Download Unit 1 - The Microbial World: Surprising and Stunning

Unit 7 Notes
Methods of Microbiology
Viewing Microorganisms
There are many different types of microscopes that are used in the field of microbiology. Below each will be
discussed.
1. Light Microscope – this is the simplest type of microscope, and one that allows you to see up to 1000
times the size of an object. We will concentrate on a new type of objective this year, called an oilimmersion objective. The purpose of this lens is to further magnify the size of the bacteria to about
1000 times its normal size. To use this objective, we place a drop of oil on the object to be viewed
under the microscope, and use this special lens. Please refer to microscope handout for the light
microscope diagram. This is the microscope that we will use in this class.
2. Dark-Field Microscope – this is a microscope that illuminates objects from the sides rather than from
the bottom. This type is used to detect the syphilis bacteria, which is spiral shaped.
3. Fluorescence Microscope – this microscope uses a beam of ultraviolet light to illuminate an object
which is coated with a fluorescent dye.
4. Electron Microscope – the most powerful of all microscopes, it uses electricity to activate the electron
source, or the specimen. The electrons bounce off of the microbes and create an image that is many
thousands of times larger than the object. There are two types of electron microscopes:
a. Transmission Electron Microscope (TEM) – takes pictures of microbes sliced into pieces.
Final magnification is 20,000,000 times the normal size.
b. Scanning Electron Microscope (SEM) – permits us to see surfaces of microbes. Images are
magnified about 100,000 times. See images below:
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The Light Microscope
Microbial Measurements
Notice viruses are
the smallest of the
microorganisms.
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Methods of Classifying and Identifying Microorganisms
Microorganisms are not necessarily identified by the same techniques by which they are classified. Because of
how hard it is to tell them apart just by looking at them, due to their small size, scientists have devised a variety
of methods to test metabolic reactions and other characteristics. Below are some ways scientists identify
microorganisms:
1. Morphological Characteristics – morphology means shape & outer characteristics. We can look at the
shape of bacterial cells, or how a particular virus is shaped. We can tell if the organism has spores, or
flagella, or other body parts that are distinguishing.
2. Differential Staining – organisms can be stained with a stain that places it, depending on how it stains,
in one group or another. And organisms stain differently based on their cell wall makeup. An example
is the Gram stain, which divides bacteria into either the Gram-positive group or the Gram-negative
group. See Gram Stain below.
3. Biochemical Tests – bacteria can be tested for the ability to ferment different carbohydrates, or whether
or not they fix nitrogen gas, or oxidize sulfur. There are MANY biochemical tests that can be performed
to differentiate bacteria. Look at the flow chart below for an enteric (intestinal) bacterium. There are
specific flow charts like this for all types of specimens received:
4. DNA Fingerprinting – this is where you look at the DNA code for the organism. More to come on this
later.
5. Many More – there are many more ways that can be used to identify microorganisms – this is just a list
of the most common ones.
In the Laboratory
There are many types of specimens that can be analyzed for microbes that come from humans. Here are a few
of the types, and why they might be cultured:
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Blood – might look for blood infections, such as HIV or Hepatitis
Spinal Fluid – might look for bacteria or viruses causing meningitis
Feces – might look for causes of food poisoning, or worms or parasites
Urine – might look for what is causing a urinary tract, bladder, or kidney infection
Throat Scraping – might look for Strep throat or another cause of sore throat
Sputum (stuff you bring up from a deep cough)– might look for a lung infection
Vomit – might look for some types of GI viruses
Pus – might look for what is causing an abscess or wound to be infected
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Growing Bacteria in the Laboratory
In order to grow bacteria in the laboratory, the culture media must meet the following criteria:
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Must contain the right nutrients for a specific microorganism.
Must contain sufficient moisture.
Must have properly adjusted pH.
Must have a suitable level of oxygen.
Media must be sterile.
Must be incubated at the correct temperature.
There are two basic types of culture media: solid and liquid. Solid is generally used to grow most medical
specimens.
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Solid Media – When we grow bacteria on a solid medium, we add agar (a solidifying agent) to the
medium. Agar is a polysaccharide derived from marine algae, and is used as a thickener in jellies. It is
good to grow bacteria on because it cannot be degraded by the bacteria and cannot be liquefied at
normal incubation temperatures. Agar is generally heated until it is liquid, and poured into a Petri dish
and allowed to solidify. There are different types of agar media:
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o Chemically-defined media: a medium whose exact chemical composition is known.
We use this when we are trying to isolate a specific type of bacteria, such as the
gonorrhea bacterium.
o Complex media: made up of a broad slate of nutrients in which the exact chemical
composition can vary – this would be used if we were trying to grow any bacteria that are
growing on our skin. This is the type we used in our micro lab, called “Nutrient Agar”.
o Reducing media: used to grow anaerobic bacteria, it is designed to deplete the oxygen
in the culture media.
o Selective media: designed to stop growth of unwanted bacteria, and encourage growth
of the bacteria you are looking for. An example is when you are trying to isolate
Salmonella out of a feces sample, which contains many types of bacteria. It kills
everything except the Salmonella bacteria.
o Differential media: a type of media that grows everything, but will make one particular
type of colony stand out from the others. Example: when you are trying to culture Strep
from a strep throat, you put it on blood agar. Most bacterial colonies look normal on this
agar, but Strep causes the agar to look clear around its colonies.
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Liquid Media – this is a broth that has nutrients added. Typically, it is used when you have small
numbers of bacteria in a specimen, and you want it to grow into larger numbers before you plate it
out on agar.
