WEBVTT 00:00:07.290 --> 00:00:08.969 Welcome to the laboratory. 00:00:08.970 --> 00:00:10.709 This video is a demonstration 00:00:10.710 --> 00:00:13.049 of the standard plate count technique, 00:00:13.050 --> 00:00:15.330 also known as serial dilutions, 00:00:15.330 --> 00:00:18.869 and a calculation of the colony forming units per mil. 00:00:18.870 --> 00:00:21.839 The aim of the standard plate count is 00:00:21.840 --> 00:00:24.539 to determine the number of living, 00:00:24.540 --> 00:00:28.289 that is viable, cells in a culture. 00:00:28.290 --> 00:00:31.558 So, in theory, one bacterial or yeast cell 00:00:31.558 --> 00:00:33.178 should give rise to one colony, 00:00:33.178 --> 00:00:35.069 and it's the same for fungi. 00:00:35.070 --> 00:00:38.309 One spore or one mycelium unit 00:00:38.310 --> 00:00:41.589 should be equivalent to one colony forming 00:00:41.589 --> 00:00:43.559 on your plate. 00:00:43.560 --> 00:00:46.139 So when performing serial dilutions, 00:00:46.140 --> 00:00:47.999 you need to use aseptic technique. 00:00:48.000 --> 00:00:50.069 So that means you need to make sure 00:00:50.070 --> 00:00:52.829 that your bench has been wiped down with disinfectant 00:00:52.830 --> 00:00:55.512 and a Bunsen turned on. 00:00:59.460 --> 00:01:02.899 You've been provided with five McCartney bottles, 00:01:02.900 --> 00:01:06.199 each containing nine mils of diluent. 00:01:06.200 --> 00:01:10.049 Diluent is just another word for dilution solution. 00:01:10.050 --> 00:01:11.999 So that is the solution 00:01:12.000 --> 00:01:15.589 that you're going to perform your serial dilutions in. 00:01:15.590 --> 00:01:18.499 On your bench, you should also have a culture 00:01:18.500 --> 00:01:20.689 of Micrococcus luteus. 00:01:20.690 --> 00:01:23.929 So you can see this McCartney bottle 00:01:23.930 --> 00:01:25.999 contains a bacterial culture, 00:01:26.000 --> 00:01:30.529 it's cloudy, and Micrococcus is also yellow in color. 00:01:30.530 --> 00:01:32.239 So this is our stock solution. 00:01:32.240 --> 00:01:35.199 And this is the solution we're going to calculate 00:01:35.200 --> 00:01:38.079 the number of viable cells for 00:01:38.080 --> 00:01:41.997 by completing the serial dilution. 00:01:41.997 --> 00:01:46.609 So we're going to perform ten to the minus one dilution. 00:01:46.609 --> 00:01:49.059 So one in ten dilutions, 00:01:49.059 --> 00:01:51.608 from ten to the minus one 00:01:51.608 --> 00:01:53.679 to ten to the minus five. 00:01:53.680 --> 00:01:56.709 So the first thing you should do is label each 00:01:56.710 --> 00:02:00.409 of the McCartney bottles with the correct dilution factor. 00:02:00.410 --> 00:02:04.129 So to do that, just grab your texter 00:02:04.130 --> 00:02:08.119 and label each of the bottles with the dilution factor. 00:02:08.120 --> 00:02:10.541 So, ten to the minus one, 00:02:12.710 --> 00:02:15.301 ten to the minus two, 00:02:18.800 --> 00:02:21.383 ten to the minus three, 00:02:23.360 --> 00:02:25.339 ten to the minus four, 00:02:25.340 --> 00:02:26.209 and finally, 00:02:26.210 --> 00:02:28.924 ten to the minus five. 00:02:31.510 --> 00:02:34.719 So if we're performing one in ten dilutions, 00:02:34.720 --> 00:02:37.899 that means we need to add 1000 microliters, 00:02:37.900 --> 00:02:40.539 or one mil, of the stock culture 00:02:40.540 --> 00:02:43.449 to our first tube to collect-- 00:02:43.450 --> 00:02:46.119 or to create a one in ten dilution. 00:02:46.120 --> 00:02:48.189 And then serially dilute that 00:02:48.190 --> 00:02:53.139 into the other bottles using one mil. 00:02:53.140 --> 00:02:55.149 So you need your pipette, 00:02:55.150 --> 00:02:58.179 which measures 1000 microliters, 00:02:58.180 --> 00:03:01.679 so make sure it's set to the correct volume. 