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Quality Procedures for Serology

One Step HBsAg Test (serum/plasma)

Principle: The One Step HBsag test is a rapid, one step


immunochromatographic assay for the detection of hepatitis B surface
antigen (HBsAg) in human serum or plasma.
Assay Procedure:
For test cards:
Bring all reagents and specimens to room temperature.
Remove the test card from the foil pouch and place on a clean dry
surface.
Identify the test card for each specimen or control.
Dispense 100ul (3 drops) of the specimen or control into the sample well
on the card.
Interpret test results at 15 minutes.
For test strips:
Bring all reagents to room temperature.
Remove the test strip from the foil pouch and place on a clean dry surface.
Identify the test strip for each specimen or control.
Apply at least 80ul of specimen to the sample pad.
Interpret test results at 15 minutes.
Interpretation of Results

Negative: Only one band appears in the control region.


Positive: In addition to the control band, a band also appears in the test region.
Invalid: Neither test band nor control band appears. The specimen should be
tested again using a new test card/strip.

Rapid Anti-HCV Test (serum/plasma)


Principle: Rapid Anti-HCV test is a colloidal gold enhanced, rapid
immunochromatographic assay or the qualitative detection of antibodies to hepatitis C
virus in human serum or plasma.

Assay Procedure:
For test cards:
Dispense 1 drop of serum or plasma to the”S” well of the test card using the plastic
dropper provided according to the figure.
Add 2 drops of sample diluents to the “D” well immediately after the specimen is added.
Interpret results at 15 minutes.
Reading the test results:
Positive: Both purplish red test band and purplish red control band appear on the
membrane.
Negative: Only the purplish red control band appears on the membrane. The absence
of a test band indicates a negative result.
Invalid: There should always be a purplish red control band in the control region
regardless of test result. If control band is not seen, the test is considered invalid.
Repeat the test using a new test device.

One Step Anti-TP (Treponema pallidum/syphilis) Test


Principle: One Step Anti-TP test is a rapid, serological, immunochromatographic assay
for the detection of antibodies to syphilis antigen in human serum or plasma.
Assay Procedure:
For card:
Bring all reagents and specimens to room temperature.
Remove the test card from the foil pouch and place on a clean dry surface.
Identify the test card for each specimen or control.
Dispense 100 ul (3 drops) of the specimen or control into the circular sample well on the
card.
Interpret the test result at 15 minutes. Do not interpret after 20 minutes.

For strip:
Bring all reagents and specimens to room temperature.
Remove the test strip from the foil pouch and place on a clean dry surface.
Identify the test strip for each specimen or control.
Apply at least 80ul of specimen to the sample pad behind the arrow mark at the bottom
of test strip.
Interpret the test result at 15 minutes. Do not interpret after 20 minutes.

Interpretation of results:
The presence or absence of syphilis antibody provides useful information on the status
of individuals with infection of syphilis.
NEGATIVE: Only one purplish red colored band appears on the control region.
POSITIVE: In addition to the control band, a distinct colored band appears on the
control region.
INVALID: Neither purplish red test band nor purplish red control band appears. The
specimen should be tested again using a new device.
ONE STEP ANTI-HIV (1&2) TEST (serum/plasma)
Principle: ONE STEP ANTI-HIV (1&2) TEST is a colloidal gold enhanced, rapid
immunochromatographic assay for the detection of antibodies to Human
immunodeficiency Virus (HIV) in human serum or plasma.
Assay Procedure:
For test card:
Bring all reagents and specimens to room temperature.
Remove the test card from the foil pouch and place on a clean dry surface.
Identify the test strip for each specimen or control.
Dispense 100 ul (3 drops) of the specimen or control into the circular sample well on the
card.
Interpret the test result at 15 minutes.

For strip:
Bring all reagents and specimens to room temperature.
Remove the test strip from the foil pouch and place on a clean dry surface.
Identify the test strip for each specimen or control.
Apply at least 80ul of specimen to the sample pad behind the arrow mark at the bottom
of test strip.
Interpret the test result at 15 minutes. Do not interpret after 20 minutes.

