Chapter 4 Microscopy and Staining 8.31.16
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Not Syncedgreetings good people of the universe welcome back to microbiology um in this chapter chapter four we're going to
discuss microscopy and staining we'll have a little bit of a discussion in classification and identification by
this point in the semester you probably already talked about or even if you've had an introductory biology class you
talked about um taxonomy and you talked about the binomial nomenclature so we'll just hit on it just a little taste but a
majority of the is going to be focused on the main tool of micros M
microbiology which is the microscope and microscopy
so and kind of giving you an understanding of exactly what it is we're working with kind of like we did
with the previous um session in chapter three where we looked at the chicken egg and compare like the pox virus
stockus um and then also looking at Like A Parasite like a protoo and the Gardia
so we're kind to kind of do the same thing except that we won't use images of different organisms we're going to talk
about units of length so I'm not going to give a full detailed lecture on the metric units of the lane except to say
that as you know in science and in biology we primarily use the metric system and the meter is the standard
unit of length the liter is the standard unit of volume for liquids so since
we're talking about size here then we're going to use the standard unit of length the metric system which is the meter so
one meter is about the length of a tapeworm and if we go a little bit
smaller and go to a tenth of a meter so at a tenth of a meter um we don't really
have anything that we look at the M microbiological application of the unit on there but a decameter is about a
tenth of that a centimeter each one of the hash marks that are in a meter stick so Envision a yard stick is what we
would call it in the US but Envision a yard stick or a meter stick and then look at as these little tiny hash marks
that we have in there and if you count them they go all the way to 100 so there are 100 cm in one
meter and um so when we calc look at that um the size of that would be like
the diameter of a mushroom cap so you're looking at about 12 cmet for the diameter of the mushroom cap millimeters
if you go back to that yard stick and that yard stick had the larger lines
that were the centimeters in between the centimeter lines are going to be millimeters in fact there are 10
millimeters in each centimeter so if there are 100 cm and 10 millimeters
within each one of those 100 cm then that tells you that there are a th000 millimeters in one meter so on that
meter stick there are a thousand of the really small hash marks now keep in
point mind that this level at the millimeter is the smallest that we'll be
able to see with the naked eye so examples of things that would be measured in millimeters would be the
diameter of a bacterial Colony so on our AER plates our nutrium AER plates and we saw those different colonies from um our
a testing of different places where we did our shoe and we did our um our cell
phones or we did the toilet seat or the IDE the toilet seat of the door handle and we looked at our place and we saw all those different colonies the average
diameter of a colony is about 2 to three Mill MIM um how thick a tick would be
would be about 5.7 millimet so if you see we're getting very very small now once we get to the point of a
micrometer a micrometer has one million it's one one millionth of a meter so um
if we were to look at that on a yard stick there'd be a million of those little tick marks on a micro on a yard s
of micrometers but remember we can't see them with their naked eye so for micrometers things that we would um
measure would definitely be that of cells bacteria included and also white blood cells and down at a nanometer
nanometers are very very very small so a
nanometer is going to be about the diameter of a PO the polio
virus so here's just another picture that's kind of showing the same thing but it's showing you different organisms
that represent those sides so what I'm asking of you now is to kind of refresh
for our exam refresh your understanding of the metric system um because we will be discussing that on your exam and then
I also want you to just kind of explore and just look at some of these organisms
so that you can have a frame of reference when we begin to talk about um why the microscope is important and um
this relative size and comparison so I'll just give you a second to kind of go over
this notice that for the compound microscope the one that we use in lab primarily the
compound microscope only has a certain range that it's able to see things things that are as large as a flea at 10
micrometers and things that are as small as um typical bacteria which is right about
here at about half a micrometer so right at this point here is where we can see
it to see anything smaller than that you're going to have to use a scanning electron microscope a transmission
electron microscope or a scanning tunneling microscope for anything smaller than that um electron
microscopes the two primary electron microscopes that are used in microbiology are transmission and
scanning because we're really not concerned with seeing molecules of water or amino acids or um any of those other
atoms or things like that so everything that we need to see we can use with a transmission or a scanning electron
microscope notice how limited the human eye is as to what we can
Microscopy
see so the general principles of microscopy is that we're looking at wavelength of radiation specifically if
we're talking about electron microscopy um also even if we're talking about light microscopy we're still talking
about wavelength A good rule of thumb is is that the shorter the wavelength the higher the resolution what resolution
means is that it's the ability to distinguish two objects as separate entities from one another so that you
