This subunit focuses on the diagnostic tests, and treatments for identifying and studying infectious pathogens. This subunit focuses on several bacterial and viral diagnostic tests used in the lab to identify and treat infections.
In both bacterial and viral infections there is typically an immunological response by the host cell. When host organisms interact with foreign agents, there is an adaptive immune response. The body will produce antibiotics targeting the agent as not-self and tagging it for infection. The presence of the antibody immunoglobulin M (IgM) produced by the immune system appears in patient serum after contact with infectious pathogens.
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Aseptic Technique
All of the techniques listed below should be completed aseptically in order to prevent contamination. Before the transfer and preparation of slides, the surface area of the work surface should be wiped down with surfactant. After, a burner is lit to prevent bacteria from settling into the environment while working. All work should be completed in the area of the burner to limit contamination. Any loops used in the transference of bacteria must be flamed until red hot and then cooled. To remove bacteria from capped tubes, pass the cap quickly through the flame and then quickly flame the mouth of the tube. This heats up the air inside of the tube and pushes out any bacteria or fungal spores that might enter during the transference process.
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Bacteria can be removed using the sterile loop and then the tube re-flamed and re-capped.
If students have been progressing through the subunits in order, they have already been exposed to simple staining technique and negative stains. These two techniques, while not in and of themselves diagnostic, allow students to gain an understanding of the basic shapes of bacterial colonies. If subunits are being completed out of order or selectively, the protocols for simple staining of slides is listed above in subunit one.
Gram Staining
Gram staining is the first type of diagnostic testing that students will be exposed to. Gram staining is a procedure that exposes bacteria fixed on a slide to a series of stains in order to characterize their cell walls. Bacterial cells with thin cell walls stain red and are considered Gram Negative. Bacterial cells with thick cell walls stain blue and are considered Gram Positive. If the facility is properly equipped, students can practice Gram Stain technique on pre-fixed bacterial cells. Otherwise, a virtual gram stain lab is available and is included in the resources portion of this document. Gram staining capitalizes on the cell wall type to differentiate between bacterial species.
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It is important to let students know that not all bacteria respond to Gram staining and that this is one of a few diagnostic techniques available to identify bacterial types.
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Media: Nutrient, Differential, and Selective
Media often refers to the substance on which a bacterial colony is grown and maintained. There are many different types of media, all with different purposes. Nutritive or general agar is the most common for students to have come into contact with. This media is usually composed of some sort of agarose gel complete with all of the nutritive elements needed to support the growth of the bacteria colony. This media allows all bacteria to grow and is often used in labs in order to maintain culture collections. While this media isn't necessarily diagnostic, looking at the bacteria colony characteristics on this type of media can yield some diagnostic evidence. For example,
Pseudomonas aeruginosa
often grows with fluorescent colonies that give off a grape odor. These preliminary characteristics are often key evidence for identifying
Pseudomonas
infections.
Altering the basic composition of the media be either adding or subtracting elements can greatly impact the way bacteria grow on the media. This principle is used to identify specific types of bacteria either by selecting for the bacteria type or differentiating the growing culture. Selective media is media that has an inhibitor agent added to prevent the growth of certain bacteria.
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Often, inhibitor agents select for either gram positive or gram negative bacteria. Common inhibitor agents used in selective media include high concentrations of salt, low or altered pH, missing amino acids, or specific antibiotics embedded into the media.
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Differential media, unlike selective media, relies upon the biochemical composition of the bacteria to interact with additives in order to cause a measurable, observable change to the media. Often times, differential media includes pH indicators and dyes that allow for metabolic reactions to be observed.
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Often, a specific sugar is added to the media. If the bacterium metabolizes that particular sugar, it decreases the pH of the surrounding area.
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This decrease in pH causes a color change
For this unit, only two selective and differential plates will be studied—Eosin Methylene Blue (EMB) and Mannitol Salt Agar (MSA).
EMB plates are selective for Gram Negative bacteria. The inhibitor agent methylene blue prevents the growth of any thick cell walled Gram Positive bacteria. There are several differential properties of EMB. Coliform bacteria like Escherichia coli grow with a metallic green sheen. Bacteria that metabolize the lactose sugar found in EMB, like
Enterococcus
species
,
grow with fisheye colonies spotted with a dark center.
MSA plates are selective for Gram Positive bacteria and differential between species of
Staphylococcus.
The high concentration of salt in the media prevents the growth of Gram Negative bacterial species and the pH indicator allows for the identification of bacteria that metabolize mannitol sugar. This agar is useful for differentiating between
Staphylococcus
epidermidis
, a resident bacterium found on the skin and in the eyes and
S. aureus,
a potentially devastating pathogenic bacteria that can have multi-antibiotic resistance.
Antibiotics
After performing differential diagnostic tests, like those listed above, pathologists are able to narrow down the infectious agent causing the disease. Antibiotics are medicines that are developed to treat bacterial infections. Antibiotics can be naturally derived, semi-synthetic drugs that are altered to increase their effectiveness against bacteria or decrease their toxicity to the host, or completely synthetic drugs.
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There are two major effects an antibiotic may have on a bacteria cell. First, antibiotics might kill the cell directly, either by preventing the formation of a vital component of the cell's structure like the cell wall as is the case with penicillin. We term these types of drugs bactericidal or bacteria killing antibiotics.
