Agglutination is the sticking together and precipitation of microbes or other cells under the action of antibodies in the presence of an electrolyte ( isotonic solution sodium chloride). Groups of glued bacteria (cells) are called agglutinate. The following components are required for the agglutination reaction:
1. Antibodies (agglutinins) that are found in the serum of a sick or immune animal.
2. Antigen - a suspension of living or killed microbes, red blood cells or other cells.
3. Isotonic (0.9%) sodium chloride solution.
The agglutination reaction for serodiagnosis is used for typhoid fever and paratyphoid fever (Vidal reaction), brucellosis (Wright and Heddleson reaction), tularemia, etc. The antibody is the patient's serum, and the antigen is a known microbe. When identifying microbes or other cells, their suspension is used as an antigen, and a known immune serum is used as an antibody. This reaction is widely used for diagnostics intestinal infections, whooping cough, etc.
Methods for staging RA
Approximate RA on glass
Deployed RA
(volume method)
Coagglutination reaction
Unfolded RA on glass (seroidentification)
Agglutination reaction on glass. Two drops of specific (adsorbed) serum and a drop of isotonic sodium chloride solution are applied to a fat-free glass slide. Non-adsorbed serums are pre-diluted in a ratio of 1:5 - 1:100. Drops must be applied to the glass so that there is a distance between them. The culture is thoroughly ground on glass with a loop or pipette, and then added to a drop of isotonic sodium chloride solution and to one of the serum drops, stirring in each until a homogeneous suspension is formed. A drop of serum without culture is a serum control.
Attention! You cannot transfer the culture from the serum to a drop of isotonic sodium chloride solution, which serves as an antigen control. The reaction takes place at room temperature for 1-3 minutes. If the serum control remains clear, a uniform turbidity is observed in the antigen control, and agglutinate flakes appear in the drop where the culture is mixed with serum. clear liquid, the result of the reaction is considered positive.
Diagnostic Physiological
serum + culture solution + culture
Detailed agglutination reaction (volume method). Serial, most often twofold, dilutions of serum are prepared. The method is called volumetric. To determine the antibody titer in blood serum, take 6 tubes. Pour 1 ml of the original serum dilution 1:50 into the first test tube and add 1 ml of saline solution into all 6 test tubes using a graduated pipette. The first test tube will yield a serum dilution of 1:100 with a volume of 2 ml. Transfer 1 ml from the first test tube to the second test tube, where the dilution becomes 1:200. So make a series of serial dilutions of the serum in the first 5 test tubes (1:100, 1:200, 1:400, 1:800, 1:1600). From the fifth test tube, pour 1 ml into the disinfectant solution. Add 2 drops of diagnosticum to all 6 test tubes. The sixth tube is a culture control, as it contains only saline solution and diagnosticum.
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serum control |
control diagnostic-kuma |
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Such control is necessary to exclude spontaneous agglutination of the culture. The tubes are shaken and placed in a thermostat at a temperature of 37°C for 2 hours, and then left for a day at room temperature, after which the results of the agglutination reaction are recorded. When performing an agglutination reaction with the sera of children in the first months of life, due to the functional inferiority of antibody formation, it is necessary to identify lower antibody titers, which is taken into account when diluting the serum. The initial serum dilution is 1:25. In the first test tube, a dilution of 1:50 is obtained, then 1:100, etc.
If the reaction result is positive, the test tubes show stuck cells in the form of grains or flakes against the background of a clear liquid. The agglutinate gradually settles to the bottom in the form of an “umbrella”, and the liquid above the sediment becomes clear. The antigen control is uniformly cloudy.
Based on the nature of the sediment, fine- and coarse-grained (flaky) agglutination is distinguished. Fine-grained agglutination is obtained when working with O-sera. Coarse-grained - when motile microbes interact with flagellar H-sera. It occurs faster than fine-grained, and the resulting sediment is very loose and easily broken.