can see that even though they're really really close together and they're really really small you can tell that they're two different objects that's a little
bit different than magnification which is another principle of microsopy magnification is basically saying how
big can we blow this image up how big can we make this image for us to see it whereas resolution is looking for more
definedness to clear up those edges and that's the job of the oil immersion objective on the microscope and then
contrast contrast helps us to be able to see the specimen against different
backgrounds and so in order to do that with our light microscopes and even with electron microscopes it's going to
require some staining to take place you'll notice when we look at the yeast cells or if you looked at yeast cells in
other classes under the microscope we didn't stain them so when we didn't stain them we couldn't see them very
very clearly um we had to actually turn the D Iris diaphragm down so that we could get some
contrast so as I said before the shorter the wavelength the higher the resolution
ution that we would have light visible light is in a certain Spectrum so right
here is that visible light spectrum so this is just the re um the representative wavelength for that as we
start to get down into electron microscopes notice how the wavelength get smaller so from Crest to Crest is a
wavelength and the shorter those Crest to Crest units are the higher the
resolution so contrast is the difference between the intensity between two objects or an object in its background
um staining helps to increase that contrast and the use of light in its space increases contrast as well so
contrast is really just the ability to have an object your specimen specifically stick out for
you now our primary tool in an introductory microbiology class is the light microscope specifically the bright
spill microscopes um we can use darkfield microscopes but we just don't use any in our class um and darkell
microscopes are really useful for St for viewing things that you don't want to stain or you can't stain um in order to
get that contrast because they'll stand out against that dark background bright fill microscopes um
are can be simple and a simple microscope means that it contains only one lens so instead of having the
objective piece and the eye pieces that we have in our microscopes which are considered compound microscopes they
just have one lens and it's very similar to a magnifying glass in fact l hook
used a simple microscope to observe many different microorganisms so they're quite effective and his microscope would
be considered a simple bright field microscope now the more advanced version
of that simple microscope is a compound microscope so that means that there are more there's more than one set of lenses
there so we know we have our ocular lenses which on our microscopes and most microscopes are 10 times magnification
and then there's various different objectives on nose or the ends of the microscope that get closest to the slide
and they range from four times magnification up to 100 times magnification if we're using the oil
emerging objective in order to get the total magnification we have to multiply
the magnification of the objective lens whatever one you're using whether it's four 10 40 or 100 and also that of the
oxid lenses um using of the oil emersion objective is what helps to increase that
resolution resolution so remember when we talk about resolution we're talking about wavelength so it helps to make
sure that the wavelength is a little bit tighter and that the light goes through
the objective and isn't scattered or refracted off into the environment that it goes straight through the objective
and it gives a nice clear picture and you've probably observed that when you put your oil emerging objective down
what was once diffused and kind of grainy you can kind of see a lot clearer now as a result of the oil and the resol
the increase in res solution because of the oil emerging objective so here's a beautiful picture
that is illustrating everything that I have just said so when we don't have the oil that's in used we're still working
in an air environment and light is one of those things that can scatter out the
light source some of it's going to be refracted and lost so not all of those light rays are going through the
objective to for you to view the image that's on your slide so some of it's going to be lost and it lost and it's
going to reduce the qu quality of the resolution for the image that you see back in the eye piece when we have a the
oil immersion on there so notice how the oil is on there that nice thick oil this is one of the reasons why oil is one of
those things in microbiology that too much of it is not usually a problem is that we don't have all of those light
rays that are refracted and M off to the environment in fact there are more light rays so there's more of a concentrated
use of that wavelength that go into the objective lens so that you can make the picture a lot clearer and easier for you
to see when you're viewing it under the microscope with your oil emerging
objective so other types of white microscopes that we're going to be interested in are florescent microscopes
now we won't use them in our class in introductory biology microbiology but I think they're important to discuss
because they are of clinical significance instead of using just regular visible white light as we use
for our brightfield compound microscopes it's going to use ultraviolet light um to Source at the specimen the specimen
radiates energy back as a longer more visible wavelength and that UV light is
going to increase the resolution and increase the contrast why I feel that micro fores of microsopy is important
because it has clinical significance we can use it for immunal fluoresence to identify various different pathogens and
also to make visible a variety of different proteins so in order to see things like syphilis and even other