HBsAb TEST

Principle: HBsAb test is a chromatographic immunoassay for qualitative detection of the


surface antibody of Hepatitis B virus (Anti-Hbs) in human whole blood samples.
Procedure:
Bring all materials and specimens to room temperature.
Remove the test from the sealed foil pouch.
Label the test strip with specimen identify by writing the ID on the top label of the strip.
Place the test strip on a flat horizontal surface.
Use the transfer pipet to draw up the sample.
Hold the transfer pipet in a vertical position over the sample pad and dispense 2-3 drops
(80-120ul) of sample onto the sample pad.
Read the result at 20 minutes after adding the sample.

ANTI-STREPTOLYSIN O (ASO)
Qualitative Test Procedure:
1. Bring the reagents and samples to room temperature.
2. Place 50ul of the sample and 1 drop of the control into separate circles on the card.
3. Resuspend the latex gently.
4.Add one drop of the latex reagent to each circle next to the sample which is to be
tested.
5. Mix with the disposable pipette/stirrer and spread over the entire area enclosed by
the ring. Use a new stirrer for each sample.
6.Rotate the cards at 100 r.p.m for 2 minutes.

Quantitative Test Procedure:

1. Using a semi-automatic pipette, add 50ul of 9g/ saline to circles 2,3,4 and 5. Do not
spread the saline.
2. Add 50ul of patient sample to circles 1 & 2.
3. Mix the saline and sample in circle 2 by drawing the mixture up and down being
careful to avoid the formation of any bubbles.
4. Transfer 50ul from circle 2 to the saline in circle 3.
5. Perform serial dilutions in the same manner until the last circle, discarding 50ul at the
end.
6.Using the pipette/stirrer, spread the diluted samples over the entire area of each circle
starting at circle 5 and working backwards to the sample in circle 1.
7.Proceed as a qualitative test from step 3.

Dilution ASO IU/ml


Neat 200
1:2 400
1:4 800
1:8 1600

Typhoid IgG/IgM Combo Rapid Test-Cassette (serum/plasma/whole blood)


Principle: Typhoid IgG/IgM Combo Rapid Test is a lateral flow immunoassay for the
simultaneous detection and differentiation of anti-Salmonella typhi IgG and IgM in
human serum, plasma or whole blood.

Assay Procedure:
Bring the specimen and test components to room temperature if refrigerated or frozen.
Mix the specimen well prior to assay once thawed.
When ready to test, open the pouch at the notch and remove device. Place the test
device on a clean, flat surface.
Be sure to label the device with specimen’s ID number.
For whole blood test
Apply 1 drop of whole blood (about 40-50ul) into the sample well. Then add 1 drop
(about 35-50 ul) of sample diluents immediately.

For serum or plasma test


Fill the pipette dropper with the specimen.
Holding the dropper vertically, dispense 1 drop (about 30-45 ul) of specimen into the
sample well making sure that there are no air bubbles.
Then add 1 drop (about 3550 ul) of sample diluents immediately.
Set up timer.
Results can be read in 15 minutes. Positive results can be visible in as short as 1
minute.

Interpretation I assay result:


Negative: If only the C band is present, the absence of any burgundy color in the both T
bands (T1 and T2) indicates that no anti- S. typhi antibody is detected. The result is
negative.
Positive:
In addition to the presence of C band, if only T1 band is developed, the test indicates for
the presence of anti- S. typhi IgM. The result is positive.
In addition to the presence of C band, if only T2 band is developed, the test indicates for
the presence of anti- S. typhi IgG. The result is positive.
In addition to the presence of C band, both T1 and T2 band is developed, the test
indicates for the presence of anti- S. typhi IgM and IgG. The result is also positive.
Invalid: If no C band is developed, the assay is invalid regardless of any color in the T
bands. Repeat the assay with a new device.
Dengue IgG/IgM combo Test
Principle: Dengue IgG/IgM combo Test is a rapid imunochromatographic assay for the
simultaneous detection of IgG and IgM antibodies to dengue virus in human whole
blood serum or plasma.
Procedure:
Bring the kit components to room temperature before testing.
Open the pouch and remove the card, once opened, the test card must be used
immediately.
Label the test card with patient’s identity.
Apply 5 ul of serum, plasma, or whole blood to the S1 area indicated by the arrow mark.
Add 2 drops of sample buffer to well marked as “S”.
At the end of 20 minutes read the results. A string positive sample may show result
earlier.