The intensity of the reaction is expressed as follows:
All the cells have settled, the liquid in the test tube is completely transparent. The result of the reaction is sharply positive;
There is less sediment, the liquid does not clear completely. The result of the reaction is positive;
There is even less sediment, the liquid is cloudier. The result of the reaction is doubtful;
There is a slight sediment at the bottom of the test tube, the liquid is cloudy. Questionable reaction result;
There is no sediment, the liquid is uniformly cloudy, as in the antigen control. Negative reaction result
The agglutination reaction is based on the specific interaction of antibodies (agglutinins) with whole microbial or other cells. As a result of this interaction, agglomerate particles are formed that precipitate (agglutinate). Bacteria, protozoa, fungi, yeast, rickettsia, erythrocytes and other cells, both living and killed, can participate in the agglutination reaction. The reaction occurs in two phases: the first is a specific combination of antigen and antibody, the second is non-specific, i.e. the formation of a visible agglutinate. Precipitation of agglutinate occurs in the presence of electrolytes, such as sodium chloride. The microorganisms in the agglutinate remain alive, but lose their mobility.
The agglutination reaction is widely used for serological diagnostics infectious diseases and determination of the antigenic structure of isolated microbes. To determine the antigenic structure of a pathogen isolated from the body of a patient or a carrier, specific immune serum is used, obtained by immunizing animals (rabbit, donkey, sheep) with certain microorganisms. Identification of the microbe is carried out in an agglutination reaction on glass with adsorbed or monoreceptor sera or in test tubes with specific agglutinating sera. Adsorbed sera contain antibodies only to antigens specific to a given microbe, and monoreceptor sera contain antibodies only to one specific antigen of the pathogen.
Species sera contain antibodies to all antigens of a particular microbe.
Whether the isolated microorganism culture belongs to this species determined by agglutination with a known serum to the antibody titer indicated on the label of the serum ampoule. The antibody titer of a serum is considered to be its last dilution, in which agglutination of the culture of microbes used to immunize the animal is still observed. Adsorbed and monoreceptor sera are usually used undiluted in the glass agglutination reaction.
When determining the presence of antibodies in the patient’s blood serum, it is diluted with an isotonic sodium chloride solution starting from a dilution of 1: 50 to 1: 800 or more. A suspension of live or killed microbes is added to each dilution. Preparations containing microbes killed by heat or formaldehyde are called diagnosticums. Diagnosticums obtained by heating microorganism cultures contain only somatic antigens. When using only formaldehyde, microbes retain their flagellar antigens.
In the presence of antibodies in the patient’s blood, the diagnostic test taken in the reaction sticks together and a precipitate (agglutinate) forms on two test tubes. In this case, the results of the agglutination reaction are regarded as positive. In the control tube, into which isotonic sodium chloride solution and diagnosticum are added, the suspension of microbes should be homogeneous (negative agglutination reaction).
The results of the agglutination reaction in some diseases, such as leptospirosis, are taken into account only microscopically in a dark field of view of a microscope (microagglutination). To make a serological diagnosis of a disease, take into account diagnostic disease. It usually corresponds to a serum dilution of 1:100 or 1:200.
Antibodies in the patient’s blood serum can be detected in case of illness using an agglutination reaction typhoid fever and paratyphoid fever (Vidal reaction), brucellosis (Wright reaction), tularemia, etc.
Castellani's reaction. For certain infectious diseases or immunization with microorganisms containing group antigens, in the blood serum, in addition to antibodies specific to this type, group antibodies also appear. In this case, related bacterial species will be agglutinated by the resulting sera.
Castellani proposed a method for the adsorption of group antibodies from immune sera, based on their removal with the help of microorganisms of related species that have group antigens but lack specific ones. A culture of such microorganisms added to the serum adsorbs nonspecific group antibodies, and after removal of the antigen-antibody complex by centrifugation, only specific immunoglobulins remain in the serum. Sera processed according to the Castellani method can be used in the agglutination reaction as highly specific.
IMMUNOMICROBIOLOGICAL STUDIES
Immunological methods are used to solve many problems:
1. Condition assessment immune system person (immune status) by determining quantitative and functional characteristics cells of the immune system and their products.
2. Determination of the composition and characteristics of human tissues: blood groups, Rh factor, transplantation antigens.
3. Diagnosis of infectious diseases and resistance to them by detecting and establishing antibody titers (serodiagnosis), identifying pathogen antigens in the body, and determining cellular reactions to these antigens.
4. Seroid identification of cultures of bacteria and viruses isolated from the body of humans and animals.
5. Detection in the human body and in external environment any substances with antigenic or hapten properties (hormones, enzymes, poisons, drugs, drugs, etc.).
