Transcribed

by Ana Sangadala

July 11, 2014

Microbiology Lectures 6 and 7-Microbial Growth and Classification and

Methods and Tools in Microbiology by Dr. Saxena

**Warning: The order in which Dr. Saxena presents his slides is different than the
order they appear in the powerpoint uploaded to NYU classes. Please compare the
title of the slide when reading the transcript so you know which slide he is referring
to. I also listed the actual slide number in parentheses, but please check if on your
own if it doesnt make sense.

Slide 1- Title
Ok. I never knew that there are so many students and now always in July first week,
we see 200-300 students and then it keeps going down. By the last lecture I give in
May in pharmacology, there are 5 students. And thats if there is no exam. If there is
an exam, it is just me. So it is great to see you all here and welcome to D2. This is the
first course you start and microbiology is generally very easy. People, those who
have done microbiology before, they may think why are we teaching this high school
in graduate fundamentals of microbiology. And you will see the same thing in my
lecture in the next 2 hours. You may think that everything is easy, but dont be
overconfident because some of the stuff in the exam you may get confused and you
will make mistakes. But if you are in this class today and if you pay attention, I think
that will be enough and you will be able to do good. At least in my 10-15 questions
or whatever will be there. Now, if you are doing online shopping, planning your
vacation or whatever you are doing on your computers, but if you are here, just use
this 2 hours and pay attention. It will be very helpful. One more thing. Last year,
every year, you have to evaluate faculties right? You must have all done this for your
D1. Before this, I never got a comment. This time I got a comment in my evaluation
that I was disrespectful to the students. 1 comment. I know the incident what
happened and why that person must have written that comment. But whenever you
write such comments, you have to write the incident. What you were doing when I
said something to you. Dont just write Dr. Saxena is disrespectful to the students.
No, its not like that. If youre talking, chatting, if you are doing cheating in the exam,
there are a lot of things which are involved in it. Just pay attention because that
doesnt say the full picture that was happening that day. Ok?

Now, in the next 2 hours, Im going to talk to you about very fundamental, basic,
microbiology. Dr. Boylan must have already covered some of the introduction in
microbiology. He must have already covered metabolism, how the bacteria grow,
and all that stuff. My job today is just to tell you some of the basic techniques which
we use in clinical microbiology or diagnostic microbiology, or microbiology,
whatever you want to call it. What are those instruments? What are those
techniques? What are those principles, based on which we can identify specific
microorganisms. Before I start, I have to bring one point. In the oral cavity, which
you are all connected to in dentistry, most of the infectious diseases or diseases
present in the oral cavity are associated with one or more infectious agents. That
means one or more pathogens, one or more bacteria, right? Initially people used to
feel that it was one bacteria involved in a specific disease but now we are seeing that

Transcribed by Ana Sangadala

July 11, 2014

there are more than one bacteria involved in it and its called polymicrobial
infections. You take the example of dental caries, periodontal disease, any of them.
There are maybe one, two, three, or some group of microorganisms involved in it. So
where are all these microorganisms coming from? Recent studies based upon
genetics or 16 S, we have come to know that there are more than 700 different
bacterial species present in our mouth. At any given time, today, if I take a saliva or
plaque sample from you, I can easily identify more than 300 bacterial species from
there. So, all these bacteria are present in our mouth as a community. Most of the
time, they are at a very unique balance that is being maintained. These are called
commensal organisms. Once they are there and if there is an ecological imbalance,
that means the present of large amounts of fermentable sugar, or there is a trauma,
or dental extractions or anything, any kind of trauma or ecological imbalance, the
balance between the good and bacteria changes. The bad comes up and the good
goes down. And then we start seeing the disease. As a clinician, you are doing
something.

Slide 3- Diagnostic Microbiology
This is general practice dentistry. You are seeing something in the oral cavity and
now you want to find out if there is any infectious agent associated with it. Ok. This
is just a basic diagnostic microbiology. If you are involved in a research project or
something like that, you want to see what is involved in that. Most of the diseases,
like I said, are associated with some kind of microorganisms in the oral cavity. We
are working on multiple diseases in my lab and we have seen that each of those
diseases are associated with some kind of pathogen from the oral cavity. If you see
here, this is what you are seeing here. After seeing here, you are deciding on how
many extractions you can do and you can plan your vacation based upon the
number of extractions right? But thats not happening here. You see there are
infections associated with it. You collect the specific samples, go to the lab, it comes
back, you do the interpretations, diagnosis, and treatment, and all that stuff is done.
Now this is important here. If the samples which you collected here are not correct,
all the interpretation which is being given to you form the diagnostic labs will be
wrong. Suppose you want to check the organisms that are responsible for
periodontal disease. You have to collect sub gingival, supra gingival plaque samples
and send it to us. Or a GCF or something like that. Close to the proximity of the
disease. If you want to study the association of oral cancer and microorganisms, you
need to have a tumor sample if you want to see. If you want to study a biomarkers in
the saliva, then you need to have the saliva samples. Thats how the lab analysis is
done and then the data is given back to you. And based upon the data, you will start
whatever antibiotic therapy you want to do. Or antibiotic treatment.

Slide 4-Diagnostic Microbiology
In the first hour, will see what are the clinical specimens we can collect. Series of lab
testing procedures, interpretations of microbiological reports, definitive diagnosis
and then how you can start an antibiotic therapy.

Slide 5- Clinical Sample Collection and Handling

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July 11, 2014

Collect from the right site because if you want a plaque sample, you can easily
collect from the right side. Saliva samples are very easy. There are 2 types of saliva
samples. One is called stimulated saliva sample. That means you are stimulating
large amounts of saliva production. How can this be done? Generally, we give a
paraffin strip or a non-flavored gum for a subject or a patient to chew for 5 minutes
and then they can give 5-10 mL of saliva. Unstimulated means at any time, no
chewing, nothing, and they can give easily 5-6 mL of saliva at any given time. You
require enough amount of sample if you want to know the good analysis. Send to the
labs for processing as soon as possible. Why this is important? In the oral cavity,
most of the microorganisms are facultative or strict anaerobes. Facultative
organisms, we will see the definition in the next few slides. Facultative organisms
are those that want to grow in the absence of oxygen, but if little amount of oxygen
is there, they can survive. Bit strict anaerobes are the organisms like Porphyromonas
gingivalis which is associated with periodontal disease. They are strictly anaerobes.
If they get exposed to any amount of oxygen or environmental oxygen, they die
immediately. If you want to do a lab analysis of the specimen which you collected
and you want to find out what are the anaerobes present there, you need to send it
to the lab as soon as possible. Most of the samples collected here in the clinics when
you go to the clinics, samples will be collected here. They go to the Tisch hospital.
Thats where all the diagnostic microbiology work has been done, so the samples
have to be shipped as soon as possible. Transport in an appropriate medium.
Generally you will be provided with a specific kit. How to collect and how to store
and then ship it. Avoid contaminations. In microbiology, there is a technique called
aseptic technique, ok? Which has to be very strictly followed. Aseptic techniques
means there is no presence of pathogens. But in this, aseptic means presence of any
other contamination. You have to protect yourself and you have to protect the
specimen which you are collecting. All the work done in the lab, in the microbiology
lab and while you are collecting the samples-of course you cannot make the person
sterile, or you cant autoclave him-the time you collect the samples from the oral
cavity and then put it in the tube. From that until the lab, everything has to be done
in aseptic conditions. The vials which you are collecting, the samples which you are
collecting cannot be left open into the environment. Air has hundreds of
microorganisms that can go inside. You cannot touch any of the stuff like your
iPhones, iPads, or whatever devices you have. If you are working with a patient, you
cannot touch those kinds of stuff because you are contaminating those things and
later you will get contaminated. Ok? So you have to avoid contamination. Remember
aseptic technique. This is a very unique technique which people use in the
microbiology lab. Transport with the proper records. We have seen multiple times.
Especially in the research labs. People just collect the samples and then they send it
to our labs and there are no records. We cannot track them back. We dont need to
know their names or social security or phone numbers. Thats okay for the
administration to work out. But at least we need the medical history. And I know
that in the labs when you collect samples or specimens in the clinics, they come with
all these bar codes so now it is more and more protected. But in old days, a lot of
these problems came about.

