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Sesión Practica 3- Micología :ESTUDIO MACROSCÓPICO Y MICROSCÓPICO DE HONGOS AMBIENTALES Y LEVADURAS — Transcript

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0:00You can see it, everyone.
0:06Yes,
0:08yes, yes. Ready? Okay. So, look, today
0:12we are going to perform the macroscopic
0:14and microscopic identification of
0:16environmental fungi and yeasts, right?
0:19If last week we saw how we can perform
0:21isolation, which can be done on
0:23different media, right? since they were
0:27placed in incubation at room
0:29temperature for 3 to 5 days, or
0:31depending, since these are
0:32environmental fungi, for 3 to 5 days,
0:35we are also going to see in this case
0:37that we will observe fungi and yeasts
0:39on these plates, right? We already said
0:43that fungi have a different appearance
0:45than yeasts; yeasts look a lot like
0:47bacteria in this case, right? So, the
0:51objective of this lab is to perform a
0:53macroscopic and microscopic study of
0:55molds and yeasts for their
0:57identification. As a specific objective
1:03, we are going to see that you will
1:05apply inoculation techniques on culture
1:07media for the growth of these fungi,
1:09right? How do we...let's see, back
1:15again. So, apply inoculation techniques
1:18on culture media for the growth of the
1:20fungi, right? And therefore, we are
1:22going to describe the macroscopic and
1:24microscopic characteristics of
1:26filamentous fungi and yeasts. Why?
1:28Because in this case, they have certain
1:30characteristics, right? Just as we saw
1:34with bacteria, they were round, lobate,
1:36rhizoid; we also have to describe both
1:39the molds and yeasts, right? So, we
1:42must also differentiate the macroscopic
1:45and microscopic characteristics of
1:47filamentous fungi and yeasts, right?
1:49Because while it is true that we can
1:51differentiate them macroscopically, we
1:53can also make the distinction
1:55microscopically in this case, right?
1:59And apply study techniques for the
2:01microscopic examination of fungi,
2:02because there are different types of
2:04ways or techniques we can use to
2:06identify them, right? And among these
2:10techniques, the microculture technique
2:12is used for the microscopic study of
2:14fungi. Therefore, the competency to
2:16develop is to identify fungi according
2:18to the macroscopic and microscopic
2:20characteristics of the fungi, following
2:23established procedures, right? So,
2:27regarding the identification part, we
2:29are going to do it in the case of
2:31Guaroto, but Guaroto isn't here. And
2:35why are you joining? Wait a second. I'm
2:37going to stop sharing because I'm...I'm
2:39getting in there. Guaroto, where is
2:41Guaroto? Guaroto, aren't you from this
2:44...from this group? I mean, you are,
2:46let's see, Piero Alexandro Guaroto,
2:51please, leave the leave the room, leave
2:58the room, Piero. Okay,
3:00professor, excuse me, my classmate
3:02Aparc, Aparcana just entered. Yes, okay
3:05. Now yes, Aparcana. Yes, it's just
3:07that I thought, uh, he is working and
3:09Guaroto entered, and I accepted him and
3:11I didn't see the other one. Yes. Okay,
3:13now he is in. But you are arriving late
3:16, Marañón. Ah,
3:18professor, excuse me, I am trying to
3:20enter, but my internet is a bit poor. I
3:23don't know if you can count me in the
3:24attendance. It is the first time that I
3:26am going to miss
3:26attendance. I will let you in, but you
3:28will be marked late. That that always
3:31goes to your to your to your to your
3:32grade that
3:35yes.
3:36Yes, yes,
3:37yes. I am marking you as present. I am
3:39marking you as present, but I am
3:41telling you that those are tardies. The
3:43tardies.
3:43No, you are not marking me as absent.
3:45No, I am not marking you as absent.
3:48Okay, okay. Okay. So, we are going to
3:54look at the identification in this case
3:56of fungi, which is studied through
3:58macroscopic study, right? When I am
4:01talking about fungal identification, in
4:03this case I am I am uh referring to
4:05molds, to those that are filamentous,
4:07right? So, what is it that has to be
4:10studied here? The shape and size of the
4:14mold, color of the surface and the
4:16reverse as well, because, I mean, we
4:18are not just going to study the
4:20characteristics I am seeing, I have to
4:22turn the plate and then I have to study
4:24the reverse. Why? Because it happens
4:29sometimes that on the obverse, as we
4:31call it, when I am looking at it, I can
4:33see a uh a granular surface of black
4:35color, but I turn the plate over and I
4:37will see in this case a white or yellow
4:40color, right? Another color, it could
4:43be red too, right? Or different types
4:45of colors. So, those characteristics
4:48that we have to put down, we have to
4:50note them because according to the
4:52taxonomic keys that is what is being
4:54asked of us, right? We can also see if
4:58the fungus has the capacity to diffuse
5:00the pigment, right? Which means that
5:02the fungus has this characteristic of
5:04presenting these secondary metabolites
5:06that I told you about yesterday, but by
5:08by producing these secondary
5:10metabolites it will produce a pigment
5:12that will diffuse into the medium.
5:13Therefore, sometimes we can see in this
5:16case that the medium turns somewhat
5:18brownish, yellowish, or reddish. So,
5:20this means it has the ability to
5:22produce, in this case, pigments, right?
5:26Regarding the texture, in this case,
5:28you have to observe if it is chalky,
5:30looking like plaster, like soil, right?
5:33Like a little bit of soil, like some
5:34dust that has been added there. It
5:36seems like they are little granules,
5:38right? Sometimes they look like, uh,
5:40little granules like pepper, right?
5:42Sometimes we can see that they present
5:44fine hairs or otherwise it looks like
5:46wool, usually coiled, right? Sometimes
5:48they can also be creamy or velvety. In
5:51this case, we also have to see, in this
5:53case, if we observe the plate, right?
5:57And we look at it with the naked eye,
5:59we can see if that colony is raised or
6:02if the colony is completely flat, or
6:04perhaps it only has a central elevation
6:06. Do you remember that we, uh, also
6:10worked on that part with bacteria,
6:12right? So, here it is very similar, it
6:15is studied, except that in this case we
6:17have to, uh, see the type of fungus,
6:19right? And also, in this case, apart
6:22from the texture we can see, the fungus
6:25has aspects it can present. If you
6:27observe and if you have seen this type
6:29of fungus, sometimes you can say, you
6:31can see the colony is somewhat rounded,
6:33which will form some small folds, right
6:36? Or otherwise they will form, in this
6:39case, like circumferences, right? Or it
6:42could be that in this case there are
6:44elevations and depressions, which looks
6:46cerebriform like a brain, or it could
6:48be crateriform when there is an
6:50elevation in the center, right? As for
6:55the consistency, we cannot determine
6:57that just by observation. In this case,
7:01we have to use a mycological loop to
7:03touch the colony to see if it is hard,
7:05soft, if it is firm, or if it comes off
7:08like a thin membrane, right? Why?
