Mounting bacteria on a slide (making a bacterial smear) — Transcript

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1. 0:01When you're preparing a sample to look at in  the microscope in this Microbiology course,
2. 0:05you're going to do it a little different than you  might be used to. So you might be used to having
3. 0:09your microscope slide, and you add a sample, and  then you put another piece of glass on top -- a
4. 0:14cover slip -- so you have a little sandwich with  your sample in the middle. We are not going to use
5. 0:18cover slips in the Microbiology lab. We will,  instead, still have our sample on a microscope
6. 0:24slide, but then we will have a special technique  to keep our bacteria from rinsing off when we
7. 0:30stain them so that we can see them in the  microscope. So I recommend labeling your
8. 0:35slide -- I actually have already labeled a couple  of these. You don't have to use all of the same
9. 0:41labels that we've been using for cultures because  the slides are not going to go in the incubator,
10. 0:45so we don't need to know like a temperature  or medium or anything like that. Probably your
11. 0:50name or your initials, maybe the name of the  stain that you're doing, if you're going to do
12. 0:54multiple stains. Basically whatever information  is going to help you stay organized. I would
13. 1:00also recommend maybe using one of these nifty  wax pencils to label your slides, rather than
14. 1:07the permanent markers, because "permanent" marker  is not actually true 100% of the time. Some of the
15. 1:13stains that we're going to teach you have an  alcohol rinse step, and alcohol will take the
16. 1:18marker off of the glass. Whereas the wax pencil --  it can still come off, but it's not going to come
17. 1:23off as easily, so that's helpful. Most of the time  you're going to be working with cultures that are
18. 1:31growing on a solid. We use a lot of slants here,  so we're actually going to have you add a little
19. 1:36bit of liquid to your slide, to help spread your  bacteria out a little more. You definitely want to
20. 1:43smear your bacteria around on the slide, because  if they're all piled on top of each other, it's
21. 1:47going to be really hard to see their individual  shapes, which is one of the main things that we
22. 1:51want to be able to see in the microscope. So the  adding a little bit of water just helps lubricate
23. 1:56that a little bit. If you were taking a sample  from a liquid culture, you would not need to
24. 2:01start with a drop of water, but that's actually  pretty rare that you're going to be doing that.
25. 2:07And starting with a drop of water, you're going  to want a *small* drop of water. So even though
26. 2:11you're going to be using your squirt bottle, I  would not recommend trying to get a drop directly
27. 2:16from the bottle onto your slide. You're going to  end up with too much. Instead, you're going to use
28. 2:20your loop. So this is a multi-purpose tool here in  the lab. Basically catch a little drop of water on
29. 2:27your loop. Maybe do this over the sink -- but it's  water. The sink is not in the camera right now,
30. 2:35so I'm just getting water on the desk. So I have  a little drop of water on my loop, and I can touch
31. 2:39that to my slide and now I have a little drop of  water on my slide. Some people have difficulty
32. 2:46kind of catching the water with the loop, and if  that is the case, it is also perfectly legal to
33. 2:52take the lid off of your water and just dunk  your loop in it. I would of course recommend
34. 2:57immediately screwing the lid back on, so that it  doesn't get knocked over and spill everywhere.
35. 3:03And that's true for really any container! Not  even just in the lab, do that in your kitchen,
36. 3:08so that you don't end up with a giant pile of salt  on the floor! Okay now you can also do multiple
37. 3:15samples per slide, so once we start teaching you  some of the differential stains, you're going to
38. 3:21have multiple samples on the same slide, which is  why this particular slide has a couple of little
39. 3:25circles drawn on it. You can draw circles for  your different samples if you want. You don't
40. 3:32have to. Again, write whatever on your slide is  going to help keep you organized. And then you
41. 3:39would just add three drops of water and you would  have a different sample to add to each drop. I'm
42. 3:44not actually going to do that on that slide,  but that's a thing that you can do. And putting
43. 3:49multiple samples on the same slide is really  great for many reasons. Let me just tell you two
44. 3:55of them. First of all, now you're doing one slide  instead of three. If you were doing three samples,
45. 4:00okay, so this one slide here I'm going to end  up staining three different types of bacteria
46. 4:05on the same slide. One slide rather than three --  yay! Less work, less materials, that's good for
47. 4:11everybody. Also, this is going to make your life a  lot easier when you go to focus, because once one
48. 4:16part of your slide is in focus, all parts of your  slide are in focus. Because getting something in
49. 4:21focus means moving your slide up and down while  the lens kind of stays in one place. And we have
50. 4:26to get that slide into the right position so that  it's in focus, and then once that takes place,
51. 4:31all you do to look at other parts of your  slide is just use those directional knobs
52. 4:34to move your slide around on the stage. This is  my imaginary lens, okay, and this is my imaginary
53. 4:40slide. You may also have noticed that I did not  sterilize my loop when I put my water on here,
54. 4:49and that's okay, because I know that this water  is not sterile. This is just deionized water;
55. 4:54it comes out of any of the taps that have a white  insert on their handle. So if you need a refill,
56. 5:01go ahead and do that. If you've never used one  of these before, basically you just squeeze it,
57. 5:06and water comes shooting out. Try not to aim it  at anybody when you do that. And this water, when
58. 5:12it comes out of the tap, is not sterile. We don't  need it to be sterile. This bottle is not going
59. 5:16to keep it sterile. This little thing is open to  the air, and probably people have been touching
60. 5:22it with things. That's okay. The microbes that are  in there are going to be vastly outnumbered by the
61. 5:28microbes that you're going to add from your actual  culture, so one or two random microbes that just
62. 5:34came from the water is not going to be a big deal.  That *would* be a big deal if you were starting a
63. 5:38culture, because one or two microbes, when it sits  in the incubator with a bunch of food, then grows
64. 5:44to a huge number, and you might not want those.  Okay, so I've got my little water droplet on my
65. 5:51slide. I'm going to move my other slide out of  the way just to keep things a little bit simpler.
