Practical Finals SS-1

Practical skills.
Step by step staging

1. Step-by-step technique for setting up an approximate agglutination reaction
1. Determine the purpose of the reaction and its meaning
Purpose: detection of complex antibody and antigen.
Meaning: the positive reaction is indication of a pathogen.
Agglutination reaction is the reaction between the insoluble antigen and soluble
antibody that results in formation of the clump, which indicates a positive reaction for
antigen.
2. Select/specify required accessories
Slide, Physiological solution( normal saline), antigen(micro), antibody (antiserum)
Method:
3. Wipe with a tissue and divide the slide. And label them as control and test.
4. From one end of the slide, apply a drop of saline solution for control
5. Apply a drop of agglutinating serum to the other end of the slide in test (1:20)
6. Before each culture, get annealed the bacterial loop
7. Add a small amount of pure culture to each drop until a homogeneous suspension of
bacteria is obtained.
After 15-40 sec.
*An antigen control by mixing s drop of antigen with normal saline should be included
to rule out false positives due to autoagglutination of the antigen and also to validate
the results.*
8. Interpret the result;
in control part : we see turbidity without any sedimentation. In false positive results we
see spontaneous auto agglutination with physiological solution and antigen. We cannot
use this test to determine the type of antigen.
In test part; sedimentation occurs in positive test but we see turbidity in negative test.
After the test, we must put the slide into the biohazard container.
9. Explain the reaction mechanism
Agglutination test is an antigen-antibody reaction in which a particulate insoluble

antigen combines with its antibody in the presence of electrolytes at a specified
temperature and pH resulting in the formation of visible clumping of particles. It occurs
optimally when antigens and antibodies react in equivalent proportions.
Extra points:
Agglutination reactions are more sensitive than precipitin reactions because of the
direct nature of the antigen/carrier/antibody interaction.
Advantages :
1. Easy and quick.
2. Sensitive.
Limitations :
1. Semi-quantitative test.
2. Agglutination can be inhibited owing to the presence of an extremely excessive

number of antibodies (prozone).
2. Step-by-step technique for setting up an agglutination reaction (in test tubes)
Purpose: detection of complex antibody and antigen.
Meaning; quantitative test. Determine the concentration of the antibody which

produce complex antigen & antibody with antigen.
7 tubes, saline solution, patients serum, diagnosticun
Step 1 ; 7 tubes , { 5 test tubes, and 2 control tubes }
3. Step 2 : Add 1.0 ml of saline solution to 7 tubes (two of them are control tubes)
4. Step 3 : Introduce the patient's serum in primary dilution (1:25) into the 1st tube
and to the first control tube. Prepare dilutions. Take 0.1 ml from the first tube and
add to second, will have next two fold dilution. Repeat the step to the next tubes.
(Serial dilution)
5. Add 4 drops of diagnosticum(antigen) to the experimental tube except control of

serum tube.
6. Incubation 2 hours at 370С
7. Leave the tubes at room temperature for 18-20 hours.
8. Interpret the result
We see sedimentation if the antigen produce with antibodies of patient serum.
We see turbidity if the antigen does not produce with antibodies of patients
serum.
The last two fold dilution minimal concentration of antibodies which produce complex
antigen and antibody with diagnostic.
Agglutination test is an antigen-antibody reaction in which a particulate insoluble

antigen combines with its antibody in the presence of electrolytes at a specified
temperature and pH resulting in the formation of visible clumping of particles. It occurs
optimally when antigens and antibodies react in equivalent proportions.
3. Step-by-step technique for setting up the ring precipitation reaction
Purpose: detection of the complex antigen and antibody.
Meaning: to determine the pathogen causing infectious disease.
Small tube with precipitate serum, percipitinogen, pasteur pipette.
Method,
3. Take a small tube with precipitating serum
4. With a Pasteur pipette, slowly layer the precipitinogen along the wall in the same
volume add to precipitate serum.
5. Carefully turn the test tube with two liquids to a vertical position.
White ring of precipitate; complex antigen and antibody is produce.
No ring formed; negative results.
Precipitation reaction is serological reaction between soluble antigen and antibody

