Today we're going to talk about Sterilization, Disinfection, & Microbial Waste Disposal,

which is the main topic of this lecture. And about the first part of non specific immunity.

So lets begin with some terms:

Sterilization is the complete destruction of all microbes, including cells, spores, and viruses.
And can be accomplished by physical methods such as radiation, heat. And can be
accomplished by chemical methods.
In contrast to sterilization, disinfection is destruction or removal of all or most pathogens
from objects by physical or chemical methods.
So sterilization is the destruction of all microbes, pathogenic or non pathogenic. While
disinfection only relates to pathogenic microorganisms.

An example of disinfection technique is basically wiping your skin with alcohol. It will
remove most of microorganisms of it.Another example of disinfection is the process of
pasteurization of liquids such as milk. So you heat the milk for a certain temperature for
several times, and you do that to remove mainly brucella. We're not going to talk about
another micro organisms here, only brucella.
So pasteurization, at the bottom line, is a type of disinfection technique.

Disinfectants are chemicals that we use to perform regular disinfections and usually
disinfectants are used for non living objects such as bench tops.Disinfectants usually can kill
most vegetative bacteria, most growing dividing bacteria, but cannot destroy regular spores
or bacterial spores.

Antiseptics, these are basically disinfectants that are safe enough to be used on living tissues.
So 70% alcohol is a type of antiseptics, whereas bleach is a disinfectant because it's very
harmful for the skin, and if u put it on metals it might ruin the metals.

Whenever you see a word that ends with –cide or –cidal, that basically means death so a
germicide agent is an agent that kills germs.
Bactericidal agent is an agent that kills bacteria, but not necessarily bacterial spores.
A sporocidal agent is an agent that kills bacterial spores.
A fungicidal agent is an agent that kills fungi including their spores.
And Algicidal agents is an agent that kills algae.
A viricidal agent destroys viruses.

We took this term for microbistatic, and microbistatic agent is an agent that inhibits growth
and metabolism of bacteria, but not necessarily kill them. So if you remove the microbistatic
agent from a microorganism, this microorganism can resume its growth.
This is obviously a contrast to bactericidal agent that will actually kill the bacteria.
Final two terms are sepsis and antisepsis.
Sepsis refers to the presence of pathogen in blood or tissues, whereas asepsis (antisepsis)
means the absence of pathogens.

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Whenever you try to prevent an infection, the procedure or whatever you are trying to do is
referred to as antisepsis. So cleaning of surfaces using disinfectants, is relate to antisepsis
technique.
Now let's discuss the various types of physical and chemical methods that are used to inhibit
bacterial growth. And we'll begin with the physical methods.
Basically we have the heat method, which is the most common method used to inhibit
bacterial growth or destroy microorganisms, and we have two main types.
1) Dry heat. Basically increase the temperature without adding moisture such as in the
case of the dry hot oven, or in the case of directly using the flame or a heating device
to burn the organism. So later on as dentists, you use the oven to basically sterilize
your various equipments. In the oven you place the items at 160 degrees centigrade or
you can use higher temperature with less time.
2) The second type of heat is moist heat. Examples include boiling and using an
autoclave.
We have two factors that determine the effectiveness of heat in killing the microbes.
1) Temperature.
2) The exposure time.
So the higher the temperature is, the more effectively sterilization occurs. And the longer the
time of exposure is, the more effectively sterilization occurs.
And each microbe has a particular property regarding its sensitivity to heat and this is called
the thermal death point.
So the thermal death point of any species is the lowest temperature that will kill all of the
organisms in a standardized pure culture within a specified time. For example, we can see
that bacteria x needs to be exposed to 70 degrees centigrade for 30 minutes in order to be
completely killed. If exposed to 65 degrees for 30 minutes, that will not ensure the killing of
all of the bacteria. Obviously if we go above 70 we will kill it. As a minimum you need 70
degrees. So 70 degrees for 30 minutes is the thermal death point. And each microbe will have
its own different thermal death point.
You probably have seen this in the lab, whenever we are trying to transfer the bacteria from
one place to another, we use an instrument called a loop so the bacteria is attached to one end
of the loop, and we use it to streak another plate. And we probably use the flame to sterilize
the loop to prevent contamination between different cultures.
We can use another method other than flame such as electrical heating device, which heats
the tip of the loop to a very high temperature.
Autoclave is another example of using heat to sterilize materials. The autoclave is simply a
large metal pressure cooker. So it’s a sealed container that contains some liquids and water.
We heat the container and the water starts boiling but since it's under pressure the boiling