spiral Keys we can use for rest of microscopy and here we have a picture of
that happening um so we're going to let you guys kind of look at this and see if you can kind of determine what's going on and then we'll discuss it in just a
sec so with this imuno fessin what we're doing is that we have these festin dyes
and then those fuesen dyes are um going to be attached to these antibodies and
in order to determine whether or not the individuals whom specimen that we have
taken as a sample to see whether or not they're actually have um this particular
um bacterial infection what we could do is we can take the antibodies that we know would be attracted to the antigens
on the surface of the bacteria and we can Dy them so that those dyed antigens
if that the bacteria is there is present then our antibodies will be attracted to them and it will s the bacterial cells
to these anti bodies curing the Dy so that we can look at it underneath the microscope and we will see it foress as
we have in this image over here now this isn't an electron microscope it's just a regular old light microscope that's what
LM stands for so by using a regular light microscope and this fluorescent D
um this uh fluoresent microscopy we're able to give a diagnosis at a much
faster rate without having to run a lot of biochemical test we'll still run biochemical tests just to be sure but
this is a great way to get a a clinical diagnosis of various different um
bacterial infections so on to the electron
microscopes electron microscopes are going to give you the greatest resolution why do they give you the
greatest resolution well what did we discuss earlier shorter wavelength greater resolution so electron
microscopes have a shorter wavelength so they have a greater resolving power they
also happen to have a greater magnification they're able to magnify objects between 10,000 and 100,000 times
their actual size so we can get a very detailed view of things like bacteria viruses the intercellular structures of
bacteria or any other type of cell molecules and even some really large atoms we can see with an electron
microscope there are two different types of electron microscopes transmission and
scanning transmission electron microscopes are going to allow you to see those internal cellular structures
and even on the inside of the inside of those structures so we could we're talking UK carotic cells we could see
the inside of a nucleus or the inside of a GGI apparatus um if we're talking about procaryotic cells we can see the
inside of a ribosome and the inside of that bacteria cell scanning electron microscopes give you a nice
three-dimensional picture of the outside of the
cell so here is a transmission electron microscope um in order to use an
electron mic microscope typically the specimen has to be killed that's different from the light microscope
where we can look at Living objects underneath the light microscope which we do in lab all the time um but with an
electron microscope in order to view those specimens and to get that short wavelength to bounce off of the um
specimen that you're viewing and give you a picture back to um the the person
that's viewing it we have to stain them with heavy metal so we have to stain them with things like silver and Mercury
um those really heavy metals and that typically is going to kill our specimen in addition for transmission electron
microscopes many of the preparations may require that we slice the specimen into
very very thin layers in order to view the internal structures so one of the drawbacks of an electron microscope is
that we have to kill our specimen and the other drawback is that they're kind of expensive so on the the The Bargain
Basement version of an electron microscope you know the one that's on super clearance and not the name brand
type is going to probably right near about 10 grand so it's about $10,000 for the least expensive electron microscope
and then the price is just going to Skyrocket from there so here are some pictures of a
scanning electron microscope so notice that the pictures of the scanning electron microscope unlike what we saw
on the previous slide we're looking at internal structures with the scanning electron
microscope same principles apply we're using shorter wavelength they're expensive machines and you have have to
stain your specimen with heavy metals um we can see a lot greater definitions and
in my opinion they're always just prettier pictures of things that we look at with a scanny electron microscope um
ASP regillus which we've looked at in La class it looks a lot nicer you can actually see the three-dimensional
quality of the ASP regillus and the spores here and than what we noticed in class um the same thing with the parium
you can actually see all of the cyia that's as a result of the increased magnification and the increased
resolution and then we can see our individual balls with strepto cacus typically when we see the term stre that
means that these balls are going to be um these cocky are going to be in Chains and stats means they're going to be in
clusters and we'll talk more about that as we go throughout the
semester probe microscopy on here um we won't be using probe microscopy on here
but it we use it typically to look at things that are very small specifically um molecules so d and enzymes or those
proteins we can use probe microsopy for that and that's also a form of an electron micro
microscope so in order to see these different Images stating is required and we talked about the limitations of
staining with electron microscopes and that we have to use these really heavy stains and that usually kills our
specimen but we also use staining with our light Sprite filled microscopes that we have in lab so in order to provide
that contrast this is why we're going to be using that staining and there are various different staining mechanisms
that are just kind of a Hallmark in microbiology one of which we've already talked about very briefly in chapter
