Microbiology Section3

7/31/2019 Microbiology Section3

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- SEROLOGICAL IDENTIFICATION OF MICROORGANISMS,

DIRECT SEROLOGICAL TESTING- LATEX AGGLUTINATION

- ENZYME IMMUNO ASSAY

- SEROLOGICAL IDENTIFICATION OF PATIENT ANTIBODIES,

INDIRECT SEROLOGICAL TESTING

- QUALITATIVE DETECTION OF INFECTIOUS MONONUCLEOSIS,

HUMAN IgM ANTIBODIES TO HETEROPHILE ANTIGEN

- ANTIBODY TITER

SEROLOGICAL IDENTIFICATION OF MICROORGANISMS,

DIRECT SEROLOGICAL TESTING

Highly specific identification of microorganisms can be obtained by serological techniques. In

vitro (that is, outside the body and in an artificial environment, such as a test tube), antigens

and antibodies react together in certain visible ways. The chemical composition of antigens

differ, and therefore, the reactions are highly specific; that is, each antigen provokes an

antibody response with that antibody only. When it provokes an antibody response, the

antigen is known as an immunogen.

In gram-negative bacilli, the carbohydrate antigens within the wall of the organism are

called somatic(associated with the soma, that is, the body of the cell) or . Each

species has a different array of O antigens that can detected in serological tests. In like

manner, those bacilli that are motile also contain characteristic flagellar protein components

called (H is from the German word hauch, which refers to motility). In

streptococci, the carbohydrate wall antigens are used to group the organisms by alphabetic

designations A through V. Many bacteria also contain antigenic carbohydrate capsules that

can be used for identification, the primary example being the pneumococci, whose capsules

permit them to be differentiated into more than 80 different types. Exotoxins and other

protein metabolites of bacterial cells are also antigenic.

The interaction of antibody with antigen may be demonstrated in several ways

Examples of these are , coagglutination, and

These tests depend on linking antibody to a particle or enzyme in order for a positive

reaction to be observed.

The is similar to the enzyme immunoassay except that

the antibody is linked to a dye that fluoresces when it is reviewed microscopically under anultraviolet light source. Fluorescent antibody tests can provide rapid diagnosis of infections

caused by pathogens that are difficult to grow in culture, or that grow slowly. Thus they have

become popular for detecting such organisms as Legionella pneumophilia (the agent of

Legionnaires disease), Bordetella pertussis, Chlamydia trachomatisand several viruses

in patient specimens. A portion of the specimen dried on a microscope slide is

treated with the fluorescent antibody reagent, rinsed to remove unbound antibody, and then

viewed under a fluorescence microscope with an ultraviolet light source.


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In a positive test, bacteria or viral inclusions fluoresce apple green. This test is used in a

similar way to identify microorganisms isolated on culture plates or in cell cultures.

A simpler test, which detects O and H antigens of gram-negative enteric bacilli (usually

Salmonella and Shigella species and Escherichia coli), is the bacterial agglutination test

When the unknown organism isolated in culture us mixed with an antiserum (prepared in

animals) that contains antibodies specific for its antigenic makeup, agglutination (clumping)

of the bacteria occurs. If the antiserum does not contain specific antibodies, no clumping is

seen. A control test in which saline is substituted for the antiserum must always be included

to be certain that the organism does not clump in the absence of the antibodies.

In this exercise, you will note how a microorganism can be identified by an interaction

of its surface antigens with a known agglutinin that produces a visible agglutination of the

bacterial cells. The test is referred to as a .

You are given two unknown gram positive, catalase positive cocci on Blood Agar

labeled A and B. review laboratory 7

Step 1. Each desk is provided with a STAPH LATEX TEST.

Step 2. You will perform a Latex agglutination assay according to the instructions provided

with you test kit. The instructor will demonstrate the Latex agglutination assay.

Step 3. Record results below.

unknown A unknown B


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The instructor will demonstrate and discuss the Abbott TESTPACK STREP A Enzyme

Immunoassay for the rapid detection and confirmation ofS. pyogenes(Group A

Streptococci).

The serology laboratory tests patients sera to detect specific antibodies. The results of such

tests may provide a serological diagnosis of an infectious disease in which antibody was

produced in specific response to the microbial antigens of the infecting microorganism

Demonstrating increasing quantities of antibody in the serum during the course of disease

form its onset (when they may be little or no antibody) and acute stages through

convalescence (when large amounts of antibody have been produced) constitutes evidences of

current active infection and indicates the natures of the etiological agent.