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July 11, 2014

Slide 6-Laboratory Analysis

So once the sample or specimen is coming to the microbiology lab, what can you do
with it? We can take an example that we want to isolate Streptococcus mutans which
is a causative agent for dental caries. We want to isolate that specific
microorganism, so what are we going to do? That is our example. So the first thing
you do when you get the specimen in the lab is you can use solid or liquid media to
grow them. Growing means you have to provide them with nutrients so if there are
10 microorganisms growing, you can use a specific media. Selective media,
differential media, or enrichment media to bring their number is such a way that we
can study them. Ok? Thats how you are going to grow into a liquid or solid media.
Once you have this, you can use a technique called streaking or isolation to obtain a
pure culture. Anybody who has worked in a microbiology lab must know what
streaking is. In nature, these microorganisms are not present as a single species.
They grow as a community, same as in the oral cavity. They grow as a community so
whenever you are collecting a specimen from the oral cavity, it will not be 1
microorganism. There will be at least 50 or 60 or 100 microorganisms coming with
your specimen. If you want to study one specific species, you want to isolate that
species from these polymicrobial specimen. TO do that, you can use a technique
called streaking isolations. Then, once you have a single isolated colony, or single
isolated bacteria which grows like a colony. You need to find out what that
microorganism is. So then you have to stain it. You know the size of a
microorganism is close to 1 micrometer, right? You cannot see it by naked eye. You
need to see under the microscope and to have a better contrast, you need to do a
staining. You need a specific dye to stain the microorganisms and then it is very easy
to visualize under the microscope. This staining property of a specific
microorganism is also a classification for microorganisms. Most of the
microorganisms, I can say 90% of microorganisms can be divided into 2 groups:
gram positive and gram negative. We will see those properties later but that is how
bacterial staining can be used to visualize bacteria under the microscope. Then,
what are the different microscopes that are there and what are the macroscopic
identifications? In the next 2 lectures, you will be listening to certain definitions like
classification, taxonomy, phylogenetics, genetics based upon the phylum is called
phylogenetics. Phenotype, the way the organism looks or appears is called
phenotype. These kinds of words, you will be listening in these lectures very
frequently. So we will see microscopic properties of an organism. We will see how
the phenotype or the pigments or analytical properties of microorganisms can be
used to identify them. If you see here, this is a definition of a pure culture. A
population of microorganisms composed of a single strain. Such cultures are
obtained through selective lab process and are rarely found in a natural
environment. So to obtain a pure culture, you are taking a saliva or plaque sample,
which has hundreds of microorganisms. You use this technique called streaking.
What you are doing is that by streaking, first you are diluting your sample and then
you are isolating a single bacteria on the petri dish. You are not going to see it, but
by doing the process correctly, youre isolating single bacteria. Now, you dont see a
single bacteria, right? So whats going to happen when you incubate that plate or
that petri dish that has the nutrient in it into the incubator. This single bacteria will

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July 11, 2014

grow, multiply, based upon their microbial growth or whatever growth period they
have. Within 24 hours, they will make a colony. That colony has 9-10 days to make
the same kind of microorganism of the same strain. And that is pure culture. We
need to have a pure culture to make a diagnosis or classification of a bacteria.

Slide 7-Laboratory Analysis
There are non-cultural methods. These were like a cultured method, how you can
grow them (referring to previous slide). In the oral cavity, fifty percent of
microorganisms are non culturable. Non culturable, we can call it today, but in my
opinion, we dont have at this time, ways to grow them so we classify them as non-
culturables. We dont have a specific media to grow those microorganisms. Now, I
told you before that each person can have 300 different microbial species in their
mouth or there are over 700 microbial species present. If you consider that you
cannot grow 50% of them, then we dont even know which organisms are associated
with disease, right? We dont even know 50% of them. TO find out and study the non
culturable microorganisms, the DNA and RNA studies are very important. If the
microorganism is present in the saliva, the DNA of the microorganism will be there
because there is autolysis and all that stuff. DNA will be there. We use that DNA to
identify specific microorganisms. How we can do that? By using DNA and RNA
probes. Then there are immunological methods. These are used in the clinics very
regularly. The best example is the triponema, syphilis, all those sexually transmitted
diseases are generally identified using the immunological method.

Slide 8-Microorganisms exist in nature as mixed populations
How can you separate a particular bacterium that is responsible for an infection out
of 100s of microorganisms that are present there? Thats exactly what it is here.
Hundreds and hundreds of microorganisms.

Slide 9-Obtaining pure cultures
Obtaining the pure culture. So if you are collecting something that from here, it has
10^8 or 10^9 microorganisms there. In that there will be hundreds of different
species. How do you get a single colony here? This is a pure culture and single
colony. When you are doing a streak plate method, you are streaking on this and one
bacteria will grow into this big colony which you can visualize by normal eyes. But
from here, we can pick this colony, which has hundreds of microorganisms of the
same strain. We can do staining, biochemical tests, genomic tests, we can do a lot of
tests to do a confirmed identification and classification of bacterial species. There
are 2 methods. Streak plate method for isolation and we can use a specialized media
to enrich the microorganism that we are interested in.

Slide 10-Streaking for Isolation
The most common way of separating bacterial cells on a solid media surface to
obtain isolated colonies. Ok? This is just an animation to show how you can do
streaking to isolate a single colony. This is your petri dish. Most of you must have
seen it. It has a specific media on that. Then, form your specimen, whatever
specimen you have, you take a loop full of the specimen and streak on a plate in one

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July 11, 2014

direction. And then, you have to sterile your loop. Sterile means there is the flame
with the Bunsen burner. The wire loop which you use to streak should be heated up
in such a way that all the microorganisms on that loop should be killed. Then you
take a little bit of specimen from here and then streak it on the other side. Now what
is happening if you have here high density, after doing the sterilization of loop, you
are taking a little from here and streaking. This is a dilution kind of a thing and then
you are isolating again. Keep doing that 4 ways, generally it is also called the 4 flame
method because you are flaming the wire loop 4 times. And then in the center, you
streak it by making a curve. If this is done properly, it will look something like this.
This is the highly dense area, which is this one. And the diluted one, and then in the
center you will get isolated colonies. This single isolated colony is a pure culture.
From any clinical specimen, if you are good in doing this technique, you can isolate
many different microorganisms. Here, this is a mixed population. If you see, there
are yellow colonies, white colonies, small colonies, big colonies and all that stuff. If
this is a polymicrobial culture, you can still get isolated colonies after streaking here
and here. You can easily pick these colonies and again do the same streaking and
again you will get the same colonies.

Slide 11-Methods used to obtain pure bacterial cultures
So these two techniques, aseptic techniques, where you have to transfer all the
specimens in a sterile condition. Avoid contamination. Then this, using the streaking
method, or an isolation plate, you can easily identify bacterial species from any
given polymicrobial specimen. The other way to do it from streaking is this called
diluted samples. You can do serial dilutions the way you do dilutions in chemistry.
Take the original specimen, do a dilution. 1/100, 1/1000, 1/10,000 dilutions. You
take the dilutions from there and you spread it on a plate with a spreader and you
get single colonies from there. Any way, whether you do an isolation plate or you do
streaking, you will get single isolated colonies.

Slide 12-Methods used to obtain pure bacterial cultures
Once you have these single isolated colonies, you can streak it again and get a
perfect pure culture. Now this is enough to do any kind of work in your lab. Then,
you can go into identification from here. You can do bacterial staining and see
whether it is gram positive or gram negative, or acid-fast, you can identify from
there.