00:03:01.680 --> 00:03:05.519 Make sure that your Bunsen flame is turned 00:03:05.520 --> 00:03:09.449 to the blue flame because we do need to work aseptically. 00:03:09.450 --> 00:03:12.599 And you also need your blue tips. 00:03:12.600 --> 00:03:14.549 So the blue tips go with the pipette 00:03:14.550 --> 00:03:18.239 that measures 1000 microliters. 00:03:18.240 --> 00:03:22.049 So pop a tip on to your pipette, 00:03:22.050 --> 00:03:25.319 and then working aseptically, we need to transfer one mil 00:03:25.320 --> 00:03:27.929 of the stock culture to our first bottle 00:03:27.930 --> 00:03:31.169 to create the one in ten dilution. 00:03:31.170 --> 00:03:34.919 So using the crook of your finger, 00:03:34.920 --> 00:03:38.274 loosen the lid of the bottle. 00:03:38.274 --> 00:03:40.859 Flame the neck of the bottle, 00:03:40.860 --> 00:03:44.249 collect one mil. 00:03:44.250 --> 00:03:46.229 Flame the neck of the bottle. 00:03:46.230 --> 00:03:49.789 Replace the lid. 00:03:49.790 --> 00:03:53.426 Grab your ten to the minus one bottle. 00:03:54.950 --> 00:03:57.349 Remove the lid. 00:03:57.350 --> 00:03:59.119 Flame the neck of the bottle. 00:03:59.120 --> 00:04:03.169 Transfer the one mil of stock culture. 00:04:03.170 --> 00:04:07.069 Flame the neck of the bottle and replace the lid. 00:04:07.070 --> 00:04:10.789 Once you've done that, you can eject your pipette tip 00:04:10.790 --> 00:04:13.879 into the pipette bucket. 00:04:13.880 --> 00:04:16.399 Before we do the next dilution, 00:04:16.400 --> 00:04:18.499 we need to make sure that our ten 00:04:18.500 --> 00:04:20.999 to the minus one dilution is mixed properly. 00:04:21.000 --> 00:04:23.429 So to do that, we use the vortex. 00:04:23.430 --> 00:04:25.889 So turn the vortex on, 00:04:25.890 --> 00:04:28.259 and then hold the bottle in the vortex 00:04:28.260 --> 00:04:31.439 until the solution swirls 00:04:31.440 --> 00:04:34.499 around for a couple of seconds. 00:04:34.500 --> 00:04:37.043 And then it should be thoroughly mixed. 00:04:38.190 --> 00:04:40.859 So to perform the ten to the minus two dilution, 00:04:40.860 --> 00:04:44.769 we need to grab a new tip on our pipette, 00:04:44.770 --> 00:04:47.529 and aseptically transfer one mil 00:04:47.530 --> 00:04:49.929 from the ten to the minus one bottle. 00:04:49.930 --> 00:04:52.449 So using the crook of your finger, 00:04:52.450 --> 00:04:54.189 remove the lid of the bottle, 00:04:54.190 --> 00:04:56.739 flame the neck of the bottle. 00:04:56.740 --> 00:04:58.989 Collect one mil. 00:04:58.990 --> 00:05:01.098 Flame the neck of the bottle. 00:05:01.098 --> 00:05:03.123 Replace the lid. 00:05:06.870 --> 00:05:09.989 Loosen the lid of the ten to the minus two bottle, 00:05:09.990 --> 00:05:13.799 flame the neck of the bottle, transfer the one mil, 00:05:13.800 --> 00:05:15.899 flame the neck of the bottle, 00:05:15.900 --> 00:05:17.769 replace the lid, 00:05:17.770 --> 00:05:22.479 and then eject your pipette tube. 00:05:22.480 --> 00:05:23.979 Again, we need to make sure 00:05:23.980 --> 00:05:26.859 this solution is mixed properly. 00:05:26.860 --> 00:05:29.889 So we use the vortex, 00:05:29.890 --> 00:05:32.259 give it a quick mix 00:05:32.260 --> 00:05:34.779 for a few seconds, 00:05:34.780 --> 00:05:36.489 and then it's ready to perform 00:05:36.490 --> 00:05:39.199 the ten to the minus three dilution. 00:05:39.200 --> 00:05:42.259 So we collect a fresh tip. 00:05:42.260 --> 00:05:44.329 Using the crook of our finger, 00:05:44.330 --> 00:05:46.399 remove the lid of the bottle, 00:05:46.400 --> 00:05:48.799 flame the neck of the bottle. 