7:10Because we do have to check the type of
7:12consistency, right? Sometimes there are
7:17even fungi that are so hard that, for
7:19us to observe them, we have to make a
7:21small cut and place it on the slides
7:22for us to see, because it is very stuck
7:25to the culture medium in this case.
7:29Apart from that, it is also important
7:31in the macroscopic study that during
7:33the time you carry out the incubation,
7:35you have to see, uh, how it has grown,
7:38right? Sometimes by the third day you
7:41will see it completely filling the
7:42plate, right? Sometimes Rhizopus muor
7:46has that property of immediately
7:48filling the plate, but there are others
7:50that you will see on the fifth day and
7:52you will see the colony is still very
7:54small, right? So, these are the
7:57macroscopic studies that can be
7:59performed, right? So, here you can see
8:02the surface. I was telling you that you
8:05have to work on the color of the
8:07surface and the reverse, right? Here we
8:09are seeing the obverse, right? It is
8:11the surface, as you can see, right?
8:13Here we are going to see it somewhat
8:15powdery, right? Here in this case as
8:18well, look, somewhat velvety, right?
8:21Here, somewhat creamy. You will see
8:23this one here, for instance, it can be
8:25hairy, as you see it, like a type of
8:28hair, right? And apart from that, you
8:32can also see its appearance in this
8:33case sometimes. Look here, a fold is
8:37formed here. Here there is a central
8:40uplift. If you observe here, look, it
8:43is forming circles, right? Look, this
8:46is like a brain type. So, first the
8:49color, later we will see the other, but
8:51I am telling you that you can also
8:53observe all that part there, right? So,
8:56this is an example of how we are going
8:58to see the obverse and the reverse,
9:00right? If you observe here on the
9:02obverse, you will see what color, sort
9:05of white, right? But if you flip it
9:07over, generally, what will we see?
9:09Actually, sort of yellowish, right? So,
9:13that is what you have to do to study
9:15first, the macroscopic characteristics
9:17of the colony, right? I told you that
9:20it has the capacity to produce pigment.
9:22Look at the sort of orange pigment it
9:24shows here. Here also somewhat red, if
9:26you see, look, pink that they might be
9:29giving, that is to say, the fungus has
9:31the capacity to diffuse in this case
9:33the type of pigment, right? And this is
9:37a secondary metabolite that they are
9:39going to diffuse. I even told you that
9:42this diffusion of pigment, which I
9:44forgot to mention yesterday in the
9:46metabolism part, nowadays these
9:48pigments are being used as dyes, right?
9:51At a student congress, a project we did
9:53with the students took second place,
9:55right? Right in the course, right?
9:58Where they isolated fungi, fungi, and
10:00saw fungi that had the capacity to
10:02produce pigment. So, these fungi that
10:05produced pigments, in this case, what
10:07they have done is extract the pigment
10:09from these types of fungi and they dyed
10:12, in this case, some cotton, right? And
10:15so nowadays, it is also being used in
10:17the industrial sector to obtain
10:19pigments. These would be natural
10:22pigments, right? What was missing in
10:24the work is simply, why? Because every
10:26time one can extract a metabolite, in
10:28this case a secondary one, one must
10:30check for toxicity, right? Because
10:32sometimes it can also be toxic, right?
10:34So we have to look at that part. But
10:36the importance of the pigment is also
10:38an important factor, right? There we
10:41can see the texture, which can be
10:43chalky, it can be earthy, it can be
10:45granular, right? They look like little
10:47granules, right? Hairy, woolly, creamy,
10:50velvety. meaning that we have to look
10:53at what texture it has in this case,
10:54right? And these are the aspects I was
10:58telling you about, that if you are
11:00going to see, look, they can produce a
11:02folded aspect. And here, highlight in
11:05this case that when one sees the color,
11:07one cannot only see a single color, but
11:09can see several colors. And so you also
11:12have to describe it, right? As an
11:15example of this colony, you can see
11:17that the color is generally orange in
11:19the central part, around this color, in
11:21this case it will be green, and it ends
11:23, in this case, with a cream border,
11:25right? So that is what you have to do
11:28in this case according to the color, in
11:30the case of the aspect you are going to
11:32see that they form folds, it is folded.
11:34Look, here yes, here also, here you are
11:36going to see that they have three
11:38colors. Look, even four, one here
11:40darker, one lighter, a dark green, and
11:43here around it also a border, right?
11:46And the aspect in this case is radiated
11:48. Look, apart from the fact that they
11:51can be mixtures of two, in this case it
11:53is radiated, but also folds, not folds
11:55very close together like this one here,
11:57but slightly open folds, but generally
11:59the radiated aspect. Look how it is
12:01forming the circumferences, right? This
12:04one here you can see a cerebriform
12:06aspect. This one is also folded, but in
12:09this one it is not cerebriform, but it
12:12is like a crater, right? Look, here you
12:15can also observe this one has a central
12:17elevation. Look, flat here around, but
12:19it has a central elevation. And besides
12:22that, it’s also important, look, this
12:24one is folded, right? This one is also
12:27folded in this case, but look here,
12:29there are some oily spots, right? There
12:32are certain types of fungi that have
12:34these exudates, and this must also be
12:36noted: they present certain types of
12:38exudates that can be yellow, can be
12:40white, can be pink, which look like
12:42little droplets that you have created,
12:44oil droplets that are dispersed, mainly
12:46the type of, and the surface, as you
12:51can see, look, is raised, this one is
12:53flat, this one has a central elevation,
12:55and this one has a generally total
12:57elevation, right? Everything is raised
12:59in this case. Now, once we perform the
13:03macroscopic study, we must perform the
13:06microscopic study, right? So, I have
13:09already done the study, so for the
13:11microscopic study, what do I have to do
13:13? So, I am going to take my sample and
13:16for the microscopic study, I have to
13:18make use, in this case, of the
13:20microscope, right? So there, in the
13:23microscopic study, I have to see, this
13:26is done with—uh, there are two
13:28techniques—that is, the wet mount,
13:30right? Or the Scotch tape technique,
13:33right? Or we will see the microculture
13:36technique. And that will allow us to
13:40observe, in this case, what—and
13:42sometimes we will have to stain with
13:44lactophenol blue. And so, once we make
13:47this preparation, which I will explain
13:50to you later, we will see the type of
13:52hyphae, and that is why we did the
13:53theoretical part, right? In the
13:56theoretical part, we said that fungi
13:59can be septate or coenocytic or
14:00aseptate, right? So you have to note,
14:05you have to look through the microscope
14:07, you have to see the type of hypha to
14:09see if it presents septa or does not
14:10present septa. What else should you
14:14have there? hyphal modifications and
14:20the hyphal modifications that we said
14:22we can see if, in modifications, they
14:25generally form chlamydospores, they
14:27will—they will form a rhizomorph,
14:29right? This is what we are going to
14:32have to see, if it will form a rhizoid,
14:34right? So we have to see if the fungus
14:37presents hyphal modifications or not,
14:39or nodular forms as I told you there,
14:41right? Now, apart from that, one can
14:44observe the type of mycelium. The type
14:46of mycelium, you see if it is
14:48transparent or if it is dematiaceous,
14:50right? We have already said that there
14:53are fungi that won't have the capacity
14:55to produce or display these dark
14:56pigments, and therefore the mycelium
14:58will be transparent. That is why
15:02lactophenol blue is used to be able to
15:04observe this much better, right? And
15:06the dematiaceous ones are generally
15:08dark, to the point that sometimes even
15:10lactophenol blue doesn't stain them,
15:12right? But we will see them as dark,
15:14right? In the case of Alternaria, for
15:16example, we will see that it is a dark
15:18mycelium. What else can we do in the
15:20microscopic study? Type of spores.