three is gr staining but we also have acid fast staining that takes place as well that we do fairly regularly but
then there's also staining of endospores and staining of fella so there are other mechanisms and and ways that we con
stain objects um in microbiology and even in just an introductory microbiology course
course so when we prepare a specimen for standing and a lot of this is probably um very intuitive to you or something
that you've seen before we're first going to want to put our culture on a thin flil film on our slide and then we
let it air dry a little now granted it's probably not going to air dry completely you may have some areas where it's still
a little bit wet but we then want to place it um through a flame to fix it so
by heat fixing and this is what this process is called by heat fixing in our specimen we're doing two things we're
allowing the specimen to stay Stu to the slide Thing One and Thing Two since we're working with bacteria we're also
killing the specimen so we don't run the risk of contamination or of of our microscopes or of anything else that
we're using so we kill it and then we make sure that it's attached from her to the
Staining
slide the basic principles of of staining is that we use some sort of dyes and these dyes are typically salts
and salts can carry a char of them whether it's a positive or A negative Char charge what we call a chromophor is
the actual colored portion of the D anytime we use acidic and let's go ahead
and bring out the old highlighter anytime we use acidic
D acidic dyes will stay in alkaline structures so the charge of these acidic
dyes is probably um a a positive charge alkaline
structures are things that are like hydroxy ions and they're negatively charged basic D on the other hand are
going to stay in acidic structures so if basic D can and remember the the old law
that you guys learned opposite the track with you have a North End of a magnet and a south end of a magnet they will
come to each other if you have a North End and a North End they repel one another so the same principles apply
here these acidic D can stain alkaline suck structures alkalines are bases
bases are negatively charged and this is just something from like a chemistry or from introductory biology course so
these acidic dieses are going to be attracted to a negative structure basic
dyes are negatively charged they're attracted to acidic structures acids are
things like hydrogen ions and they carry a positive charge to them basic D are
more common because the cell surface of a cell is more negatively charged
if we go back to what we talked about in chapter three when we looked at the um membrane potential the cellular membrane
potential of a bacterial cell of a eukariotic cell and we said that their resting potential is atga 70 molts well
there's that negative number that we talked about so the outside of the cell is negatively charged so we want to have
a d That's positively charged that will be attracted to that negative charge of the cell so it can coat it so that's why
basic D are much more common simple stains mean that we just use one
stain so we're not using you know a series of stains an examples of simple stains um and there usually a basic D
Crystal Violet which we've used saffrin which we're going to use in our gram staining and methylin blue we use that
to stain our cheek cells so we just use one stain to stain our cheek cells they're very useful in determining
relative size shape and arrangement of the cells themselves in order to differentiate between different classs
of bacteria we want to what's called differential staining so here's just a picture of
some simple staining on here so this is um looks like Mite green for endospore staining and then it looks like we have
Crystal Violet that we have to stain these bacteria notice that we're just able to visualize them we can see that
we have some billus there we have some coxide there but we're not really able to tell who's gr negative who's Grand
positive um we're just looking at the um just looking at the realtive side size
and shape of these different cells that we stain with the crystal violets now differential staining is
where we're going to use more than one die so in simple standing we just use one die just like a simpal microscope is
just one lens with differential standing we're going to use more than one die and in the word differential what word do
you see different right so you want to be able to detect differences between
the cells that you've Stained on your slide so you're going to be able to distinguish between different cells or chemical structures we're more
interested in staining between different distinguishing between different cells common types the differential staining
our gram staining which we will do in class endospore staining and acid FAS
and histological stain um we won't do either one of those in class but those are just examples of it histological
stains are where you're staining with various different um stains to enhance
different structures so we use that more in um at our level in community in um anatomy
and
physiology so here is the first thing that we're going to talk about which for us is the most important standing for
cedure in an introductory microbiology class and that is gram staining so gram stainings generally takes place in four
steps now there are some other steps that you'll have to to do with rinting the slide letting the slide air dry and
that sort of stuff um and that's all outline in your your lab book but the four basic parts of gram standing are
right here in front of you and what gram standing allows us to do is it allows us to differentiate between gram positives
and gram negatives if you remember from chapter three gram positives are more
susceptible or they're more likely to be killed by antibiotics whereas gram negatives are less susceptible to be
killed by antibiotics they're more resistant to them they have this outer layer that kind of protects them from