Slide 13- Use of specialized Media
Ok. Use of specialized media. In the oral cavity like I said, there are a number of
microorganisms present. Our example is streptococcus mutans. If you know
streptococcus mutans what it is and what it does. It has a glucose transferase gene.
When the fermentable sugars are present, the gene will get activated and they will
produce large amounts of polysaccharide or glucain and it becomes sticky and all
that stuff. That is how it sticks to the tooth surface and it causes dental caries and all
that stuff. If you want to isolate streptococcus mutans from there, so much work has
been done on that so we know there are special media. That means there are
selective media. In this selective media, there are certain chemicals added. Either to

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support the growth of one organism or inhibit the growth of lots of other organisms.
We can enrich it. Streptococcus mutans are resistant to bacitracin which is an
antibiotic so if you add bacitracin into the medium, most of the time, you will see
streptococcus mutans growing because all other microorganisms will be inhibited.
Thats a selective media. Differential media is when you take a chemical or a
carbohydrate into the media, and you have polymicrobial specimen. You then streak
it on a plate and if one of the organisms can utilize that carbohydrate or chemical, it
will produce colonies in a different fashion or it will produce a pigment and the
other will not. Immediately you will know it is a differential step because it is
fermenting. Based upon the fermentation, the metabolism, which Dr. Boylan must
have covered. Not all microorganisms can utilize all the sugars, right? Like lactose.
Some organisms can use lactose and some cannot. The organism which can produce
lactose will produce different colonies and those that cannot produce lactose will
produce different colonies and thats how you can differentiate and you can use
differential media. Enrichment media can be a combination of selective media with
antibiotics. So you are enriching a specific kind of microorganism in that. Then this
is a very nice example here. It is called EMB again. Eosin Methylene Blue agar, just
one example. It is a combination of selective and differential media. If you see here,
this produces this green sheen color colonies on that. Now, this media has
methylene blue. Which inhibits lots of other microorganisms. And then it has lactose
as a carbohydrate. The bacteria like E. coli which can utilize lactose and can survive
methylene blue will produce this kind of green colonies on this petri dish, this plate.
This is a very nice example of differential and selective media. It is also called
combination selective plates. The example is EMB agar plates. There are many more
examples, if you go to the book. There are MacKonkeys agar. Dr. Boylan must have
talked to you about MacKonkeys agar. There are other examples, it is used for
Streptococcus mutans is MSB media which has bacitracin in it.

Slide 14-Selective Media
So what are the selective medias? There are different kinds of selective medias. The
best is chemical defined. It permits the growth of most cells of the sought after
bacterial species. That is how we define. You add a certain chemical. Starting low pH.
This is interesting because most microorganisms want to grow at neutral pH. But
some of the bacteria that are present in the oral cavity like aciduric bacteria, like
lactobacillus and some species of streptococcus mutans. They love to grow at a low
pH close to 6 or 5. Some of the bacteria grow at 3 and 4 pH. Now that is low starting
pH that allows only the growth of aciduric bacteria involved in the decalcification
process. That is very selective because other organisms will be killed because of the
acidic nature of the media. Right? Then antibiotic containing media inhibits the
growth of all or most organisms except for the one we are interested in. Like I talked
about the media MSB media for streptococcus mutans, which has bacitracin. It is
most useful when the desired organism is present in low number, like specifically in
the plaque samples which has streptococcus mutans. You want to enrich them. You
use this antibiotically defined media and then you can get large number of that
microorganism.

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Slide 15-Microscopic Principles and Applications

Ok. We will go through this. Any questions about the media or anything? No? Last
year there were 3 lectures for me. This year, they gave me 2, so I may be skipping
certain things which you have in the slide. But whatever is covered here will be in
your exam. Whatever is not covered in this lecture will not be there, ok? And last
year we had conferences and this year we dont have conferences. So a lot of the
stuff that was covered in conferences are there in some of these slides. If I am going
too fast, let me know. We can modify a lot of stuff. We dont have to cover
everything. We dont have to cover everything, but that doesnt mean we finish
lecture in 1 hour and go. Ok?
Now we have a single isolated colony. What are you going to do with it? You need to
find out how they look, how the specific microorganism looks. You cannot see with
the naked eye so you have to use some device and the best device is a microscope,
right? So youre going to use a microscope for the detection of microbes in clinical
specimen and making a definitive identification of microbes. You have a single
colony so you can make a smear on a glass slide and see under the microscope. You
may not see anything because the contrast is very bad. There are different kinds of
microscopes. There are bright field microscopes, there is a dark field microscope,
there is a phase contrast microscope, there are fluorescence microscopes, and there
is an electron microscope. Each of these microscopes are used for a specific purpose.
We are first going to talk about bright field microscope, how it can be used, and
what are the things we can do to have a better contrast.

Slide 16-Microscopic Methods
These are the 4 basic microscopes.

Slide 17-Bright-Field Microscopy
This is a bright field microscope. You must have seen in your high school, middle
school, undergraduate, this microscope. This is a simple student microscope. It is
also called a compound microscope. It has a light source here. Then you have a
platform where you can put your slide or stage. Then you have objective lenses and
ocular lenses. This is a 2 lens system microscope because you have 2 lenses:
objective lens and ocular lens. Any magnification which you are seeing is a
multiplication of this number and this number. Correct? If this is 10X and this is 10X,
you are seeing 100X. 2 Years ago, I had a question that if you have an objective lens
of 10X and ocular lens of 15X, what will be the final magnification? And I got 25
people gave a wrong answer. But this is simple. In any microscope, the objective and
ocular lens, you multiply and thats your magnification. Okay, so objective lenses can
be of many type. If you see here low power, thats 10 X, if you have high dry, thats
40X. And then you have an oil immersion. Oil immersion is almost like 100X and if
you see the ocular lenses, most of the time, in compound microscopes, they are
either 10 or 15 X. Even if you use the highest magnification of objective lens and this
15, you cant get much magnification. Thats one of the drawbacks of this. But this is
a very quick and dirty way of looking at the microorganisms immediately when you
have something. This is an easy, cheap microscope. And we can use it in a regular

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microbiology lab. It has a light source, stage, condenser, two lens system and is a
very nice microscope.

Slide 18-Bright-Field Microscopy
Now this is a bright-field microscope, which is now available. Do you see how
complicated this is? Its doing the same thing but now it has different kinds of
halogen lamps, different light source, and it is highly, highly complicated. But you
get some benefits out of it. You have a better contrast, you have a bright background
and all that stuff using this complicated microscope.

Slide 19- Bright-Field Microscopy Oil Immersion
This is oil immersion. Ok? Why do you use oil? If you see here, the refractive index.
The refractive index is the way that the transmission of light is being explained in
physics. The refractive index of lens is 1.5-2 and the refractive index of oil is 1.5-2,
the refractive index of slide is 1.5-2 and the refractive index of air is 1. So when you
are using a high magnification and the light has to pass through the air, if there is no
oil here, the image is distorted because of different refractive index so scientists
have developed this immersion oil, which has a similar kind of refractive index. So
now the image is seen very clearly with better contrast and there is no distortion of
the image. Thats why oil immersion is used. This is a simple immersion, 100X. The
limitations of bright field microscopes: we know the resolution of the image is no
that great and the angle of the light entering creates a bigger problem. Thats why
we use oil. We cannot visualize viruses from there. To visualize any virus, you need
to have an electron microscope.

Slide 20- Calibrated Ocular Micrometer
You can calibrate. You can see the size of microorganisms in the oil immersion. This
kind of scale is present in ocular eyepieces there. If you see each of these units, it is 1
micrometer. This is 10 micrometers, 20 and 30. Using oil immersion, you get almost
1000X and you can visualize the size of the microorganism. This is streptococcus. A
strep, a chain of coccus. And thats a streptococcus here and you can see that 1 cocci
is almost like 1 micrometer.

Slide 21- Bacterial Morphology
In lab, bacterial morphology may be examined in 2 ways by observing living
unstained organisms. Its called wet mount. By observing killed strain, its called
heat fixed or a stained specimen. Now we have a bright field microscope, we have
single isolated bacteria that we have isolated from a single colony. Now we want to
see it under the microscope but to have a better contrast, we need to do something
to the organism.

Slide 22-Bacterial Stain
For that, we do bacterial staining. Ok? To visualize bacteria clearly as with greater
contrast, you need to stain it. You need to color it, ok? So you can see it in great
details. To differentiate and categorize various morphological types according to the
staining properties, that is gram negative and gram positive and to observe certain

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structures such as capsules, endospores, flagellas and all that stuff, you need to do
staining.