15:23Whether it presents sexual spores or
15:25asexual spores, right? We still need to
15:29look at reproduction in this case, and
15:31you are mainly going to learn to
15:33recognize if they are sexual or asexual
15:35spores. What else should we look at?
15:38And this is very important, right? Here
15:41regarding the aspect of the
15:42sporangiophore or conidiophore. Fungi,
15:47as I was telling you, have a vegetative
15:49mycelium that enters the substrate to
15:51feed, but there is a mycelium that
15:53emerges, which is the aerial one, and
15:55there we will have the reproductive
15:57structures, and therefore, in those
15:59reproductive structures, a sporophore
16:01will be produced. This sporophore can
16:06be a sporangiophore or it can be a
16:08conidiophore. When do we call it a
16:11sporangiophore? When this mycelium—
16:15and that's why there is a
16:17differentiation here—usually the
16:19hypha emerges and forms a vesicle, and
16:21inside that vesicle, we will have
16:23spores. That type of sporophore is
16:29called a sporangiophore. And when we
16:34talk about a conidiophore, this hypha
16:36no longer makes that differentiation,
16:38it doesn't form that rounded shape that
16:41we will observe; instead, the conidia
16:43will emerge not enclosed, but
16:44externally. And those conidiophores
16:48will also adopt different types of
16:49shapes depending on how the spores are
16:51generally grouped, right? And so we
16:53will have sporangiophores or
16:55conidiophores. And this is an important
16:57factor because in taxonomic keys, if
16:59you look at an Aspergillus, you see it
17:02looks a bit rounded, and we will see
17:04there are phialides with little spores
17:06that look like a sun, it seems. That is
17:09a conidiophore; the spores are external
17:11. But if you observe, let's say, Mucor,
17:14you will generally see a vesicle that
17:17encloses them, and inside those
17:19vesicles are, in this case, the spores,
17:21right? That is in the drawings in the
17:24taxonomic keys. That is why fungal
17:27taxonomic keys allow us to perform
17:29identification based on these aspects
17:31that fungi produce. Why? Because if you
17:36only look at the type of hyphae, you
17:37won't be able to identify the fungus
17:39for me. If you see hyphal modifications
17:43, well, possibly if you find a rhizoid,
17:45you can say, "Ah, it could be a
17:47Rhizopus or maybe a Rhizomucor, right?"
17:51If you only see that it is transparent
17:53or dematiaceous, you can't tell me what
17:56it is. If it is the type of sexual
17:59spores, many fungi will present the
18:01same sexual spores or the same asexual
18:04spores that are very similar, right?
18:06Although, well, with sexual spores we
18:09can in this case tell the difference,
18:11right? Because there are ascospores,
18:13zygospores, but it doesn't identify it
18:14for me. I can say it is an ascospore,
18:17but I cannot say which fungus it is. No
18:19, I can say it is a zygospore, but I
18:21cannot say which fungus it is. But this
18:24aspect of the sporangiophore can give
18:26me, therefore, an idea of the genus,
18:28right? An indication of the genus. I
18:30can say, this is a Mucor, or this is a
18:33Rhizopus, this is a Rhizomucor. I can
18:36suddenly say this is a Penicillium,
18:38right? This is a Fusarium, right? This
18:41is an Alternaria because of the
18:42characteristics of the conidiophore
18:44that I will see, right? But it is also
18:46important to look at the aspect and
18:47size of the conidia. The aspect and
18:50size will allow me, if I already see
18:52the sporangiophore or conidiophore, the
18:54aspect and size of the conidia can give
18:56me an index of what species it might be
18:59. Why? Because conidia can generally be
19:02different, right? One could say there
19:05are Aspergilli whose conidia can be
19:07small and round, smooth, and others
19:09that will be somewhat spiky. So that
19:11will be in the taxonomic keys as well,
19:13they will tell you Aspergillus
19:15such-and-such will present this, right?
19:19And we will see if they present
19:21specialized structures, right? If they
19:27are going to produce, in this case, the
19:29types of perithecia or apothecia, we
19:31will see that in the microscopic study,
19:33right? So, what do we have to look at?
19:38Type of hypha. Yes, we have already
19:40said, this is a septate hypha. And this
19:42is a coenocytic or non-septate hypha,
19:44right? If you look here through the
19:47microscope, I am only seeing hyphae,
19:49right? And so I am going to see the
19:51septa, right? So I can observe that
19:53this one has a septum. If I look at
19:55this part here, I will see that it
19:57doesn't have septa, right? Even though
19:58it appears to have septa here, right?
20:00Actually, a hypha has just crossed over
20:02here, right? But this is a coenocytic
20:04hypha; it does not present the septum
20:06that in this case generally separates,
20:09like, a compartment. Not here; here it
20:12is completely, in this case, without
20:14circulation. Generally, the cytoplasm
20:18circulates completely, right? If we
20:20look at the type of mycelium, we are
20:22going to have a hyaline type of
20:24mycelium. You can even see here if the
20:26type of mycelium is microsiphonous or
20:28macrosiphonous, right? The
20:29microsiphonous one is thin, whereas the
20:32macrosiphonous one is wide, right? For
20:34example, this is a microsiphonous
20:35mycelium and this is a macrosiphonous
20:37one, but in terms of color, this one is
20:39generally transparent and this one is
20:40dematiaceous. Look at me, you see it is
20:42dark; even here it is no longer
20:44necessary. Well, it is not stained here
20:46, but here you will see that none of
20:48these are stained; they are directly
20:50like this so you can differentiate that
20:52this is generally transparent. Look,
20:54even the spores themselves that you can
20:56see. On the other hand, you put this
20:58here and you can already tell mainly
21:00what it is; sometimes staining is not
21:02necessary, right? However, the
21:03techniques and procedures tell us that
21:05we have to stain with lactophenol blue,
21:07right? Look, and here you are going to
21:09observe, right? You will even see that
21:12the septate mycelium they form with
21:14this is—this is a conidiophore, but
21:16the conidiophore, let's see, look at
21:18how the spores come out in this case,
21:20right? That is what we have to look at.
