Serology: Agglutination Techniques andBlood CellIdentification

erology is a branch of immunology dealing with techniques toidentify and measure antigens, and to detect serum antibodies.Agglutination is a serological reaction in which antibodies

react with antigens on the surface of particles and cause the particles toclump together, or agglutinate. This reaction generally results in a visiblemass that can be seen with the unaided eye. In the diagnostic laboratory, theagglutination reaction is a valuable aid in the identification of an unknownbacterium, because the unknown can be pinpointed by its agglutination withknown antibodies.

Although serology has become highly automated, this exercise illustrateshow agglutination reactions can be studied without sophisticated or special-ized equipment. In Part A, the slide agglutination technique will be performedto learn the identity of an unknown bacterium. In Part B, the tube agglutina-tion technique will be performed to determine the amount of antibodies in asolution of serum. In Part C, the agglutination of blood cells will be studied.

Slide Agglutination

The slide agglutination technique is used to determine which of severalbacteria agglutinates a preparation of selected antibodies. Emulsions ofunknown bacteria are mixed with drops of known antibodies on a micro-scope slide, and the mixture is observed for agglutination. This process is essentially a trial-and-error method because different combinations of bac-teria and antibodies are tried until agglutination is observed. In this exer-cise, Salmonella antibodies will be combined with various bacteria todetermine which, if any, is a Salmonella serotype.

pecial Materials

• Polyvalent Salmonella antiserum in dropper bottles• Unknown broth cultures of various bacteria

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A.

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PURPOSE: to identify a bac-terial species using a poly-valent antiserum.

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• Wooden applicator sticks or toothpicks• Beakers of disinfectant• Saline solution in dropper bottles• Nutrient broth culture of Salmonella

• Tube of nutrient broth

rocedure

1. Obtain a clean glass slide and, using a wax pencil, draw a circle for each ofthe unknown broth cultures available. Aseptically deposit two loopfuls ofeach broth culture into their respective circles.

2. Add one drop of the Salmonella antiserum (a solution of Salmonella anti-bodies) to each circle. Mix the bacteria and antiserum well, using a woodenapplicator stick or toothpick. Use different sticks for each sample, andplace the stick in the beaker of disinfectant after its use.

3. As a positive control, take a clean glass slide, draw a circle, and mix a dropof Salmonella antiserum with two loopfuls of a known broth culture ofSalmonella. An agglutination reaction should be evident. As a negativecontrol (to show the absence of agglutination), mix a drop of saline solu-tion with two loopfuls of the known Salmonella culture in a second circle.Also as a negative control, mix a drop of antiserum with two loopfuls of ster-ile, bacteria-free nutrient broth in a third circle.

4. Observe the various unknowns and determine which agglutinated with theSalmonella antiserum by comparing the mixtures to the control reactions.Enter your results in Table 30.1 of the Results section, using (�) to indicateagglutination and (�) for nonagglutination. Discard the slide in the beakerof disinfectant after use. From your data, determine the identity of theunknown organism. If this is not possible, indicate why.

Tube Agglutination

In the tube agglutination technique, the object is to use the agglutinationreaction to determine the titer of antibodies in a serum sample. The titer isa measure of the amount of antibodies in the sample. It is determined by find-ing the most dilute concentration of antibodies that gives a detectable reac-tion with an antigen. Various dilutions of antibodies are prepared, afterwhich a standard amount of antigen is added. Following an incubation period,the titer is determined. This section will demonstrate the tube agglutinationtechnique and the method for ascertaining the titer of antibodies.

pecial Materials

• Polyvalent Salmonella antiserum• Salmonella antigen solution• Saline solution (0.85% NaCI)• Serological tubes with racks

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PURPOSE: to determine anantibody titer in a serumsample.

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• 1-ml pipettes• 5-ml pipettes• Mechanical pipetters• Beakers of disinfectant

rocedure

1. Select ten clean serological tubes, number them, and set them up in a rackon the desk. Obtain samples of coded Salmonella antiserum, Salmonellaantigen, and saline solution with which to work.

2. Using a mechanical pipetter and a sterile 5-ml pipette, place 0.9 ml ofsaline solution in tube #1, and 0.5 ml of saline solution in tubes #2 through#10, as indicated in Figure 30.1.