Interpretation of results
Negative: only control line appears.
IgM Positive: Both control line and the test line (the higher test line) appears. It indicates
the possibility of primary infection.
IgM and IgG Positive: control line and both test lines appear. It indicates the possibility
of acute secondary infection.
IgG positive: Both control line and the second test line (the lower test line which is
closer to the sample well) appear. It indicates the possibility of secondary infection or
past infection.
Invalid: If after 20 minutes no control line appears, the result is invalid. The test should
be repeated with a new device.
H. pylori

Principle: This assay is a double antigen chromatographic lateral flow immunoassay.

Procedure:
Remove the test device from the foil, and place it on a flat, dry surface.
Transfer 10 ul of serum or plasma ( or 20 ul whole blood) ti the sample well (S) of the
test device, then add 3 drops of assay diluent (approx. 110 ul) and start the timer.
As the test begins to work, you will see purple color move across the result window in
the center of the test device.
Interpret test results at 10 minutes. A positive result wil not change once it has been
established at 10 minutes. However, in order to prevent any incorrect results, the result
should not be interpreted after 10 minutes.

Interpretation of the test:


Negative Result: The presence of only one purple color band within the result window
indicates a negative result.
Positive Result: The presence of two color bands (T band and C band) within the result
window, no matter which band appears first, indicates a positive result.
Invalid Result: If the purple color band is not visible within the result window after
performing the test, the result is considered invalid. The test should be repeated with a
new device.
BLOOD TYPING
Slide method

Principle: The slide test detects the A and B antigens on red cells by using the principle
of agglutination.

Procedure:

1. Divide a slide by marking through the center with a pencil. Mark “A” on the left and “B’
on to the right.
2. Place 1 drop of Anti-A serum (blue) in the square marked “A” and 1 drop of Anti- B
serum (yellow) in the square marked “B”.
3. Using either finger, clotted, citrated or oxalated blood, transfer a small amount of
blood to each serum. With the aid of applicator stick, mix well and observe for
agglutination.

INTERPRETATION:

If only the A antigen is present on the red cells, the cells will agglutinate with ant-A but
not with Anti- B.
If only the B antigen is present on the red cells, the cells will agglutinate with ant-B but
not with Anti-A.
Group O blood will show no agglutination with either Anti-A or Anti- B.
Group AB blood will agglutinate with both Anti-A or Anti- B.

POSITIVE= AGGLUTINATION
NEGATIVE= NO AGGLUTINATION

RH TYPING ( D TYPING)
(SLIDE TECHNIQUE)

1. Use a finger pricked blood or venous blood.


2. Place 1 drop of Ant-D (Rh) antiserum on a slide.
3. Add 1 drop of capillary or venous blood.
4. Mix with the use of applicator stick.
5. Read for agglutination.

INTERPRETATION:
POSITIVE= AGGLUTINATION
NEGATIVE= NO AGGLUTINATION

DIRECT ANTIGLOBULIN TEST


Principle:

Polyspecific (broad spectrum) anti-human globulin (AHG) reagents contain antibodies to


IgG and various complement components which react with red blood cells (RBCs)
sensitized in vivo with these globulins causing agglutination of the RBCs. Monospecific
anti-human globulin reagents contain antibodies to IgG only or complement components
only.

Purpose:

The direct antiglobulin test (DAT) is used to demonstrate the sensitization of red blood
cells in vivo with IgG antibodies and/or complement components (C3b, C3d, C4). This
test is useful in the investigation of hemolytic transfusion reactions, hemolytic disease of
the fetus and newborn, autoimmune hemolytic anemia, and red blood cell sensitization
due to drugs.

Procedure:
1. Label three tubes- Test, Positive Control, and Negative Control
2. Place two drops of each of the test cells, Rh positive sensitized control cells, and
negative control cells in their corresponding tubes.
3. Wash the cells in the three test tubes by adding at first a small amount of saline
solution, mixing well to suspend the cells, and then adding more saline saline solution,
washing it down the slides of the test tubes.
4. The cells are washed three times and each time after washing, centrifuge at 1000rpm
for 1 minute. THE supernatant is decanted and the saline solution drains each time.
5. After the last wash, drain the saline solution well so as not to dilute the anti-globulin
serum by inverting the test tube over a piece of clean gauze.
6. Add 1-2 drops anti-globulin serum to the cell bottom in the three test tubes. Mix.
7. Centrifuge the three test tubes at 1000 rpm for one minute.
8. Gently resuspend the cells, dislodge the cell button and observe for agglutination
both macroscopically and microscopically.