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temperature will reach 121 degrees centigrade. And the internal pressure of the water vapor
will reach 15 pounds per square inch, or 15 psi.
So that high temperature will kill many bacteria and that high pressure that is generated will
push the vapor very deep within the material insuring proper sterilization of the bacteria. So
you can use an autoclave at that temperature of 121 degrees and at pressure 15 psi for at least
15 minutes and you insure killing of all microbes that are present in the sample. And you
have to memorize these numbers (121 at 15 psi for 15 minutes). If you go longer obviously
that’s fine but if less than any of those numbers you cannot insure the killing of all the
microbes present.
A simple example of an autoclave is a pressure cooker and we have also other professional
machines that can perform autoclaving and they can take a very large volume. And they have
computers system to control the pressure, temperature and duration of autoclaving.
In order to make sure that you have properly autoclaved and sterilized the material, you can
add some type of indicator with the material you try to autoclave in order to see if the
temperature and pressure were reached.
Examples of these indicators are specific type of tape or specific types of vials .
Here's an example of a very large autoclave (slide #8) so basically you place the material
inside and you close the lid and press start and the machine itself will heat the material and
insure that the heat and pressure were reached, and we'll run the machine for a specific time
(at least 15 minutes).
Here's an example of the autoclave tape (slide #9) so you sterilize the cloth and you add this
indicator tape, when you started the tape was completely clear, it did not contain these black
stripes but exposure to the high heat and the high temperature and pressure of the autoclave
will change these white stripes into black. So the black stripes indicate that the autoclaving
was successful.
Another method which we can use is basically introducing these vials which contain bacterial
spores that are highly resistant to heat. So we place the vial in the autoclave, and after the
autoclave we take it out and incubate it at 37 degrees, which would allow any surviving
spores to germinate and grow, and the metabolism of the microorganisms will change the
color of the tube from purple to yellow. So if you did the autoclave and incubated the tube
(vial) at 37 degrees and the color stayed the same that means that you've killed all the
microorganisms in that sample. But, if the tube changes from violet to yellow, that means
that the autoclaving was not successful.

Other physical methods that inhibit bacterial growth;

1) Cooling, or freezing the sample will not kill all the bacteria in the sample, in fact it will
preserve the bacteria. So once the sample returns to the room temperature or 37
degrees, it will resume its growth.

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 2) Desiccation or basically drying does not necessarily kill the bacteria, many organisms
can easily survive drying, but if they dry up they usually stop multiplying, and
temporarily stop their metabolism.
3) Radiation is commonly used in labs and in factories to sterilize many things. UV lights
can be placed in rooms to sterilize bench tops and large areas in addition to air
sterilization. Gama rays and Beta rays are used in the food industry and in various
factories to sterilize disposable equipments, plates and swabs.
4) Ultrasonic waves themselves do not necessarily kill organisms but they can help in
cleaning of contaminated equipments. So in dentistry we may use many tools with
debris stuck on them, so we place them in an ultrasonic bath and the waves in the bath
will help dislodge the bacteria stuck to that instrument. So after the ultrasonic bath we
rinse the tool and that will make sure it's clean and after that we usually place the tool
in an autoclave or a dry heat oven.
5) Filters or filtration can be used to sterilize certain solutions, such as vaccines that
cannot withstand high temperature, so by filtration we remove the bacteria. Most filters
cannot filter out viruses but can easily filter bacteria.
6) Gaseous atmospheres can be altered to inhibit growth, some bacteria are strict aerobic
bacteria, so if you change the atmosphere surrounding them to anaerobic then they will
stop growing and may die and vice versa.
Chemical agents used to inhibit microbial growth:
Chemical disinfection refers to the use of chemical agents to inhibit the growth of pathogens
either temporarily or permanently and whenever you are trying to disinfect a surface there are
many factors that affect the effectiveness of this infection.
1) prior cleaning of the object or surface, if the object is visibly dirty then the pathogens
might be hiding within these debris and on the surface of the thing you are trying to
disinfect, so basically the disinfectant will not reach all the microbes.
2) The organic load on the material. Any material that has protein such as feces, blood,
pus, can inhibit the activity of many disinfectant solutions. So the less contamination
by these organic materials the more effectively the disinfectant will be.
3) The bioburden. Meaning the types and numbers of microbes present on the material
you are trying to disinfect. Various microbes have different susceptibility to different
antiseptics and different disinfectants. So if you have a susceptible organism, the
disinfectant will be very effective. But if we have high resistant organism such as
pseudomonas, the disinfectant would not be as effective. Also the number of microbes
present determines the effectiveness. If you have very little number of microbes then
obviously the disinfection will be more effective than if you had the opposite situation.
4) Concentration of disinfectant. Contrary to what you might think, using the highest
concentration of any chemical is not always the best thing to do. There is usually an
optimal concentration you have to use for each disinfectant, in the case of alcohol it is
70%, not 100%, so very high or very low concentrations aren’t as effective as the
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 optimal concentration. In the case of alcohol we said that the optimal concentration is
70%, and they found that 70% alcohol has the most penetrating power regarding
tissues, so it can reach the most amount of bacteria, and is just as effective as 100%
killing power(70% alcohol has 100% killing power).
5) Physical nature of the object being disinfected. If you have a flat, smooth surface, it is
going to be very easy to disinfect the surface. In contrast, if the surface is very rough
and has many groove or cracks, the bacteria and viruses can be hiding in those cracks
and will not be easily disinfected.
6) Temperature and PH. they can directly affect the nature of the disinfectant and they
can change it from the active form to the inactive one.