Universal Precautions – The “Rules”
Universal Precautions are a set of “rules” that you follow when collecting specimens and working with
microbes that help you keep from being infected with any organisms present. The underlying premise is that
you treat all people and/or specimens as if you KNOW they are contaminated. Here are the rules:
1. Treat all bodily fluids as if they were contaminated.
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2. Use disposable non-latex gloves when exposure to bodily fluids is possible. Hands should be washed
after gloves are removed and gloves discarded in a plastic bag or lined trashcan, secured, and disposed
of daily.
3. Use protective clothing when anticipating spattering of fluids.
4. All personnel, regardless of job, should use bloodborne precautions when cleaning and disinfecting.
Aseptic Technique
Aseptic technique is a method that we use in the laboratory when working with microorganisms that prevents
the introduction of unwanted organisms into an environment. Microorganisms are everywhere in an
environment, and you must handle specimens in such a way that they are not contaminated with environmental
microbes. Here are some practices we use in the microbiology lab:
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Always use sterile instruments when culturing specimens.
Never leave the lid off of a Petri dish unless you are actually plating the specimen.
Do not set the lid of test tubes down on the counter when you are putting microbes in the tube.
Always have clean hands or wear gloves.
Any other practices that help keep your culture uncontaminated by outside microbes.
The Gram Stain
Developed by Danish bacteriologist Christian Gram in 1884, the Gram stain is the most fundamental test
used to differentiate bacteria. This simple, rapid stain separates most clinically important bacteria into 2
broad groups: gram-positive bacteria, which appear blue or purple, and gram-negative bacteria, which
appear pink or red. The Gram stain is used both to characterize bacteria growing on culture media and to
directly examine specimens submitted for culture.
The differential staining seen with the Gram stain occurs because the two types of bacteria differ
significantly in their cell wall composition. It is the way the cell wall stains that separates the bacteria into two
groups. The cell wall, which is composed of a peptidoglycan macromolecule, is much thicker in gram-positive
bacteria than in gram-negative bacteria. Also, the cell walls of gram-positive bacteria contain teichoic
acids and mycolic acids which fortify the cell wall. The thin cell walls of gram-negative bacteria, on
the other hand, contain lipopolysaccharides which are extracted during decolorization.
In the Gram stain procedure, the slide is flooded with crystal violet (the primary stain), followed by Gram’s
iodine (the mordant), which chemically bonds the alkaline crystal violet to the cell wall. The slide is then
decolorized with acetone, absolute alcohol, or a mixture of the two. The decolorizer substantially damages the
thin cell walls of gram-negative bacteria and allows the crystal violet-iodine complex to wash out, whereas the
thicker cell walls of gram-positive bacteria are more resistant to damage and therefore retain the stain complex.
In the final step, the slide is flooded with safranin (the counterstain), which stains the decolorized gram-negative
bacteria pink or red.
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Gram Stain Procedure
1. Using either heat or methanol, fix the specimen onto the slide. Heat fixation: Gently pass the slide through a
flame until all moisture is evaporated.
2. Flood the slide with crystal violet. Allow to remain 30 seconds. Rinse with water.
3. Flood the slide with Gram’s iodine. Allow to remain 30-60 seconds, then rinse with water.
4. Decolorize the slide with acetone, alcohol, or a mixture of the two. Rinse with water as soon as the purple
color disappears. Caution: Avoid prolonged decolorization or excessive rinsing. These can wash the
crystal violet-iodine complex from gram-positive cells.
5. Flood the slide with safranin for 30-60 seconds. Rinse gently with water.
Laboratory Equipment
Following is the main laboratory equipment you will be using in this microbiology laboratory:
Disposable inoculating loop
Petri Dish w/Agar – a shallow plastic flatbottomed dish with a lid, contains agarose (a
gelatin-like substance with nutrients), and is
used to culture bacteria on top of.
Disposable Inoculating Loop – a rod with a
tip: the tip is dipped into the microbial
specimen you wish to put on the Petri dish,
then it is smeared on top of the agar in the
dish, and then thrown away. Single use only.
Incubator – used to place inoculated Petri
dishes into so that the microbes can grow.
Usually set at 37oC, which is the same
temperature as body temperature.
Petri dish with agar
** Petri dishes are incubated lid side
DOWN so that, as moisture collects in the
dish as it is incubated, it does not drip
down onto the growing microbes on the
agar.
Incubator
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Culturing of Bacteria
When a specimen is ready to be grown in the laboratory, there are two methods we use to plate the specimen on
the Petri dish, depending on what you wish to accomplish.
1. Smear Method – if you just wish to grow bacteria, perhaps just to get a colony count, but you don’t
need to identify the individual colonies, you will use this method. For instance, if we wish to see how
many bacteria grow out of a particular water specimen, we would use this method. To accomplish this,
we just use either an inoculating loop or a sterile cotton swab to smear the specimen over the whole
surface of the agar in the Petri dish. Here is what this looks like on a plate:
2. Streak Plate Method – let’s say you wish to culture a spit specimen, and then identify each of the kinds
of bacteria growing in the specimen. To accomplish this, we will need to use the streak plate method so
that we can first obtain a pure culture. A pure culture is a culture that contains only ONE kind of
microorganism. An individual colony on a plate is a pure culture – it starts out with one bacterium, and
through the process of binary fission, it creates many clones of itself so that it can be seen on the agar.
Each of the cells in a colony is identical to all other cells! By using this method, we can isolate
individual bacteria, and they can grow into isolated colonies so we can re-plate each different type of
colony and have multiple pure cultures to work with. Here is what this looks like on a plate:
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