antibiotics and deter detent so gr negatives are generally more difficult to kill and this is the very first
process of our identification um things so when we're trying to
identify bacteria this is the first thing one of the first things that we'll do is that we'll gramp in it so first
things first we want to flood our entire slide so you're going to make a a smear
you're going to make a fixed um a fixed lie so that means that you put your specimen on there you smear it you stick
it through the let it air dry stick it through the buns and burner to um fix it on there and then the next thing that
we'll do is that we'll flood it with Crystal Violet so we put all that Crystal Violet on there we rinse it off with water and then what we notice if we
were to stop at this point we would see that that would just be simple staining that everything on this slide is all
purple okay that's kind of like duh because we just use crystal Violet the
next thing that we want to do is we want to use iodine and while using that iodine fling that slide with iodine is
that we're now going to make sure that that purple stays fixed onto those cells that it can stay fixed onto so for gram
positives remember they only have that cell wall that very thick cell wall gram negatives they have that extra LPS layer
that's surrounding them so because they have that extra LPS layer that means that Crystal Violet and that iodine is
going to be attached to that LPS layer so what iodine does is it act to what we call a
it helps to make sure that all of those cells on that outer surface remains purple so after this point if we were to
stop our gram standing procedure at this point everything is still going to be purple now we're going to start to get
some differences here so the third step that we have is that we're going to use
ethanol and acetone or we just call it an alcohol wash we're going to rinse our
solution with an alcohol wash what that alcohol wash will do alcohols are
notorious for um dissolving lipids so just as something that you might be
familiar with when you go out a night of bins drinking with your friends not that any of you guys do this but you've
probably heard of people that do this that they typically tell you that you should eat some greasy foods have
something greasy in your stomach that's going to make sure that your hangover is not so horrible and it's also going to
slow down the process of your body um absorbing that alcohol through your stomach so you can drink more and have a
merrier time again not that you do that but other people that may do it so having those greasy Foods in there the
alcohol actually breaks apart those greasy foods and it has something to work on before it actually crosses um
into your bloodstream so alcohol is actually going to remove anything that
has a phospholipid or lipid layer to it and we know that gram negatives have
that LPS or lipop polysaccharide po phospholipid layer associated with with
them so those phospholipids are going to be disintegrated and washed away by the
alcohol so as a result what happens is that if you are gram negative we're to
look at your slide after you rinse it with alcohol if you were gram negative then notice that it seems like you
disappeared Noti they had like all these slides all these cells here and it's like whoa what the heck happened look
like they disappear they didn't disappear just their LPS that outer layer of those gram negatives have been
disappeared so the gram positives are still there and they're still purple but the gram negative cells are now
colorless so in order for us to see those colorless cells we have to now stain it with saffran so the final step
is to stain this the slide with saffran and then rinse it with water and allow it to dry and notice what we have now we
have these pink cells or these red cells these are our gram negatives these red cells so these gr negative cells are
pink or sort of reddish and we will be gram staining a lot in
lab and as I said before is this is a very very important process in
identifying bacteria and especially for the students in my class where we'll have two unknowns which means I'm going
to give you a test tube with some bacteria in it and you're going to tell me what that bacteria is using this standing procedure and other biochemical
tests we'll talk a lot more about it as we get closer in the semester but just to kind of started to plant those seeds
in your head that probably the most fun and interesting part of microbiology is
the identification process and it requires that we have a very intimate understanding of gram
staining so um another type of differential stain is the acid fast
stain and we primarily use acid fast stains to stain things like
microbacterium microbacterium tuberculosis is a great example of that so in GR staining our colors were purple
and our colors were um pink and in acid fast staining we have red and blue as
our colors so those would be blue and those would be
red in just for staining is another staining mechanism um we will actually use this in class it helps to stain the
endosport um with malachite cream we use stee to drive that malachite green stain
into the inside of the cell and we can actually see whether or not these billas which many species of billas um form
endospores and what we're particularly looking at right now is billas anthesis which is the disease the bacteria that
causes the disease Anthrax but these Green jobbies in here these are those endospores that we talked about in
chapter three so the histological stains the two
common stains for that are the GMS stain and the H stain um and those are just
for histological specif mens like tissues red blood cells um not the red blood cells but white blood cells you
don't stain red blood cells you can see them without they have already had that color for contrast um but for white BL
cells and you con stain um the granules in them so that's why when you look at the slides and amps sometimes the um red
the neutrophils for white BL cells look different from one flly to the next as far as color is concerned that's why we