Characteristics of an ideal chemical antimicrobial agent:

1) Ideally the agent should have a broad antimicrobial spectrum.
2) Needs to be fast action.
3) Not affected by the presence of organic matter.
4) Nontoxic to human tissues and noncorrosive to metals.
5) Should leave a residual antimicrobial film on surface, so it would
be active it evaporates.
6) Soluble in water and easy to apply.
7) Inexpensive and easy to prepare.
8) Stable as both a concentrate and a working solution.
9) Odorless
Antiseptics, we mentioned this before, these are basically disinfectants which are safe to be
used on living tissue. So antiseptics usually reduce the number of organisms on the surface of
the skin, but not completely eliminate all the microorganisms. And usually these antiseptics
cannot penetrate sweat pores or sebaceous glands and cannot reach the hair follicles, so the
bacteria will remain in those areas.
In the case of surgeons, they use antiseptic soaps and scrubbing methods to remove as much
bacteria and microbes off their skin as possible.
This is the universal symbol for infectious medical waste (slide # 17).
Very simply, infectious medical waste is waste that has infectious material and can lead to an
infectious disease.

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So what qualifies as infectious medical waste?!
It is medical waste that is contaminated or has slightly been contaminated. It does not have to
be completely contaminated; the fact that it could possibly have been contaminated makes it
infectious medical waste. So contaminated or possibly contaminated medical items with
organisms that are serious pathogens that are not usually present in the environment and in
the population. And usually considered an infectious waste if there's a chance for them to be
contaminated with enough numbers of viruses, bacteria or fungus to cause infection once
another healthy person is in contact with these items. So it needs to meet these criterions.
So the plan to deal with these infectious diseases is very simple.
You basically sterilize all items. You sterilize anything with bacteria, fungi, viruses or
parasites on them.

Examples of infectious medical waste:

1) Cultures of microorganisms, so the plates you see in the microbiology lab are usually
collected and placed in an autoclave
2) Human blood and blood products, so blood or any substance derived from blood is
potentially infectious waste.
3) Pathological waste from histology labs, as needles plates and so on.
4) Contaminated animal carcasses (dead bodies), usually used in research, such as body
parts, bedding materials for the animals and related wastes.
5) Materials (such as soil, water or other debris) which result from cleaning up of a spill
of any infectious medical waste.
6) Waste contaminated by or mixed with infectious medical waste. So anything that
comes in contact with any infectious material is automatically considered to be an
infectious medical waste.
Slide# 21 is an example of a culture that we saw in the lab.
Slide # 22 is an example of a blood unit.
Slide# 23 is an example of some body organs and tissues.
Sharps is a general term used for any item that can puncture the skin or a bag or anything,
for example: needles, syringes, scalpels, blades, razors and forceps.
Sharps should be discarded or put in a specially labeled puncture resistant container.
Packaging of disposable infectious waste: we have to place all items that need to be
sterilized, in a special type of bag which is usually orange in color or sometimes red.
Usually we place the items in a bag, and we place the bag in another bag (so we actually
have 2 bags) just in case one of them is, or gets punctured. The bag should be used up to