3. With the mechanical pipetter and a sterile 1-ml pipette, transfer 0.1 ml ofthe antiserum solution from the stock supply to tube #1. This yields a 1:10dilution of antiserum. Mix the contents by drawing them up and expellingthem from the pipette, and then transfer 0.5 ml from tube #1 to the salinein tube #2. This forms a 1:20 dilution, as shown in Figure 30.1. Mix the con-tents of tube #2, and transfer 0.5 ml to tube #3, as shown in figure. Mix andtransfer 0.5 ml to tube #4, and continue this process on through to tube #9.Remove 0.5 ml from tube #9, and discard it into a beaker of disinfectant.Tube #10 will be a control tube and will not receive antiserum. Discard thepipette as directed by the instructor.

4. Using a mechanical pipetter and a sterile 5-ml pipette, transfer 0.5 ml of Sal-monella antigen solution to each tube. This doubles the dilutions to the finaldilutions shown in Figure 30.1. Mix the contents, and set the rack aside toincubate at 37º C. A water bath is helpful for the incubation step.

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0.1 mlantiserum

1

Saline (ml) 0.9

Initial dilution 1:10

Antigen (ml) 0.5

Final dilution 1:20

2

0.5

1:20

0.5

1:40

3

0.5

1:40

0.5

1:80

4

0.5

1:80

0.5

1:160

5

0.5

1:160

0.5

1:320

6

0.5

1:320

0.5

1:640

7

0.5

1:640

0.5

1:1280

8

0.5

1:1280

0.5

1:2560

9

0.5

1:2560

0.5

1:5120

10

0.5

Control

0.5

Control

0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml0.5 mldiscard

F I G U R E 3 0 . 1Preparation of a dilution series for the tube agglutination test.

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5. After an hour or longer at the instructor’s suggestion, observe the tubes forevidence of agglutination by tapping the bottom of the tube to suspendthe sedimented material. Large masses should be observed in the lowerdilutions (e.g., tubes #1 and #2), and the amount of agglutinated materialshould decrease as the dilution becomes higher. Eventually there shouldbe a tube that shows the last evidence of agglutination. The dilution ofthis tube is the titer of antibodies. Note that tube #10, the control tube,should show no evidence of agglutination since it does not contain anti-serum. Overnight refrigeration will enhance the results.

6. Enter your results in Table 30.2 of the Results section, using (���) toindicate maximum agglutination, (��) to indicate moderate agglutina-tion, (�) to indicate minimum agglutination, and (�) to indicate no agglu-tination. Determine the titer of antibodies in the serum sample by noting thedilution of the tube where the last evidence of agglutination appeared.Write your answer in the Results section.

Hemagglutination: Determination ofBlood Type

Hemagglutination is a type of agglutination that involves blood cells. Inmicrobiology there are many clinical uses for this reaction, including thediagnostic procedures for measles, mumps, and influenza. The hemag-glutination reaction is also important in blood banking because it is used todetermine the blood type of an individual prior to using the blood for trans-fusion. Most textbooks contain accounts of the importance of compatibleblood types in transfusion.

In this exercise, the blood type will be determined by hemagglutina-tion. The blood used in this exercise will not be taken from the studentunless specified by the instructor. Instead, a human blood substitute will beused. This blood substitute is commercially available and consists of animalblood or microscopic beads bonded to blood antigens. It will give reac-tions identical to those of human blood.

Four major blood types are recognized. Type A individuals possess Aantigens on the red blood cells, while type B persons have B antigens. Both Aand B antigens occur in type AB individuals, and neither is found on the redblood cells of type O persons. The antigens may be detected by reactions of theblood with antisera containing A or B antibodies, as shown in this exercise.

Another antigen, the Rh factor, is found in 85% of Caucasians, who are saidto be Rh-positive. It is absent in the remaining 15%, who are Rh-negative. InAfrican-American populations, the percentages are 93% positive and 7% neg-ative. This factor is an important consideration in hemolytic disease of the new-born. Its presence also may be determined by a hemagglutination reaction.

pecial Materials

• 70% ethyl alcohol and gauze or cotton pads• Anti-A and anti-B typing sera

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PURPOSE: to determineABO and Rh blood types.