never ever ever in& just use color as a mechanism or a a rule by which we're
able to determine one tissue type from another because it just depends on what type of staining mechanism that they
use so some special stains um negative stains will highlight capsules and
flagel stains are going to highlight fagell and then fuesd stains we already talked about those with fuesd microscopy
um are to get things to Glow for us so that we can get a positive or A negative
diagnosis or confirmation for the presence or absence of various different types of bacterial
infections we will do a capsule stain and we will actually use that capsule stain with clal and ammonia um what
we're doing with this negative stain or capsule stain is that we can take India ink and actually stain the background so
we stay in the background so that the capsule surrounding the bacteria this is pretty much exactly what it looks like
except ours is black we use NBA um um we stain the background so
that we can have the bacterium and its capsule be shown for us so we can see
The Capsule that's surrounding it and remember the capsule falls into the category of a glycocalix and it's nice
and tightly organized helps to protect the bacteria um we believe and it also
helps the bacteria to adhere to different surfaces and we're just highlighting that
capsule and then for flagel stain we won't be doing a flagella stain um but this is a picture of proteus volar which
is a organism that we will work with in La quite frequently and we can see that it has fella that are all around it so
we would consider Proteus vulgaris um to be mo motile which means it can move do
that fella and we would also consider protus fella arrangement of fella to be per trixis because they're all around so
um we can use iodine or other um Morant stains to help stain those fella so that
they're show more contrast and you're able to see them so here are those different stains
that we um looked at and we talked about with simple stains differential stains
and those three different types of them um endospore staining acid fast staining so um acid fast cells are red and non-
acid fast staining cells are are blue and we use that for microbacterium and noard and some other types of bacteria
and then we had our special stains here as we we have with every other time I put a table in your notes this is good
to make a note card off of or it's good to have memorized because it gives you an a glance of a comparison um and a
contrast of these different types of stains and also what they're used for and what their results would look
like now staing for an electron microscope very important as I said before we have to
stay with heavy metals the stains bind to the molecules in the specimens or to
the background in order to give um something for the electrons to bounce that wavelength off of in order to give
us an image so we have to stay with these heavy metals so typically our specimen is going to die as a result of
electron microscopy all right so the last little portion of this chapter is going to talk
Classification and Identification of Microorganisms
about classification and identification of microorganism we understand that taxonomy consists of classific ation
nomenclature which is naming and identification that's important for us in microbiology because we want to be
able to classify organize and name these various different microbiological
specimens even if we're just talking about bacteria so we want to organize this large amount of information and we
also want to be able to make predictions about what how these organisms or how an organism in one class is going to behave
most likely if we do our nomenclature correctly in our our taxonomy correctly actually we'll find that many of these
um bacterium that are in the same class will behave in many of the same ways another reason that we have taxonomy and
there's an entire discipline that's devoted to this is to understand evolutionary connection so we have
evolutionary philogenetic trees and evolutionary phog geneticist that are always looking at the relationship um
between different organisms and trying to understand those connections and how organisms are related to one
another so caros L as you're very familiar with he's kind of the father or the grandfather if you will of tax
taxonomy his classification system was based on very common characteristics that he could actually see so it was
more morphologically based so if you had fur you went into mammals if you laid eggs you went into you know a different
classification on there so his classification scheme although it's very archaic and sort of r
um he was able to group organisms successfully and he also was able to
develop this idea of binomial nomenclature binomial means two nomenclature means naming so it just
means two-part naming system so your specific scientific name as a human would be a homo sapien you have the
genus of homo and then you have Sapien as your specific epit on there um same
process can also work for bacteria so we have stakus orius St is going to be the
genus and then orius is a specific epit so although Carlos leas his his
taxonomic um categories were very broad they still
have a really good solid foundation and what we're able to do and been able to um enhance on his
work so for caral L he follows two different kingdoms so he said you read a plant or you an animal keep in mind that
at this time we consider bacteria to be plant so we put it in you know Flora so
you've probably heard those terms before flora and fauna fauna were animals Flora were were plants and everything else
later on we decided to expand that into five different kingdoms and those five different kingdoms that we had were
animals plants Fung protus and procaryotes on there at this point in
time and actually this is how I learned it in high school and in college at that point in time we hadn't really teased
out Arcadia from bacteria so we just kind of grouped them all together as