forming an insoluble product, the precipitate. (Ring)
4. Step-by-step technique for setting up the hemagglutination reaction in paired sera
Purpose: detection of complex antigen and antibody.
Meaning: to determine the pathogen causing infectious disease.( influenza virus)
6 experimental tubes, 3 control tubes(serum, antigen, RBC)
Method,
3. Add 0.5 ml of saline to a line of holes of a polystyrene plate (three of them are
control holes: CS, CA, KRBC)
4. Introduce the patient's serum into the 1st hole of the first line and prepare
dilutions(serial dilution)
5. Add 0.5 ml of the patient's serum to the first control serum tube only.
6. Add viral antigen to the experimental and second control antigen tube of the first
line.
7. Add 4 drops of chicken RBC to the experimental and the control tubes.
8. Incubation 60 min at room temperature
9. After 7-10 days, repeat the experiment (this will be the second line of test tubes)
In first line of tube; antibodies are able to inactivate the influenza virus in the
antigen containing tube. And there is no agglutination where normal concentration of
antibodies are present, which we detect as button.
In the second line to tubes, it increases by 4 times. If it is <4 then we can as its a cross/
false positive reaction.
In control serum, no antigen so its button.
In antigen control, no antibodies and no serum , so hemagglutination occurs ( shield

like pattern)
In RBC control, only physiological solution and RBC is present.(normal sedimentation

of rbc.)
Hemagglutination is a reaction that causes clumping of red blood cells in presence of
some enveloped viruses, such as the influenza virus. A glycoprotein on the viral
surface, namely hemagglutinin, interacts with red blood cells, leading to the clumping
of red blood cells and the formation of a lattice.
5. Determination of bactericidal activity of the skin
Purpose: to detect the level of non specific protection of the skin against the
pathogen.
Meaning: to determine the pathogen.
E.coli suspension 5X10*4 ( coloniform ), 4 slides consistent and agar.
Method,
3. Apply daily culture of Escherichia coli, diluted 5x10* 4 times on the skin of the
anterior surface of the forearm of the patient with a sterile swab
4. Prints are made on slides covered with a thin layer of Endo medium from different
areas of contaminated skin immediately after application, as well as after 5, 10 and
15 minutes
5. Incubation 24 h at 37°С
Calculate,
Bactericidal index = k1-k2/ k1 x100%
k1= slide incoulate immediately
K2= slide incoulate after 15 mins.
Normal bactericidal index, 90-100%
Nonspecific type of reaction to the pathogen. ( first line of immune defence)
6. Step-by-step technique for microscopy of a preparation with incomplete

phagocytosis (calculation of phagocytic number and phagocytic index)
1. Determine the purpose of microscopy
Determination of of morphological tinterial properties of microbe for choosing the

correct nutrient media for culture.
Phagocytic number= total number of phagocytic microbes/ total number of

phagocytes
Normal: 6-14
Phagocytic index= % active phagocytes
Phagocytic index= number of phagocytes/ number of leucocytes X 100%
Normal: 65-100%
Microbes, slide, phagocytes
Method;
3. Turn on the microscope. Adjust lighting
4. Apply a drop of immersion oil to the preparation
5. Put the preparation into the grooves of the object stage and fix it with the holder
6. Adjust the interocular distance so that only one circular image is observed through
both eyepieces.
7. Select and install lens x 100
8. Adjust the stage with a smear in the center according to the objective
9. Slowly move up the stage with a smear by macro screw until the lens is immersed
in oil under the control of lateral vision
10. Use a macro screw to make a rough adjustment of the image
11. Set the image clarity with a micro screw
Calculate the phagocytic number and phagocytic index.
13. Explain the stages of phagocytosis
Chemotaxis
Adhesion
Formation of phagosome
Fusion of phagosome and lysosome
Killing.
7. Step-by-step technique for setting up a complement fixation test
Test tube, serum control, antigen control), antigen, patients serum, complement,
saline solution.
Method,
3. Add 0.5 ml of antigen to the 1st and 3rd tubes( antigen control)
4. Add 0.5 ml of the patient's serum to the 1st and 2nd tubes( serum control )
5. Add 0.5 ml of complement to the 1st, 2nd and 3rd tubes
6. Add 0.5 ml of saline solution to the 2nd and 3rd test tubesи ( control tubes)
7. Incubate all tubes for 30 min at 370C.
8. Add 1.5 ml of hemolytic serum and 3 % of ram erythrocytes in all the tubes.
9. Incubate three tubes for 30 min at 370C
Postive : complement cannot absorb on the rbc and cannot lysis.
Negative; free complement gets absorb and causes hemolysis.
Positive reaction : delayed hemolysis (test tube)
Negative reaction: hemolysis, ( control tube)
Positive reaction: complex antigen and antibody absorbed at the surface of the
complement of this complex.
Negative reaction: antigen possible, antigen possible antibodies and complement