Slide 23-Bacterial Stain
So what are bacterial stains? These are simple dyes. Ok? Those who have worked in
the cell biology lab or microbiology lab or molecular biology lab, or you must have
done in undergraduate. It is simple staining: crystal violet, methylene blue, eosin,
safranin, all those are dyes. They have a color component there. Like methylene
blue. And dissociate water into a positively charged methylene blue ion, which is
blue in color, and negatively charged chloride ion, which is colorless. So this blue
part will stain the cells. It is the positively charged methylene blue. Why will it stain
the bacterial cell? The cytoplasm in the microorganisms is slightly negatively
charged. If the cytoplasm is negatively charged, you use a positive chain that is blue
in color, opposites attract. Thats the reason the microorganism is stained blue.

Slide 24-Bacterial Stain
Its also called basic dye. The color portion lies in the positive ion, thats methylene
blue. And acid dye, which we can also call negative dyes, the color portion is in the
negative ions. Some examples are nigrosin and congo red.

Slide 25-Bacterial Stain
Now this is an example here. If the cytoplasm is negatively charged, you use a
positive dye. The whole organism will be colored. But if you are using a negatively
charged dye, like congo red, nigrosin, and those kind of stuff. What will happen? The
cytoplasm is negative, the dye is also negative and they repel each other. The
specimen or the bacteria will not get stained. The surrounding will get stained.
Thats called indirect staining. Direct staining is when the bacteria gets stained or
colored. In indirect, the surrounding gets colored. You see a clear specimen in a dark
background. Ok?

Slide 26-Bacterial Cell Wall
Once we have the dye, we know that the microorganisms can be divided into gram
positive and gram negative, right? This property is uniquely based upon of the
content of the cell wall of the microorganism. What are gram positives and what are
gram negatives? If you see here, the structure of a gram positive cell wall. This is
generally seen in the electron microscope. The gram positive bacteria has large
amounts of peptidoglycan. Multiple layers of peptidoglycan. One, two, three, four,
and five. In this peptidoglycan, they have different amounts of lipoteichoic acids,
teichoic acids, surface proteins, and thats how all antigens and characters are based.
For this gram staining, what we are concerned with most is the peptidoglycan. So
when Dr. Boylan covered metabolism, he must have covered how the peptidoglycan
is being synthesized. In gram positive, you have high amounts of peptidoglycan and
then you have a cytoplasmic membrane. Whereas if you see in the gram negative cell
walls, you have an outer membrane, you have a very thin layer of peptidoglycan and
then you have a cytoplasmic membrane, same as like this. So here, there are only
two layers of peptidoglycan. Here there are more than 5 or 6 layers of

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peptidoglycan, and then you have this outer membrane which is made of proteins
and lipids, lipoproteins here. It has LPS, polysaccharides, and all that stuff is present
there. This is the major different between gram positive and gram negative bacterial
cells. This is very significant in understand gram positive staining and gram negative
staining.

Slide 27-Staining Methods
So here when you are doing a gram staining, to look at these microorganisms under
the microscope, you have a gram positive here. Both of them are colorless. You use
the crystal violet, which is a positive stain. Everything will get stained in both gram
positive and gram negative. Then you use an iodine, which is called Grams iodine.
Ok? What this iodine does is it goes and interacts with crystal violet and makes a
very tight complex inside the peptidoglycan. Now both the organisms, gram positive
and gram negative look purple. Then comes the differential stage. You use a
decolorizing agent. Its called, its made up of alcohol and acetone. When you use
that, what happens is that the thick layer of peptidoglycan retains the purple color.
In the case of gram negative, the outer membrane which is covered in lipoproteins
and lipids get solubilized into that alcohol and acetone. And now this very thin layer
of peptidoglycan gets exposed and because its exposed, the purple color is not
retained. The purple color is not retained and it becomes decolorized, same as like
this. But here, it is retaining that purple color because of the thick peptidoglycan
layer. Now you use a counterstain again, thats a positive stain, its called safranin
and it is red in color. Now you stain it, both the organisms at the same time. This will
not retain any color because it has already been saturated by crystal violet, whereas
this will be colorized in red. Thats how you differentiate between gram positive and
gram negative microorganisms. Once this classification is done, you have already
divided microorganisms into two different categories. Whether the organism is
gram positive or gram negative. This technique can be used even without isolating a
single bacterial cell. If you have somebody that has a serious infection in the clinic,
you may not see it, but generally in the emergency room. If somebody comes with a
serious cough or serious stuff, they can take the sputum or the saliva of that person
and straight away go and do the gram staining. The ratio of gram positive and gram
negative will tell them what kind of interaction it will be and they can start the
treatment immediately again if they want to.

Slide 28-Bacterial Identification
This is how you will see a gram positive cells and this is how you will see E. coli or
gram negative. If it is a mixed species, or sorry mixed culture, you will see a lightly
stained pink gram negative and a dark stained gram positive microorganisms.

Slide 29-Staining Methods
There are different kinds of staining methods. It is called capsule staining
procedures. These are generally used to stain the capsule and the main ingredient
here is malachite green. Where is it? Anyway, you can use the malachite green and
do the capsule staining or the endospore staining. In capsule staining, sometimes
you can use a negative stain and you can see the organism.

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Slide 30-Capsule Stain
These are the capsule staining using a negative stain. The bacterial cells are not
stained and the surrounding is colored.

Slide 31-Endospore Staining
Endospore staining. This is generally done by malachite green. Some of the bacteria
or the bacillus produced produce endospores. Endospores are the organs or the
system generally used by microorganisms to survive in extreme environments. If
there is no food, extreme heat, they have to survive. What do they do? They produce
endospores. Endospores can survive in an environment for years. At this stationary
phase, when you see the microbial growth. From the stationary phase, to the death
phase, they start producing these endospores. These endospores, if you want to see
the specimen to see if you have endospores or not, you have to do a malachite green
or endospore staining. This is clostridium tetani, which you will see in infectious
diseases in November. This is a very unique pathogen. It has racket like structures
and they have terminal spores. You can use endospore staining to see how they look.
This is Bacillus anthracis, a very beautiful chain-like structure here. You see like
these bead-like structures, these are all endospores.

Slide 32- The Acid-Fast Stain
Acid fast stain. So I said 90% of microorganisms can be stained and classified into
gram positive or gram negative. Some of the microorganisms cannot be classified by
doing gram staining because they have very unique properties. The examples are
Mycobacterium or Nocardia. Those microorganisms have a very thick, waxy
substance over their cell wall. They have a high amount of glycolipids and mycolic
acids. Because of that, these organisms cannot be differentiated by using gram
staining. So then, people do acid fast staining. Acid fast means they cannot be
decolorized by acid fast treatment. It resists decolorization so it remains stained red.
The stain here is carbol fuchsin. Just remember the staining and the acid fast is used
for which organism. Last year there was a question to identify mycobacterium or
differentiate mycobacterium from other bacterial species, what kind of staining you
are going to use? The answer is acid fast staining. There was a question on the
boards, same kind of question. To differentiate mycobacterium, what stain are you
going to use? ACID-FAST STAINING. Here, if you see here, this is acid fast staining.
This is from a sputum or saliva sample from a patient. You will see this red or
filamentous thing and those are the mycobacterium.

Slide 33-Dark-Field Microscopy
Ok. Dark-field microscope is only important to see a very thin specimen. Ok? It has a
special condenser and the light source doesnt directly illuminate the specimen. It
passes from the side of the specimen. It is a very unique microscope that is only
important when you are visualizing a very thin microorganism like spirochetes.

Slide 34- Dark-Field Microscopy

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Here you have a condenser lens that has been specially put here. And then the light
passes through here, and this is your stage specimen. It doesnt pass through the
specimen, it passes from the sides. So you will see a bright specimen with a dark
background.