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 2/3 of its volume then we grab the bag by its top and place some autoclave tape on the top
of it, we do not tie it, because we want the autoclave steam to enter and exit the bag. So
once we place it in the autoclave and perform the autoclave cycle the material becomes
disinfected by destroying all the microbes, and now we can dispose the material as any
other trash. We place the orange bag into another black trash bag and throw it in the
dumpster.
Items that do not need to be autoclaved or handled this way:
1) Bubble wrap.
2) Paper towels.
3) Wrappers for scalpel blades
4) Needle wrappers.
5) Packaging materials such as cardbroad, Styrofoam
6) Paper.
7) Food wrappers
8) Pop cans. (aluminum cans).
All of these are obviously not infectious and can be thrown in the regular trash in the lab.
Blood and blood borne pathogens:
Any microbes that is present in the blood or body fluids that can cause serious diseases,
including hepatitis B virus and HIV.
All hospitals and all labs should have an exposure control plan regarding blood borne
pathogens. So if a lab technician or a physician was struck by a needle during drawing
blood or handling a sample, then there are certain procedures and certain steps that he
needs to do in order to prevent himself from contraction one of the bloodborne pathogens.
So each institution should have an established exposure control plan.
Exposure control plan include steps such as, proper cleaning of the site using disinfectants
and maybe taking certain types of drugs that prevent establishment of diseases.
So Universal Precautions whenever dealing with body fluids and tissues, is that to treat all
human blood and byproducts as if it was contaminated even if you know that that person
does not have HIV or hepatitis virus. You have to treat it as if he has these conditions or
other conditions. So you have to be absolutely carful all the time.

Very helpful guidelines include:

1) No eating, drinking, smoking, applying cosmetics or contact lenses in the labs.
2) No mouth pipetting. Use pipetting device.

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 3) Proper disposal of any fluids by adding bleach to them and later on autoclaving the
solution that you have.
4) Using protective equipment such as face masks and goggles for the eyes to prevent
splashes in your face and using a body gown to protect yourself and clothes from
contamination.

That was the first part of this lecture. I'll go briefly over the first part of nonspecific
immunity.

Nonspecific immune system

Your body has basically three lines of defense against pathogens. So this is a pathogen trying
to invade your tissue and trying to cause a disease (slide# 4).
Your body has 3 lines of defense against that pathogen.
First and second lines of defense are called nonspecific and the third line of defense is called
specific immune system which includes the formation of antibodies and cytotoxic T
lymphocytes.
This pathogen, in order to cause disease it needs to go through these three lines of defense.
The first line of defense include skin and mucous membranes, so if it goes through these, it
has to deal with the second line of defense which is the inflammatory and phagocytic cells. If
it survives these two lines of defense then it needs to survive the third line of defense in order
to cause the disease.
Here's another slide illustrating the three lines of defense (slide# 5). We have the first and
second lines of defense which are considered to be nonspecific defense mechanisms and the
specific line of defense which is the third.
So nonspecific host defense mechanisms are general and serve to protect the body from many
harmful substances.
The skin and mucous membranes protect us at all times from pathogens like bacteria, fungi,
viruses that don’t normally pass through the skin and mucous membranes. That’s why we
call it nonspecific because it doesn’t matter what type of organism is trying to invade, the
first and second lines of defense will prevent it.
Examples: the innate immune system, which we'll talk about later on.
Other examples: the mechanical and physical barriers of the skin and mucous membranes,
chemical factors which are present in the skin and mucous membranes, the presence of
normal flora which leads to microbial antagonism, the fever response, the inflammatory
response, and phagocytic white blood cells.

