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Triple Sugar Iron (TSI) Agar — Transcript

by Catalyst University · 3,255 words · 440 segments · language en · Watch on YouTube

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0:04[Music]
0:08welcome back to microbiology lab my name
0:11is Kevin tow cough please make sure to
0:13like this video and subscribe to my
0:14channel for future videos and
0:16notifications in this video we're gonna
0:19discuss a test that seems on the surface
0:21really complicated but I hope I can
0:23break this down and make it easily
0:25digestible and that is the triple sugar
0:27iron test or I'll just refer to it from
0:29here on out as TSI okay because it's
0:32easier to say now on the surface
0:35TSI seems like there's a lot to memorize
0:38I mean just looking down here we've got
0:40all these results here how do I possibly
0:43memorize all of this but there's really
0:45no memorization or very little
0:47memorization instead you should approach
0:50interpreting the results like a strategy
0:52okay and you're gonna ask yourself three
0:54questions and actually you don't even
0:56have to do them in this order if you
0:57don't want but there's three questions
0:59you're gonna have to ask yourself you
1:01have to look at what I've always called
1:02the color scheme then the gas and then
1:05hydrogen sulfide okay
1:06we'll go into those questions in a
1:08minute right now I'd like to discuss the
1:10theory behind this and then this is a
1:13test where it's gonna be best to do some
1:14practice problems we'll do that next but
1:17let's talk about the theory all right so
1:20first of all the name triple sugar iron
1:23triple sugar would imply there's three
1:25sugars there are okay now one of them I
1:27don't have shown here but it's sucrose
1:29the other two are lactose and glucose
1:32okay there's also pep tones I'll briefly
1:35talk about that in just a minute okay so
1:38the three sugars are gonna be
1:40metabolized by bacteria potentially that
1:43you inoculate in the agar with your
1:45needle okay remember this is one of the
1:48tests where you actually stab the agar
1:50with the needle and as you're pulling
1:53that out you inoculate the slant okay
1:57again you can look in your manual with
1:59respect to that now let's suppose for a
2:02minute we have bacteria that metabolize
2:04lactose right now it turns out that for
2:09whatever reason we won't go into it but
2:11if your bacteria metabolized
2:13tose they're gonna be producing a ton of
2:16acid and I and I I color-coded this the
2:19acid is yellow okay
2:21very similar to what we saw in the
2:23previous video on carbohydrate
2:24fermentation remember yellow was acidic
2:28okay like you see over here on the Left
2:30yellow still acidic here and lactose it
2:33turns out is metabolized or fermented we
2:35could say into a ton of acid a lot of
2:38acid make sure you understand it's a lot
2:40okay now some bacteria do not metabolize
2:45lactose instead they only metabolize
2:47glucose and if they metabolize glucose
2:51they don't produce a lot of acid they
2:54dis a moderate amount okay so not as
2:56much acid as it were with lactose
2:59fermentation so with that just
3:01understand if the bacteria ferment
3:03lactose they make a ton of acid lots of
3:06it lots of yellow but if they only
3:09metabolize glucose it's just a moderate
3:11amount of yellow a moderate amount of
3:13acid okay now hold that in mind and also
3:17embedded in the agar are molecules
3:19called pep tones now we consider all
3:22bacteria to metabolize pep tones and if
3:25they metabolize pep tones they are
3:26converted to peptides and the peptides
3:29are actually going to appear red okay
3:31now first of all let's let's talk about
3:34the color scheme and how we actually
3:36named it this is the hardest part in the
3:39agar slant the top part which is where
3:42the slant is is aptly named the slant
3:44down at the bottom at the base bottom
3:48base B it's the butt okay so that can
3:51help you remember the difference slants
3:52up by the actual slant up here and the
3:55butt is on the base at the bottom BBB
3:58okay so hopefully that makes sense to
3:59you and when we name the color scheme
4:02you can see here we have like an a slash
4:04a here we have an A / K we always
4:08indicate the slant first then you put a
4:11slash and then the butt and in this
4:15context yellow is always the letter A
4:18okay if it's yellow that's a if there's
4:21red like here at the bottom of this agar
4:23red is either given the symbol K
4:26or in our class we use the term ALK so k
4:30or ALK okay and so for example if you
4:34were naming this one right here where my
4:37mouse is you would say a / K or a / ALK