Slide 35-Dark-Field Microscopy
The resolving power is significantly improved in this compared with bright field
microscopy. You can see the specimen which are very thin and the disadvantage is
you cannot see the internal structures, cannot be studied with a dark-field
microscope. Ok? The thing which you have to remember, in this, is why do we use
dark-field microscopes? To study thin microorganisms. What are the thin
microorganisms? Spirochetes. Ok?

Slide 36- Dark Field Microscopy of Spirochetes
These are spirochetes and this is a dark field microscopy. You can see here and this
is a very unique microorganism that we will see in the next lecture hour or so.

Slide 37- Phase-contrast Microscopy
Phase Contrast Microscopy, we can skip it. Let it go.(Dr. Saxena then skips a bunch of
slides on the powerpoint).

Slide 38-Electron Microscope (41)
Electron microscope. Ok, this is important because when you study the biofilms, oral
biofilms. Sometimes you want to study the real oral biofilms, how they are
connected to each other, how the cells are connected to each other, and a lot of
things you can do with an electron microscope. You can use an environmental
microscope where you can use a real specimen, wet specimen, to see in detail what
it is and how it looks. And here, you dont have lens and all that stuff. These are
made up of magnetic coils, and you have electronic beam passing through it. It gives
very good magnification. The magnification can be improved. You can see a
specimen that is 0.001 micrometer, which can be easily seen. There are 2 different
kinds of electron microscopes typically used. I think we have here, both of them.
Transmission electron microscope and scanning electron microscope, ok?

Slide 39- Electron Microscopy (42)
In the transmission electron microscope, it works exactly the way the slide projector
works. So, if you have a specimen and that is like a slide, any object on a slide. The
light passes through it and you can see an image. Whereas in the scanning electron
microscope, you can see the surface and the texture of the microorganisms, it
bombards the electrons on the surface in a zigzag fashion and the image is formed
on the surface of the microorganism. The transmission and scanning, they both have
different function. But they are both important to the clinical microbiology.

Slide 40- Electron Microscopy (43)
This is a typical biofilm here. This is what you see in 100X like in a bright field
microscope, 1000X, again if you are using an oil immersion you can see it. But if you

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go beyond that, you see the cells. You can see how they are, how they are connected
to each other. These are different kinds of cells here. These are bacilli and oval
shaped structure. And you can really see the cell and the real community in the oral
cavity by using SEM scanning.

Slide 41- Electron Microscopy (44)
This is just a comparison. If you see TEM, transmission electron microscope, you can
see what is inside, what is the cell wall made up of, how the cell division is
happening, what are the chromosomes and divisions that are happening inside the
cells. If you see the scanning, you can see the outer surface, exactly how they look.

Slide 42- Microscopes Summary (45)
This slide is just a comparison of all of them. You can go through it. It is good for an
exam point of view.

Slide 43- Summary Staining (46)
And this is the staining properties again.

Slide 44-Summary (47)
And ok, do you want to take a break for a few minutes? And then we will start this.
Any questions? You can come here, you can send me an e-mail.

Slide 45- Bacterial Classification and Microbial Growth

Slide 46- Microbial Metabolism and Growth (48)
Okay. So in the first hour we saw some of the tools which can be used for
identifications and classifications of microorganisms. How you can grow them, how
you can get single colonies, how you can use the microscopes, what kind of staining
can be done, and all that stuff. Its very basic and fundamental microbiology. Now,
once you have a single isolated colony, what can you do to find out what are these
microorganisms? If they stain gram positive, and you look under the microscope,
you will see a specific structure there. You can see if they have flagella, they have
endospores, and all that stuff, right? Now we have to classify them. Classification is
based on identifications and similarities. There is a field called taxonomy is science.
This field is generally being used to categorize all the living beings. Which phylum,
class, genus, species we are. Most of the living beings or the living structures have
been classified into some kind of a group or a class or a species or genuses. So before
we do that, we need to understand some of the microbial metabolism and their
growth. So metabolism is I think covered by Dr. Boylan in the previous 2 lectures or
something like that. What I am going to do is very briefly cover the growth curve of
microorganisms which is very important or the growth cycle of microorganisms.

Slide 47- Metabolic Requirements (88)
So whenever the microorganism or the living being or the living structure or the
living organism has to multiple. Why do they multiply? They multiply to survive,
right? They also multiply to produce or reproduce. And then, to do that, they require

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a carbon and nitrogen. They require an energy source, they require water, and they
require various kinds of inorganic salts, organic stuff, and all that stuff, right? So all
those stuff combine as nutrients for the microorganisms which have all this stuff.

Slide 48- Essential Elements (90)
Now, this is a Patrick Murase book which is on Vitalbook. And you can see how
many different kinds of elements and cofactors and stuff are required for
microorganisms to grow and multiply. If anybody has free time, they can go and
memorize this table.

Slide 49-Prokaryotic Reproduction (91)
Prokaryotic reproduction. Here if you see this, most prokaryotes reproduce by
binary fission and cells double in mass. At any given time, if you have 2 bacterial
cells, in the germination time. Germination time is the time required for bacterial
cells to double. It is called doubling time or germination time. There are different
ways we can call it. The cells double in mass, so if you have 2 bacterial cells, within
20 minutes, if their doubling time is twenty minutes, these 2 cells will become 4 and
in the next twenty minutes, they will become 8, 16, and you keep on going on. Thats
how microorganisms do, they just double at a specific doubling cycle. So if you have
2 or 4 microorganisms, then by tomorrow at the same time, they will be in the
billions and trillions. How do they divide? They have a cell wall, a plasma
membrane, and a DNA molecule. The DNA molecule has to replicate. And again, it
was done by Dr. Boylan, how the DNA replication takes place. And once the DNA
replication has happened, then the division or the cell wall and plasma membrane
begins to divide the bacterial cells also. Once that happens, it will divide into 2
bacterial cells and it will have the same number of DNA copies that was present in
the parent cell and you have 2 daughter cells. Now they separate and the cycle starts
again. This whole process in most of the microorganisms takes 15-20 minutes. Ok?
Now in that 15-20 minutes, the DNA is being replicated and they are using all the
metabolic requirements, all the iron and minerals and everything. They produce the
2 strands of DNA, which replicates and separates out. The septum formation starts
and 2 daughter cells are produced. This is the whole process of bacterial division.
Once this division is happening, it can happen in a very slow fashion, which is called
a lag phase. And then there is a log phase in the bacterial cycle when these cells are
multiplying in a faster phase. And we can have the bacterial growth in a much faster
way.

Slide 50-No Title (92)
So if you see here, this chart, this is from a book. This phase of any microorganism,
the first few hours of their life is called the LAG phase. The lag phase is the time
when microorganisms are trying to adjust to the environment to which they have
been added to. They will find out what are the food stuff present here? What is the
food? Can I eat this? Stuff which is harmful for them, they will find out there. They
are trying to adjust to the environment here. Then, after a certain period of time,
this is called the log phase. There is an exponential growth of the microorganisms
and this is an exponential growth phase. Now, if you are having here, zero time, you

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have one bacterial cell and by this hour like 7 hours, the number of bacterial cells in
any given suspension. That is bacterial growth. They grow in such a high fashion.
Now the germination or doubling time is the interval of time between successive
binary fission. Some have 20 minutes, some have 40 minutes. Some of the
microorganisms like triponema or spirochetes have doubling time of 16 hours but
most of the organisms like E. coli and all that have a doubling time of 20 minutes. In
pathogens, a shorter doubling time means a shorter incubation period of disease.
Remember this: if you have a pathogen and it has a shorter doubling time, that
means they can produce a large number of their organisms in a very short period of
time. That means the incubation time for a disease is very short. So you will start
seeing clinical symptoms of infection very quickly.

Slide 51- Bacterial Growth (93)
If you see here, this is a typical example of how E. coli can germinate in 20 minutes
when you can start with a specific number of cells. If you see here, the equation
here. In this you have the total number of bacterial cells, which is N (zero) here, how
many cycles, if there will be 6 cycles. And thats the value here. If you put all the
values, this is simple math, if you put all the values here then you will know how
many bacteria will be present after a specific period of time.