Agglutination indicates a positive anti-globulin reaction, proving thet the red cells are
sensitized.

INDIRECT ANTIGLOBULIN ( COOMBS) TEST


A test to demonstrate the presence of free antibody in the patient’s serum.
USES:
1. Cross matching to detect incompatibility.
2. Use in typing blood with antibodies in the serum which are only reactive by the
antiglobulin method.
3. Use in cases where the antigen is not detectable by ordinary methods requiring the
increased sensitivity of the anti globulin technique.
4. Use in gamma globulin neutralization.
Procedure:
1. Place 1 drop of donor’s saline washed RBC to a test tube, add 1 drop of patient’s
serum and mix.
2. Incubate the mixture in the tube for 3 minutes in a water bath at 37 degree Celsius.
3. Immediately upon removal from the water bath centrifuge for 1 minute at 1000 rpm.
Examine for hemolysis and or agglutination using an optical aid.
4. Wash the RBC three times in a large amount of saline. Decant each wash as
completely as possible. After the third wash, invert the test tube over a piece of gauze to
drain the saline completely.
5. Add 1 drop of anti globulin to the washed cells. Mix.
6. Centrifuge at 1000 rpm for 1 minute.
7. Examine for agglutination using an optical aid. If negative, check microscopically
INTERPRETATION:
Agglutination indicates a positive anti globulin test and therefore the presence of
antibodies in the test serum capable of acting with the test cells.
STOOL EXAMINATION PROCEDURE

DIRECT FECAL SMEAR:

Macroscopic Examination:
1. Age
2. Consistency: Formed, soft, loose, watery
3. Abnormalities: Excessive mucus blood or worms. Visible evidence of interfering
substances such as barium or water
Microscopic Examination:
1. On a clean glass slide, place a drop of saline (NSS, 0.85% NaCl) on the left side of
the slide
and a drop of iodine on the right.
2. Using an applicator stick, take a small portion of the stool. If the stool is formed
semi- formed, take the portion from well inside and from the surface. If it contains
mucus
or is liquid, take the portion from the bloodstained mucus on the surface or from the
surface of the liquid.
3. Mix each preparation.
4. Place a cover slip over the mixture (exclude bubbles).
5. Examine the preparation under the microscope from LPO to HPO. Be sure to search
in all
areas of the mount/smear.
6. Report parasites seen either in no. of eggs per cover slip or in plusses for
helminthes or as
positive or negative for protozoans.

KATO THICK SMEAR

Cellophane thick smear technique:

1. Cellophane 25x35m – soaked in glycerine – solution for at least 24


hours
2. With an applicator stick, around 100mg of sample is transferred to
ordinary microscopic slides.
3. Cover with soaked cellophane then pressed with rubber stopper to
distribute the smear evenly.
4. Examine right after smearing or up to 5 hours if stored in 10 C refrigerator (2 hours
without
refrigeration
ERYTHROCYTE SEDIMENTATION RATE ( ESR)

Measures the rate of fall of red blood cells over a given period of time ( usually one
hour) at room temperature. It is a method of detecting inflammatory process.

PROCEDURE:
1. Draw 2.0 ml venous blood into syringe and add immediately to 3,8% citrate (Blue
Top
Tube).
2. Mix gently but thoroughly.
3. Fill the Westergreen tube exactly to the 0 (zero) mark and place upright in a
special
Westergreen rack.
4. Allow tube to sit at room temperature, undisturbed for exactly one hour.
5. Read the distance in mm that the red blood celss have fallen over a one hour
period.
6. Report the mm reading as the results

Normal Value:
Male: 0 – 15 mm
Female: 0 – 20 mm

HEMATOCRIT DETERMINATION PROCEDURE

Micro Method:

1. Blood is collected into a heparinized capillary tube by finger puncture


2. The tube should be at least two- thirds (2/3) full.
3. One end of the tube is sealed with clay or other suitable material.
4. The tubes are centrifuged in a micro-hematocrit centrifuge for five minutes at a
minimum of 12000 G.
5. After centrifugation, the capillary tubes are read on a microhematocrit reading device.
Normal Values:
Male 0.42 – 52%
Female 0.37 – 0.47%
Infant 0. 45 – 65%
MANUAL PROCEDURE FOR ERYTHROCYTE (RED CELL) COUNT

1. Anticoagualated whole blood is drawn (EDTA is preferable)up to the 0.5 mark of the
red cell pipette. If the blood is just slightly above this mark, the excess is absorbed or
“blotted”out with the use of a tissue until the blood level is exactly on the 0.5 mark.
2. When the pipette is properly loaded with blood, the specimen is diluted to the 101
mark with red blood cell diluting fluid and mix the specimen completely,.
3. Place the pipette on a pipette shaker.
4. Allow the dilution to mix thoroughly for approximately 2 t0 3 minutes on the shaker.
5. Remove the pipette and allow approximately one- third of the fluid to run out onto a
tissue and load to hemacytometer. Make sure the fluid flows evenly between the
hemacytometer and the cover glass. Avoid bubbles, overfilling, or underfilling. The fluid
should evenly reach the edge and each side without spilling over or without leaving
empty spaces around the edges of the chamber.
6. The cells in the chamber are permitted to settle for several minutes. Then check with
the low – power objective to see whether they are evenly distributed.
7. Count the cells in 80 small squares – five groups of 16 and multiplied by 10000 ( or
simply add 4 zeros) to the number of red cells counted in 1/5 sq mm ( 5 small squares
of the red cell area.
Calculation: Total RBC/cumm = Number of erythorcytes counted
1/5 (sq mm counted) x 200 (dilution) x 10 (depth of counting chamber
Normal Value:
Male 4.6 – 5.2 x 10 12/L
Female 4.0 x 10 12/L

LEUCOCYTE (WHITE BLOOD CELL) COUNT:

1. Anticoagulated whole blood (EDTA preferred) is drawn to the 0.5 mark of


2. Blood is diluted to the 11 mark of the Wbc pipette with white blood cell diluting fluid.
3. Mix the specimen completely and place on pipette shaker.
4. Allow the dilution to mix thoroughly for approximately 2 t0 3 minutes on the shaker.
5. Remove the pipette and allow approximately one- third of the fluid to run out onto a
tissue and load to hemacytometer. Make sure the fluid flows evenly between the
hemacytometer and the cover glass. Avoid bubbles, overfilling, or underfilling. The fluid
should evenly reach the edge and each side without spilling over or without leaving
empty spaces around the edges of the chamber.
6. The cells in the chamber are permitted to settle for several minutes. Then check with
the low – power objective to see whether they are evenly distributed.
7. Count the number of white blood cells in the corner squares ( 4 square millimeters)
of the counting chamber.
Calculation:
Number of white cells counted in 4 sq mm x 1/volume in which the cells are counted x
dilution
Or
Number of white blood cells counted in 4 sq mm x 2.5 (volume) x 20 (dilution) = number
of white blood cells/cu mm
Or
Number of white cells counted in 4 sq mm x 50 = number of white blood cells/cumm

DIFFERENTIAL STAINING PROCEDURE

1. A small drop of blood is placed at one end of a clean microscope slide.


2. With a second slide, the drop of blood is spread across the entire end of the slide
and evenly
spread over at least two – thirds (2/3) the length of the slide and have a thick end at
the
beginning of the smear, gradually getting thinner as the blood is spread and finally
ending
with an even feathered edge
3. The film should be stained as soon as they have been dried in the air.
4. Place the slide on solution A quantity for fixing.
5. After 2 minutes, stained the slide with solution B (eosin). Allow the slide to remain
for 3 to 4 minutes. Then stained with Methylene Blue.
6. Wash the film with water and let it dry.
7. Observe under the microscope and count different leukocytes (WBC)
8. Red blood cells should be studied and any abnormalities must be reported
9. Platelets should be evaluated and reported.
ROUTINE URINALYSIS

1. Urine sample must be collected in a clean dry container and should be examined as
soon as possible. Refrigeration and use of toluol or thymol as preservatives may retard
the changes in the urine.
2. Mix the specimen thoroughly, color and appearance are recorded and 5 ml is
transferred to test tubes (appropriately numbered).
3. Dip urine chemistry reagent strip to the test tubes and remove immediately. (to
determine the chemical properties of urine ph, specific gravity, protein, glucose,
bilirubin, urobilirubin, blood, nitrite, leukocyte esterase ). Touch strip against edge of
container to remove excess urine. Compare resulting color of each test area with
appropriate color in corresponding color chart.
4. The specimens are centrifuged for 5 minutes at 2000 r.p.m.
The clear portion is removed and the sediment is saved for microscopic examination.