6. Identification of immunopathological conditions, allergies, transplantation and antitumor reactions.
The process of interaction between antigen and antibody in serological reactions occurs in two phases:
1) specific- the interaction phase in which a complementary combination of the active centers of antibodies (paratopes) and antigen epitopes occurs. Typically this phase lasts a few seconds or minutes;
2) nonspecific- manifestation phase, characterized external signs formation of immune complexes. This phase can develop from several minutes to several hours.
The optimal specific interaction of antibodies with antigen occurs in an isotonic solution with a pH close to neutral. The antigen-antibody reaction in an in vitro system can be accompanied by the occurrence of several phenomena
· agglutination,
· precipitation,
· lysis.
External manifestations reactions depend on physical and chemical properties antigen (particle size, physical condition), class and type of antibodies (complete and incomplete), as well as experimental conditions (medium consistency, salt concentration, pH, temperature).
The polyvalency of antigens and antibodies ensures the formation of aggregates visible to the naked eye. This occurs in accordance with the theory of network formation, according to which other antibody and antigen molecules are sequentially attached to the resulting antigen-antibody complex. As a result, network structures are formed, which turn into aggregates that precipitate. The nature and severity of the reaction depend on the quantitative ratio of antigens and antibodies. Reactions are most intense when the reactants are in equivalent proportions.
Prerequisite lattice formation (networks) - the presence of more than three antigenic determinants for each antigen molecule and two active centers for each antibody molecule. Antigen molecules are lattice nodes, and antibody molecules are connecting links. The region of optimal ratios (equivalence zone) of antigen and antibody concentrations, when neither free antigens nor free antibodies are detected in the supernatant after sediment formation.
Aggregates that can precipitate are formed when antigens combine with full antibodies. Incomplete antibodies (monovalent) do not cause the formation of network structures and large aggregates. To detect such antibodies, use special methods based on the use of antiglobulins (Coombs reaction).
Serological tests, due to their high specificity and sensitivity, are used to detect and quantification antigens and antibodies. The amount of immunoreagents in reactions is expressed by titer - the maximum dilution of serum or antigen at which a reaction is still observed.
Serological reactions in microbiological and immunological laboratories are used for two purposes:
1) for seroidentification of microorganisms, toxins, antigens in general using a known antibody (immune diagnostic serum),
2) for serodiagnosis - determining the nature of the antibody in the patient’s blood serum for bacterial, viral, and less often other infectious diseases using a known antigen (diagnosticum).
To determine the generic, species and type of antigen, known immune tests are required. diagnostic sera. They are obtained by repeated administration animals (usually rabbits) in increasing doses of killed or live microorganisms, their decay products, neutralized or native toxins. After a certain cycle of immunization of animals, massive bloodletting or total bleeding of the animal is performed. Blood collected in a sterile container is first placed in a thermostat at a temperature of 37°C for 4 - 6 hours to accelerate clotting, then in an icebox for a day. The resulting transparent serum is sucked into a sterile container, preservatives are added, the antibody titer is determined, checked for sterility and poured into ampoules.
Used non-adsorbed And adsorbed diagnostic sera. Unadsorbed serums have high titers antibodies, but are capable of giving group (cross) reactions.
Adsorbed sera are characterized by strict specificity of action (they react only with a homologous antigen). Sera containing antibodies to only one specific antigen are called monoreceptor.
They also produce serums labeled with fluorochromes, enzymes, and radioisotopes, which allow high degree accurately detect even traces of antigen.
Suspensions of living or killed bacteria, their breakdown products, toxins, and viruses are used as antigens (diagnosticums) in serological reactions. In some cases, extracts or chemically isolated antigens from microorganisms and animal tissues are used.
All immunomicrobiological methods can be divided into 3 groups:
1) based on direct interaction of antigen with antibody(phenomena of agglutination, precipitation, hemagglutination, immobilization, etc.);
2) based on mediated interaction of antigen with antibody(reactions of indirect hemagglutination, coagglutination, latex agglutination, carbon agglomeration, bentonite agglutination, complement fixation, etc.);
3) using labeled antibodies or antigens(fluorescent antibody method, enzyme-linked immunosorbent and radioimmunoassays and other methods).
AGGLUTINATION REACTIONS
These reactions involve antigens in the form of particles (microbial cells, red blood cells and other corpuscular antigens), which are glued together by antibodies and precipitate.
To perform an agglutination reaction(RA) three components are needed: 1) antigen (agglutinogen);
2) antibody (agglutinin)
3) electrolyte (isotonic sodium chloride solution).