localisation separately without any fixation and connection.
8. Step-by-step technique for setting up ELISA
Antigen, patients serum, antiglobulin serum, peroxidase enzyme, hydrogen peroxidase,

diamine benzodiaze.
96 well plate with holes
Method:
3. Adsorb the antigen into the wells of a polystyrene plate.
4. Add patients sera consisting antibodies.
5. Incubation 30 min at 370С
6. Wash off unbound immunoglobulins with wash solution
7. Add antiglobulin serum with peroxidase enzyme.
8. Incubation 30 min at 370С
9. Wash off unbound immunoglobulins with wash solution
10. Add substrate (hydrogen peroxide) and indicator (diamine benzidine).
Calculated by 4+, 3+, 2+, 1+ (positive)
This method is based on targeting antigen capture in samples using specific

antibody using an enzyme reaction with its substrate.
9. Step-by-step technique for setting up a hemagglutination reaction
6 Test tubes+ rbc control, physiological solution, patients serum, chicken rbc.
Method;
3. Add 0.5 ml of saline to a line of holes (two of them are control holes: CA, CRBC)
4. Add 0.5 ml of test material from the patient to the 1st hole and prepare dilutions
(serial dilution)
5. Add 0.1 ml of chicken erythrocytes to the resulting dilutions
6. Incubation 45-60 min. at room temperature
No agglutination: button
Agglutination: shield like pattern. (Umbrella)
Depends on the structure of influenza virus. Influenza virus consist of hemaggultinin in

the form of spike on the surface of the virus. It also consist of receptor destroying
enzyme that is neuraminidase, which leads to release of the virus from the red cell
surface called illusion.
10. A step-by-step technique for setting up a hemagglutination inhibition reaction to

determine the type of virus
Polystyrene plate, patients serum
Method:
3. Add 0.5 ml of saline to 5 holes of a polystyrene plate
4. Add 0.5 ml of test material from the patient to each hole
5. Add type-specific serum A0 to the 1st hole, A1 to the 2nd, A2 to the 3rd, B to the
4th, and C to the 5th.
6. Add 0.1 ml of chicken erythrocytes to each hole
7. Incubation 45-60 min. room temperature
Umberlla : agglutination
Button: no
Depends on the structure of influenza virus. Influenza virus consist of hemaggultinin in