Slide 52- Phases of Bacterial Growth (94)
This is a growth curve of microorganisms. Again, this is the lag phase, when the
bacteria is adjusting to the environment. Then, you have the log phase. Then you
have a stationary phase. In this stationary phase, most of the microorganisms have
already utilized most of the resources that are present in the medium. Now, they are
dying, or they are trying to survive by producing endospores in this phase. And then
there is the death phase or the decline phase, where most of the microorganisms
start dying. Why? Because there is a toxic waste that may be produced into the
medium OR, there may be certain things like there are no nutrients lift, maybe the
temperature isnt favorable, maybe there is no carbohydrate left in the media. And
now, the living cells, the live bacterial cells will be less than the dead cells. Here, it is
your inoculum, the cells that you have inoculated. Here, the live cells are in much
higher number than the dead cells. Here, the live and dead cells are equal. In this
phase, the living cells are much less as compared to the dead cells

Slide 53- Growth curve of bacteria (95)
Again, same thing here. Same growth curve. You have a lag phase, then exponential
or a log phase, then you have a stationary phase, then you have a decline. If you see
here, there are very few cells. More of the live cells, less of the dead cells. Same here
at the end, you have more dead cells and very less of live cells. This is the typical
bacterial growth curve. And if I give you an exam of mark A, B, C, or D, of this
growth curve, you should be knowing it. Right? This is in the lag phase, log phase,
stationary, and death phase. Ok?

Slide 54- Bacterial Classification (96)

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So once we have the cells we saw, we have the microscopes, we have staining
properties, we have the media in which we can grow, we know the doubling time.
And now we want to classify them. If you see under the microscope, we see
elongated cells, we see circular cells, we see a bunch of cells together, we see a chain
of microorganisms. What does this all mean to us? Thats how we are going to see
the net few slides. How can we use different methods to classify these
microorganisms?

Slide 55-Cataloging Microorganisms (49)
Okay. The first classification was given here if you see in 17th century. And until that
time, there was no classification there. They didnt even really know how to classify
the human: which class, which phylum, genus we are present? The first
classification was given in this year okay?

Slide 56-Cataloging Microorganisms (52)
Here, something is important because this is a microbiology class. If nobody has told
you, I should tell you that in each name, there are 2 words. 1 is representing the
genus, the other is representing a species. Now, in most microorganisms, if you see
there are 2 names, and at the end there is a subtype or a subspecies. Lets take the
example of streptococcus mutans. Streptococcus is the genus. Why? Because it is a
strep of coccus, we will see in detail why it is called streptococcus. And then, at the
end, it ends in mutans. Streptococcus mutans. Thats the scientific name of the caries
causing organism. Streptococcus mutans. Mutans is the species. The name of the
species is given based on the metabolic properties of the organism or the type of
disease they cause. Or from the place where they have been isolated, something like
that. Take the next example. Mycobacterium tuberculosis. Mycobacterium is the
genus and tuberculosis is the disease so that becomes the species. So the organism is
called mycobacterium tuberculosis. The other example is Bacillus anthraciis.
Bacillus is the genus and the last one, anthrax, is the disease. Thats why it is given
the name Bacillus anthraciis. Thats how all the bacterial names have been classified.
The first one is the genus to which they belong and then you have the species which
is based upon the metabolite or the disease or the infections they cause.

Ok, this we can skip. (skips slide)

Slide 57- No Title (53)
Ok. The first and the most accepted 3 domain system was given by Carl Woese in
1971. He gave this 3 domain system to classify all the living beings. And in that, he
put bacteria and archaea and prokaryotes and eukaryotes. It is similar to the old
classification, but he put in place of, instead of having prokaryotes and eukaryotes as
the 2 domains, he made it 3 domain. In prokaryotes, he divided it into bacteria and
archaea and then you have eukaryotes. Its very easy to differentiate between
eukaryotes and prokaryotes. Single cell, multiple cell, cell wall, cell membrane, there
are lot of other stuff which you can use to differentiate which you have studied
before. In this, in the 3 domains, he put bacteria and eukarya, and archaea. What was

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his basis of this classification? Why did he differentiate? What structures did he base
this?

Slide 58-Three Domain System (54)
Okay, he used 3 main characteristics. The first one is the difference in the sequences
of nucleotides in the cells ribosomal rRNAs or ribosomal RNA. If you see, what is
the function of ribosomes? Protein synthesis. If you see in bacteria, if you see in
fungi, if you see in humans, animals, anywhere you go, in all the living species,
ribosomes are responsible for protein synthesis. It is highly conserved, highly
protected. So any changes happening in that will be very drastic. If we can
understand the sequence of this rRNAs in different groups, we can easily identify
what are the differences. Can we use those differences to classify? He used that and
he made this 3 domain structure. 3 characteristics based on rRNA. The cell
membranes lipid structures and sensitivity to antibiotics. This is very important.
Sensitivity to antibiotics. But this is very simple. Why havent people thought this
before him? The bacteria are sensitive to a specific antibiotic. Human cells are
sensitive to specific antibiotics. Prokaryotes and eukaryotes have different
sensitivity pattern to antibiotics. The same is with the archaea. He used that. Then
cell membranes, lipid structures. Our cells have different structures, and bacterial
cells have peptidoglycan. And please note, archaea DONT have peptidoglycan. So he
differentiated archaea and bacteria very easily because bacteria have peptidoglycan
and archaea dont. So he said no, they are not they same. They have to be in a
different structure, a different pool, different group. Then, he used the antibiotics.
The antibiotics which kill bacteria does nothing to archaea. So he said oh yeah, they
are different, so he created another category. Thats how he used the 3 domain
structures and he used these 3 characteristics: rRNA sequence, cell membrane
structures, and then sensitivity to antibiotics and he created this whole new
classification system.

Slide 59 (55)
We can skip this.

Slide 60-Bacteria who are they?? (56)
Okay. What are the bacteria? They are single cells, prokaryotic are very much
smaller than eukaryotic cells. They are very much complex despite their size and
they can be distinguished from one another by their size, shape, staining
characteristics. Thats the morphology. Thats the phenotype. The way they look.
The way they look, we can classify. We use this kind of stuff in our day to day life.
How a person looks? We can easily say, this person is from Asia, this person is form
here. The way they look, thats how we classify them. Right? Exactly. The way you
see under the microscope. You see the shape and size and the properties of the
microorganisms, you can classify them. This belongs to this group, this belongs to
that group. Its an easy way of classifying things.

Slide 61- Bacteria on human epithelial cells from the mouth (57)

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Okay. These are just the comparison between the epithelial cells and the
microorganisms. The size comparisons. You can see that one mammalian cell can be
infected by multiple bacterial cells.

Slide 62- The four Major Categories of bacteria (58)
So there are 4 major categories of bacteria. It is gram positive, eubacteria that have
cell walls. 50-90% of their cell wall is peptidoglycan. Then you have gram negative
bacteria which have an outer membrane and a thin layer of peptidoglycan. The
other one is eubacteria lacking cell walls that are called mycoplasma. There was a
question about this: the bacterial cells which doesnt have a cell wall is known as
mycoplasma. Ok? Remember that. Archaeobacteria, terrestrial and aquatic microbes
in hypersaline and extreme environments. Now, they are present at high
temperatures, deep in the ocean, and these archea are totally different from normal
bacteria. The 3 characteristics: their rRNA is different, their antibiotic patterns are
different, they have different cell walls structures and they can survive in extreme
environmental conditions.

Slide 63-Bacterial Classification (59)
Ok. Any of this can be used to classify a bacterial cell. Gram staining, yes, thats the
most important one. Then the genome, or genotyping, or the genetic classification of
microorganisms can be based upon different components of nucleic acids that are
present there. The ratio of nucleic acids that are present there can be classified.
Where they survive: high temperature, low temperature, you can use to classify
them. Ability to form heat stable spores. If they can produce endospores which can
survive in extreme conditions can be used to classify microorganisms. What is the
respiration, what is the requirement, what is the carbohydrates and nutrients they
require? Are they motile? What is the cell shape? Can they use various carbon and
nitrogen sources? Does it require special nutrients? Some microorganism cannot be
cultivated in the lab on normal media. You need to provide blood with it. The sheep
blood. Ok? So thats a special nutritional requirement.