The interaction of a patients antibody with a specific antigen may be demonstrated in

one of several ways. Descriptive terms for antibodies refer to the type of visible reaction

produced.

are antibodies that produce agglutination, a reaction that occurs when

the bacterial cells or other insoluble particles are visibly clumped by antibody combined with

antigens on the cell surfaces.

are antibodies that produce precipitation of soluble antigens (free in

solution and unassociated with cells). When antibodies combine with such antigens, the large

complexes that result simply precipitate out of solution in visible aggregates.are those that, in combination with their

antigens, bind or fix complement, a normal component of human or animal serum.

are antibodies produced in response to antigenic toxins. Since toxins

are soluble antigens, in vitro interactions with antitoxins are seen as precipitation.

are antibodies that coat the surfaces of microorganisms by combining with

their surface antigens. This coating on the bacterial cell makes them highly susceptible to

phagocytosis by white blood cells. (The word opsonin has a Greek root that means to

relish food)

Serological tests may be performed in vitro (in the test tube) or in vivo (in the body of

an animal or human). In in vitro tests, such as those just described, quantitative methods are

often employed. An in vivo test may employ experimental animals or cell cultures to

demonstrate neutralization of an antigen by its antibody. Depending on the nature of the

antigen injected intradermally, a humoral or cellular (delayed hypersensitivity) immune

response may occur. Patients immune to diptheria experience no reaction at the site of

injected diptheria toxin (Schick test) because their circulating antitoxin neutralize this

antigen. Conversely, when persons who are (or have been) infected by tubercle bacilli are

injected with a purified protein derivative of this microorganisms, the response is a reddened

area of induration at the injection site. This reaction results

from vasodilation and infiltration of lymphocytes.


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Infectious mononucleosis (often called simply "mono") is a common viral infection that causes

fever, sore throat, and enlarged lymph nodes. The most common complaint is a sore throat. It

is commonly caused by but can be caused by other viruses such as

cytomegalovirus (CMV). It is diagnosed most frequently among teenagers and young adults.

The illness may last weeks to months. Treatment mainly is to releive symptoms and can

nearly always be done at home with plenty of rest. By adulthood, 90-95% of men and women

have already been infected. Mono usually occurs between the ages of 15-25 and is highly

contagious; 1-3% of college students contract mono each year. Infection is spread through

exposure to body fluids containing the virus. It is most often transmitted via saliva (hence the

name "kissing disease"). However, mono can also be spread through blood and genital

secretions.

A blood test that detects the presence of (antibodies that non-

specifically react against different proteins and are useful in the diagnosis of infectious

mononucleosis) may be employed about 1 week after the onset of the disease. The antibodiespeak at weeks 2 to 5 and may persist for several months to 1 year.

The instructor will demonstrate and discuss the COLOR SLIDE II Mononucleosis Test for the

rapid detection of of Infectious Mononucleosis.

To quantitate antibody in serum, serial dilutions of the serum are made by setting up a row of

test tubes, each containing the same measured volume of saline diluent. A measured quantity

of serum is added to the first tune and mixed well. The dilution in this tube is noted (1:2, 1:4

1:10). A measured aliquot of this first dilution is then removed and placed in the second

tube, containing measured saline. Material in the second tube is mixed, and an aliquot is

removed and placed in the third tube. The procedure is repeated down the line of tubes, so

that a graded series of serum dilutions is obtained. (This procedure is analogous to preparing

antimicrobial dilutions). The antigen is then added in a constant volume per tube. After

allowing time (at the right temperature) for antigen-antibody combination to occur, the tubes

are examined for visible evidence of such combination. The reciprocal of the last (highest

dilution of serum that produces a visible reaction is reported as the titerof the serum because

it indicates the relative quantity of the antibody present. If two sera are compared for

reactivity with the same antigen, the one that can be diluted furthest and still show reactivity

is said to have the highest titer, that is, the most antibody.


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In serological diagnosis of infectious disease, it is almost always necessary to test two

samples of the patients serum:

. The

reason for this is that the antibody production takes time to begin and to build up to

detectable concentrations during the course of active infection. The first sample may show no

antibody, or a low titer that could either reflect past infection or previous vaccination with

the microbial antigen in question. If the second sample shows at least a fourfold or greaterincrease in titer as compared with the first, it is evident that current active infection had

induced a rising production of antibody. Such laboratory information is of great value both

in diagnosis and in evaluation of the immunologic status of the patient with respect to any

antigen tested.

The instructor will demonstrate and discuss procedures involved in pooling blood serum

saline-serum dilution and agglutination assays to measure an antibody titer.

ANTIGEN (Ag)

O ANTIGEN

H ANTIGEN

ANTIBODY (Ab)

HOMOSPECIFIC REACTION

AGGLUTINATION

BLOOD SERUM

ANTISERUM

ANTIBODY TITER


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1. Name the components of microbial cells that are antigenic (immunogenic).

2. What is the purpose of the control test run in parallel with the bacterial agglutination?

3. What is the principle of serological identification of microorganisms?

4. What is the value of serological identification of a microorganism as compared withculture identification?

5. If two separate species of bacteria share the same antigenic chemical group, what would bethe result when each is mixed with antibody prepared against one of them?

6. What is a fluorescent antibody?

7. Describe a doubling serial dilution of six tubes, beginning with a serum dilution of 1:2 inthe first tube.

8. Define serum titer.


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9. What are acute and convalescent sera? Why must they both be tested in making a

serological diagnosis of infectious disease?

10. Define toxin, antitoxin, and toxoid.

11. What is the difference between an agglutination and a precipitation test?

12. How do immunological tests for detecting microorganisms or their antigens in patient

specimens differ from serological tests to detect antibodies in patient sera? Describe direct

and indirect testing.

13. Why is immunity to tuberculosis detected by a skin test rather than by a test for thepatients serum antibodies?

Microbiology Section3

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