21:23So, we have to look at the types of
21:25sexual and asexual structures. If you
21:27observe here, there are different types
21:29of structures that produce sexual
21:31spores. The first three that you are
21:34going to see here, which you see here,
21:36are sporangiophores, right? And the
21:38others, and these here, are
21:40conidiophores. Look at this one here,
21:42where is the spore? Internally. Here,
21:45also internally. Here, internally,
21:47right? But here, the spores are
21:49generally on the external part. And you
21:51will see that these sporangiophores are
21:53also different, they are also different
21:55. There are some elongated ones, some
21:57rounded ones, but we have to look at
21:58that. But here you can differentiate.
22:01This is a sporangiophore and this is a
22:03conidiophore. These are the ones that
22:06will produce asexual spores, right? And
22:08the sexual spores here are also
22:10unicellular, they can be multicellular
22:12depending on the fungus and the
22:14taxonomy. If we look at the bottom part
22:17, they are actually sexual spores,
22:19right? In this case, it is an oogonium,
22:23this is a zoospore, right? This is an
22:26oogonium that you will see will form
22:28its spore here. Here this is an
22:31aeciospore, this is a basidiospore.
22:34Look here that they are—sorry, this
22:36is an ascospore that is inside an ascus
22:38and here we will see the ascospores
22:40that are internal and this is a
22:42basidiospore that are external, right?
22:45This is the basidiospore, right? And
22:47here in this case it is born from a
22:49basidium, right? So there we can, in
22:52this case, determine. So there we have,
22:55look, the different types of structures
22:57that we can observe, right? If I ask
23:00you there, are these conidiophores or
23:02sporangiophores? Hm. What do you think
23:08there? Are there internal spores or are
23:12there external spores? And there are
23:17also
23:19I can't hear you. Sure. Let's see, a
23:22bit slower so I can hear you. Well, I
23:25observe external and internal ones. It
23:28is also observed.
23:29Let's see, which one would be internal?
23:31The one here, the one here, the one
23:33here. Let's see, 1, 2, 3. Is it in the
23:36first row or in the second row that is
23:37internal? Let's see,
23:38in the second row there is an internal
23:40one and in the middle of the first row
23:42is the internal one. There are also
23:44four internal ones.
23:45It is internal, this is one, two, three
23:48, four. No, if you observe there, they
23:50are all spores. They are all spores,
23:53look. Ah, they are spores and they are
23:56external spores. Let's see here. Let's
23:59let's change here. Look, this is a—
24:03here these are spores. Apparently it
24:06seems like there is something inside,
24:08right? But these are macroconidia. It
24:12is a macroconidium that has this
24:14elongated shape, but it is in clusters.
24:17This is also a macroconidium and this
24:19that you observe here is the
24:21conidiophore. Look, it's the
24:23conidiophore. From this conidiophore
24:26one, two, three conidia will emerge and
24:28these are macroconidia. This is the
24:31same, it's a macroconidium. That is,
24:34each one of these that forms a cluster
24:36is a macroconidium. This is from
24:37Fusarium, for instance, right? This is
24:40a macroconidium too. Look,
24:41macroconidium. Which one is the
24:43microconidium? This one here, these
24:45little dots. This fungus, for example,
24:47presents—there are fungi that present
24:49macroconidia and microconidia. This one
24:52here is also microconidia and this is
24:54the sporophore, the conidiophore, the
24:56conidiophore itself. So, this
24:59characteristic that you have to look
25:01for is important for genus
25:02identification. I’ve shown you this
25:06here so you can see that these are
25:07asexual spores. Asexual spores can be
25:11microconidia, or they can be
25:13macroconidia. In this case, there are
25:15fungi that will present both micro and
25:17macroconidia. There are others that
25:19only present macroconidia and sometimes
25:21don’t present microconidia, right? So
25:24I also have those characteristics. But
25:26these are asexual spores, right? And
25:29these are sexual spores. I was telling
25:32you that fungi can produce ascospores.
25:34So, if you look here, this is an ascus
25:36and inside this ascus we will have the
25:38ascospores, right? So these are asci.
25:41If you look here, look, asci. And
25:43inside here you will see the ascospores
25:45. This is sexual reproduction. The
25:50basidiospore also has a basidium here
25:52and its sterigmata, and inside these
25:54sterigmata you will see the
25:55basidiospores coming out. This is a
25:58sexual spore. Here I will also observe
26:01it. Look at the basidium here and the
26:03sterigmata are here, and here are the
26:05basidiospores. This quantity of spores,
26:07how the basidium type is formed, also
26:10helps me in species identification,
26:12right? For example, Agaricus bisporus,
26:15which is the mushroom, has the ability
26:17to produce this type of basidium with
26:20only two basidiospores; there are
26:22others, generally there are four
26:24basidiospores, but there are some that
26:26can present two in this case, right,
26:29Professor?
26:29And this is the zygospore. Yes, I’m
26:31listening.
26:32Is there a possibility of finding
26:34basidiospores and ascospores at the
26:36same time?
26:36No, no, no.
26:38Only
26:39one of
26:39it’s one type of sexual reproduction.
26:42For example, a fungus can form oospores
26:45, basidiospores, ascospores, or
26:47zygospores, but not both types. That is
26:50why they have been classified; that is
26:52why there is this classification in
26:53this case. So there you can observe
26:56this is a zygospore, for instance here
26:58is the zygospore, right? And this is
27:01the way it originates, right? In the
27:03reproduction class we will have next
27:05week, we will see it. And this is the
27:08appearance of the sporangiophores and
27:10the conidiophores. Look at the
27:12sporangiophores. Look, here is the
27:14hypha, and there is also a
27:15differentiation here, and here is a sac
27:18, and inside this sac we will have
27:20spores; these are called
27:21sporangiophores and these are
27:23conidiophores. Look here, right? Here
27:27we generally have the hypha where we
27:29will see phialides, and here are the
27:31conidia. These conidia that are in this
27:35case are also asexual. Yes, asexual,
27:38look, asexual this whole part, right?
27:41So, it is important that we are able to
27:44know this. Why? Because in taxonomic
27:46keys these diagrams will appear, and
27:48you have to look, even to say, "It's a
27:51Penicillium, but what type of
27:52Penicillium is it?" There are different
27:55species. So, you will have to see if
27:57they are monoverticillate,
27:58diverticillate, due to these whorls
28:00that they will present. No, there are
28:02some that are just long, others are
28:04more leafy. It is the same as
28:06Aspergillus, right? Aspergillus niger,
28:08for instance, is like a like a like a
28:11sun, right? Why? Because these
28:13phialides will surround the entire
28:14vesicle. On the other hand, Aspergillus
28:17fumigatus only has like a little tuft
28:19here, and that will allow me to
28:20differentiate the types of Aspergillus,
28:22right? That is why I told you that here
28:24one must have a lot of expertise
28:26generally to be able to identify them.
