely orange flame. 80 00:04:08,990 --> 00:04:10,910 So this is known as our safety flame. 81 00:04:10,990 --> 00:04:13,590 I can see this, I know it's that my hair 82 00:04:13,710 --> 00:04:17,310 is tied back. People in my environment 83 00:04:17,390 --> 00:04:19,470 can also see this flame, to hopefully 84 00:04:19,590 --> 00:04:22,070 reduce that risk of someone being hurt. 85 00:04:24,870 --> 00:04:26,070 So we're going to do the lawn spread 86 00:04:26,150 --> 00:04:27,990 then with this aseptic technique, 87 00:04:28,070 --> 00:04:29,790 making sure it's only the E.coli 88 00:04:29,910 --> 00:04:31,350 I'm going to be spreading on my plate 89 00:04:31,470 --> 00:04:33,470 and nothing within my environment. 90 00:04:34,270 --> 00:04:36,270 So first thing, I'm going to take 91 00:04:36,350 --> 00:04:38,990 my agar plate and I need to put 92 00:04:39,070 --> 00:04:41,270 some information so I know for this 93 00:04:41,390 --> 00:04:44,510 particular experiment, the date, 94 00:04:44,630 --> 00:04:46,590 who's performed it and what it's 95 00:04:46,670 --> 00:04:49,630 actually consisted of. So with my marker 96 00:04:49,750 --> 00:04:52,070 pen, I'm not going to write all along 97 00:04:52,190 --> 00:04:55,150 my lid. I'm not going to write all 98 00:04:55,270 --> 00:04:57,310 on the bottom, because when I get 99 00:04:57,430 --> 00:04:59,630 my results, I can't see them properly 100 00:04:59,750 --> 00:05:01,470 because I've written all over it. So I'm 101 00:05:01,550 --> 00:05:03,830 going to write along the edge 102 00:05:04,190 --> 00:05:07,630 of the plate. So, for example, I'm going 103 00:05:07,750 --> 00:05:16,590 to put DG my initials, the date, 104 00:05:16,670 --> 00:05:29,350 and this is a lawn spread and I'm using 105 00:05:29,430 --> 00:05:33,950 E.coli. So for this particular 106 00:05:34,070 --> 00:05:36,070 experiment, I feel that's enough 107 00:05:36,150 --> 00:05:39,350 information, it may be that I'm changing 108 00:05:39,430 --> 00:05:42,110 my nutritional content as a form 109 00:05:42,230 --> 00:05:43,470 of looking at different environments, 110 00:05:43,590 --> 00:05:45,070 so I might need to put that. I might be 111 00:05:45,190 --> 00:05:46,910 changing temperature, so I might need 112 00:05:46,990 --> 00:05:48,630 to put where this one's going to be 113 00:05:48,750 --> 00:05:50,750 housed. But for now, that's all I need. 114 00:05:52,910 --> 00:05:54,390 You'll notice I'm always keeping the lid 115 00:05:54,510 --> 00:05:57,430 closed, because ensuring I'm maintining 116 00:05:57,510 --> 00:05:58,670 that sterilized environment, 117 00:05:58,790 --> 00:06:04,030 not introducing anything new. I do now 118 00:06:04,150 --> 00:06:07,630 want to introduce my E.coli. So first 119 00:06:07,750 --> 00:06:11,550 thing need to remove the lid, so it's 120 00:06:11,670 --> 00:06:14,390 loose. I'm going to turn my Bunsen 121 00:06:14,510 --> 00:06:18,790 burner onto the blue flame and you can 122 00:06:18,870 --> 00:06:20,910 hear it's more aggressive; if you can 123 00:06:20,990 --> 00:06:23,630 see on the flame, we've got this blue 124 00:06:23,750 --> 00:06:25,950 triangular point. The point 125 00:06:26,070 --> 00:06:27,550 of the triangle, then this 126 00:06:27,670 --> 00:06:29,790 is the hottest part of the flame. So I'm 127 00:06:29,910 --> 00:06:34,550 going to now just pass through the top 128 00:06:34,670 --> 00:06:38,790 of the bottle. So I'm heat sterilizing. 129 00:06:38,870 --> 00:06:42,190 For if there are any microbes otheer 130 00:06:42,270 --> 00:06:45,030 the E.coli on this surface, I'm not 131 00:06:45,110 --> 00:06:47,110 keeping the glass in the flame 132 00:06:47,510 --> 00:06:48,630 because I don't want it going too hot 133 00:06:48,750 --> 00:06:56,470 and breaking. Back to safety flame, 134 00:06:56,550 --> 00:06:57,110 everyone can see 135 00:06:57,230 --> 00:06:58,790 where it is and everyone's safe. 136 00:06:58,910 --> 00:07:02,990 I'm going to take my sterilized pipette. 