Slide 64- Bacterial Classification (60)
When youre doing a classification of a bacterial species, you can use 3 different
ways. One is the phenotypic classification which is once again, the way the
microorganism looks. The shape and size of the microorganism. If they produce any
flagella or cilia or anything like that. You can use to classify them. If you have a
microorganism that produces a pink pigment when you grow them, you can
immediately find out that this is Frashia (??sp?). Streptococcus mutans produces
large amounts of polysaccharide, the sticky substance. As soon as you grow them
onto the glucose or sucrose plates, they immediately start producing the
polysaccharides, so you can immediately make out that it is Streptococcus mutans.
Klebsiella pneumonia, which is present in most of the hospitals, that organism when
you grow on the petri dish produces mucoid colonies. If the plate has 10-15 colonies
of Klebsiella pneumonia, the next day by the time you come in the lab, half of the
plate will be of the sticky substance, with these mucoid colonies. You can easily do
phenotypic classification just by seeing how they are, how the colony looks,

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classification, staining properties, and all that stuff. And then there is an analytic
classification where you can use different kinds of proteins which are present, cell
membrane structures, lipid content of the cell membrane. Now there are more and
more advanced techniques to label like chromographic techniques which you can
use to find out what is present in the cell wall, in the cytoplasm, and all that stuff.
Nobody used this until the research lab today to do classification. People used
phenotypic classification. Most of the diagnostic labs use phenotypic classifications.
They grow it in different medias, they see how it looks, they do biochemical tests.
Whether this utilizes citrate positive, catalyze negative, all that stuff which you will
see or hear in the next few lectures can be used in phenotypic classification. People
use it in regular diagnostic microbiology lab. The field which is gaining tremendous
amount of interest now in diagnostics is genotyping or genetic classifications. There
was a case, I love giving this example all the time, there was a case of a drummer. He
was performing in Philadelphia I think a few years back and he collapsed on the
stage while performing. There were no signs or symptoms what happened to him.
He was immediately was moved to the hospital, they took his blood, and they found
out he has elongated bacillus with endospores immediately. They suspected he had
a bacillus anthrax infection. They cannot wait for doing a lot of tests because bacillus
anthrax, if you dont treat it immediately will kill the person, and it was in his lungs.
They took the spinal fluid, lung fluid and they saw light cells there. Elongated
bacillus with endospores. Immediately they thought it was bacillus anthrax. Plus he
has clinical symptoms. What they did is, immediately, they did the genotyping or
genetic classification. They took his blood and take the specific probes for bacillus
anthrax. They did the PCR, which is polymerase chain reaction. They did the PCR,
they got the positive signal within 45 minutes. They started the treatment and the
guy survived. If they couldnt have done this, it would take 2-3 days to make a
confirmed diagnosis of this organism and by that time, he would already be in coma.
The result would have been different. Nowadays, more and more diagnostic labs are
trying to do this genotypic classification or genotyping of microorganisms.

Slide 64-Bacterial Classification (61)
Ok. Phenotype, if you want to see the phenotypic classification, what can you see?
You can see the microscopic morphology, you can see macroscopic morphology. In
microscopy, you can see the shape, size, arrangement, and gram staining. In
macroscopic, you can see hemolytic properties, pigmentations, size and shape of
colonies, how colonies are produced. If you see a streptococcus mutans on the petri
dish specifically on MSB media, you will see colonies are like grains of salt. Sprinkled
grains of salt. They dig inside the medium. When you see the in the microscope,
these colonies dig inside the medium. Whereas if you see an E. coli colony, which is
growing on the surface. Flat surface, big colony, pale in color, you can easily
differentiate based on the colony morphology. Ok? Then the biotyping. Biotyping is
the fermentation properties. So what carbohydrates or sugars can they use? Like
streptococcus mutans, if you give them glucose, sucrose, they are happy. Right? You
can immediately make out. Like catalase test, all those stuff can easily be done by
doing biotyping and easily be used for phenotypic classification. And then
serotyping. Antibodies, antigen reactions can easily be used to identify subspecies.

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The best example for this, how the serotyping became famous. For salmonella and
typhi, it was very difficult to identify. For salmonella typhi, the causative agent for
typhoid fever. But by doing serotyping, they can easily find out the subspecies of
salmonella typhi and immediately start treatment. And this takes like 15-20 minutes
to get it done. Not all the tests are based upon serotyping. Some are very well
developed, some of them are not developed at all.

Slide 65-Bacterial Classification (62)
Ok. Then comes the analytic classification by using different chromatographic
methods. You can check cell walls for fatty acids, different compositions of cell walls,
proteins, and you can do multi locus enzyme electrophoresis. This is a very
sophisticated technique where you take the cytoplasm from the bacterial cells and
you run everything on a gel. And you see a pattern there. Based on the pattern, you
can identify many different microorganisms.

Slide 66- Bacterial Classification (63)
Ok. Genotypic classifications which are very significantly used within the research
lab nowadays. It can be based upon the G and C content of the chromosome based
upon hybridization, nucleic acid sequence analysis, plasmid analysis, ribotyping, and
all that stuff. Now, in this, this one is very important because in dentistry, the ratio of
guanine and cytosine is very regularly used to classify microorganisms. All the
pathogens, or all the microorganisms in the oral cavity which can be cultivated and
some of them which cannot be cultivated, they already have the G/C content of them
and they can classify based on that. DNA hybridization is a very unique technique.
Some of you must have already used it in your research projects. You have a known
strand of DNA and then a radioactively labeled strand and then you combine them
together and if they are 99.9% the same, they will hybridize. You can deduct that
signal on a radiograph. Then you have a nucleic acid sequence analysis. This is very
commonly done in my lab, we do a lot of this. We can take the samples, do the
sequencing, and based upon that, we can identify microorganisms. And then lets
see, there are a few examples on the next few slides.

Slide 67-Bacterial Morphology (64)
Bacterial morphology here, there are 3 different kinds of bacterial shapes. Coccus,
rods, and spirals. A spherical or oval shaped bacterium. Bacillus are usually
cylindrical shaped bacteria but it is much more elongated. And then you have spiral
or helical structures.

Slide 68-Arrangement of Cocci (65)
In the next few, we have some examples of that. So in case of this coccus here, you
have a spherical shape. There are different categories here. You have single cells
called coccus. When you double, two here, they are called diplococcus.
Streptococcus is a chain of coccus. This is streptococcus mutans. If you have a
streptococcus mutans colony and you see under the microscope, you will see exactly
like this. There is an immediate structure. Then there is another group called tetrad
when 4 of them are combined together. Then you have sarcina when you have 8

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July 11, 2014

cells. They form 8 cells together, separate out. 8 cells together, separate out. Then
you have this staphylococcus. They divide and they form irregular structures but
when you see under the microscope, they look like a bunch of grapes. It exactly
looks like that. So all the cocci or coccus can be divided into different groups and you
will see in your infectious disease class diplococcus, tetracoccus, streptococcus, and
single coccus. You will see different names and generally lots of genuses, genuses
and species I talked about streptococcus mutans. The genus is based upon the
similarities in morphology, how they look. So the division of this takes place. If it is a
coccus, it divides in the center into 2 cells becoming diplococcus. Ok, this is like 4
cells, a tetrad. This is sarcina, where there are 8 cells. And if you see the irregular
division, this is how irregular division takes place. This is a unique form of division.
Whatever you do, if the bacteria is there and belongs to any of these categories, they
will divide in this fashion. They dont adjust, they will just have single division,
double, triple, and then multiple divisions and thats how the classification can be
based.

Slide 69-Diplococcus: pair of cocci (66)
This is diplococcus if you see here, streptococcus pneumonia. This is streptococcus
pneumonia. Sometimes there were two and then sometimes they start multiplying
and forming a smaller chain.