4:44depending on how you want to talk about
4:45that
4:46and the reason is because the slant is
4:48first and the slant is yellow so that's
4:50a and the butt is red so it's K or ALK
4:54so it's a / k as you see right here okay
4:58so hopefully that makes sense to you
5:00over here on the first one this one's
5:02all yellow the slant is yellow so that's
5:05a the butt is yellow that's a as well so
5:08it just be a slash a okay over here on
5:11the far right this one's all red so the
5:14slant is red so that's gonna be a K or
5:16an ALK and at the bottom this but is
5:19also red so it's gonna be k or a okay so
5:22it says k / k or you could put as i have
5:25here a lk / a okay and so hopefully that
5:28color scheme makes sense this next part
5:32i'm gonna go over i just want to discuss
5:33very briefly the theory behind that
5:35you're welcome to ignore it or skip skip
5:38past this part but i just want to make
5:40sure I cover my basis so remember how I
5:43said that if bacteria metabolize lactose
5:45you get a ton of acid a ton of yellow if
5:49you have a case where the entire agar
5:51turns yellow such as a / a that
5:55indicates that you produced a lot of
5:57acid because the whole thing turns
5:59yellow and so this one over here would
6:01be this would indicate that these
6:03bacteria ferment lactose and that's just
6:06because the entire thing turned yellow a
6:08/ a and so that's lactose metabolism
6:12over here on the far right k / k or al k
6:18/a this is all red and so remember I
6:22mentioned that peptone metabolism
6:24created peptides which cause it to turn
6:26red and so over here these bacteria are
6:30only metabolizing pep tones and that's
6:33why this is all red now in any of these
6:37cases we won't see this kind right here
6:40but any case where there's a mixture of
6:41red and yellow and typically we're gonna
6:43see the red at the top and the yellow at
6:45the but this is a mixture this is
6:47indicative of glucose fermentation
6:49remember I said it's a moderate amount
6:51of acid and so we're not getting as much
6:54yellow we're getting some yellow but not
6:56all of it turns yellow so we're gonna
6:58have some red there and the red that's
7:00there such as this red at the slant at
7:02this one Roy Mao says that red is
7:04actually due to peptone metabolism okay
7:07remember at all times these bacteria are
7:11metabolizing pep tones so they're always
7:13producing these peptides the reason that
7:17you see this red here even though
7:18there's some yellow at the butt is
7:20because this is glucose metabolism it's
7:22only producing a moderate amount of acid
7:25a moderate amount of yellow so only some
7:28of the red is able to be masked by the
7:30yellow okay so down here there's still
7:34peptone metabolism but the yellow kind
7:37of overpowers the red and so you don't
7:39see the red down here you only see it at
7:41the top because there's only enough
7:43yellow to cover what's at the bud okay
7:46over here on the far left this a /a case
7:49it is fermenting lactose it's also
7:53metabolizing pep tones so why isn't any
7:56of it red well yellow in this test
7:58always masks red so there is peptone
8:02metabolism here but you can't see it
8:04because the yellow covers up the red in
8:07other words it overpowers the red okay
8:09so always peptone metabolism when it's
8:12all yellow such as these first four
8:14right here all yellow that's lactose
8:16metabolism all the peptone metabolism is
8:19being covered up over powered you don't
8:21see it when you have a mixture of red
8:24and yellow such as from here the fifth
8:25one to this one I think it's the ninth
8:27one that means glucose fermentation
8:30because you're producing some yellow
8:33some acid but not enough to cover up all
8:36the peptide production okay or peptone
8:38metabolism over here on the far right
8:41this one that's all red
8:42that's only peptone metabolism so
8:45there's no acid production whatsoever so
8:47that's why it's all red okay again I'm
8:50not gonna really hold you super
8:52responsible for that
8:53the exam but I just wanted to make sure
8:55to cover the basis on it so hopefully
8:58now you understand how to classify the
9:00color scheme that's the first thing you
9:02ask the second thing this is easy is
9:05there gas so the way the gas manifests
9:08itself it manifests itself in two ways
9:10one ways you can see these little
9:12bubbles right here that are inside the
9:14agar just somewhere inside here they
9:17will probably you'll probably see them
9:19more as inconsistencies with whom the
9:20agar they're fairly easily recognizable
9:23but they're little pockets of space
9:25inside the agar itself okay now what