Approximate agglutination reaction (RA)
An indicative, or plate, RA is placed on a glass slide at room temperature. To do this, use a Pasteur pipette to apply a drop of serum at a dilution of 1:10 to 1:20 and a control drop of isotonic sodium chloride solution separately onto the glass. Colonies or a daily culture of bacteria (a drop of diagnosticum) are introduced into both bacteriological loops and mixed thoroughly. Reactions are taken into account visually after a few minutes, sometimes using a magnifying glass (x5). With a positive RA, the appearance of large and small flakes is noted in a drop of serum; with a negative RA, the serum remains uniformly cloudy.
Detailed agglutination reaction in order to identify the titer of specific antibodies in a patient.
Full-blown RA for serodiagnosis is made in the serum of patients. It is also diluted in isotonic sodium chloride solution from 1:50 - 1:100 to 1:800 or 1: 1600. Since lower serum titers may contain normal agglutinins found in healthy people or patients with another diagnosis (diagnostic titer). As an antigen in this reaction, diagnosticums are used - known suspensions, usually of killed bacteria.
1 ml of isotonic sodium chloride solution is first poured into agglutination tubes. 1 ml of serum diluted 1:100 is added to the first of them, and after mixing it, 1 ml is transferred to the second, from the second to the third, etc. 1-2 drops of a bacterial suspension containing 3 billion microbial bodies in 1 ml are added to the resulting two-fold dilutions of sera (from 1:100 to 1:1600 or more). The tubes are shaken and placed in a thermostat at 37°C for 2 hours, then kept at room temperature for 24 hours.
The detailed agglutination reaction is taken into account by evaluating each test tube sequentially, starting with the control ones, with gentle shaking. There should be no agglutination in control tubes. The intensity of the agglutination reaction is marked with the following signs: ++++ - complete agglutination (agglutinate flakes in an absolute transparent liquid); +++ - incomplete agglutination (flakes in a slightly opalescent liquid); ++ - partial agglutination (flakes are clearly visible, the liquid is slightly cloudy); + - weak, questionable agglutination - the liquid is very cloudy, the flakes in it are difficult to distinguish; - - absence of agglutination (the liquid is uniformly cloudy).
The serum titer is taken to be its last dilution, in which the intensity of agglutination is assessed as no less than two pluses (++)
in microbiology
"Agglutination reaction and its types (RA)"
Plan:
1. Introduction………………………………………………………………………………..3
2. RA on glass……………………………………………………………………………….4
3. Test tube RA……………………………………………………………………………………….5
4. Literature used…………………………………………………………………..7
1. Introduction.
Interaction microbial antigen and antibodies are strictly specific in nature and are aimed in the animal body at neutralizing the pathogen and its toxins. The interaction of antigen and antibodies in vitro, under certain conditions, is accompanied by visible phenomena (agglutination, precipitation, immune lysis), which allows the use of AG-AT reactions, called serological (from the Latin serum), for practical purposes. Biofactories produce antigens and immune sera (antibodies) of a known specific nature (diagnostic). Using such sera in serological reactions, it is possible to identify an unknown microorganism or, using a known antigen, to detect in the body antibodies synthesized in response to the introduction of a pathogen, and thus make a diagnosis (serological diagnosis). In addition, serological reactions can be used to assess the intensity of the immune response after vaccination or an infectious disease.
Agglutination reactions, such as indirect agglutination and Coombs, are based on the in vitro interaction of corpuscular antigens with antibodies and the ability of the resulting complexes to precipitate. Bacterial cells or soluble antigens extracted from microorganisms and sorbed on carrier corpuscles: red blood cells, latex particles, etc. are used as corpuscular antigens.
Antigenic determinants of corpuscular antigens specifically interact with homologous antibodies (specific, invisible phase of the reaction), and then antigen-antibody complexes form large conglomerates visible to the naked eye, which precipitate - an agglutinate (non-specific, visible phase of the reaction). Flagellate-free forms of microbes (Brucellae) produce granular agglutinants, while flagellated forms (Escherichia, Salmonella) produce large-cotton agglutinants, which settle to the bottom of the test tube in the form of an inverted umbrella and easily break when shaken. Antigens and antibodies interact only in the presence of an electrolyte (0.8% sodium chloride solution). The course of the reaction is influenced by the salt concentration in the electrolyte, the number of microbial cells in the suspension, serum concentration, pH, temperature and other factors.