66. 5:56The second thing I need to do, after my drop of  water, is to add add the bacteria. I'll get my
67. 6:01burner going, get my loop all flamed and cooled  down. Remember to cool your loop before you use
68. 6:08it! Also, as a reminder while I'm waiting for  my loop to cool, that when you are picking up
69. 6:16bacteria from your culture, you don't need to dig  up a whole bunch. Okay? Don't scoop a whole bunch
70. 6:24in here, don't swipe along the whole surface,  don't dig in there. Just *touch* your loop to the
71. 6:28surface where you can see the bacteria growing.  It'll either have color, or if it's not colored,
72. 6:33it will at least be cloudy. And once you've  touched, you've gotten tons and tons and tons of
73. 6:37bacteria. Remember, they're microscopic. You can't  see them. So as long as you made that contact,
74. 6:43I guarantee you have lots and lots and lots  of bacteria. So I'm going to add my bacteria,
75. 6:52on my loop, into that drop of water, and then  I'm going to smear this out. Okay, if you're
76. 6:57only doing one sample, be free with your smearing!  This is literally called preparing a smear, and
77. 7:05you're smearing it around, so use lots of space.  If you have multiple samples, then you'll want
78. 7:10to make sure not to overlap between the samples,  but if you're just doing one, like a simple stain,
79. 7:15use lots and lots of space. Again, the reason we  added water here is to help spread the bacteria
80. 7:21out from each other, and that just kind of  lubricates things. You can do this without water,
81. 7:26but the bacteria really do not spread out very  well. And then they're all piled on top of each
82. 7:31other, and you can't see their shapes in the  microscope. So we need them to be separated out
83. 7:35from each other. Also, we're not trying to fit  everything under a cover slip, so we can use as
84. 7:41much space as we have available. And of course,  the thinner this smear is, the faster it's going
85. 7:47to dry. Because the next thing I need to do is let  this thing dry. And I have to just let it sit. Do
86. 7:54not blow on it to dry it out, because now you're  adding maybe some spit -- not a lot of microbes,
87. 8:01but still maybe some microbes that you didn't want  in your sample. And blowing it may take some of
88. 8:06the microbes that are down here, on your slide,  and send them out into the air. Also something
89. 8:11that we don't want. So this is the worst part of  of staining, is waiting for this to dry. Now mine,
90. 8:17because I'm pretty experienced with this, and so I  know not to add a ton of water and to smear it out
91. 8:22a lot, it's dry now. It's air dried, and I can get  a little reflection, make sure there's no water
92. 8:28drops. Let's say that this wasn't dry, and I was  getting impatient. Again, you're not going to blow
93. 8:33on it, you're not going to heat it up to dry it.  The one way that you can make this dry a little
94. 8:39faster -- other than starting over and using less  water -- is resterilize your loop and cool it down
95. 8:46again. So that it's basically pristine, and then  make sure you cool it down -- the other annoying
96. 8:55part is just waiting for your for your loop to  cool -- then you would just go back to your smear,
97. 9:00and smear it out some more. You can go over places  that you've already gone over, assuming that it's
98. 9:05that one particular sample. Just stay within that  particular sample. There's only one sample on this
99. 9:10slide, so I don't have to worry. If I was doing  multiple samples on my slide, I'd have to keep
100. 9:15them in their own little sections. And basically  the thinner it is, the faster it'll dry. Then once
101. 9:23this is all dry, the last thing I need to do is  I need to heat this up a little bit. I definitely
102. 9:30recommend using a holder for heat fixing. We  have a bunch of clothespins in the lab. Keep
103. 9:38your fingers away from the flame a little bit.  They are either plastic or wood, so keep in mind
104. 9:43that these are not heat resistant, but they're  more heat resistant than your fingers are!