Slide 70- Streptococcus: chain of cocci (67)
Streptococcus pyogenes. Here you see a chain of them. Streptococcus pyogenes you
see a chain and that is the name of that. These are gram positive. You see the color?
Gram positive.

Slide 71- The cocci: tetrad and sarcina arrangements (68)
Then you see here the arrangement of tetrad and sarcina here. The multiple of
eights here and the 4 cells if you see: one, two, three, four. Exactly, they look like this
under the microscope.

Slide 72: Staphylococcus (69)
This is staphylococcus aureus, a bunch of grapes. Ok? See here, these cells if you see
here look like a bunch of grapes. And this is a very irregular fashion of division but
this is how they look. This is how they look under the electron microscope.

Slide 73- Arrangement of bacillus (70)
Ok, the second group is bacillus. They divide only in 1 plane. Their singles are called
bacillus. If they have a chain, it is called streptobacillus and sometimes they have an
irregular or oval shape, a little bigger than coccus. And they are called bacillus. This
is the bacterial cells in the electron microscope. See the size. They are sometimes 4
micrometers long. They are much more elongated. They divide in only 1 plane.

Slide 74-Arrangement of Bacillus (71)
Ok, this is streptobacillus arrangement. You see the chain? Elongated cells. Again
this is gram positive and you have some of this coccobacillus arrangement.

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Slide 75-The Spiral (72)
The spiral. Most of them are spirochetes, very thin bacteria. We use the dark field
microscopy to see spirochetes. This vibro is a deadly pathogen. It is called vibro
cholera and in developing countries, this organism is responsible for most of the
waterborne diseases. If you see this structure under the electron microscope, you
will see exactly the same comma shaped structures. If you have on a blood slide or a
sputum slide or any kind of a biospecimen and you stain it, and you see these kinds
of structures, it is very easy to differentiate. You have to see it if you are interested
in lab work, you should go to the lab and see this. You will not miss this. They look
just like this microorganism. You will see and say oh my god, these people have
spirochete infection. They are not supposed to be there. If they are there in the
blood stream or the spinal cord, or spinal cord, or plaque samples or something like
that, you immediately know this person must have a specific disease because they
organisms should not be there in high number. So you cannot miss them when you
are seeing under the microscope.

Slide 76-The Spiral (73)
Again, these are different kinds of spirochetes and this is how you see under the
electron microscope-spirochetes.

Slide 77-Bacterial morphology (74)
Some microorganisms dont have any shape. They are called pleomorphic shapes
and they are called pleomorphic microorganisms. They adapt to the shape based
upon the environment sometimes. But they look like filamentous bacteria like this
so people get confused that they are not fungus, but fungus. But based upon their
RNA and antibiotic sensitivity, they classify them again in the bacterial group.

Slide 78- Bacterial Classification (75)
I will cover this part, response to oxygen, because thats important. Lets see the
definition of obligate anaerobes. Obligate aerobes are the organisms that require a
high amount of oxygen to multiple and survive. If they are put in an anaerobic
condition, where there is no oxygen, they will die. Obligate anaerobes are just the
opposite to them. These are the microorganisms which require no oxygen-no
oxygen at all. If you open the vial of these organisms, here, they will start dying. The
example, the best example is porphyromonas gingivalis-strictly obligate anaerobe.
The example for obligate aerobes is bacillus anthrax. Then there is a third category.
We always say there is black and white, right? But there is always a gray area. This is
facultative-that gray part. A lot of microorganisms, they love growing in any of these
conditions but its okay. If I dont get any of those conditions, I will not grow that
fast, but I will still survive. Right? Thats how it is. Obligate aerobes require oxygen.
Obligate anaerobe doesnt require oxygen at all. Facultative can survive in either of
these conditions or in less amount of oxygen.

Slide 79-Biological Classification (76)

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This is in a streptococcus classification where we saw it. Here there are less
similarities because all the classification are based upon similarities. At the domain
level, the bacteria are based upon 3 properties. They are there. If you keep on going
down, and come here, this is genus. Genus is based upon morphology, the way they
look. Streptococcus. And then the species is streptococcus mutans because they
produce an antibody called mutacin. That is a metabolite of metabolic product.
Mutacin. Thats the reason the name is given streptococcus mutans. Then if you go
by doing serotyping, you can get subspecies of C, E, and F. And then you get an ATC
number. This is what you get if you go doing ribotyping and all those techniques.

Slide 80-Bacterial Classification-Streptococcus mutans (77)
There is another example here of..we can skip this.

Slide 81-Biological Classification-Staphylococcus aureus (78)
Ok here, staphylococcus aureus. Its in a cluster, irregular clusters. Gram positive,
contains peptidoglycan and everything. If you see here, the difference can be made
here. First time when you see them, you have a chain of coccus here. That are
arranged in chains, Streptococcus mutans. Now if you see here, cells are arranged in
irregular clusters (referring to staphylococcus aureus). Just by seeing that under the
microscope, you can see the difference between streptococcus and staphylococcus
aureus.

Slide 82- DNA Base Composition Varies Among Different Bacterial Species (79)
I was talking about the GC content. So these are the DNA base components of
various bacterial species if you see here. They all have their own GC content. Mutans
has 40-41, whereas Fuso bacterium nuceatum, which is present in oral cavity, has
27-28. So that can be easily used and they all are associated with different kinds of
oral diseases. So this is how you can use genomic or the percentage of GC for
classification purposes.

Slide 83- Ribotyping (82)
Ribotyping. Ribotyping is the, like I said the ribosome is the most conserved stuff in
the living system, so any changes happening there will give an idea of what is
happening to the organism. So in RFLP, you take an rRNA gene and you do
restriction digestion. You take different kinds of restriction enzymes, you cut that
gene, and then you run it on a gel. And you will see a specific pattern. Based upon
that pattern, you can easily find out which bacterial species are there. Now, for this
lecture, you have to remember that ribotyping is also called RFLP. And this is
restriction fragment length polymorphism. Ok? Restriction fragment, restriction
means you are using an enzyme to cut the gene into smaller pieces. Length
polymorphism, polymorphism is the mutation. The changes in the bases of the
nucleic acid. If it is ATCG, and if you replace one of the base with another base, like
ATCG, instead you have 2 As in the beginning. Thats called polymorphism.
Polymorphism is associated with a lot of microorganisms, but it doesnt affect the
survival of the bacteria or anything else. Even if there are changes, it doesnt change
anything in their normal cycle. But when you have a mutation, the protein which

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July 11, 2014

that sequence is producing may change. So, what we are detecting is the shift that is
happening in the nucleic acid sequences by doing restriction fragment length
polymorphism.

Slide 84- Ribotyping (83)
Ribotyping is very commonly used. Nothing important there.

Slide 85-Bacterial Ribosome RNA (84)
16 S rRNA is very highly conserved. Everybody, all the labs, are using 16S. This is
just for knowledge purposes. Know that 16S ribosome sequencing is used for
classification. Very few labs are using other stuff.

Slide 86- Differentiation of S. mutans and S. sobrinus using CDF (85)
This is a last, or one or two slides. This is called CDF, chromosomal DNA
fingerprinting. Chromosomal DNA fingerprinting. Ok? What we do here, for
classification purpose, is we take the chromosome of the bacteria. Exactly the same
as we do in ribotyping. In ribotyping, we are taking the rRNA gene. Here, you are
taking a chromosome. You take the chromosome of any unknown microorganism,
ok? You do the restriction digestion using a specific restriction enzyme and you run
it on a gel. If there is a polymorphism, if there is a mutation, there will be changes in
the restriction site, right? Where the enzymes can work. If there are changes in the
restriction site, it will cut that DNA at a different level. When it cuts at a different
level, the fragments are of different length. If the fragments are at a different length,
when you run it on a gel, you will see different bands. And based upon that different
bands, you can easily identify and classify these microorganisms. Ok?

Slide 87- PCR amplification of representative oral bacteria (86)
This is PCR, polymerase chain reaction, generally used in diagnostic labs, but we can
let it go.

(skips a few slides)

Slide 88-Summary (96)
Ok. Any questions? I know this last part was fun, but it should be ok. Its not that
important. Ok? Thank you.