137 00:07:03,110 --> 00:07:04,910 I'm going to try my best not to touch 138 00:07:04,990 --> 00:07:07,910 the edges of the glass. And I'm just 139 00:07:07,990 --> 00:07:10,630 going to collect a few drops 140 00:07:10,710 --> 00:07:16,990 of my E.coli. put the lid back on. 141 00:07:17,070 --> 00:07:19,070 Again, it's maintaining safety 142 00:07:19,550 --> 00:07:20,910 if it falls over. I'm not going to have 143 00:07:21,030 --> 00:07:25,310 a spillage. My E.coli then I'm going 144 00:07:25,430 --> 00:07:30,310 to place onto the surface of the agar, 145 00:07:30,390 --> 00:07:32,310 I'm not going to lift the lid open waft 146 00:07:32,390 --> 00:07:34,470 about because everything's coming down 147 00:07:34,590 --> 00:07:41,030 onto my agar. Ever so slightly introduce 148 00:07:41,110 --> 00:07:45,510 and in the center of the agar, one drop 149 00:07:45,630 --> 00:07:53,670 of the E.coli. Close the lid. This then 150 00:07:53,910 --> 00:07:59,190 can be disposed of in my bleach. 151 00:07:59,310 --> 00:08:01,750 Now going to get my lawn spreads and I'm 152 00:08:01,870 --> 00:08:03,870 going to do both chemical and heat 153 00:08:03,950 --> 00:08:06,390 sterilization, so I'm going to take 154 00:08:06,470 --> 00:08:11,670 my alcohol. Place the lawn spread 155 00:08:11,790 --> 00:08:15,750 inside. Now, it's very important that 156 00:08:15,830 --> 00:08:18,070 when you remove the lawn spread, 157 00:08:18,510 --> 00:08:20,950 don't tip it upside down because you can 158 00:08:21,070 --> 00:08:23,750 see the alcohol running towards your 159 00:08:23,830 --> 00:08:25,670 fingers and if I now put that 160 00:08:25,790 --> 00:08:27,710 in my flame, it's going to set me 161 00:08:27,830 --> 00:08:35,030 on fire. So we must ensure that once 162 00:08:35,150 --> 00:08:38,390 it's gone in the alcohol. We're keeping 163 00:08:38,470 --> 00:08:42,950 it point down; safety flame and just 164 00:08:43,030 --> 00:08:46,070 pass it through, to use the flame 165 00:08:46,190 --> 00:08:50,350 to burn off the alcohol. If I introduce 166 00:08:50,470 --> 00:08:52,110 the alcohol to my E.coli, it's going 167 00:08:52,190 --> 00:08:55,030 to kill the E.coli, so I must use 168 00:08:55,150 --> 00:08:57,150 the heat to burn the alcohol off, 169 00:08:57,550 --> 00:09:08,470 which sterilizes my lawn spread. Now I'm 170 00:09:08,630 --> 00:09:11,150 going to spread my E.coli, so the corner 171 00:09:11,230 --> 00:09:13,910 of the lawn spread goes into the center 172 00:09:14,030 --> 00:09:17,750 of my agar plate, I'm going to then roll 173 00:09:18,270 --> 00:09:19,950 so the edge of the lawn spread 174 00:09:20,070 --> 00:09:22,470 is on the outer edge of my plate. 175 00:09:23,350 --> 00:09:25,670 You should see the E.coli work along 176 00:09:25,790 --> 00:09:30,310 that surface. This is experience 177 00:09:30,390 --> 00:09:32,390 you need to make sure that you are not 178 00:09:32,510 --> 00:09:36,310 pressing down hard on the E.coli. 179 00:09:36,430 --> 00:09:40,830 So corner, in the middle, lay it down 180 00:09:40,910 --> 00:09:43,270 and I can see my E.coli running 181 00:09:43,390 --> 00:09:46,670 to the edge of the plate. All the time 182 00:09:46,750 --> 00:09:49,190 I'm keeping my lid slightly open 183 00:09:50,070 --> 00:09:53,630 to protect the environment and then 184 00:09:53,750 --> 00:09:57,390 going to rotate the lower section 185 00:09:57,510 --> 00:10:00,190 of the petri dish. Which will be 186 00:10:00,310 --> 00:10:03,830 spreading my E.coli along that surface, 187 00:10:03,950 --> 