In Vitro Phagocytosis: Principle, Materials and Procedure

Principle

A drop of whole blood is mixed with a drop of a bacterial culture and incubated at room temperature to demonstrate engulfment of bacteria by leukocytes.

Materials

• Test tubes, 12 75 mm 
• Broth culture of Staphylococcus epidermidis 
• Lancets for finger puncture 
• Heparinized microhematocrit tubes 
• Microscope slides 
• Wright stain

Procedure

1. Take two 12 75 mm test tubes and label one 0 minutes and the other 5 minutes. If there is enough blood available, a 10-minute tube can also be set up.
2. Do a finger puncture and fill a heparinized microhematocrit tube about three-quarters full of blood. Note that blood drawn in an EDTA tube within the last 10 minutes can also be used.
3. Using a black rubber bulb, expel one drop of blood into each labeled test tube.
4. Add one drop of Staphylococcus epidermidis culture to each tube, using a disposable Pasteur pipette. The culture should be no more than a 0.5 McFarland standard in concentration. Dilute in an additional broth tube or in sterile saline if necessary.
5. Shake the tubes to mix, and make a blood film of the 0 tube immediately. Let the other tubes incubate for 5 minutes and 10 minutes, respectively, at room temperature before making blood films of them. 

Method for Making Blood Smears

1. Obtain two clean glass slides, one of which will be used as a spreader slide.
2. With a Pasteur pipette, carefully place a small drop of blood at one end of a microscope slide.
3. Holding the spreader slide with the thumb and forefinger, place the spreader slide slightly in front of the drop of blood on the other slide, maintaining a 25-degree angle between the slides.
4. Move the spreader slide back toward the drop of blood. As soon as the slide comes in contact with the drop of blood, the blood will start to spread along the edge.
5. Keeping the spreader slide at a 25-degree angle, push it rapidly over the length of the slide. There should be a feathered edge on the end of the smear. 

Wright Stain

1. Allow the blood smear to air dry.
2. Stain according to typical laboratory protocol for staining blood smears.
3. Blot the back of the slides to remove excess stain and let them air dry.
4. Use immersion oil and look for engulfment.

Interpretation/Analysis

These blood smears will be more watery than usual due to the addition of the broth culture with bacteria; therefore, it may be difficult to get a blood smear with a feathered edge. The feathered edge is not essential, as long as the blood cells are spread out on the slide. There should be a noticeable difference between the 0- and the 5-minute slide. The 0-minute slide will probably not show much engulfment, but bacteria may be seen in contact with leukocytes. The 5-minute slide should show bacteria within the cell as small purple dots. Neutrophils will be the predominant phagocytic cells, but an occasional monocyte may be seen. If lymphocytes are the only white blood cell seen, the bacterial suspension was too heavy, and the phagocytic cells destroyed themselves in attempting to engulf the bacteria present.

Immunofixation Electrophoresis: Principle, Procedure and Interpretation

Principle

Immunofixation electrophoresis (IFE) is a two-stage procedure using agarose gel high-resolution electrophoresis in the first stage and immunoprecipitation in the second. Proteins are first separated by electrophoresis. Then, in the second stage, soluble antigen in the gel reacts with specific antibody, and the resultant antigen–antibody complexes become insoluble (as long as the antibody is in slight excess or near equivalency) and precipitate. The precipitation rate depends on the proportions of the reactants, temperature, salt concentration, and the solution’s pH. The unreacted proteins are removed by a washing step, and the antigen– antibody complex is visualized by staining. The bands in the individual lanes are compared with the precipitin bands obtained with a normal control serum. 

Sample Preparation

Fresh serum or urine is the specimen of choice. Evaporation of uncovered specimens may cause inaccurate results, so specimens must be handled carefully. Plasma should not be used, because the fibrinogen may adhere to the gel matrix, resulting in a band in all patterns across the gel. If storage is necessary, samples may be kept covered at 2°C to 8°C for up to 72 hours.