28:27You have to be at the microscope
28:29looking to be able to work, right? And
28:31these are the aspects of the colonies,
28:33right? They are oval, rounded,
28:35spiculate, right? They take the form
28:38that they are grouped in chains, others
28:40are all alone. So, this also has to be
28:43worked on. That is why in mycology one
28:46must use uh these these these these
28:48little rulers that go on the microscope
28:50, right? For us to be able to generally
28:55measure the sizes of the spores, and
28:57even the size in this case of the hypha
28:59to say if it is microsiphonous or
29:01macrosiphonous. I told you that those
29:05greater than 1 micrometer are generally
29:07macrosiphonous and smaller ones are
29:09microsiphonous, right? So, that will
29:12allow us to see the size that the
29:13conidia or in this case the hypha can
29:16generally have, right? Well, and these
29:18are aspects and sizes of ascospores,
29:20right? These are reproductions. Look,
29:22even the ascospores, the ascospores
29:25generally uh we will see that it is
29:27inside an ascus, right? Let's add more
29:30here. Let's No, if we are going to
29:34assume that we are going to have that
29:36this is an ascospore, right? This is an
29:39ascospore, and inside these ascospores
29:42they can generally come like this, so
29:44to speak, right? And these are the asci
29:46, this is an ascus, and inside this
29:48there can be different ascospores,
29:50right? Let's assume it's this type, LV,
29:53look, right? So inside this, they are
29:55going to come like this. The ascus has
29:58to break open so that they can, so that
30:00the fungus can reproduce in this case,
30:02because fungi reproduce by spores,
30:04right? So they can come like that, or
30:06they can be inside this ascus, and they
30:08have morphologies like Saturn-shaped,
30:11lemon-shaped, or urchin-shaped, but
30:13it's inside the ascus. These ones here
30:15that you are seeing in this case are
30:17ascospores, right? These are ascospores
30:20, the different forms of ascospores
30:22that can be determined. And this is
30:24when this ascus, we are generally going
30:26to break it, right? And these
30:28specialized structures I was telling
30:30you about can be, uh, they produce
30:32pseudoparenchyma, it can be a
30:34prosenchyma, it can present a rhizoid,
30:36a rhizomorph, or a haustorium, right?
30:39An appressorium. You have to determine
30:41that part, right? For example, there is
30:44a haustorium, right? Which is here
30:46inside, right? You make a cross-section
30:48, and these are mycelial aggregations.
30:51What happens is there are, uh, the
30:53hyphae generally, or also the
30:55conidiophores, right? Or the spores
30:59generally, uh, sometimes asexual ones
31:01can also, uh, aggregate, right? As a
31:05way for these spores to protect
31:07themselves, and they form sclerotia,
31:09right? Which is, is, is the filament,
31:13in this case, the union of fungi. Do
31:16you remember when we did, uh, uh, the
31:19sclerotia or sclerotium? Those are
31:22macroscopic, right? What it means is
31:24the union of hyphae, right? And it
31:27generally forms like small nodules, but
31:29something more that one can see, right?
31:32It is not, it is not, it is not that we
31:34need a microscope. This here we can
31:36observe, right? Uh, we can see that
31:39sometimes the conidiophores that we
31:40have seen here can join together, right
31:42? And form a coremia, right? So it is
31:46the, the coremia is the union of
31:48conidiophores, right? Or it could be
31:51that the spores, in this case asexual
31:53ones, can be placed inside a pycnidium,
31:56right? And they are generally protected
31:58there. Well, this is a pycnidium.
32:01Generally, if you see a pycnidium,
32:05Right? Now, in the case of yeasts, I
32:08was generally telling you that it is
32:10different in this case than for...than
32:12for...than for bacteria, right? Oh,
32:16sorry, different from molds, right?
32:18They are very similar to bacteria,
32:20right? We can determine the shape, we
32:23can determine the appearance, what
32:25elevation, what margin or what pigment,
32:27right? The shape is rounded, sometimes
32:30medium or irregular, it can be, right?
32:32What is the appearance? Right? They can
32:35be creamy in appearance, right? Uh, if
32:38it is flat, crateriform, or concave,
32:41right? Uh, the margin, right? If it has
32:44an entire margin, an undulate margin,
32:47and if it has the capacity in this case
32:49to produce pigments or not. So there we
32:52can see microscopic characteristics,
32:54right? As I was telling you, yeasts are
32:57unicellular, generally with creamy
32:59growth on culture media, very similar
33:01to bacteria. Reproduction can also be
33:04sexual or asexual, and asexual
33:06reproduction generally occurs by
33:08budding, right? So you will see that,
33:11uh, it will form a little bud. It is a,
33:13uh, that is a blastospore, right? In
33:17this case, but it can also have sexual
33:19reproduction which will be by
33:21ascospores or basidiospores, right? So
33:25we will also see that for, yes, yeasts
33:28generally, we will see here, uh, here
33:30that sometimes the yeasts that have
33:32this rounded shape, uh, when there is
33:34sexual reproduction, they will form
33:37ascospores inside. Yes, here they will
33:41form inside this. Why? Because this is
33:45the ascus, and inside this we will see
33:47ascospores, right? So there we will see
33:50if it is going to form or not form
33:52ascospores, right? So that is what we
33:55have to look at in this case, right?
33:57But look here, let's see for a moment,
34:03right? If you observe here, look here,
34:05these are ascospores. Here they are.
34:07Here, look, this one is inside. Yes, it
34:10is forming ascospores. Here it is also
34:12forming ascospores. And if you observe,
34:15the shapes are different, right?
34:16Elongated, rounded; for instance, this
34:19one here is forming, let's say,
34:21pseudomycelium, right? These are
34:23examples of yeasts that we might have,
34:25right? Look, there are the different
34:27elongated shapes. Look, here are
34:29ascospores. These are ascospores. Look
34:32inside here. This one is budding. Look,
34:34here is its little bud. It is sexual
34:36reproduction. Or, in a yeast, both
34:38sexual and asexual reproduction can
34:40occur. Ah, that’s right, but not an
34:42asexual reproduction where we will have
34:44a basidiospore or ascospore, right? It
34:46is either an ascospore or a
34:47basidiospore. Yes, here too, look, here
34:50you will see, they will even generally
34:52form a certain type of nodule. Look
34:55over here, they can be elongated here.