00:10:05,590 and you will be able to see that 188 00:10:05,710 --> 00:10:10,470 spreading evenly. You don't want 189 00:10:10,590 --> 00:10:12,150 to press on too hard, so you're not 190 00:10:12,230 --> 00:10:16,830 breaking the agar. But it needs to be 191 00:10:16,910 --> 00:10:18,550 firm enough that you've always got 192 00:10:18,630 --> 00:10:21,990 contact with the surface. If you do 193 00:10:22,070 --> 00:10:24,910 break the agar, try again with a new 194 00:10:25,030 --> 00:10:29,750 one. So you may want to do several 195 00:10:29,830 --> 00:10:32,710 rotations like I'm doing, just ensuring 196 00:10:33,590 --> 00:10:35,390 you have covered the entire surface 197 00:10:35,510 --> 00:10:38,390 area. I know I'm talking all the time 198 00:10:38,510 --> 00:10:40,750 above it, but when you were practicing 199 00:10:40,830 --> 00:10:43,950 it. You don't want to be talking at all 200 00:10:44,030 --> 00:10:45,550 because you could be introducing new 201 00:10:45,630 --> 00:10:49,510 pathogens. You may want to wear a mask. 202 00:10:53,190 --> 00:10:54,390 So when you're confident and happy 203 00:10:54,510 --> 00:10:56,590 you've covered that entire surface area, 204 00:10:56,710 --> 00:11:00,710 remove the lawn spreads, close the lid, 205 00:11:00,790 --> 00:11:07,750 once again, get your alcohol. Pop that 206 00:11:07,870 --> 00:11:13,430 in. Burn off the alcohol with the safety 207 00:11:13,510 --> 00:11:19,470 flame and that is once again sterile 208 00:11:19,550 --> 00:11:21,190 and we're confident we've not got any 209 00:11:21,310 --> 00:11:24,030 E.coli on there that could potentially 210 00:11:24,670 --> 00:11:27,430 cause an illness to anybody. Going back 211 00:11:27,550 --> 00:11:29,790 to health and safety if there 212 00:11:29,910 --> 00:11:31,390 was something on there, and we've laid 213 00:11:31,470 --> 00:11:34,070 it on this table. Someone else comes 214 00:11:34,150 --> 00:11:35,990 to use this space they don't know what's 215 00:11:36,070 --> 00:11:37,510 on the surface. So that's another reason 216 00:11:37,630 --> 00:11:39,630 why we never eat or drink within a lab. 217 00:11:42,150 --> 00:11:43,630 Now, on mine for this particular 218 00:11:43,750 --> 00:11:45,630 experiment, I've got my E.coli 219 00:11:45,710 --> 00:11:47,750 evenly distributed on that surface 220 00:11:47,950 --> 00:11:49,630 and with my experiment, I want to look 221 00:11:49,710 --> 00:11:52,150 at how effective different antibiotics 222 00:11:52,270 --> 00:11:56,110 are in killing E.coli. So I'm going 223 00:11:56,190 --> 00:11:58,150 to use one of these guys called a mast 224 00:11:58,230 --> 00:12:01,750 ring and a mast ring. You will see 225 00:12:01,830 --> 00:12:05,470 on the box you've got a key - different 226 00:12:05,590 --> 00:12:08,710 letters which are representing different 227 00:12:08,790 --> 00:12:14,630 antibiotics And when we look at the mast 228 00:12:14,750 --> 00:12:20,790 ring. We will see we've got different 229 00:12:20,910 --> 00:12:25,070 coloured circle tabs. All with a letter. 230 00:12:26,070 --> 00:12:28,830 Each circular tab is an antibiotic, 231 00:12:30,070 --> 00:12:33,590 so we can see the name of each of those 232 00:12:33,710 --> 00:12:43,430 antibiotics using this key. I want 233 00:12:43,550 --> 00:12:45,910 to place the mast ring in the middle 234 00:12:45,990 --> 00:12:48,470 of my agar plate, so when my E.coli 235 00:12:48,590 --> 00:12:52,190 is growing, I can see what we call zones 236 00:12:52,270 --> 00:12:56,310 of inhibition. So where I see no growth 237 00:12:56,390 --> 00:12:59,510 has occurred around my antibiotic, 238 00:13:00,190 --> 00:13:02,070 I could see the antibiotic is effective 239 00:13:02,190 --> 00:13:06,070 in killing E. coli. The larger that zone 