105. 9:47You're only going to use this clothespin for heat  fixing. Do not use this while you're staining,
106. 9:52or while you're rinsing the stain, or anything  like that. Because notice that this clothes pin
107. 9:56has a bunch of stain on it, from people who did  not follow this direction -- and let's say that
108. 10:02I was all ready to stain this and -- what color  is this? This might not be the color that I want,
109. 10:07in the stain that I'm about to put on this slide.  So only use the clothespin for heat fixing,
110. 10:12and then don't use it for the rest of the process.  So I have my clothespin here, and I'm going to
111. 10:16pass this through the flame fairly quickly. You're  not going to hold it in there and toast it like
112. 10:22a marshmallow. You're not trying to sterilize  this. Basically this is going to kill most of
113. 10:27your bacteria, and we don't don't actually care  if they're living or dead at this point, we just
114. 10:31want to know what size and shape they are, maybe  what their cell walls are like. But it's going to
115. 10:35help stick them to that slide a little bit more.  Now one thing that I do -- and I amazingly did not
116. 10:40do it just by habit because it's a habit -- when  I was taught this, I was taught you go through
117. 10:46the flame and then you touch it *very*carefully*  to the back of your hand. Again, you don't want
118. 10:50to burn yourself. And then you just do that  over and over until it feels *uncomfortable*.
119. 10:55Not until it's burning and melting through your  glove, but just until it's uncomfortable. You
120. 11:00don't have to do that if you're not comfortable,  most of the time I do it just because it's habit.
121. 11:06Again three or four times, just quick through  the flame, that will heat fix your slide, and
122. 11:11then you can remove your clip, and you're ready  to go. This slide cools off pretty fast. It's a
123. 11:16little warm as I hold it, but it'll cool off and  it's not a problem. There, okay. So now that this
124. 11:24is heat fixed, it's ready to stain. Don't forget  to heat fix, because without that step most of
125. 11:32your bacteria are going to rinse off when you  stain your slide. Also make sure that your slide
126. 11:36is completely dry. Even if there's a little teeny  tiny drop of water you have to wait for it to dry,
127. 11:41okay? I'm sorry, it's just a thing. You will get  used to it. If there's any water that's left on
128. 11:47your slide when you go to heat fix it, that water  heats up and it changes into steam, and if that
129. 11:55has bacteria with it, now there's live bacteria  floating around in the air. Don't want that.
130. 11:59We also don't want it to explode your bacteria,  which can happen if either you heat it too hard
131. 12:05--okay so again, don't hold it in the flame --  I'm actually going to turn the flame off because
132. 12:08it's really warm and loud. You don't want to hold  your slide in the flame for a long time because
133. 12:13that can destroy your bacteria. Or again, if  they're still wet when you try to heat it -- this
134. 12:19is why you shouldn't heat it up to get it to dry  faster -- it has to be dry because you don't want
135. 12:24to explode your bacteria. We're trying to see  what the bacteria look like. If they exploded,
136. 12:28that's not what they're supposed to look like.  Now that this is all heat fixed it's ready to
137. 12:33go on to whatever staining procedure I'm going to  do next. There's a whole bunch of different ones.
138. 12:38Almost all of the time you're going to do this;  you're going to make your bacterial smear before
139. 12:43you do whatever staining procedure is appropriate.  The only time you're not going to heat fix is with
140. 12:49the capsule stain, which I'll show you in a  different video. And that one has a couple of
141. 12:53different components so it'll be pretty obvious  that that one is different. Just to refresh the
142. 12:59steps of preparing your bacterial smear: First,  you're going to add a drop of water -- a *small*
143. 13:04drop of water! Then you're going to add your  bacteria to that water using aseptic technique.
144. 13:10Then you are going to smear them around as much  as possible on your slide, so you get a thin film.
145. 13:15Then you're going to let that film completely air  dry, which is usually the hardest part, waiting
146. 13:21for it to dry. Then, finally, you're going to heat  fix your slide. You're going to pass it through
147. 13:25the flame of your Bunsen burner three or four  times -- hold it with the clothespin if you wish.
148. 13:31And then once you've done all of those steps,  you're ready to move on to the staining process.