34:58Look, this is almost not—it is also
35:00elongated, they are yeasts, right? This
35:03is a pseudomycelium, for instance, look
35:05, a pseudomycelium, and these over here
35:08that you see are also chlamydospores,
35:10right? There are yeasts that will
35:12produce chlamydospores as well. So, you
35:16have to recognize that morphology,
35:18right? Now, apart from that, both molds
35:21and yeasts can undergo biochemical
35:24study, right? I told you that for the
35:28most part, what we generally observe is
35:30the microscopic and macroscopic study,
35:33but there are some that do require it,
35:35mainly yeasts, just as bacteria require
35:37a biochemical study in this case, but
35:39molds do too. So, the use of—it is a
35:45—it is a biochemical study that is
35:48used, right? Here, it is used the same
35:51way as it has been used for bacteria in
35:53Christensen’s urea agar, where we
35:55will see that the culture medium has
35:58urea and a pH indicator, right? And
36:02when we inoculate it, throughout the
36:04incubation period, if the microorganism
36:06is urease-positive, because there are
36:08fungi that will have this urease enzyme
36:11. Then, the final products will be
36:16basic, right? And since it has a color
36:19indicator there, we will generally see
36:21that it will turn—in this case, what
36:23color does it turn?—red, right? It is
36:26urease-positive. Another medium that is
36:30also used in this case is nitrate
36:32reduction. So, here you can use a
36:35nitrate broth or a nitrate agar, right?
36:38And if the microorganism has nitrate
36:41reductase, it will reduce nitrate to
36:43nitrite, and for that, reagent A and
36:45reagent B are used, right? Review that
36:48because we have already done it in
36:50microbiology. Eh, now there are other
36:53tests that in this case are used for
36:55molds—sorry, for yeasts, right? Which
36:59are the auxanogram and the zymogram.
37:04The auxanogram means the fungus's need
37:06to use carbon or nitrogen sources,
37:08right? So, look up what an auxanogram
37:14is and what a zymogram is, okay? I will
37:18ask you about it in the practical
37:19session next class. For an auxanogram,
37:24I will ask what carbon or nitrogen
37:26sources a fungus generally needs for
37:28its growth. On the other hand, a
37:34zymogram generally involves the
37:36utilization of carbohydrates with the
37:38formation of acid, so a medium is used
37:40where we determine if it is utilized or
37:43not, right? Let's use an example that
37:49we might have. Let's suppose, right?
37:56Let's assume we have a plate—well,
37:58not a petri dish, there has to be a
38:00special medium here, sometimes agar is
38:03used, right? And for an auxanogram,
38:08let's say I want to know, and here I
38:10will have my little disks, my
38:12carbohydrate disks, which could be
38:14glucose, lactose, sucrose, right? This
38:20is a medium that is free of carbon
38:22sources, right? And so, what you will
38:26do is take each little disk—it could
38:29be glucose, lactose, or sucrose, right?
38:34These disks are soaked, right? And this
38:38is the medium. And then, what you will
38:41do is inoculate the yeast you want to
38:43evaluate to see what it needs for its
38:45growth. Then, you will place the disks
38:49just like an antibiogram. An
38:53antibiogram. You place one disk here,
38:56one here, and one here. Right, I'm just
39:01giving an example; I can't draw well
39:03with this thing, but anyway. So, here
39:06we have the sugars, right? And I take
39:09it to incubate. If the microorganism
39:12needs—let's assume this one is
39:14lactose, this is sucrose, this is, well
39:17, maltose, let's put that here. If
39:25around these, say, you see growth
39:27forming—meaning you see the
39:29microorganism has moved here and grown
39:31—it means this microorganism needs
39:33lactose as a carbon source, right? And
39:39that is how it is tested, right? It
39:42could be that a yeast can use two
39:44carbon sources, right? But if it hasn't
39:48grown, this is called a lack of growth.
39:51Notice that when we talk about an
39:52antibiogram, we look for an inhibition
39:54zone where we didn't see growth, right?
39:58On the other hand, a growth halo forms
40:00here, meaning that around the disk,
40:02since the yeast needs that carbohydrate
40:04to grow, the yeast will grow around
40:06that little piece of filter paper
40:08soaked in sucrose. So, the auxanogram
40:12is used to demonstrate, in this case,
40:14the carbon and nitrogen requirements
40:17that the yeast needs to grow, right?
40:20And the zymogram is for fermentation,
40:22right? Here you can use a medium that
40:26has, let's suppose, glucose. Glucose,
40:31right? There are liquid media and there
40:33are also solid media, right? Glucose is
40:35used, and it also has a pH indicator
40:38here. If the microorganism uses the
40:41glucose, the final product will be
40:43acidic, and therefore, this medium will
40:46turn yellow, right? Always depending on
40:48which indicator is used, right? That is
40:50one example that can be done. And also
40:52the vitamin requirements. I told you
40:54yesterday that there are microorganisms
40:56that need vitamins, right, to be able
40:58to grow. And so, for yeasts, a series
41:01of vitamins is also placed to know
41:03which vitamins, in this case, they need
41:05for their growth. Well, here you are
41:10generally seeing examples of this is,
41:13uh, this is urea, this is a urea
41:15positive, and this is, ah no, excuse me
41:17, this is a urea positive and this is a
41:20negative one, right? Here, this currant
41:25red color gives me a urease positive,
41:27and this is a urease negative. Here is
41:31an auxanogram, generally where you see
41:33in this case, I don't know, it doesn't
41:35look clear that the Well, but around
41:37this you will see that look, the colony
41:39has grown, right? In these, on the
41:41other hand, it hasn't. These are disks
41:43that are soaked with sugars, right? And
41:45this is a nitrate broth, right? This is
41:48nitrate positive and this is a negative
41:50broth, right? They are nitrate positive
41:52, nitrate negative. What other studies
41:55can be done? I told you that
41:56physiological studies can be done,
41:58right? And to be able to observe if
42:01they are growing, physiological or
42:03chemical studies, if they can be
42:05growing in culture media in liquid
42:06media, uh, you can see at different
42:08temperatures and if they are resistant
42:11to chemical agents, right? So there,
42:15you can also determine if they grow in
42:17liquid media, and in liquid media we
42:19will see if the growth is superficial
42:21or at the bottom, right? That can also
42:26be observed, done at different
42:27temperatures to determine what
42:29temperature these fungi tolerate, right
42:31? And there is resistance to chemical
42:34agents, right? To be able to determine,
42:37uh, if it is resistant, say, even to
42:39antibiotics, right? If they are
42:42resistant to chloramphenicol, to
42:44cycloheximide, it is determined. Now,
42:48apart from these, these, uh, these
42:51tests that we work with, in this case,
42:53semi-automated methods are used, right?