procario um later on we decided to take animals plants fungi and protest put
them in ukaria domain and then the procaryotes were broken down into the Arcadia and in the bacteria domain so
what L's goal was was just to categorize these um animals um and these plants in
order to um categorize them to classify them and to catalog them now our modern
goal is that we want to understand the relationships among these organisms we
want to see how this philogenetic hierarchy is established so what cells
came first and how did some of their structures Go off into the Next Generation to really show on a molecular
level The evolutionary or a DNA or RNA level the relationship between these VA
very vast different types of organisms and how they all fit with one another so
at this point we have a much greater emphasis on the genetic material and that's what has led us to this domain
system of Aria bacteria and
ukaria so Carl Loi was the one um one of the people that helped to determine
these um uh domains and he was looking at nucleotide sequences of ribosomal RNA
and as a result of his research we have those three domains there and the cells in these three domains also differ from
respect to one another in many other characteristics not just by looking at the RNA
sequences so when we're ready to classify microorganisms so so far those first two slides are kind of like a
history of where we've gone and what we've talked about and how we've gotten to where we currently are now so as this
taxonomy and this identification and classification that we just talked about how that relates to microbiology is that
you have all of these classes of bacteria and they all kind of molecular or I'm sorry morpholog morphologically
look the same so the shape and the size and some of those very basic characteristics are pretty much the same
it's not like with animals or plants where we can say ah this plant has three broad leaves and this plant has two broad leaves now there's a lot more
similarities to the bacteria so we can't just rely on physical characteristics although physical characteristics are a
big part of our identifyed different types of bacteria um and we use those
those physical characteristics we just talked about in staining whether it's gram staining or acid fast staining or
endoor staining we can also run biochemical tests so we can see what
types of chemicals can these bacteria or these cells use for energy or used for
various different purposes what kind of byproducts do they have this is also an important feature of your unknown in
fact Grand staining and running biochemical tests are the only two things that we're going to use to figure
out how to identify this bacteria more sophisticated and advanced ways of
identifying and classifying bacteria microorganisms are to run theological
tests to see what antibodies they make and what type of antigens they are studied on top of them so we're looking
at protein sequences we can use Fage typing on there what types of um
bacterial phages can affect this bacteria cell there are certain phases that certain um in a phase is just a
virus virus um certain viruses that can affect only certain types of cells so we
can use that as a way of identifying and then also as an advanced method of identification and classification is
analysis of nucleic acids none of these things at the bottom are we going to be doing in an infector microbiology class
but I think it's important that you are aware that these are more advanced ways
to identify and classify microorganisms we're going to stick right here between physical characteristics and bio
chemical test so physical characteristics are
oftentimes used and we talked about how we can use gr staming as a physical characteristic endospore or acid fast
staining we can also just physically look at under a microscope what protoo and fungi algae and parasitic worms look
like and we can do a lot of really good classification for these cells um based Along on their morphology or what their
shape is or um whether or not they have cyia whether or not they have a fagella
what type of vaces do they have so um what we're going to do in last class is that you're going to actually look at
things like a pami and a ukina which are both considered protozoan and see how they're different from one another and
how they would fit in different classifications we can also do the same thing with bacterial colonies bacterial
colonies like those that were studied on your augur plates when you did your different samples from the environment
um they also have distinct appearances and you'll remember some colonies were glossy and white some some were kind of
Ashen and yellow others had nice round edges some others had you know irregular
edges The Next Step that we use is biochemical testing so with biochemical
teses were able to distinguish procario by their ability to use or produce certain chemicals so what kind of
byproducts do they have what kind of chemicals can they or can they not use
in order to get energy um use biochemical test um to identify
pathogens and we're going to use biochemical test in our class just to identify that unknown microscope that
I'm went or unknown micro that I'm going to give you throughout the
semester so here is an example of two different biochemical tests that we're going to use um we have hydrogen sulfide
test and we also have um fermentation test not all organisms can ferment
sugars so if they can ferment sugars not only do we have sugar in these test tubes that
start off red we have sugar in the test tub but we also have a pH indicator that
pH indicator is usually seen all red and the red color so if the bacteria can use
those sugars and get energy out of it they're going to produce a byproduct that is an acid that acid is going to
turn this red test tube into this yellow color if they can break down or ferment
that sugar they also may be able to produce a gas so they might have a