There are specific agglutination, which is based on the interaction of the antigen With homologous antibody , contained in the animal’s body to which this antigen was introduced (immunoagglutination); nonspecific (chemical), arising from changes in the pH of the environment, the concentration of electrolytes; spontaneous, which is observed when bacteria (in R-form) are suspended in saline solution and when heated, which is associated with a change in the colloidal state of the bacterial cell. Antigen , involved in RA is called an agglutinogen, the antibody is called an agglutinin, and the resulting precipitate is called an agglutinate. When an agglutinate is formed, the quantitative ratio of antigen and antibodies is important (the optimum phenomenon). With an excess or deficiency of antibodies, A delay occurs.
The agglutination reaction (RA) is one of the first immunological reactions used in microbiological practice. For the first time (1895), F. Vidal used RA to diagnose typhoid fever. Later (1897), A. Wright used the same reaction to diagnose brucellosis in humans. RA has also found application in the diagnosis of chicken pullorosis, leptospirosis, infectious abortion of mares, as well as for typing unknown microbial cultures using a known agglutinating serum. RA is highly sensitive; it can be used to detect 0.01 μg of antibody protein nitrogen in 1 ml.
Several variants of the agglutination reaction have been developed, differing in methodological execution and purpose of the study.
In this variant of RA, the test subjects can be either serum or antigen, but most often this option is used to identify microorganisms.
1. To identify the microorganism (m/o), a drop of a known agglutinating serum, for example Salmonella serum, and a drop of physiological solution (control) are applied separately to a fat-free glass slide. Then, using a bacteriological loop, the bacterial mass of the culture being studied is taken from a colony in a Petri dish or from the surface of a slanted MPA in a test tube and suspended separately in immune serum and physiological solution until a homogeneous suspension is obtained. The result is taken into account after 2...4 minutes.
Accounting for results: there should be no changes in the control sample. If the bacterial culture specifically matches the immune serum, agglutinate flakes appear (positive result); if there is no agglutination phenomenon, it is concluded that the bacterial culture under study does not correspond to the immune serum.
2. Let us consider the detection of anittel in the blood serum under study using the example of the rose bengal test used in the serodiagnosis of brucellosis. 0.3 ml of the test animal blood serum and 0.03 ml of brucellosis antigen (rose-Bengal-stained Brucella cells) are applied to a glass slide. The components are thoroughly mixed by shaking the glass and the result is taken into account after 4 minutes.
Recording of results: if the reaction is positive, pink flakes of agglutinate appear. A serological reaction of this type is classified as qualitative, since it can be used to detect antibodies to the pathogen in the animal’s blood serum, but it is impossible to assess their quantitative content.
Agglutination reaction Agglutination reaction
(RA) is a method for identifying and quantifying Ag and Ab, based on their ability to form agglomerates visible to the naked eye. In the department of infectious diseases. diseases or for other purposes is used to identify unknown microbes and cells, to determine the presence and amount of antibodies in blood and other liquids. The determination principle is based on the specificity of the interaction between Ag and Ab and consists in finding the known from the unknown. There are many options for RA: quantitative and qualitative, test tube and glass, volumetric and droplet, conventional, accelerated and express methods. To stage RA you need: 1) s-ka blood. In the variant with determining the type (var) of bacteria, industrial agglutinating tests are used, produced by immunizing rabbits. In the variant with determination of the type of Ab, a blood sample is taken from the test. people or animals. The solution must be sterile and free of suspended particles. Prepare the basic dilution in saline solution. It should be 2-4 times lower than the diagnostic titer for this disease; 2) Ag. In the version of the reaction with determination of the Ab type, industrial diagnostic kits are used; in the variant with the determination of Ag, diagnosticums are prepared themselves in the form of a 1-3 billion suspension in a saline solution of 18-20-hour agar (less often broth) test. microbe inactivated by heating in a water bath at 70°C for 1 hour or by 24-hour incubation at 37°C with formaldehyde (final concentration 0.2%); 3) electrolyte in the form of saline solution. Staging technique volumetric serial tube RA to determine the Ab titer in s-ki: several rows of working dilutions are prepared from the main dilution of s-ki. The number of rows depends on the number of