00:05:48.800 --> 00:05:50.899 Collect one mil. 00:05:50.900 --> 00:05:52.609 Flame the neck of the bottle, 00:05:52.610 --> 00:05:55.519 replace the lid, 00:05:55.520 --> 00:05:59.869 and transfer it by removing the lid 00:05:59.870 --> 00:06:01.789 of our ten to the minus three bottle, 00:06:01.790 --> 00:06:03.169 flaming the neck of the bottle, 00:06:03.170 --> 00:06:05.449 transferring one mil, 00:06:05.450 --> 00:06:08.642 flaming the neck of the bottle, and replacing the lid. 00:06:11.270 --> 00:06:14.699 Eject your pipette tip. 00:06:14.700 --> 00:06:17.250 Give it a mix on the vortex. 00:06:20.310 --> 00:06:22.169 And then keep performing 00:06:22.170 --> 00:06:24.082 the dilutions 00:06:25.380 --> 00:06:29.905 from ten to the three right up to ten to the minus five. 00:06:34.050 --> 00:06:36.809 So it's really important when you are performing 00:06:36.810 --> 00:06:39.149 a serial dilution 00:06:39.150 --> 00:06:42.359 that it is a one-person operation. 00:06:42.360 --> 00:06:45.809 So one person in your group 00:06:45.810 --> 00:06:50.180 should be serially diluting the solution, 00:06:50.180 --> 00:06:53.979 just to ensure that it stays nice and sterile and clean, 00:06:53.980 --> 00:06:56.749 and that nothing becomes contaminated, 00:06:56.750 --> 00:06:58.249 and you don't get mixed up 00:06:58.250 --> 00:07:00.779 with what solution has been transferred 00:07:00.780 --> 00:07:02.660 to which tube. 00:07:02.660 --> 00:07:04.489 [no monologue] 00:07:34.120 --> 00:07:36.099 When you've completed the dilutions, 00:07:36.100 --> 00:07:40.029 make sure that you do give your last bottle 00:07:40.030 --> 00:07:43.194 the ten to the minus five dilution, a mix as well. 00:07:48.114 --> 00:07:51.359 If you have a look at your dilution series, 00:07:51.360 --> 00:07:53.239 you can note two things. 00:07:53.240 --> 00:07:56.929 You will see that the ten to the minus one dilution 00:07:56.930 --> 00:07:58.669 is still quite cloudy. 00:07:58.670 --> 00:08:01.609 And then from ten to the minus two onwards, 00:08:01.610 --> 00:08:05.558 it's very difficult to see cells suspended in there. 00:08:07.010 --> 00:08:10.699 The other thing you might note is that each of the tubes, 00:08:10.700 --> 00:08:12.919 ten to the one to ten to the minus four, 00:08:12.920 --> 00:08:15.649 contain nine mils of solution 00:08:15.650 --> 00:08:18.709 because we were transferring one mil. 00:08:18.710 --> 00:08:22.459 And then our final ten to the minus five bottle 00:08:22.460 --> 00:08:25.779 contains ten mils of solution. 00:08:25.779 --> 00:08:29.669 So it is okay to leave that extra one mil remaining 00:08:29.670 --> 00:08:31.559 in the ten to the minus five dilution. 00:08:31.560 --> 00:08:35.239 It shouldn't affect your actual spread plate 00:08:35.239 --> 00:08:37.153 or the number of colonies that will grow 00:08:37.153 --> 00:08:40.169 for the ten to the minus five dilution. 00:08:40.170 --> 00:08:41.699 But if you are a perfectionist, 00:08:41.700 --> 00:08:44.489 you can remove that one mil. 00:08:44.490 --> 00:08:46.289 So what you need to do is, 00:08:46.290 --> 00:08:49.529 using a nice clean fresh tip, 00:08:49.530 --> 00:08:52.139 aseptically remove one mil 00:08:52.140 --> 00:08:53.999 from the ten to the minus five dilution. 00:08:54.000 --> 00:08:57.059 So using the crook of your finger, 00:08:57.060 --> 00:08:59.669 remove the lid of the bottle, 00:08:59.670 --> 00:09:02.429 flame the neck of the bottle, 00:09:02.430 --> 00:09:06.809 collect one mil. 00:09:06.810 --> 00:09:09.269 Flame the neck of the bottle, replace the lid, 00:09:09.270 --> 00:09:12.089 and then inject that tip with the one mil of solution 00:09:12.090 --> 00:09:16.679 into your pipette container. 00:09:16.680 --> 00:09:18.333 So now... 00:09:18.333 --> 00:09:21.930 your serial dilutions are complete. 00:09:21.930 --> 00:09:25.944 You will be able to move on to spread plating them.