Lab Exercise 17
Specific Immunity and Microbial Immunological Testing
Objectives
1. Name and describe the functions of each cell of the non-specific (innate) and specific
immune systems.
2. Describe the nature, origin and function of antigens and antibodies.
3. Describe each component of a Complete Blood Count, detail normal values for each, and
explain likely causes for values outside of the normal range for each component.
4. Distinguish among natural active and natural passive immunity, artificial active and
artificial passive immunity.
5. Accurately perform an immunological agglutination test, and correctly interpret both
negative and positive results.
6. Explain the immunological reaction that occurs in an agglutination test.
7. Explain the importance of Staphylococcus aureus as a pathogen, and enumerate the
diseases it can cause and the symptoms of each.
Introduction
The vertebrate immune system has developed a multi-pronged mode of attack against
foreign invaders. 1. Non-specific or innate immunity, which involves phagocytosis,
inflammation, and cytotoxic responses. The non-specific cytotoxic response is mediated by
Natural Killer (NK) lymphocytes. All of these responses attack without regard to the
specific identity of the foreign invader. 2. Specific immunity involves responses by T-and
B-lymphocytes against specific foreign invaders.
Foreign Antigen (ag)
Fungus, Bacteria, Parasite, Virus,
Allergen, Toxin, etc
Non-specific or Innate Immunity
Phagocytosis, inflammation,
cytotoxic response
Humoral Immunity =
B cells and antibodies (abs)
Specific Immunity
T and B Lymphocytes
Cellular Immunity =
T cells
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Specific immunity has two arms. The first is humoral immunity, which involves
antibody production by B-lymphocytes, plasma cells and memory B-cells. After
stimulation by an antigen, B-cells become activated, which involves proliferation (clone
production by rapid cell division into thousands of daughter cells) and differentiation
(conversion into active, antibody-secreting cells). Most activated B-cells become the
antibody-producing factories known as plasma cells. Plasma cells are found primarily in the
lymph nodes, spleen, tonsils and mucosa-associated lymphoid tissue (MALT), such as
Peyer’s patches of the intestines. A small percentage of activated B-cells become memory Bcells, which produce small amounts of antibodies, but mostly lie dormant, waiting to be
activated in large numbers, should the antigen be encountered in a subsequent infection.
Antibodies are blood proteins, also known as immunoglobulins, which attach
themselves to antigens, and target them for destruction by other immune system cells. When
an antibody attaches to its antigen, it forms an antigen-antibody complex or immune
complex. Immune complexes protect the body in a number of ways:
a. agglutination or clumping of cells with the antigen on their surface,
b. precipitation of soluble antigens,
c. chemotaxis (attraction of phagocytes, such as neutrophils, monocytes and macrophages),
d. degranulation of basophils and mast cells (which secrete inflammation-promoting
substances), and
e. opsonization (enhancement of phagocytosis).
The leukocytes known as eosinophils, have among their functions, phagocytosis of immune
complexes, direct attack on fungal, protozoan and multicellular parasites, and moderation of
inflammation.
Cell-mediated immunity involves T-lymphocytes of various types that orchestrate
the immune response by secretion of lymphokines (protein messenger molecules, which
activate or deactivate immune system cells). In order to do their job, T-cells must be
sensitized by contact with a partly digested or processed antigen, which is presented to
them by antigen-presenting cells, such as tissue macrophages, dendritic macrophages
intestinal epitheliocytes, or B-lymphocytes. Antigen-presenting cells secrete the
lymphokine, interleukin-1, which attracts T- and B-lymphocytes and stimulates them to
examine presented antigens.
T-lymphocytes include helper T-cells, which activate themselves upon contact with
their antigen, then secrete the lymphokine, interleukin-2 that enables other T-cells and Bcells to be activated upon exposure to their antigen. Aiding the helper T-cells are the inducer
T-cells, which secrete additional interleukin-2. Still other T-cells carry out direct attacks as
cytotoxic T-cells, which secrete perforin, a protein molecule that punctures cell membranes
of foreign, abnormal and parasitized cells, causing them to die from cytotoxic lysis.
