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Figure 1.3: Directions to count cells Figure 1.4: Empty hemocytometer grid at 100x
power
RESULT:
Total RBC count of the given blood sample is ______________ million/mm3
a) Age: At birth total leucocytes count is about 18,000/mm3, later it drops to adult level.
b) Pregnancy: At “full term” the total count tends to be about 12,000-15000/mm3. It rises
soon after delivery and then gradually returns to normal.
c) High temperature
d) Severe pain
e) Muscular Exercise
Causes of leucopenia:
Certain viral and bacterial infection (typhoid) leads to leukopenia rather than leucocytosis.
Infection
a) Bacterial (Typhoid, Paratyphoid, Tuberculosis etc.)
b) Viral (Hepatitis, Influenza, Measles etc)
c) Protozoan (Malaria)
REQUIREMENTS:
1. Neubauer Haemocytometer slide with cover slip
2. WBC pipette
3. WBC diluting fluid
4. Microscope
WBC Pipette:
This is a bulb pipette having a long stem with a capillary bore and a pointed tip. The bulb
contains a red bead inside. A small rubber tube provided with a mouth piece is connected to the
small narrow portion above the bulb for sucking blood and fluid into the pipette.WBC pipette is
like RBC pipette but here the bulb has a white glass bead in place of red and the third graduation
mark which lies just above the bulb is „11‟ instead of „101‟, this means the WBC pipette has 11
volumes from the tip up to „11‟ mark.
WBC diluting fluid (Truck’s fluid):
1. 1ml of 3% acetic acid, 1% gelatin violet- 1 or 2 drops or methylene blue.
2. The function of diluting fluid is to destroy RBC and stain the nucleus of WBC to increase
the visibility.
PROCEDURE:
1. The blood sample is taken up to the „0.5‟ mark, then the diluting fluid is taken up to the
„11‟ mark, this gives 1:20 dilution.
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RESULT:
Total WBC count of the given blood sample is ___________ /mm3.
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Basophils 0 - 1%
PROCEDURE:
1. A thin smear is prepared by spreading a small drop of blood evenly on a slide
Making the film:
a. Take clean, dry (grease free) slide.
b. Transfer a small drop of blood near the edge of the slide.
c. Place the spreader slide at an angle of 300 to 350. Pull back the spreader until touches the
drop of blood.
d. Let the blood run along the edge of the spreader.
e. Push the spreader forward to the end of the slide with a smooth movement.
f. Dry the blood smear at room temperature.
g. Adequate drying is essential to preserve quality of the film.
SOLUTION – B
Eosin (Yellow, Water soluble) -1.0g Potassium dihydrogen phosphate
Disodium hydrogen phosphate (anhydrous)-6.25g
(anhydrous) -5.0g Distilled water-500ml
Preparation of the solution:
1. The phosphate salts are dissolved first in distilled water and afterwards the stains are
added.
2. Azrure I ground in a mortar with phosphate solution.
3. Keep the solutions for at least 24 hours and filter before use.
4. Store it in coping jars.
Staining Procedure:
1. Dip the fixed smear in Field Stain „B‟ for 5 seconds.
2. Wash in the tap water.
3. Dip now in Field Stain „A‟ for 5 seconds.
4. Wash in the tap water.
5. Drain the water.
6. Place it vertically against a rack.
7. Examine the dry smear under oil immersion objective exactly in the same way as
described earlier (Figure 1c.1).
RESULT:
CONCLUSION:
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PROCEDURE:
1. Surfaces sterilize the finger for pricking.
2. Prick the finger and put 2 drops of blood on 1 slide & 1 drop on the other slide.
3. Add 1 drop of antisera „A‟, antisera „B‟ & antisera „D‟ separately on drop of blood.
4. Mix well with pricking stick individually and observe for agglutination after few seconds/ minute.
5. Blood group is indicated as agglutination of RBCs with specific antisera.
6. Agglutination of patient is as shown in figure.
RESULT:
The ABO blood group of the given sample is _____________.
The Rh typing of the given blood sample is ___________.
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(a) (b)
Figure 2.2:
(a) Membrane is in close contact with polyacrylamide gel containing proteins for electro blotting.
(b) At the end of electro transfer, all proteins migrate to nitrocellulose (NC) membrane.
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Figure 2.4: Notch Base plate & Spacers Assembled for casting gel
IMMUNODETECTION:
The transferred proteins bound to the surface of nitrocellulose membrane are detected
using immunological reagents. This process is known as immunodetection. All the unoccupied
sites on the membrane are first blocked with an inert protein, a detergent or any other suitable
blocking agent. The membrane is then probed with a primary antibody specific to the protein of
interest. The Ag-Ab complex formed on the membrane is then identified using an enzyme-
labeled secondary antibody and a suitable substrate to the enzyme, which results in a colored
band on the nitrocellulose membrane, referred to as blot development.
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Materials required:
1. Equipment: Gel rocker (optional)
2. Glassware: Conical flasks, Measuring cylinder, Petri dishes, staining tray.
3. Distilled water
4. Other equipment: Micropipettes, Tips, Water bath.
Note:
• Read the entire procedure before starting the experiment.
• Wear gloves while handling the gel and membrane.
• Prepare 1X TMB/H2O2, 1X secondary antibody, blocking buffer just before use.