9:28I've seen actually most of the time is
9:30actually the gas production occurs at
9:32the bottom of the agar okay and actually
9:35what it'll do is it'll actually lift the
9:37agar actually off of the bottom of the
9:39test tube like a rocket I'll skip to the
9:42examples just for a second just to show
9:44you it's actually this third one what we
9:46see right here notice that the bottom of
9:48the agar has actually been lifted off of
9:50the bottom of the test tube and that
9:52physical space right there is the gas it
9:55actually shoots it off like a rocket so
9:56to speak that's normally how I've seen
9:59the gas but you can see these as these
10:00little bubbles inside the agar itself
10:03okay and if there's gas you just kind of
10:07tack that on so for example this one
10:09right here the second one if we look at
10:12the color scheme first it's obviously a
10:15slash a because the slant is yellow a
10:18the book is yellow a so a slash a but
10:22then there's also gas so we just kind of
10:24tack on everything after that it's a
10:27slash a then we put gas so usually what
10:30you'll see is a slash a and then they'll
10:32put a comma or a semicolon and then gas
10:35and sometimes they'll just put a G for
10:37gas I don't care how you do it as long
10:39as you indicate a slash a and gas
10:42alright and we got another one right
10:44here let's look at this this color
10:47scheme would be K / a or a lk / a
10:50because the slant is red so K or ALK the
10:55butt is yellow a so that's where you get
10:57this k / a and then clearly there's some
11:00gas in here so you just tack on a gas
11:02you just keep tacking them on okay
11:05the second question that one's pretty
11:07simple the third one is also pretty
11:09simple this is hydrogen sulfide so
11:11hydrogen sulfide has the chemical
11:13formula h2s and when you're looking at
11:18the agar sometimes you'll see what looks
11:21like a black precipitate okay it'll
11:24actually look more similar to what you
11:25see right here on number four on the
11:26examples this black precipitate and the
11:30reason that forms is the iron in TSI the
11:34iron adèle actually be ferric iron and
11:37whenever the iron reacts with hydrogen
11:40sulfide it produces a compound called
11:43ferrous sulfide okay and the ferrous
11:46sulfide is the black precipitate which
11:49is black now normally the black
11:52precipitate will be found towards the
11:54bottom of the tube either you'll see it
11:56like this or sometimes the black will
11:59actually extend all the way down to the
12:02bottom of the tube but the point is is
12:04that the the black precipitate will be
12:06near the bottom okay usually about the
12:08bottom two-thirds as I've seen okay and
12:10that black precipitate indicates that
12:13you do have hydrogen sulfide so let's
12:16look at this example right here this one
12:20let's look at the color scheme first
12:22well this one it's land is yellow that's
12:24a the but is also yellow a so that's
12:27where you get this a slash a and then
12:30there is black precipitates so I can
12:32just tack on a hydrogen sulfide now for
12:34me I don't care if you write out this
12:35whole the whole word hydrogen sulfide or
12:38you can just put h2s that's fine okay
12:40but again you're just adding on each
12:42thing okay here's a more complicated one
12:45here's the the fourth one the color
12:48scheme is still a slash a because the
12:50slant is yellow and the butt is yellow
12:52there's also gas because I have these
12:54pockets so gas and then there's also
12:57black precipitate so h2s or hydrogen
13:00sulfide so this one actually it's answer
13:02has three components but again you don't
13:04need to memorize it you just look at
13:06these three things separately all right
13:09and then let's look at this one listed
13:12is number three right here I don't know
13:13why I have that three there but the
13:15color scheme is k /a or al k /a the
13:19reason for that the
13:20slant is red the butt is yellow so k /a
13:23and then this clearly has some black
13:26precipitates so you just tack on
13:28hydrogen sulfide or h2s alright so again
13:32hopefully this process makes sense again
13:36I wouldn't worry so much about studying
13:39all this stuff
13:41I would mainly focus on how to interpret
13:43the results because that's the more the
13:46more practical part and that's the part
13:47you'll actually see on the exam now
13:50let's actually look at some practice
13:51problems let's you know work with this
13:53let's look at number one right here how
13:56would you classify this TSI reaction
13:59well it appears to me that it's all red
14:01the slant is red and the butt is red so
14:06that to me says k / K or if you're doing