42:57For example, in the case of yeast, uh,
43:00there is a semi-automated system which
43:02is the API system, right? Which is a
43:05gallery of 20 little tubes, right? Uh,
43:08little tubes or cups, as they are
43:10called, that have dehydrated substrates
43:13and allow for, look, 19 assimilation
43:15tests. Right? So, let's suppose that I
43:18found a type of yeast and I want to see
43:20what type of yeast it is. So, what I am
43:22going to do is dilute this colony that
43:24I have isolated. Previously, one must
43:26isolate the colony and then perform
43:28these biochemical tests, right? So you
43:31inoculate with a minimal medium that is
43:33a semi-solid medium and, uh, the yeasts
43:36will reproduce if they use these
43:38substrates, right? These, these
43:40semi-automated methods allow for
43:43identifying, look, 34 different species
43:45of yeast, right? It is much faster than
43:47doing the whole process we might have,
43:49but these methods are much more
43:51expensive, right? So, these readings,
43:54uh, these reactions are compared with a
43:56growth control and the identification
43:58is obtained through a numerical code,
44:00right? So, these already come with a
44:03type of code one-two, and so one,
44:05according to this reading, goes reading
44:07; we go to an analytical catalog and
44:09you can identify it. But these
44:12semi-automated ones also have a
44:14software program, right? That
44:16immediately as you type, it starts
44:18showing, uh, what identifies the type
44:20of yeast it is, right? And nowadays,
44:23chromogenic media are also being used,
44:25right? Mainly for types of Candida and
44:28there is another one for Prototheca
44:30that has been used, right? In this case
44:36, these are media that generally have
44:38within their components a substance
44:40that causes the yeast that is going to
44:42act to form different colors, and that
44:44difference in colors will allow me to
44:46identify what type of yeast it is,
44:48right? These are, uh, rapid methods,
44:53much more expensive, yes, but they are
44:55providing very good results for
44:57identification, right? So, we can see
45:00Candida albicans, which is green;
45:02Candida tropicalis is blue; krusei is a
45:05pinkish, slightly rough color; and
45:07parapsilosis is a medium lilac-pink,
45:10right? And the Prototheca species are
45:12generally cream-colored, right? In fact
45:15, there's even a new yeast being worked
45:17on today, which is a type of Candida.
45:20I’ve forgotten the species right now,
45:23but this company has also released it,
45:25mainly to be able to identify it using
45:27a chromogenic method. There are other
45:33enzymatic systems that are very similar
45:35to what are called cassettes, just like
45:37when you want to test for COVID, you
45:39put a sample in, and it produces a
45:41result, right? So there is also a VITEK
45:45system for Candida to determine the
45:47type of Candida, right? So you place
45:49the sample here, and here you will see
45:51if it is Candida or if it is not
45:53Candida, right? These are the methods
45:55being used today, and these are the
45:57molecular methods currently in use,
45:59such as PCR, right? So, you already
46:02know the type of PCR where you
46:04generally have to denature the strands,
46:06then anneal, and then extend, right? So
46:10those are the cases that can be worked
46:12with, and in this case, molecular tests
46:14. I was saying that today these
46:16molecular methods are being used quite
46:18a bit for fungi to be able to work. I
46:20hope we can have this type of PCR to be
46:22able to identify things much faster,
46:24right? So, those are the techniques.
46:28Now, if we want to do a type of
46:30inoculation, and we have already
46:31isolated it and are going to inoculate
46:33it, what loops are we going to use?
46:36Here you can use a ring or circular
46:38loop; a non-calibrated one can be used
46:40for streak plating and general
46:42inoculation. This is used much more in
46:45yeasts because it is very similar to
46:47bacteria, right? So these loops are
46:49used for yeast inoculation, for example
46:52, right? We can use the L-shaped thick
46:55wire loop. If you look, this wire is a
46:59little thicker than the normal
47:00bacteriological loop we are familiar
47:02with. You have seen that we have
47:05pointed loops and round loops, right?
47:07Which are very thin. For fungi, we use
47:09slightly thicker ones, right? And this
47:13must be at a 90-degree angle in an
47:15L-shape because this mainly allows us
47:17to pluck the mycelium. We can't just
47:20use a normal one for the mycelium, or
47:22it will get tangled. But not here, here
47:24we can take it out, right? This even
47:27allows us, as I was saying, when the
47:29fungi are hard, to make a small cut in
47:31the agar to place it between a slide
47:33and coverslip and press it down to
47:35observe it, right? But there is also a
47:39sharp-pointed inoculation loop that
47:41serves to purify small colonies and
47:43distribute fresh fungal preparations.
47:46This one here is a type of
47:47sharp-pointed needle that generally has
47:49some little spikes. It's not, it's not
47:52smooth. It is a bit rough, which also
47:56allows me, in this case, to be able to
47:58take the sample, right? You can even
48:01use two of these because sometimes it
48:03curls up, and I was saying that it
48:04allows us, sometimes there's one here
48:06that looks like a spatula, right? A
48:08small spatula that also allows us, in
48:11this case, to take the type of sample.
48:15So, for the microscopic study, we are
48:17going to look at the technique using
48:19transparent adhesive tape, which is
48:21called the tape mount, right? Why?
48:25Because we are going to use a type of
48:26uh adhesive tape. Nowadays we can do it
48:31with Scotch tape, but today these
48:33adhesive tapes are already available;
48:35they even come pre-cut in laboratories,
48:37right? You just pull one off, and it
48:41looks, uh, like the size of a, of a
48:44coverslip, right? And you place that,
48:47although this is an adhesive tape that
48:49allows, in this case, uh, generally
48:51adhering to the colony, right? So, it
48:54says here, "Press the adhesive side of
48:56a piece of transparent tape onto the
48:59surface of a colony." Place the tape
49:01stretched out over a drop of
49:02lactophenol blue or aniline blue, then
49:05place it on a microscope slide, right?
49:07So, what we are going to do is take
49:08this here, we are going to put it on,
49:10right? I will explain it to you later,
49:12right? So, the transparent tape
49:14preparation allows us to observe under
49:15the microscope how the microorganism
49:17develops in the culture. Generally, the
49:19spores remain intact because, uh, the
49:21problem with the bacteriological or
49:23mycological loops we have is that
49:25sometimes we even tear off the
49:27conidiophores, and they cannot be
49:29observed. Right? So, the spores also
49:32remain intact, and identification is
49:34carried out more easily. One of the
49:39disadvantages is that if we don't press
49:41firmly enough, the tape might not
49:43adhere properly, and sometimes we won't
49:45be able to identify it. So, there must
49:49also be a methodology to be able to
49:51work, right? So look here with the
49:53bacteriological loop, you can see there
49:55is a tape that isn't visible, and it's
49:57being pressed; you press it here to be
49:59able to observe it, right? Once it's
50:02taken, usually with the scotch tape,
50:04you place it on your glass slide, right
50:06? And we have the other technique,
50:09which is the wet mount. You already
50:10know that one. It uses a slide and a
50:13coverslip, generally with those loops I
50:15told you about, the fine needles, right
50:17? Or sometimes, if we can't do it with
50:20one, we use two, right? Um, what you
50:22should try to do is pull it from the
50:24base and place it carefully in the
50:26center of the drop of lactophenol blue,
50:28right? Sometimes, if it's not possible,
50:31you use two loops and you lower the
50:33mycelium so that it can fall, right?