the form of spike on the surface of the virus. It also consist of receptor destroying
enzyme that is neuraminidase, which leads to release of the virus from the red cell
surface called illusion.
11. Step-by-step technique for color test to determine the virus titer
1. Determine the aim of the reaction and its meaning
Purpose: detection for the polio virus in virus containing material.
Meaning: to determine the polio causing viral disease.
2. Select/specify required components
Medium 199, cell culture, test material, phenol red.
Method:
3. 1st component: Add medium 199, cell culture and phenol red into 10 test tubes (1
of them is control)
4. 2nd component: Add 0.25 ml of the test material [we add virus consisting material
(in tenfold dilution) and not in cell control tube.] from the patient into the 1st tube and
prepare dilutions (serial dilution)
5. 3rd component: to protect from atmospheric air we add vaseline oil and Incubate for
72 h at 37*C
Colour change to Yellow : the serum consisting antibody aganist antigen was able to
block the metabolism of the bacteria and invade them. (Change the acidic condition,
high PH)
No Colour change: no reaction.
The reaction mechanism is based on the neutralisation test.
12. Step-by-step technique for color test to determine the type of virus
Purpose: detection for the polio virus in virus containing material.
Meaning: to determine the polio causing viral disease.
Method,
3. Add medium 199 and cell culture into 3 test tubes with phenol red.
4. Add 0.25 ml of test material from the patient to each tube
5. Add type-specific serum I to the 1st tube, II to the 2nd, III to the 3rd
6. Incubation 72 h at 37*C
Colour change to Yellow : the serum consisting antibody aganist antigen was able to
block the metabolism of the bacteria and invade them. (Change the acidic condition,
high PH)
No Colour change: the antibodies don’t activate the virus.
The reaction mechanism is based on the neutralisation test.
13. Step-by-step technique for a color test in paired sera
Aim: For detection of antibodies in paired sera.
6 test tubes, 3 control tubes. Serum, virus, cell culture
Method
3. 1st component: Add medium 199 (phenol red - indicator of ph ) and cell culture (3
test tubes) to the first lime of tubes (9 tubes)
4. Add 0.25 ml of the patient's serum into the 1st tube(control) of the first line of tubes
and prepare two-fold dilutions. Add vaseline oil.
5. Add viral antigen to each tube
6. After 7-10 days, repeat the experiment (this will be the second line of test tubes)
7. Incubate for 7-8 hours, at 37*C
1st line of tubes: positive result is when the antibodies inactiavte the virus
14. Step-by-step PCR technique and interpretation of results
Purpose: to determine the DNA of the pathogen.
Meaning: to determine the causative agent of infectious disease.
Method,
3. Total PCR consists of three steps:
1) Isolation of DNA from biological material or pure culture of a microorganism;
2) PCR itself:
- denaturation, or "melting" of DNA, when double-stranded DNA passes into a single-

stranded state under the influence of high temperature;
The double-stranded DNA template is heated to 94–96°C for 0.5–2 min to separate the
DNA strands because the hydrogen bonds between the two strands of DNA are
broken.
- annealing (binding) of primers on template DNA;
When the strands are separated, the temperature is degreased to 50-65°C to allow the
primers to bind to the single stranded DNA template. This stage is called annealing.
A primer is a short piece of nucleic acid (oligonucleotide) that is complementary to the

target DNA or RNA. It serves as a primer for the synthesis of a complementary strand
by DNA polymerase for DNA replication/synthesis. The time of the annealing stage is
30 sec.
- elongation - chain elongation.
Stage of new DNA fragment synthesis. The polymerase starts the synthesis of the
second strand from the 3'-end of the primer, which has bound to the template, and
moves along the template, synthesizing a new strand in the direction from the 5' to the
3' end. The elongation temperature is 72 °C. Stage duration – 7-10 minutes.
3) Prepare electrophoresis of the amplified fragment in agarose gel to separate the

DNA fragments by length.
PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires

DNA primers designed specifically for the DNA region of interest. In PCR, the reaction
is repeatedly cycled through a series of temperature changes, which allow many
copies of the target region to be produced.
15. Step-by-step technique for complement titration reaction and interpreting the its
results
Purpose: to detect the level of non specific protection of human body aganist
antibody.
5 tubes, physiological solution, patients serum, indicator system and haemolytic

system.
3. Add 1.0 ml of saline solution to 5 test tubes
4. Add 1.0 ml of patient's serum (1:10) to the 1st tube and prepare two-fold dilutions
5. Add 1.0 ml of the indicator system (hemolytic serum + 3% sheep erythrocytes) to all
5 test tubes
6. Incubation 45 min at 370C
Destruction of RBC by complement: hemolysis
Low concentration of complement/ its absence (unable to destruct the RBC): no