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• Anti-Rh typing serum• Toothpicks or applicator sticks• Vials containing 0.5 ml of 0.85% sterile saline solution• Sterile disposable lancets

rocedure

1. Wash and dry two glass slides. Divide one slide into sections “A” and “B”by marking the underside of the slide with a wax pencil or felt pen. Theunderside is used to prevent wax or ink from interfering with the aggluti-nation reaction.

2. Obtain the tube of blood to be used for this exercise. The instructor willspecify the source of the blood. The type of this blood will be determined.If the student’s blood is to be used, the directions for securing the blood willbe explained by the instructor.

3. Place a drop of blood in sections A and B on the first slide and a drop inthe center of the second slide. Also, place a small drop in the vial of salinesolution.

4. Place a drop of anti-A serum next to the blood in section A, and a drop ofanti-B serum next to the blood in section B. These sera contain A and B anti-bodies, respectively. Place a drop of anti-Rh serum next to the blood on thesecond slide. Anti-Rh serum (Rh antibodies) detects the D antigen, whichcauses the Rh-positive condition.

5. Mix the drops of blood and respective antibodies with clean toothpicks orapplicator sticks, being sure to use a fresh one for each mixing. Agglutina-tion with anti-A and anti-B sera is observed by the presence of large, grainy,dark clumps on the slide. Agglutination with anti-Rh sera is determined byrocking the slide back and forth for approximately 2 minutes over verymild heat. A warming tray or warming box at 45º C may be used. Small, finerclumps will be seen on close examination. It should be pointed out thatdetermination of the Rh factor by this method is questionable at best and thatfurther testing is necessary for a definitive conclusion.

6. Record results in the Results section using the following:

Agglutination with anti-A serum ................................................. type A blood

Agglutination with anti-B serum ................................................. type B blood

Agglutination with both anti-A and anti-B sera ..................... type AB blood

Agglutination with neither anti-A nor anti-B sera ................... type O blood

Agglutination with anti-Rh serum ................................................. Rh-positive

No agglutination with anti-Rh serum ....................................... Rh-negative

7. To verify your results, repeat the above process using blood from thesaline vial. The test should be performed only for A and B antigens, sincedetection of the Rh antigen will not work by this method. Cover the blood-antiserum mixtures with coverslips, and observe the presence or absenceof agglutination under the lower power (10x) objective of the microscope.Clumps of red blood cells indicate that agglutination occurred with theantiserum. Unclumped, free-floating cells reflect nonagglutination.

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!When taking your ownblood, be certain that thelancet is taken from a sealed package and is sterile.

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Blood Smear

A blood smear provides the opportunity to view red blood cells and var-ious types of white blood cells that function in body defense. Noting whichcells are present in unusual numbers may provide insight to the nature ofthe disease. For example, the number of abnormal lymphocytes is unusu-ally high in patients with infectious mononucleosis, and the monocytecount is elevated in cases of listeriosis. In this exercise, the different bloodcells will be observed, and their percentage may be determined.

pecial Materials

• 70% ethyl alcohol and gauze or cotton pads• Sterile disposable lancets• Wright’s stain• Dropper bottles of distilled water• Buffer solution (Giordano) if available

rocedure

1. Clean two glass slides and dry them thoroughly. One slide will be used forthe blood smear, the second for spreading the blood. Obtain a large drop ofblood as specified by the instructor, and place it at the end of one slide.

2. Spread the drop of blood across the face of the slide, using the second slideas a spreading slide as follows: Place the spreading slide at a 45º angle, andbring it back into the drop as shown in Figure 30.2A. Allow the blood tospread out to the slide’s edge, and then drag the blood the length of the slideone time only, as shown in Figure 30.2B. Lift the spreading slide at theend of the smear in order to “feather” the end. Repeat the process with afresh drop of blood and a new slide if a successful smear is not obtained thefirst time.

3. Thoroughly air-dry the slide . Cover the smear with Wright’s stain whilecounting the number of drops added. Permit the stain to remain for 2 minutes. Without washing the slide, add an equal number of drops of dis-tilled water or buffer solution to the slide, and mix the stain and waterthrough gentle rocking. Allow the mixture to remain 2 additional minutes.

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A BA clean slide is drawn back into the drop of blood.