50:37And in this case, the coverslip is
50:39placed while avoiding the formation of
50:41bubbles. That is indeed important,
50:44right? So there it is too, look at the
50:47bacteriological loop at a 90-degree
50:49angle. You pull it mainly from here,
50:52place, look, a portion, there it is
50:54with its lactophenol blue, and you are
50:56going to place it. Now, those are two
51:01methods that are generally used most
51:03frequently, but sometimes it happens
51:05that those methods can help us identify
51:07; but sometimes it happens that we only
51:09take spores or hyphae and we can't see
51:11them, so we can't identify the fungus,
51:14and for this case, the microculture
51:15technique or what is called slide
51:17culture is used, right? So, here we are
51:22going to need, generally, to have a—
51:24we are first going to prepare where we
51:27are going to culture. We are going to
51:31need a Petri dish. In the Petri dish,
51:35we are going to place a piece of filter
51:37paper or it could be a piece of cotton.
51:39Small glass rods are placed, cut to the
51:43appropriate size, and a slide and a
51:45coverslip are placed on top. All of
51:49this is taken to be sterilized because
51:51on that slide, I am going to place my
51:54culture medium, it's called
51:55microculture, but it must be sterilized
51:57beforehand. Now, apart from that, you
52:03can work with agar blocks, right?
52:06Sabouraud, or whatever I need for my
52:08culture medium for others, right? This
52:11must be poured into a plate, and when
52:13it has solidified in this case, it
52:15should be approximately 4 mm deep—
52:18small, not too thick. Then, with a
52:23scalpel loop, I will make—you can
52:25make them rounded or square—and with
52:27a sterile scalpel, what I am going to
52:30do is place this small piece of agar
52:32onto my coverslip. Right? So, I have it
52:37here now, in this case, uh, for me it
52:39is much more...uh, easier to have a
52:46little tube with agar, like PDA agar,
52:48let's say, which is in a liquid state
52:50and warm, and with a sterile
52:52micropipette, I take and place one or
52:54two little drops of agar on the slide
52:56instead of cutting. So I place one or
53:01two drops, and since they are tiny, it
53:03solidifies very quickly. So, now I have
53:07my culture medium on the microscope
53:09slide, and then from the slide, with a
53:11sterile needle, I generally go to the
53:13colony I want to identify, take the
53:15sample, and on that little piece of
53:17culture medium, you are going to place,
53:20in this case, uh...or you are going to
53:22inoculate, right? Uh, sometimes you can
53:26inoculate in four quadrants, in the
53:28corners, or just in the center, and
53:30then you place the coverslip on it.
53:31That is why the coverslip must also be
53:33sterile, right? So, after the
53:35inoculation, the coverslip is placed.
53:38And then, what is that paper or the
53:40piece of cotton that is placed for? Why
53:42? Because with a sterile pipette, uh,
53:45what you are going to do is soak it
53:47with water, in this case, sterile water
53:49, right? Which means you are going to
53:52moisten it to provide the necessary
53:54humidity for the fungus to grow. Why?
53:56Because if you place it as is, since
53:58the medium is so thin, it will dry out
54:00in two or three days, and then there
54:02will be no water activity for the
54:03fungus to grow. So, you generally have
54:05to soak it a little, usually the bottom
54:08part, in order to work, right? And it
54:10is incubated, and then you will see it,
54:12right? Then, the colony will grow
54:14underneath the surface of the coverslip
54:16. The assembly is examined periodically
54:18with the naked eye to determine if that
54:20little piece of agar has grown, right?
54:23And then, after you have waited well,
54:25from three to five days, you are going
54:27to remove the coverslip and take
54:29another slide; you will put lactophenol
54:31blue on it and place that coverslip on
54:33top, and on the other piece, where a
54:35little bit of the medium remains, you
54:37also add lactophenol blue and place a
54:39coverslip. That is to say, you can
54:41obtain two slides to be able to observe
54:44what is around it. You have to apply
54:47nail polish, you have to seal it, and
54:49those microculture slides can last
54:51forever if you keep them well, right?
54:53So, this is a way of how you can
54:55properly remove the little pieces. It
54:57can be in a round shape, right? You
54:58place it there. Here they are
55:00inoculating, right? If you see here, it
55:02is at the four distant points, but you
55:04can also put it in the center, right?
55:06And then you place the coverslip. And
55:08after the coverslip, look, you have
55:09already observed that it has grown. You
55:11remove this here, add lactophenol blue,
55:13and it can also be observed here. Look,
55:15this is a little square. Look here,
55:17here is the medium. Here it is, look at
55:20the base, which is the paper. They have
55:22already placed the culture medium. Here
55:24is the fungus they want to inoculate,
55:25they are going to inoculate it. Look
55:27that here it is with this, this is
55:28called, this is a mycological loop,
55:30right? Spatula-shaped. They place it,
55:32they are inoculating it, and then with
55:34the coverslip, they generally place the
55:36coverslip here. You are going to see
55:38that here they are already adding water
55:40with the wash bottle, right? The water
55:42to moisten it. You take it to incubate.
55:44Look, it has already grown here. You
55:46are going to remove this part here.
55:48Look, and here you have your slide with
55:50lactophenol blue and you place this
55:51coverslip here. But I can also use what
55:54remains there, I add the lactophenol
55:56blue in this case, and what I am going
55:58to do is observe, right? So that is
56:00what is done in this case, right? So,
56:03you are going to have to carry out the
56:05study of the following fungi. Here you
56:07are going to see the different fungi,
56:08it is an Aspergillus, which is what you
56:10have to study, right? That is why your
56:13task here is that, right? To perform
56:16the study macro and microscopic of the
56:19fungi. Look, here you have, you are
56:22going to see, I am already putting the
56:24genus and the species in this case so
56:26that you can see them and you can
56:28determine the characteristics for me,
56:30right? First, try to do it yourselves
56:33and then go to your bibliography to
56:35corroborate. What is it that you are
56:37seeing, right? What do you see? Do you
56:38see a conidiophore, a sporangiophore,
56:41do you see septate or coenocytic hyphae
56:43, is it macro-siphonate,
56:44micro-siphonate, do you see sexual or
56:47asexual spores? You have to look at
56:49that microscopically, and
56:50macroscopically I am only showing you
56:52the obverse, I am not showing you the
56:54reverse, only the obverse, right? Look
56:56here, right? So you are going to tell
56:58me, it is as if you were in the microsc
57:00—you have arrived at the laboratory
57:02and this fungus has grown for you. So,
57:04you perform the study. Later you will
57:06perform the study with lactophenol blue
57:08, which you won't actually do, but I am
57:10providing you with the figure of what
57:12possibly came out, right? Here you also
57:15have what came out, right? Here you
57:18also see this part, right? Here too,
57:21look. So I am placing different types
57:25of fungi here in this case for you to
57:27observe, right? What do you see, is
57:30there hyphal modification, is there no
57:32hyphal modification, right? What shape
57:34does it have? If it has sexual or
57:36asexual spores, likewise here, what
57:38modification does it have? And you can
57:40put it there. So, these fungi are the
57:42ones I am giving you for your
57:43assignment so that you can identify
57:45them.

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