hemolysis
Reference: 40-80 CH on 1ml.
It has been shown that the "fixation" of complement is an adsorption by the

aggregates so formed; whether these aggregates are visible as a flocculent precipitate
(e.g., sheep serum vs. anti-serum) or concentrated as a surface film on a cellular
antigen (sensitized cells; agglutinated bacteria), the reaction is fundamentally the same.
16. Step-by-step technique for lysozyme titration reaction and interpreting the its
results
Purpose: to detect the level of non specific protection of human body aganist
antibody.
Saline solution, saliva, micrococcus lysodectius (gram positive bacteria)
3. Add 1.0 ml of saline solution to 5 test tubes
4. Add 1.0 ml of saliva to the 1st tube and prepare dilutions
5. Add 1.0 ml of M.lysodecticus (M.luteus) (1 billion suspension) to all 5 tubes
6. Incubation 3 hours at 370С
Transparent solution: high concentration of lysozyme are able to destroy the gram
positive bacteria.
Stimulation: low concentration of lysozyme will not able to destroy the bacteria.
Lysozyme catalyzes the hydrolysis of glycosidic bonds in bacterial cell wall

peptidoglycan.

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Practical skills.

Step by step staging

1. Determine the purpose of the reaction and its meaning

Purpose: detection of complex antibody and antigen.

Meaning: the positive reaction is indication of a pathogen.

2. Select/specify required accessories

Slide, Physiological solution( normal saline), antigen(micro), antibody (antiserum)

6. Before each culture, get annealed the bacterial loop

After 15-40 sec.

8. Interpret the result;

9. Explain the reaction mechanism

Agglutination test is an antigen-antibody reaction in which a particulate insoluble

1. Easy and quick.

2. Agglutination can be inhibited owing to the presence of an extremely excessive

2. Step-by-step technique for setting up an agglutination reaction (in test tubes)

1. Determine the purpose of the reaction and its meaning

Purpose: detection of complex antibody and antigen.

Meaning; quantitative test. Determine the concentration of the antibody which

2. Select/specify required accessories

7 tubes, saline solution, patients serum, diagnosticun

Step 1 ; 7 tubes , { 5 test tubes, and 2 control tubes }

5. Add 4 drops of diagnosticum(antigen) to the experimental tube except control of

6. Incubation 2 hours at 370С

7. Leave the tubes at room temperature for 18-20 hours.

8. Interpret the result

We see sedimentation if the antigen produce with antibodies of patient serum.

9. Explain the reaction mechanism

Agglutination test is an antigen-antibody reaction in which a particulate insoluble

3. Step-by-step technique for setting up the ring precipitation reaction

1. Determine the purpose of the reaction and its meaning

Purpose: detection of the complex antigen and antibody.

Meaning: to determine the pathogen causing infectious disease.

2. Select/specify required accessories

Small tube with precipitate serum, percipitinogen, pasteur pipette.

3. Take a small tube with precipitating serum

6. Interpret the result

White ring of precipitate; complex antigen and antibody is produce.

No ring formed; negative results.

7. Explain the reaction mechanism

Precipitation reaction is serological reaction between soluble antigen and antibody

4. Step-by-step technique for setting up the hemagglutination reaction in paired sera

1. Determine the purpose of the reaction and its meaning

Purpose: detection of complex antigen and antibody.

Meaning: to determine the pathogen causing infectious disease.( influenza virus)

2. Select/specify required accessories

6 experimental tubes, 3 control tubes(serum, antigen, RBC)

8. Incubation 60 min at room temperature

10. Interpret the result

In control serum, no antigen so its button.

In antigen control, no antibodies and no serum , so hemagglutination occurs ( shield

In RBC control, only physiological solution and RBC is present.(normal sedimentation

11. Explain the reaction mechanism

5. Determination of bactericidal activity of the skin

1. Determine the purpose of the reaction and its meaning

Meaning: to determine the pathogen.

2. Select/specify required accessories

E.coli suspension 5X10*4 ( coloniform ), 4 slides consistent and agar.

6. Interpret the result