Cytotoxic T-cells also secrete tumor necrosis factor, a protein that causes abnormal and
parasitized cells to “commit suicide” through a process called apoptosis. Delayed
hypersensitivity T-cells secrete lymphokines, which promote inflammation, and attract,
retain and activate macrophages to superior size, activity levels and killing power. Tregulatory cells (T-reg cells) secrete lymphokines, which moderate, control and eventually
shut down the immune response after it has done its job.
It was long recognized that those who had contracted a disease and recovered were
immune to subsequent attacks of the same disease. This type of immunity is referred to as
naturally-acquired active immunity. Artificial stimulation of this protection
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(Immunization) is an important aspect of present-day healthcare which protects a person
without requiring them to actually catch the disease. Immunization has been used since
ancient times to activate the immune system and induce immunity to disease; artificiallyacquired active immunization was used by traditional healers in China, India and Europe to
hopefully infect a person with a “mild” case of smallpox by injecting them with the exudate
from active smallpox lesions. In actual practice, this was a crap shoot, because as often as
not, the patient would end up with a severe form of the disease. In the late 1700s, Edward
Jenner in England used cowpox (vaccinia) to induce immunity to smallpox (variola), thus
introducing the practice of vaccination, or artificially-acquired active immunity.
Vaccination uses a dead or attenuated germ, or even a portion of a germ or its secretions, to
induce an immune response without producing serious disease. Artificially-acquired
passive immunity can be conferred by injecting a non-immune person with blood plasma or
gamma globulin from an immune person. Naturally-acquired passive immunity is
transferred with antibodies from the mother’s blood to the baby’s blood in utero, and in the
mother’s milk to the nursing infant. In each of these cases, active immunologic responses
provide longer term protection from re-infection, measured in years, whereas passively
acquired immunization provides only short term protection from infection usually effective
only for a few months.
The young science of immunology started during the 1800s, when it was recognized
that blood transfusion reactions sometimes killed patients, but sometimes did not. Using the
agglutination reaction, it became possible to type blood for safe transfusion as early as
World War I. A host of other diagnostic immunological tests have been developed to
identify the presence of microbial antigens and to identify the presence of antibodies in a
patient’s blood.
A vast number of in vitro tests have been developed, which involve testing clinical or
cultural samples in the laboratory. These include agglutination tests (such as the blood
typing tests referred to above), in which suitable ratios of antibodies are present with
antigens. Either the antigen or the antibody may coat red blood cells or latex spheres, which
will normally form a suspension when dispersed in aqueous liquid. When the complementary
antibody or antigen is added, the suspension particles will stick together to form clumps,
which are visible to the naked eye, or by low-power magnification.
Staphylococcus aureus is a member of normal skin and nose flora, but it is a virulent
opportunistic pathogen, producing pimples, boils, abscesses, osteomyelitis, toxic shock, toxic
food poisoning, impetigo and “scalded skin syndrome.” Its ubiquity makes it a common
cause of nosocomial wound and indwelling appliance infections.
Some strains of S. aureus have long been identified by the production of a clotforming enzyme called coagulase, which may be either free (secreted into and soluble in
body fluids), or bound (attached to the surface of the bacterial cell). However, the free
coagulase test requires precise timing, with repeated observations of anticoagulant-treated
blood plasma in contact with a suspected Staphylococcus culture for up to 24 hours, looking
for clot formation. Cell-bound coagulase can be identified by a slide test, within a couple of
minutes, by agglutination of S. aureus cells in plasma.
Some S. aureus strains do not produce coagulase, but all types can be quickly
identified with the StaphtexTM test. In the StaphtexTM test, suspended latex beads are coated
with IgG antibodies to Protein A of S. aureus cell walls, and with fibrinogen, the fibrin clot
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precursor from blood plasma. Either Protein A, cell-bound coagulase, or free coagulase will
cause agglutination of the latex beads within 20 seconds.
CLASS ACTIVITIES
Your activities for this lab are divided into 3 parts:
1. Preliminary Activities Complete the definitions, questions and tables before coming to
class on the day this lab begins.
