EXERCISE 5

PHAGOCYTOSIS
Block 5
Batch Tritonos
 What is phagocytosis?
• Phagocytosis:
- a specific form of endocytosis by which
cells internalize solid matter, including
microbial pathogens.
- Macrophages, Neutrophils and Dendritic
cells
- In these cells, phagocytosis is a
mechanism by which microorganisms can
be contained, killed and processed for
antigen presentation and represents a vital
facet of the innate immune response to
pathogens, and plays an essential role in
initiating the adaptive immune response.
Materials:

Freshly drawn blood for buffy coat

sample using heparinized tube

Buffy
coat
Materials:
Broth cultures of Staphylococcus
aureus and Escherichia coli

Staphylococcus aureus

Escherichia coli
Materials:
c. Pasteur pipettes d. Wright stain set-up
Procedure:
1. Transfer 5 ml blood into an evacuated tube with heparin. Separate
plasma by centrifugation and discard.

2. With a Pasteur pipette extract the buffy coat

Buffy
coat
Procedure:
3. Place 4 drops of buffy coat into an empty tube.

4. Add 2 drops of assigned broth culture to the tube with buffy coat.
Blocks 1,3,5,7: Staphylococcus aureus broth
Blocks 2,4,6,8: Escherichia coli broth

5. Incubate the inoculated tubes at 37 degrees Celsius for 30 minutes.

Procedure:
6. Place 1 drop of the incubated specimen onto a slide and make a smear.

7. Air dry the smear.

Procedure:
8. Stain the prepared smear with Wright’s stain in the following manner
a. Place the slide in a horizontal position on a rack and add Wright’s
stain covering the surface of the smear completely. Allow the stain to
stand for 1-3 minutes
b. Add an equal amount of drops of buffered water to bring the
meniscus to the margin. Leave the mixture for 8-10 minutes. The
presence of a green metallic sheen is indicative of proper staining.
c. Wash the stain off with running water. Do not pour off the stain before washing
because this will leave a precipitate on the smear.

d. Wipe the bottom of the slide to remove the dried stain.

DO NOT BLOT DRY
Results:
• Block 1
Results:
Block 2
Results:
Block 4
Results:
Block 5
Results:
Block 6
Results:
Block 7
Results:
Block 8
Phagocytic cells

macrophage neutrophil dendritic cell

Steps in phagocytosis:
CHEMOATTRACTANTS
• Bacterial products
• Activated complement factors (c3a,c5a)
Step 1: Activation of the Phagocyte • Arachidonic acid metabolites (LTB4)
• Cytokines: such as IL-1, IL-8
Step 2: Chemotaxis of Phagocytes
(for wandering macrophages, neutrophils, and eosinophils)
Step 3: Attachment of the Phagocyte to the Microbe or Cell
Step 4: Ingestion of the Microbe or Cell by the Phagocyte
Step 5: Destruction of the microbe or cell
Phagocytosis
Question

Give the significance of finding only

lymphocytes in the smear.


Thank you!