The spreading slide is pushed across the face of the sample slide, thereby spreading out the blood.

F I G U R E 3 0 . 2Preparation of a blood smear.

PURPOSE: to identify bloodcell types and perform a dif-ferential white blood cellcount from a blood smear.

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4. Gently wash the slide with water for 30 seconds and blot it dry. Examine theslide under the oil immersion (100x) lens, beginning at the feathered end.

5. Note the presence of red blood cells, which should appear as pale orangedisks if the residual stain has been washed free. Red blood cells (eryth-rocytes) do not stain with Wright’s stain, but if the dye has not been re-moved, the cells may appear pale to dark blue. Try to locate an unclutteredarea where the cells are standing alone and may be observed individually.Enter a representation of several red blood cells in the Results section.

6. Note the presence of white blood cells (leukocytes), which shouldappear larger than red blood cells. Stained white blood cells will exhibit ablue nucleus in an unstained or pale blue cytoplasm. Neutrophils arethe most common white blood cells observed (Figure 30.3). These cells have

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F I G U R E 3 0 . 3Formed elements of stained blood smears.

Name

Thrombocytes(platelets)

Microscopicappearance

Approximate percentage of white blood cells Description

Erythrocytes

Monocytes:

Nogranules

2–8

Lymphocytes:

Nogranules

20–30

Basophils:

Bluegranules

<1

Eosinophils:

Redgranules

2–4 Red granules,blue nucleus

Blue granules,blue nucleus

Large oval orspherical blue nucleus

Largest cell;irregular blue nucleus

Neutrophils:

Pale pinkgranules

50–70Most abundant WBCs;pale pink granules,multilobed blue nucleus

Pale pink disks

Fragmentlike cells

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dark blue nuclei divided into two, three, or more lobes. Lymphocytesare the next most common white blood cell. These cells have large blue nu-clei that take up almost the entire space of the cytoplasm and make the en-tire cell appear dark blue. Monocytes are relatively uncommon. Theyare large cells, each with a large blue nucleus characteristically indentedon one side. Eosinophils and basophils are the least common whiteblood cells. They appear as multilobed, nucleated cells with distinctivered and blue granules, respectively. Small groups of cells also will be ob-served as clusters of blue fragmentlike bodies. These are the thrombocytes(platelets) used in blood clotting. Enter representations of the types ofwhite blood cells and the thrombocytes in the Results section.

7. A differential white blood cell count may be obtained by counting 100white blood cells and determining the percentage of each of the five types.To perform this step, begin at the feathered end and move the smear ver-tically while recording each type of white blood cell as it is observed.When the end of the smear is reached, move horizontally to the next plane (Figure 30.4) and once again move the smear vertically, continuing the tally. Determine the percentage of each type of white blood cell, and enter itinto Table 30.3 of the Results section. The slide may be labeled and retainedif necessary.

uestions

1. Explain the importance of positive and negative control preparations in theslide agglutination procedure.

2. Why is 0.5 ml of material removed and discarded from tube #9 in the tubeagglutination procedure?

3. What is meant by titer, and why may the titer be a significant factor in thediagnosis of disease?

4. Consider the blood type you have determined in the laboratory. What typeor types of blood could successfully be transfused to this individual underemergency conditions? Explain carefully.

5. Name several factors that contribute to a successful blood smear and sev-eral that contribute to a poorly executed blood smear.

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F I G U R E 3 0 . 4Counting procedure for a differential whiteblood cell count.

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Table 30.1. Determination of Unknown Organism

Organism 1 2 3 4

Agglutination

Name

Date Section

Exercise Results

Agglutination Techniques

A. Slide Agglutination

30

B. Tube Agglutination

Observations and Conclusions:

Titer of antibodies:

Serum sample code:

Identity of unknown organism:

Table 30.2. Determination of Titer of Antibodies

Tube number 1 2 3 4 5 6 7 8 9 10

Dilution 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 Control

Agglutination

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C. Hemagglutination: Determination of Blood Type

D. Blood Smear

Observations and Conclusions:

Blood type:

RhBA

Magnif.:

Table 30.3. Differential White Blood Cell Count

Type of WBC Neutrophil Basophil Eosinophil Lymphocyte Monocyte

Number in blood

Percentage in blood % % % % %

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