2. Laboratory Activities Complete these activities during the first half of lab class.
3. Body Defenses Video View the video and answer the questions during the second half of
lab class.
1. Preliminary Activities
A. Define the following terms:
Protein A
Free coagulase
Bound coagulase
Agglutination test
B. Name four diseases caused by Staphylococcus aureus, and describe the typical signs and
symptoms of each.
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C. A Complete Blood Count, or CBC, is completed in a clinical lab by a trained technician,
using a microscope and stained blood smear, or is completed by an automated cell counting
machine. A CBC counts the total number of White Cells, Red Cells, and Platelets in a cubic
millimeter of blood. The Differential White Cell Count calculates the proportion of each
type of White Cell, as a percentage of Total White Cells.
COMPLETE BLOOD
COUNT (CBC)
NORMAL
VALUE
WHAT THESE
CELLS DO?
WHAT DOES A HIGH
OR LOW VALUE
MEAN?
Total White Cell Count
(Total Leukocyte Count)
Differential
White Cell
Count
Neutrophils
Band Cells
Monocytes
Basophils
Eosinophils
Lymphocytes
Red Blood Cell Count
(Erythrocyte Count)
Platelet (Thrombocyte)
Count
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D. Distinguish between the different types of immunity:
Type of Immunity Description
How is it produced?
INNATE
How long does it last?
SPECIFIC
NATURALLY
ACQUIRED
ACTIVE
ARTIFICIALLY
ACQUIRED
ACTIVE
NATURALLY
ACQUIRED
PASSIVE
ARTIFICIALLY
ACQUIRED
PASSIVE
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2. Laboratory Activities
A. Materials
StaphTexTM Latex Reagent
StaphTexTM Positive Control Reagent
StaphTexTM Negative Control Reagent
Wooden sticks for mixing
Disposable, white slide cards with reaction circles
Overnight cultures of Staphylococcus aureus and Staphylococcus epidermidis on blood agar
plates
B. Procedure
1. Remove the StaphTexTM Latex Reagent from the refrigerator and allow it to adjust to room
temperature for 10 minutes prior to use.
2. It is normal for the StaphTexTM Latex Reagent to settle during prolonged storage. Resuspend the latex particles by inverting the vial several times, until the reagent
appears as a homogeneous milk-white suspension.
3. Place one drop of suspended Latex Reagent into each of two designated circles on a slide
card.
4. Place one drop of Positive Control Reagent onto its designated circle on the slide card.
5. Place one drop of Negative Control Reagent onto its designated circle on the slide card.
6. Using separate wooden sticks, mix each drop of Control Reagent with its corresponding
drop of StaphTexTM Latex Reagent.
8. Gently rock the slide card back and forth for 20 seconds.
7. If the StaphTexTM Latex Reagent is still good, you will observe agglutination (clumping)
of the Latex beads with the Positive Control Reagent, but not with the Negative
Control Reagent. If you observe any reaction, other than that expected, consult your
Instructor.
8. Procure a fresh slide card and place a drop of StaphTexTM Latex Reagent into each of the
two designated circles on the card.
9. Use a fresh wooden stick to transfer a two-millimeter fresh colony of Staphylococcus
aureus to a designated circle on the slide card.
10. Use a new wooden stick to transfer a two-millimeter fresh colony of Staphylococcus
epidermidis to a designated circle on the slide card.
11. Using the appropriate sticks, separately mix the bacterial colonies with their drop of
StaphTexTM Latex Reagent. Read results within 20 seconds. Do not read results after
20 seconds.
12. Most strains of Staphylococcus aureus should agglutinate the StaphTexTM Latex Reagent
within immediately. This indicates the presence of either Protein A, or coagulase, or
both.
13. Staphylococcus epidermidis should not agglutinate the StaphTexTM Latex Reagent. This
indicates the absence of both Protein A and coagulase.
14. Dispose of the card in containers of disinfectant in the disposal area.
15. Gram stain both Staphylococcus aureus and Staphylococcus epidermidis.
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C. Answer these questions about Staph Tex lab procedures.