carbon dioxide gas come as a result of this fermentation process so this will be considered a
positive test for fermentation if we have a yellow color and we would say not
only is this tube positive for fermentation but it's also positive for gas production this tube on the other
hand is positive for fermentation so we do have acids that were made so these bacteria that we place into this test
tubes they were able to use the sugars that we put in there um but it didn't make any gas this test tube is inert so
if we put bacteria in there then it couldn't use this sugar um so it didn't give you a gas
byproduct for hydrogen sulfide we're looking to see um if hydrogen sulfide is
produced and if hydrogen sulfide is produced it gives you this nice black color if not it gives you this color
over here hydrogen sulfide test we don't just use them by themselves we actually
use them with um we call them sulfur um and we use indol um which is the test
the presence of a protein that was made and also whether or not the microorganism could move for motility so
this will be considered a stem tube we're only looking at one aspect of that Sime and that's the ability to produce
hydrogen sulfide um the other two things we just aren't looking at here but there are three different tests that can be
run in this one too so as I said before we can use
theological tests to identify bacteria um many microorganisms can trigger an immune response that results in antibody
production um and the antibodies can be used to identify an
organism and here's a good picture of that um using the utenation test it's one type of serological test so we use
this for blood typing so that if you have type A blood and want to know if your type um whether it is type A blood
we can put um a antibodies into the serum of your blood and look for a
glutin and since that utenation takes place and we say yay you have type A blood because we put anti-a antibodies
in there if you have type B blood we put the anti-a antibodies on there those antibodies aren't going to attach to
your red blood cells so they won't be a glutin so that's a good example of how they could use it um we use it for blood
typing but we can do the same procedure with bacteria
dichotomus keys so once we've done our biochemical testing um we can use a
dichotomus keys which is basically a series of statements where only one of the two choices is correct so it's an
either or as it applies to particular organism what a dichotomus key will do
and we're going to build some dichotomus keys here in microbiology is it helps direct us to a um to name an organism so
it helps us to kind of Windle down and get to what this organism is based on
the information that we know whether it's physical how it gram stains or or
acid fast stains and also the results of those biochemical test can it ferment sugar leted to produce hydrogen sulfide
and so forth so here is a beautiful picture of an example of a dichotomous key so first
things first we Grant St and then we ask the question are the cells Grand
positive yes or no if the answer is yes then we know we're working working with
gr positive bacteria so if we look over here on this side gr positive cells gr
positive bacteria so if we go to 1B gram negative cells then we have to go down
to another question so if they're gram negative cells we have to ask a few more questions to figure out what we're
working with so if we said they're no they're not gr positive they're not purple then we asked are they Rod shaped
yes or no so if they are Rod shaped yes then we have to ask another series of
questions if the answer is no they're not Rod shaped then we say that they're coxide shaped they're so if they're not
Rod then there can be circular or they're pleomorphic bacteria so if it's no then we just stop here same thing
here non Rod shaped we just stop there if it is Rod shaped we have to go down
to the series of questions for three so at this area can it tolerate oxygen yes
or no if it can tolerate oxygen we have to ask another series of questions does
it ferment lactose if it doesn't tolerate oxygen then we they don't they're allig get anoro but if we have
to keep going further does it ferment lactose or will it give you an acid if
you put it into a test tube with um that lactose sugar in there will it give you
a yellow color to it if no then they're considered non- lactose fermentors or if
we put them in a manaal salt plate do we get a yellow color change to it all of
these things we will talk about in lab class in further detail um but um if it
does let ferment lactose then we go do another biochemical test and we say can we use citric acid as a sole source of
carbon if no then we have to ask can it produce gas from from glucose if it can
produce gas from glucose then it's um eoli aeria if not then it's chela if it
can use if it can use H citrate and gives us a a nice color change in that
citrate too then we have to do a hydrogen sulfide test when we do that hydrogen sulfide test we want to see
does it produce um aceton or does it give us that black color if yes then
it's salmonella if no then we have to um give us do another test to we have to do
an endle test and so for that Endo test we say does it produce anonin if yes
then it's calaor if um if yes it's interactor and if not then it's caor so
this is just a very simple example of a dichotomus key we will work with
dichotomous keys in our class in any microbiology class usually it's going to have a discussion or working with
dichotomous Keys especially as you're trying to identify your unknown so there'll be more discussions and
sessions on how to properly construct and to um go over um the use of your
dichotomous key and how to Pro L um lay it out all right so that is the end of
this session I hope you all are having a fantastic day wherever you are in the universe and I look forward to see you
soon bye
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CaptioningWorkspace edited English subtitles for Chapter 4 Microscopy and Staining 8.31.16 | Jun 5, 2025, 10:05 PM |