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So the skin and mucous membranes are basically physical barriers to the entry of pathogens,
and in fact you only experience disease after the skin and mucous membrane are cut. So cuts
abrasions and trauma are important for entry of pathogens.
The skin and mucous membranes also contain other mechanisms than being a physical
barrier. They contain various chemicals that inhibit the growth and penetration of the
bacteria.
For example the skin has a PH of approximately 5, and most microbes prefer a PH of 7, so
the skin PH will inhibit the amount of bacteria that can survive.
Also we have the temperature of the skin which is not actually 37 degrees, it is slightly less,
so that also makes it harder for bacteria to grow rapidly.
Perspiration and sweating is another mechanism that serves in washing away the bacteria.
Cilia which are present in the upper respiratory tract as you know, basically, serve to sweep
away the pathogens that are trapped in the respiratory tract up to the throat, where you'll
either spit it out or swallow it, if swallowed, the sputum enters the stomach where it's bacteria
is destroyed by the stomach's acidity.
Various enzymes in secretions such as lysozymes are present in the sweat, mucous and tears
of the eye. Their function is digesting the bacterial cell wall leading to cell lysis, thus
inhibiting the bacterial invasion.
Microbial antagonism which is mentioned before, we said that the presence of microbial flora
on the skin and their usage of all the nutrients and receptors available on the skin and their
ability to produce antimicrobial substances make it harder for bacteria to establish itself on
the skin.
That was all for the first line of defense.Now lets talk about the second line of defense.
Most bacteria, have an actual need for iron (free iron ions), so one way in which the second
line of defense operates is in taking away the iron from the pathogens is by producing a
molecule called transferrin (which is an iron binding protein). So level of these glycoprotein
are raised in response to systemic bacterial infection, which lead to the decrease of iron in the
circulation and thus not allowing the bacteria to easily replicate.
The fever response is another component of the second line of defense, induced whenever we
have a pyrogenic substance in our body.
We said that the gram negative cell wall has endotoxins and lipopolysaccharide and more
specifically lipid A that will interact with macrophages and induce macrophages and
monocytes to produce interleukin 1 (IL-1). IL-1 will interact with the hypothalamus and lead
to an increase in the body temperature.
So the increase in body temperature will activate WBC's which become more active in the
site of the infection. And high temperature apparently (the doctor said he doesn’t know
exactly how) will reduce free plasma iron levels which make it hard for bacteria to survive
there.

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Interferons, a very important part of the innate immune system.
There are three types of interferons: alpha, beta and gamma. And these are produced by
different types of cells in different scenarios, but the most important scenario regarding our
top today is viral infections.
So let's say that this cell was infected by a virus. Now this cell will try to warn the adjacent
cells by producing interferons. So it produces interferon alpha and beta mostly. And these
interferons will go outside the cell, attach to the receptors on adjacent uninfected cells, and
these uninfected cells will now know that a virus is very close to them, so they will induce
certain pathways that will make those cells resistant to viral replication. So basically
interferons warn uninfected cells about the presence of viruses and will allow these cells to
resist viral replication.
But obviously this is an important mechanism in fighting viral infections because it limits
viruses to local spread. But interferons have side effects, they produce nonspecific flue-like
symptoms. So whenever you have influenza, you will experience high temperature, body
aches, back aches and joint pains, all these are side effects to the interferons in your system.

The complement system:

Is a group of more than 30 different proteins. And when you'll take immunology, you will
find that there are various ways to activate the complement system. But regardless to the path
that is activated, all paths have a various activation steps (Factor III might activate factor IV,
IV activates factor V and V activates VI, VII and VIII……….). So that’s why we call it
complement cascade because we have a cascade of events.
The complement pathway accomplishes two things, first it will lead to the formation of mac
on the surface of the bacteria, mac= membrane attached complex. Basically it forms a pore
inside the cell wall or the membrane of the bacteria leading to the killing of the bacteria.
Second, certain components of the complement system will attach to the surface of the
pathogen and basically will kill or induce phagocytosis of the pathogens, so any pathogen
coated with some of the complement special components will be easily phagocytosed by
neutrophiles and macrophages. We call this property of the complement system as
opsonization. So when we opsonize a pathogen that means that we flag it for phagocytosis by
neutrophiles and macrophages. Another example of oxidizing proteins are antibodies,
whenever antibodies attach to the surface of a pathogen, that pathogen will be recognized by
the neutrophiles and macrophages and they will phagocytose them. So a certain complement
componants and certain antibodies are considered opsonins.
Acute phase proteins are proteins that are mainly produced by the liver, in very high numbers
whenever you have an infection.
Example is C-reactive protein.
So these proteins are basically helper proteins, they help increase the immune status and help
in the repair and recovery of damaged tissue.

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Cytokines: are chemical mediators released from many different cell types in the body and
enable cells to communicate with each other within the immune system and between
different systems in the body.
So whenever we have a pathogen in our skin, cells in our skin will release cytokines, and
these cytokine will induce WBC's to come to the site of the splinter and then we'll have
something called local inflammation in that specific site. These chemical substances that are
released from skin cells or any cell type of our body and from WBC's help these cells
coordinate their efforts in order to accomplish a successful immune response.
Chemokines: are certain types of released cytokines, so called because they are chemotactic
for WBC's. So a damaged tissue will produce chemokines that will induce WBC's to come to
that site. So these are chemoattractants for WBC's.

Done by:
Mohammed Tafesh and Fares Nasralla

“a big thanks to Mohamad Tafesh and Fares Nasralla for

their effort , correction team”

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