OCCULT BLOOD DETERMINATION


Benzidine Filter Paper Method (Modified)

Materials: filter paper, Reagent A, Reagent B

Procedure:
1. Smear a small particle of feces on a piece of filter paper with wooden applicator stick.
2. Add 3 drops of reagent A.
3. Add 3 drops of reagent B
4. Read within 1 minute.

Result:
Positive- any trace of blue on or at the edge of the smear indicates a positive test for
occult blood.
Negative- no detectable blue on or at the edge of the smear indicates a negative test
for occult blood.

MALARIAL SMEAR
For routine malaria microscopy, thin and thick blood films are made on the same slide.
The thin film is used as a label but, if well prepared, is also available for species
confirmation. Examination should be done in the thick film.

Materials: clean and wrapped slides, sterile lancets, 70% ethanol and water, absorbent
cotton wool, examination gloves, clean, lint-free cotton cloth, slide box (or cover to
exclude flies and dust), record form or register, soft leads pencil, ball point pen.

Procedure:
1. Holding the patient’s hand, palm upwards, select the third finger from the thumb. The
big toe can be used with infants. The thumb should never be used for adults or children.
Clean the finger with a piece of cotton wool lightly soaked in 70% ethanol, using firm
strokes to remove grease and dirt from the ball of the finger. Dry the finger with a clean
cotton cloth using firm stokes to stimulate blood circulation.
2. Puncture the ball of the finger with a sterile lancet, using a quick rolling action. Apply
gentle pressure to the finger to express the first drop of blood and wipe it away with a
dry piece of cotton wool. Make sure that no strands of cotton remain on the finger to be
later mixed with the blood.
3. Working quickly and handling clean slides only by the edges, collect the blood as
follows. Apply gentle pressure to the finger and collect a single small drop of blood on
the middle of the slide. This is for the thin film.
Apply further pressure to express more blood and collect two or three larger drops on
the slide, about 1 cm from the drop intended for the thin film. This is for the thick film.
Wipe the remaining blood away from the finger with a piece of cotton wool.
4. Thin film. Using a second clean slide as a “spreader” and. With the slide with the
blood drops resting on a flat, firm surface, touch the small drop with the spreader and
allow the blood to run along its edge. Firmly push the spreader along the, slide keeping
the spreader at an angle of 45°. Make sure that the spreader is in even contact with the
surface of the slide all the time the blood is being spread.
5. Thick film. Always handle slides by edges or by corner to make the thick film as
follows.
Using the corner of the spreader, quickly join the drops of blood and spread them to
make an even, thick film. The blood should not excessively stirred but can be spread in
circular or rectangular form with 3-6 movements. The circular thick films should be
about 1cm (1/3 inch) in diameter.
6. Label the dry thin film with a soft lead pencil by writing across the thicker portion of
the thin film the patient’s name or number and the date. Do not use a ball-point pen for
labeling the slide. Allow the thick film to dry with the slide in a flat, level position,
protected from flies, dust and extreme heat.
7. Wrap the dry slide in the patient’s record form and dispatch it to the laboratory as
soon as possible.

GIEMSA SATINING (RAPID METHOD)


10% GIEMSA stain preparation/slide: 3ml buffer solution + 9 drops Giemsa stock
solution)
1. Air dry smear before fixing the thin smear with methnol. Do not fix or expose the thick
smear to methanol vapor.
2. Flodd thick and thin smears with 10% giemsa solution for 10-15 minutes.
3. Gently wash and air dry.

