EXERCISE 1

NON-SPECIFIC HOST DEFENSE

I. INTRODUCTION

Non-antibody humoral factors, enzymes, and phagocytosis are part of the NON-SPECIFIC antimicrobial systems which assist in defense against infection and disease. Examples include intact skin, ciliary action in the lung, antibacterial substances in secretions, opsonic factors in serum and phagocytosis.

A host's initial response to the introduction of foreign material is the acute inflammatory response. When this involves bacteria, and the numbers are small, the ensuing inflammatory response may resolve the infection; involvement of the ACQUIRED resistance mechanisms - humoral and cellular immunity - may not be necessary.

External secretions and neutrophil granules contain many substances with antibacterial activity, including lysozyme. Lysozyme (or muramidase) can cleave the mucopeptide layer of most non-encapsulated Gram-positive bacterial cell walls resulting in cytoplasmic blebbing of the bacterial cell through the wall defect. If the external osmolality is sufficiently low, direct lysis can occur, or hydrophobic proteins with detergent/pore forming capacity derived from neutrophil granules may act on the exposed bleb and cause lysis.

The complement system comprises about 10% of normal serum proteins with C3 component present in the greatest amount. A number of important protective/ inflammatory functions are mediated by activated complement factors, in particular, lysis. The mechanism of complement cell lysis is not clearly defined. Activation of complement fragments on the cell surface may lead to the assembly of the terminal membrane attack complex (C5-9) and the formation of a transmembrane pore that leads to Na/K pump dysfunction and osmotic lysis. Both eucaryotic and procaryotic cells are sensitive to this method of killing.

Enhanced phagocytosis is often the ultimate effector mechanism in both nonspecific and specific immunity, whether humoral or cellular. Whole blood contains cells (neutrophils, monocytes) that are potentially phagocytic. The native rate of phagocytosis by these cells is low. Treatment of a foreign particle with normal serum opsonin (C3b derived from alternate complement pathway activation by bacterial cell surface polysaccharides) normally promotes phagocytosis.

II. LAB WORK

A. COMPLEMENT ACTION

• 1 tube of COMPLEMENT on ice (green cap)
• 1 tube of HEAT-INACTIVATED COMPLEMENT (red cap)
• 2 tubes of Gram-negative BACTERIA (brown caps)
• 2 cotton swabs
• 4 pipets, 1 ml
• 2 TS Agar plates (TSA)
• 1 PipetAid (lab drawer)

1. Use the blue PipetAid (lab drawer) to pipet 0.2 ml of complement (green cap ) into 1 tube of bacteria. Label it and one TSA plate, "Active C".
2. Use a new pipet to pipet 0.2 ml of heat-inactivated complement (red cap ) into the other tube of bacteria. Label it and the other plate, "C Control".
3. Incubate the two bacteria tubes in a WATERBATH at 37¡C for 30 min. While this incubates, complete Part B. Lysozyme Action.
4. After the incubation, retrieve the bacteria tubes from the waterbath, and agitate each tube, mixing well. Pipet 0.2 ml from "Active C" tube and deposit it on the center of the first TSA plate. Discard the pipet directly into the pipet jar. Use a sterile cotton swab to spread the liquid evenly over the surface of the TSA plate. Discard the swab directly into the small, red Biohazard bag. Throw the swab wrapper into the trash.
5. Use another sterile pipet to remove 0.2 ml from the "C Control" tube and deposit the liquid on the center of the second TSA plate. Discard the pipet directly into the pipet jar. Use a sterile cotton swab to spread the liquid evenly over the surface of the TSA plate. Discard the swab directly into the small, red Biohazard bag.
6. Invert the plates, label with your name, and place them in the 37C air incubator. At the next lab period, compare the number of colonies on the two plates and note the non-specific bactericidal effect of complement.

B. LYSOZYME ACTION

• 1 tube of LYSOZYME on ice (yellow cap )
• 1 tube of SALINE (black cap )
• 2 tubes of BACTERIA (blue caps )
• 2 pipets, 1 ml
• 1 tube of DETERGENT (purple cap )
• 1 Pasteur pipet
• 1 PipetAid (lab drawer)
• 1 rubber bulb (lab drawer)

1. Label one tube of bacteria, "Control". Use a 1 ml pipet and the blue PipetAid, to transfer 0.1 ml of saline to this tube.
2. Label the other bacteria tube "Lysozyme". Use the other 1 ml pipet to transfer 0.1 ml of lysozyme to this tube.
3. Mix each tube well and allow the lysozyme to react for 3 minutes. Warm the tubes between your hands.
4. Use a Pasteur pipet and rubber bulb to put 1 drop of detergent into both tubes. Mix.
5. Examine for the clearing of turbidity. This clearing indicates lysis due to enzymatic digestion of the bacterial cell wall. Use the saline tube for comparison.