• Resuspend an aliquot of APS in 1 ml of distilled water. Store at 4°C. Use within 2 months.
• Destaining step is not required on staining the gel with Ezee blue stainer (Coommassie brilliant
blue).
Prepare the reagents as indicated below before starting each experiment:
Preparation of 1X Assay Buffer: To 2 ml of 10X assay buffer add 18 ml of distilled water to
get 20 ml of 1X assay buffer.
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DAY: 2
IMMUNODETECTION:
27. Discard Blocking Buffer.
28. Immerse blot in 10ml of Primary Antibody Solution & mix gently for 30 minutes. Discard the
Primary Antibody Solution
29. Wash the blot by immersing in 10ml Wash Buffer for 3-5 minutes. Repeat the wash two times.
Discard the buffer each time.
30. Immerse the blot in 10ml of 1X HRP labeled Antibody. Mix gently for 30 minutes. Discard the
HRP labeled Antibody.
31. Wash the blot by immersing in 10ml Wash Buffer for 3-5 minutes. Repeat the wash process four
to five times. Discarding the buffer each time.
32. Immerse the washed blot in 10ml of Substrate Solution, mix gently for 5-10 minutes, within this
time colored band will appear.
33. Remove the blot; wash with distilled water, discard & dry.
Note:
Although the colored band fades with time, the rate of color loss can be retarded if the blots are
kept in dark.
34. Compare the SDS-Polyacrylamide gel with the developed NC membrane.
OBSERVATION:
35. On staining SDS-Polyacrylamide gel, different proteins will appear as dark blue bands against a
light blue background.
36. On immunodetection, a single blue band will be observed on NC membrane.
INTERPRETATION:
On electrophoresis of bacterial lysate on SDS-PAGE, many bands indicating the different
proteins present in the crude sample are seen. A predominant band among these is that of GST
fusion protein corresponding to 26 kD protein of the marker. Following transfer &
immunodetection, one observes a predominat band corresponding to GST protein bound to anti-
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RESULT:
CONCLUSION:
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Observation table:
Sr. No. Std Conc. mg/ml Ring Diameter mm Test Conc. mg/ml.
1 0.25 6
2 0.5 8
3 1.0 10
4 2.0 12
5 Test-1 ?
6 Test-2 ?
RESULT:
CONCLUSION:
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1. The glass plates should be wiped grease free with cotton for the even spreading of the
agarose.
2. Ensure that the moist chamber has enough wet cotton to keep the atmosphere humid.
RECONSTITUTIONS:
PROCEDURE:
2. Cool the solution to about 55-60°C and pour 5ml of the solution onto grease free glass
plate that had previously been set on horizontal level surface. Allow the gel to set for
20-30 minutes.
3. Serially dilute the test antisera up to 1:32 dilution. Take 20 μl of 1 X assay buffer in
five eppendorf tubes. To the first tube add 20 μl of test antiserum mix well. The
dilution of antiserum in the first tube is 1:2. From the first tube take out 20 μl of 1:2
diluted antiserum and add to the second tube. The dilution in the second tube is 1:4;
repeat the dilution up to the fifth tube as shown in Figure 1. Add 20μl test antiserum
in first well.
4. Keep the gel plate on the template provided. Punch wells in the gel with the help of a
gel punch corresponding to the marking on the template with gentle suction so that the
wells are cleanly formed as the resultant agarose plugs are sucked out.
5. Add 20 μl of the Ag into the centre well and 20 μl of each of neat, test serum, 1 :2,
1:4, 1:8, 1:16, 1:32, dilutions into the surrounding wells as shown in the following
figure.
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INTERPRETATION:
2. The titter of the antiserum is the highest dilution showing the precipitin line. For example
if the line of precipitation is seen up to 1:16 dilution, the titter of the antiserum is considered
as 1:16.
RESULT:
DISCUSSION:
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INTRODUCTION:
PRINCIPLE:
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REQUIREMENT:
PRECAUTIONS:
Reconstitute test samples with 0.5 ml distilled water and store at 4 - 8˚c.
Prepare 1x assay buffer by diluting 10x assay buffer 10 times with distilled water.
Use the diluted buffer on the same day.
Do not contaminate reagents with each other.
Dilute only required amount of reagents.
Do not leave the reagents at room temperature.
Ensure all three zones on strip are immersed in solution.
PROCEDURE:
1. To one drop (50μl) of the sample add 1ml of 1x assay buffer and insert an ELISA strip.
Keep for 20 minutes at room temperature
2. Wash the strip by dipping it in 1 ml of 1x assay buffer for about 5 minutes and replace the
buffer. Repeat two more times.
3. In 1ml 1x assay buffer add 10μl antibody-HRP conjugate and dip the strip. Keep for 20
minutes.
5. In a fresh tube take 0.1 ml TMB/H2O2, 10X concentrate, 0.9 ml distilled water and dip the
strip.
6. Observe the strip in 10-20 minutes for the appearance of blue/gray spot on the strip.
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INTERPRETATION:
1. A spot on the lower portion and no spot on the upper portion indicate the proper
performance of the test.
3. Intensity of the spot is proportional to antigen concentration in the sample, i.e. human
IgG.
RESULT:
CONCLUSION:
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