240 00:13:06,190 --> 00:13:08,510 of inhibition, the more effective it is, 241 00:13:09,350 --> 00:13:11,070 the smaller the zone of inhibition, 242 00:13:11,150 --> 00:13:14,390 the less effective it is. Unless there's 243 00:13:14,950 --> 00:13:18,270 no zone of inhibition at all, we can see 244 00:13:18,390 --> 00:13:19,790 it's not effective at all and not 245 00:13:19,870 --> 00:13:21,270 something you would choose for your 246 00:13:21,390 --> 00:13:24,310 patient. So I'm going to take 247 00:13:24,430 --> 00:13:28,670 my forceps. Once again, using 248 00:13:28,790 --> 00:13:34,510 my alcohol. Dip them in, make sure 249 00:13:34,630 --> 00:13:36,630 they're always pointing down so gravity 250 00:13:36,910 --> 00:13:38,230 doesn't allow the alcohol to come down 251 00:13:38,310 --> 00:13:42,310 to my hands and burn me. Through 252 00:13:42,390 --> 00:13:45,710 the safety flame. Burning off that bit 253 00:13:45,790 --> 00:13:50,710 of alcohol. So I'm confident that these 254 00:13:50,790 --> 00:13:53,550 are now sterile and I'm going to very 255 00:13:53,670 --> 00:13:55,070 carefully because they're only paper 256 00:13:55,190 --> 00:13:58,190 really, they're not that strong. 257 00:13:58,270 --> 00:14:01,910 I'm going to tease away One of the mast 258 00:14:01,990 --> 00:14:03,830 rings try not to break them, 259 00:14:03,950 --> 00:14:05,990 they're very expensive mast rings, 260 00:14:06,670 --> 00:14:10,590 one pound per ring. So when this 261 00:14:10,670 --> 00:14:13,430 is being conducted in research 262 00:14:13,510 --> 00:14:15,910 or hospital labs, that accumulates very, 263 00:14:16,030 --> 00:14:20,670 very quickly. I'm going to place my mast 264 00:14:20,750 --> 00:14:23,030 ring into the center of the agar plate 265 00:14:23,110 --> 00:14:25,750 as much as possible. Again lifting 266 00:14:25,830 --> 00:14:29,390 it ever so slightly, try and get it as 267 00:14:29,510 --> 00:14:33,750 central as I can. And then I'm just 268 00:14:33,870 --> 00:14:37,510 going to ensure delicately, using 269 00:14:37,630 --> 00:14:40,790 my forceps that each antibiotic as 270 00:14:40,870 --> 00:14:44,990 complete contact with the agar, 271 00:14:45,110 --> 00:14:46,990 not stabbing it down too hard, so I'm 272 00:14:47,070 --> 00:14:51,550 damaging in the Matrix, just gentle, 273 00:14:52,110 --> 00:14:54,430 making it a fair experiment to see 274 00:14:54,510 --> 00:14:58,310 which antibiotic is the most effective 275 00:14:58,590 --> 00:15:09,070 against the growth of E.coli. Lovely. 276 00:15:09,150 --> 00:15:12,150 And once again, because it's been 277 00:15:12,230 --> 00:15:14,150 in that environment to make sure we're 278 00:15:14,230 --> 00:15:19,670 all safe out here, burn off the alcohol. 279 00:15:26,550 --> 00:15:31,390 Finally. Simple sellotape over the top 280 00:15:31,470 --> 00:15:33,750 of the lid, attaching it to the bottom 281 00:15:33,990 --> 00:15:36,710 of the petri dish, and that will then go 282 00:15:36,830 --> 00:15:39,230 into the incubator at thirty seven 283 00:15:39,350 --> 00:15:41,990 degrees C, and after 48 hours 284 00:15:42,110 --> 00:15:45,030 of incubation, you will successfully see 285 00:15:45,430 --> 00:15:47,030 the even distribution of growth 286 00:15:47,110 --> 00:15:49,950 of E.coli and hopefully some zones 287 00:15:50,070 --> 00:15:51,710 of inhibition where the E.coli 288 00:15:51,830 --> 00:15:53,910 has failed to grow around each 289 00:15:54,030 --> 00:15:56,790 antibiotic. The larger the zone 290 00:15:56,870 --> 00:15:58,710 of inhibition, the more effective 291 00:15:58,830 --> 00:16:00,950 the antibiotic, the smaller a zone 292 00:16:01,070 --> 00:16:03,430 of inhibition, the least effective that 293 00:16:03,430 --> 00:16:05,030 particular antibiotic.