EXAMINING THICK FILMS


With the slide on the stage, position the x100 oil immersion objective over the blood
film. Add a drop of immersion oil to the slide and lower the objective until it touches the
oil. Focus and ensure that you have selected the most appropriate part of the film with
15-20 WBC’s per field. Examine a minimum 100 fields before calling the slide negative;
if you are uncertain, or parasites have been found, then examine a further 100 fields to
confirm the species and to ensure the infection is not mixed. A tally counter will help you
in counting the fields examined. Examining 200 oil immersion takes about 10 minutes.
Examining THIN FILMS
As with the thick film place the drop of immersion oil on the film. Using the x100 oil
immersion objective confirm the portion of film is appropriate and examine.

DIRECT SMEAR EXAMINATION

1. Numbering the slide. Write down the yearly serial number and order number of
sputum specimen on an end of the slide.
2. Sputum smearing. Fish out one loopful of yellowish particle of sputum. Spread one
loopful of the sputum evenly on a clean labeled glass slide, approximately 2cmx3cm in
size. The rough end bamboo splinter or coconut mid-rib stick can be used instead of
wire loop to take sputum to smear by using the rough end. The used sticks are burnt
after day’s work.
3. Removal of adherent sputum. Dip the wire loop in a washing bottle and remove the
excess sputum from the wire loop by moving it up and down in sand.(* washed sand or
small glass beads and 90-95% spirit or 5% cresol are contained in the bottle).
4. Sterilization of the wire loop. Heat the wire loop in a flame till red hot. Adequate flame
should be colorless or blue.
5. Drying and fixation of the smear. Allow the smear to dry completely at room
temperature. Do not place it under heat of the sun or over a flame at this stage. And fix
it by passing through the flame 2 t0 3 times, about 2-3 seconds cach. Flame at the back
of smeared surface of the slide. Never scorch the smear.
6. Arrangement of the slide. Place the slide on a staining bridge. Remember to leave
enough space between slides to prevent the transfer of material from one smear to
another and the solution running off from the slides.
7. Staining with Ziehl’s solution. Pour Ziehl’s (carbol fuchsin) solution to cover the whole
surface of the slide.
8. Heating of the slide. Heat the slide till steam comes off from the stain. Do not boil and
do not allow the slide to dry. Leave it for 5 minutes.
9. Removal of excess stain. Tilt the slide to drain off excess stain.
10. Washing of the slide. Wash the staining solution off with a gentle stream of running
water.
11. Draining off excess rinse water. Tilt the slide to drain off excess rinse water.
12. Decolourization. Cover the whole slide with 3% hydrochloric acid-ethanol and leave
it until solution runs clear.
13. Washing of the slide. Wash the slide with gentle stream of running water.
14. Draining off excess rinse water. Tilt the slide to drain off excess rinse water.
15. Counterstaining. Pour 0.1% methylene blue to cover the whole surface of the slide
and leave for 10 seconds.
16. Removal of methylene blue. Pour off0.1% methylene blue.
17. Washing of the slide. Wash slide with gentle stream of running water.
18. Drying the stained slide. Tilt and place the slide on the slide rack to dry in the air.
Don’t place under the sun to dry.
19. Application of immersion oil. Put one drop of immersion oil on the stained smear. Do
not touch the smear with the dropper to avoid transfer of AFB from one smear to
another.
20. Observation of stained smear. Examine the smear under 100x objective with 10x
eye piece lens. Acid Fast Bacilli appear red or pink coloured and the background is
stained in blue colour.
21. Smear scanning. Read at least 300 visual fields to give a report as negative. Record
the result on the laboratory logbook.

NATIONAL STANDARD REPORTING SCALE


0 No AFB seen in 300 visual fields
+n 1-9 AFB/100 visual fields
1+ 10-99 AFB/100 visual fields
2+ 1-10 AFB/OIF in at least 50 visual fields
3+ more than 10 AFB/OIF in at least 20 visual fields

GRAM’S STAIN
Procedure:
Filter stain daily. Heat fix smear to be stained.
Reagent/Direction Time Involved
Crystal Violet 1 minute
Wash gently in tap water
Gram’s Iodine 1 minute
Wash gently in tap water
Gram’s Decolorizer until no more pink comes off
Wash gently in tap water
Safranin 1 minute
Wash gently in tap water

Result:
Gram positive organism – purple
Gram negative organism - pink

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