14:10it the way that we do it at our
14:11university it's al k /a okay either one
14:14of those reactions if you want to put k
14:16/ k that's fine okay and actually just
14:20for reference this color scheme
14:22indicates peptone metabolism if you were
14:25asked that because it's all red you
14:27don't have any acid production no yellow
14:30production to mask any of the red so
14:32this is only peptone metabolism in fact
14:35i can go ahead and put only there let me
14:36put that in caps but can actually spell
14:39okay there we go
14:40only peptone metabolism all right now
14:44let's look at the second example right
14:46here here we have on the slant we've got
14:48red and the butt is clearly yellow
14:51alright so the slant would be K or ALK
14:57and the butt would be yellow so a so k /
15:01a or ALK / a now if you were asked which
15:06sugar is metabolized here we only have
15:09some yellow it's not all yellow so
15:12therefore this is actually glucose
15:14metabolism because we're only producing
15:16a moderate amount of acid just a
15:18moderate amount of yellow okay so this
15:21would be glucose metabolism for number
15:23two all right what about number three
15:26now this one's all yellow okay that's
15:28the colors sland is yellow but is yellow
15:32so that's clearly a /
15:33and there's gas down here at the bottom
15:37now again it looks to me like you
15:39actually it's hard to tell with just
15:41with this low resolution picture but you
15:44can kind of see relative to so these
15:46others over here there's some
15:47inconsistencies within the agar I would
15:49actually have to go in and look at it in
15:51person these might actually be little
15:53gas bubbles within the agar but clearly
15:56there's a gas a giant gas bubble down
15:58here where it actually was so massive so
16:01much pressure it lifted the agar right
16:03off the base of the test tube that is
16:05clearly gas and so our answer would be a
16:09slash a and we can put kama gas we can
16:12put semi colon gas or just a g4 again
16:15however you want to do it that's fine
16:16with me but this is typically the way we
16:18do it and this a / a since it's all
16:22yellow indicates we got a ton of acid so
16:25therefore that's lactose metabolism but
16:28we also have gas production all right
16:33now number four here let's look at the
16:36color scheme now there is some black
16:39hiding some of it but we can see red at
16:41the top at the slant and yellow at the
16:43bottom at the butt so this color scheme
16:46would be K / a or ALK / a because the
16:50slant is red k the bud is yellow a but
16:54we have this black precipitate right
16:55here so that's going to be indicative of
16:58h2s or hydrogen sulfide and so if I
17:01wanted to write this out as an answer I
17:03would put K / a comma or semicolon h2s
17:06alright now this is indicative of
17:10glucose metabolism similar to number two
17:12because we only have some yellow it's
17:14not all yellow so it's glucose
17:17metabolism now the the black precipitate
17:21means we either have sulfate reduction
17:23or hydrogen sulfide production
17:25technically these two terms mean the
17:27same thing because in order to produce
17:30hydrogen sulfide the process is actually
17:33called sulfate reduction in fact these
17:35would actually be considered sulfate
17:37reducing bacteria the probably depends
17:40on your course as to which term you use
17:42but this sulfate reduction just note is
17:45the same thing as hydrogen sulfide
17:46production
17:47where I teach we we determine hydrogen
17:49sulfide production but hopefully these
17:52examples give you a good understanding
17:54of triple sugar iron agar and just make
17:58sure that at least for the purpose of my
17:59class you majorly focus on interpreting
18:02was the results there's a few other
18:06things to understand very minor things
18:08such as the pH indicator for this test
18:10is going to be phenol red you should
18:13know that in fact that's also the pH
18:15indicator for carbohydrate fermentation
18:17and then also urease which we're going
18:19to cover in the next video but please
18:21make sure that you at the very least
18:24make sure you've got how to interpret
18:26the results
18:27take it stepwise a strategy color scheme
18:31first then ask if there's gas and then
18:34ask if there's hydrogen sulfide don't
18:36memorize all of these cases don't do it
18:39it'll be way too hard learn the strategy
18:43of how to do this and hopefully it makes
18:45sense so please make sure to like this
18:47video and subscribe to my channel and in
18:49the next video we're gonna discuss the
18:51urease test see you then

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