diagnosticums taken into the experiment; the number and dilution factors are determined by the diagnostic titer of the suspected disease. The series must at least contain a dilution corresponding to the diagnostic Ab titer, two dilutions below and two dilutions above it. for example, if the diagnostic titer is 1:100, then with the volumetric method of staging RA the following dilutions should be prepared: 1:25, 1:50, 1:100, 1:200, 1"400; with the drip method, the first dilution (1:25) is not necessary, but another higher dilution is required - 1:800 B. scientific research s-ku is titrated to negative reaction. It is diluted as follows: 0.25 ml of saline solution is poured into all test tubes, except the 1st one, when the reaction is carried out in a volume of 0.5 ml, and 0.5 ml when the reaction is carried out in a volume of 1 ml. Pour 0.25 (0.5) ml of the main dilution into the 1st and 2nd test tubes, from the 2nd test tube, into the cut volume and breeding s-k and increased 2 times, 0.25 (0.5) ml is transferred to the 3rd, from the 3rd to the 4th, etc. to the last, from the cut 0.25 (0.5) ml is poured into everything to balance the volumes. Each dilution is carried out using a separate pipette. If several diagnosticums are taken into experiment, then for each of them its own dilution series is prepared in the same way. Diagnosticum is added to each dilution of the test tube in a volume equal to the volume of the test tube, as a result of which the dilution in each test tube is doubled. The experiment corresponds to the s-ki control (0.25 - 0.5 ml of the main dilution of s-ki and the same amount of saline solution) and the Ag control (0.25 - 0.5 ml of diagnosticum and the same amount of saline solution). If several diagnosticums are used in the experiment, then each has its own antigen control. The rack with test tubes is shaken well and placed in a thermostat at 37°C for 4 hours, and then left at room temperature until next day, after which the RA is taken into account, based on the amount of sediment and the degree of clearness of the liquid. Determination of these indicators, depending on the nature of the agglutinates, is carried out with the naked eye against a dark background, in an agglutinoscope or over the concave surface of a microscope mirror. Accounting begins with controls: control C should be transparent, Ag should be uniformly cloudy (after shaking the tube). If the controls are good, establish the presence and degree of agglutination in all test tubes, which are designated by pluses: large sediment and complete clearing of the liquid - 4 pluses; large sediment and incomplete clearing of the liquid - 3 pluses; noticeable sediment and noticeable clearing of the liquid are 2 pluses. After this, the titer is determined: the highest dilution with an agglutination intensity of at least 2 pluses. Titre research s-ki are compared with the diagnostic titer for this disease. If the titer is examined. s-ki is 2 times lower than the diagnostic value, the reaction is assessed as doubtful; if the titer is equal diagnostic - how weakly positive; if it is 2-4 times higher, it is considered positive; if it is 8 or more times higher, it is considered sharply positive. When Ab is widespread in healthy people, an increase in Ab titer is used to assess RA. To determine the type of Ar in serial RA, the number of rows must correspond to the number taken for identification diagnostic tests. From the main dilution of the diagnostic test, a series of successive two-fold dilutions are prepared in the same way as in RA to determine the Ab titer. Dilution factors depend on the titer of the agglutinating test. In the experiment, it is necessary to have a dilution equal to the titer of the test, as well as 2, 4, 6, 8 times lower than it. For example, if the titer of the diagnostic test is 1 3200, then you should use dilutions 1 3200, 1 1600, 1 800, 1 400, 1 200 The same volume of tested Ag is added to the dilutions of the test, as a result, the dilution of the test increases by 2 times. 2 controls of test and Ag are added to the experiment. If in the experiment several s-k are involved, then each of them needs its own control. Upon completion of the reaction, the stand is shaken vigorously and placed in a thermostat at 37 ° C. The results are taken into account as described above. Evaluation of the reaction has features. In order to draw a conclusion about the compliance of the study. Ag taken into the experiment, the titer of the reaction must correspond to at least half the titer of the standard diagnostic test. Titers of 1 4 and below are considered as a group reaction Drip md staging of RA differs from volumetric in that s-ku is diluted in a volume of 1 ml, Ag is used in a higher concentration (10 billion/ml) and it is added 1 at a time - 2 drops into a test tube Dilution of the drug after the addition of Ag is considered unchanged. Otherwise, the method of formulation, recording and evaluation is similar to the volumetric method
(Source: Dictionary of Microbiology Terms)