1. Describe the size, staining, morphology and grouping of cells for each species of bacteria.
2. Draw pictures of both species of bacteria.
3. Perform a catalase test on both species of bacteria and record the answers.
Note: Other tests, such as anaerobic acid production on Mannitol Salts Agar, and
carbohydrate fermentation tests, will be performed on these bacterial species in the
Gram Positive Cocci Lab 19.
Note: Other species of bacteria, such as Gram negative E. coli and Gram positive
Streptococci may have positive StaphTexTM Latex agglutination tests, so this test
should only be performed on Gram positive, catalase positive cocci. Other
Staphylococcus species, such as S. hyicus and S. intermedius also agglutinate
StaphTexTM Latex Reagent, but they are rarely associated with human disease.
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D. Blood typing - An individual’s blood type is determined by specific proteins
(glycoproteins) known as antigens, on the surface of RBC’s. If a person’s RBC has
glycoprotein A on its surface, that person has type A blood. The specific proteins or antigens
on blood cells are determined by their genetics. However, there are many antigens on the
surface of red blood cells; the main ones are the A, B, O, and Rh antigens, but others, such as
Duffy and Kell, also exist on the RBCs of some specific populations. Our bodies produce
antibodies against the antigens that are not present on our own cells because they are
considered foreign. It is this same immune response that causes our body to produce
antibodies against other foreign proteins, such as viruses and bacteria. There is a short
tutorial about blood types at the Nobel Prize Website.
http://nobelprize.org/medicine/educational/landsteiner/readmore.html
Fill in the chart indicating the antibodies and antigens found in each of the following blood
types.
Antigen on RBC
Antibody circulating in
Blood type
plasma
A
B
AB
O
Using the blood specimens and reagents test the blood type of each patient below. The blood
and reagents are simulated and can not transfer any disease, but treat them with the same
precautions that you would treat human blood and plasma products. When an antibody and
antigen bind, such as those found in Rh and A, B, O blood types, agglutination occurs,
which is the chemical bonding of an antigen and an antibody. As the antibodies attach to
several cells at once, the cells stick together forming an aggregate or clump. A positive test
for blood type A is when the anti-A reagent causes the specimen to agglutinate.
In your groups, perform the following tests using the artificial blood and antisera.
1. Take a clean slide and clearly label the patient’s name. Drop two drops of the patient’s
blood in each well.
2. Follow this by adding two drops of the antisera into the correctly labeled wells. Be very
careful to put the correct antisera in each well or you will confuse your results.
3. Use a clean end of the tooth pick to thoroughly mix the blood and antisera. Using one
toothpick to touch different wells may give you a false positive result.
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4. Record the patient’s blood types and answer the questions below.
Patient
Ms Jones
Blood Type and Rh
Mr Green
Mr Smith
Ms Brown
2. Which of the above patients could donate blood to all the other? (Universal donor)
3. Which patient is the universal recipient? (Could safely receive any blood type for
transfusion.)
4. If patient Brown and patient Green are planning on getting married would they need to
worry about an Rh reaction should they become pregnant? Explain your answer.
5. A man with blood type A marries a woman who is blood type O. What are the possible
outcomes for their children?
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4. Body Defenses Video
Key Concepts
I. Three main Lines of defense
A.
Barrier – blockading the portal of entry
B.
Non-specific response -
C.
Specific Response – detailed and precise response to pathogens that “make it
through the lines.”
II. Vaccinations
A.
Attenuated
B.
Dead organisms
C.
Bioengineered subunits
Video Concepts
1. List the major barriers (first lines of defense) microorganisms must overcome to invade the
body.
2. What cells are responsible for the second line of defense? What action do they take against
foreign objects? What happens to the invaders?
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3. The second line of defense actually initiates a response from the third line. It identifies the
specific antigen that must be attacked. What cells are important to the third line of defense?
What is the strategy used here?
4. Normally the body is not capable of both identifying and specifically attacking an invader
before symptoms of the assault (illness) occur. Explain the theory behind the use of vaccines
and the type of immunity found in vaccinated people.
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