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The second way an antibiotic can be used to clear an infection is to be bacteriostatic—or to prevent the bacteria cell from multiplying.
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For example, the antibiotic tetracycline prevents the protein synthesis needed to make proteins required in binary fission. Sulfa drugs prevent the formation of new bacterial DNA.
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As a result, the unmitigated growth found in bacterial infections is now stymied, allowing for the host's immune system to remove the bacteria cells that are already present without needing to deal with new growth.
It is important to know what type of infection the patient has in order to select the correct antibiotic to treat it. Some antibiotics are broad spectrum and treat many different bacterial infections.
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This might be a good course if the particular agent causing the infection is unknown and intervention is required to save the patient's life while doctors work to identify the pathogen. However, over use of broad spectrum antibiotics may kill other, healthy, bacteria and may actually increase the amount of antibiotic resistance seen in certain species.
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Specialized antibiotics, called narrow spectrum antibiotics, work on specific types of bacteria—for example, vancomycin is a narrow spectrum antibiotic prescribed to treat
S. aureus
infections, but it is ineffective against Gram negative infections. An excellent laboratory exercise to study antibiotic sensitivity in different bacteria species is listed in the lesson plan section for subunit four.
The previous set of diagnostic tools and treatments were specific for bacterial infections. Viruses have a different mode and means of infection in their hosts. As a result, the diagnosis and treatment options for viral infections differ greatly than those of bacterial infections. Below is a general explanation of how viral pathogens are diagnosed and treated.
ELISA
Enyme-linked immunosorbent assays (ELISA) are commonly used to measure the titer of either antibodies or antigens in a solution. This assay was used to screen for HIV and other emerging infectious diseases like West Nile Virus. ELISA tests coating the wells of the microtiter plate with the antigen that researchers are testing for. The patient's serum is introduced.
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If that patient has been exposed to the viral factor in the recent past, they should have IgM antibodies for that factor. If it has been a long time since exposure IgG antibodies may be the ones to react. Antibodies that are responsive to the antigens bind together. After, the remaining serum can be removed from the well, leaving the antigen-antibody complex behind.
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At this point, animal antibodies that have been conjugated with an enzyme are added. These antibodies bind to the human antibody-antigen complex.
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A second rinse removes excess animal antibodies from the well. Finally, a color substrate is added, which interacts with the attached enzyme.
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As a result of this cascade, there is a color change that can be measured, indicating that the patient has come into contact with that viral substance. This protocol is for an indirect ELISA—an ELISA test that looks for the presence of antibodies against a specific antigen instead of looking for the antigen itself in the patient's serum.
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Detection with Nanowire Tubes
Similar to ELISA, but much more sensitive, is a possible immune response detection based on identifying changes in current flow using semi-conducting nanowires or carbon nanotubes.
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The way this process works is by lining a conducting tube with antibody receptors. If the corresponding antigen is found in the patient's serum sample, the antigen will attach to the antibody. At this point, there is a decrease in the conductance of the tube, similar to when a person stands on a running hose.
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As long as the antigen ligand attaches, there is a measurable result that can indicate presence of the antigen in the patient's blood serum.
Vaccines
Vaccination has become a staple of preventing infections. Vaccines themselves are designed to prime our immune response in order to respond quickly to an infection. Most vaccines are made up of three parts: an antigen of interest, an immune potentiator, and finally a delivery carrier.
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These three compounds work together to create an immune response in the body. Current vaccines are used to prevent a wide variety of diseases, including polio and pertussis. However, as mentioned in subunit three, there are often factors that impede the development of vaccines. First, vaccines are expensive to create and host through clinical trials.
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In the case of the Ebola vaccine, not only is the vaccine expensive to manufacture and put through trials, but there is a relatively small number of clients who would use the vaccine. This limit on returns decreases the attractiveness of the vaccine to manufacturers. Vaccines are also difficult to deliver to underdeveloped countries where they are needed most. Finally, the delivery method of vaccines and the current adjuvants needed to invoke immune response are very limited.
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Currently, a new nanoparticle delivery method using biodegradable polymers such as poly (lactic co-glycolic acid) and poly (glycolic acid) are being examined as a more effective drug delivery method. Nano-particles are small enough to be able to interact with cells and have the advantage of activating two types of immune response—antibody and cellular response. This ensures that there is a stronger immune response and increased response to the vaccine.
There is a lot of controversy over the use of vaccines in children. According to current vaccination schedules, most children should receive about nineteen injections in their first two years of life—a number that parents think is too many. The number of injections, coupled with the drastic reduction or complete eradication of measles, pertussis, and polio outbreaks in the United States have many questioning the need for these vaccines anymore.
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Vaccines can also have damaging side effects. Some case studies have been made that link vaccination with the rise in autism or other chronic diseases. The polio virus discussed in subunit three was linked to causing vaccine-associated paralytic polio in a handful of children yearly.
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This infrequent side effect was deemed acceptable when more than 16 thousand children were suffering from the crippling effects of the poliovirus. However, since polio is now considered to be eliminated from the United States, many feel that this risk is no longer acceptable.
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It is very likely many students will have their own opinions about vaccination. Students should be allowed to explore this topic and potentially clear up any misconceptions they have. A recommended teaching strategy for this subunit is to allow students to create either a position paper about vaccination or to host a debate on the topic.
