November 28, 2001

Experiment 21: Immunity against Bacteria

 

In this lab we will be studying the effects of phagocytosis and bacterial agglutination. 

 

Phagocytosis is the internalization of particulate material by macrophages or neutronphils (PMNs).  Phagocytes are used as a primary line of defense against infection and is responsible for presenting antigens to the immune system via MHC I and II. Phagocytes may engulf debris, microorganisms etc, via direct phagocytosis (e.g. “recognizing” antigenic components) or indirect phagocytosis (opsonization of antigenic components by antibodies or complement)

 

PMNs are circulating in the peripheral and migrate to the sites of infection, while the macrophages are strategically places all throughout the body.  PMNs engulf debris as well as function as APCs.  Ags are carried to macrophages via the lymphatic and circulatory system.

 

Four step ingestion process:

1. Adherence: directly via bacterial adhesions or viruses attaching to cell-surface proteins or indirectly via phagocyte’s Fc receptors attaching to antibodies (opsonization). 
2. Engulfment: invagination of cell membrane to form a vacuole (phagosome)
3. Fusion of lysosome  (containing hydrolytic and proteolytic enzymes)
4. Digestion: many digestive enzymes destroys the ingested material. 

However not all ingested microorganisms and viruses are destroyed by the phagocytic cell. 

 

Bacterial Agglutination.  There are different antibodies that can be labeled to bacteria.  Cell surface: O

Capsule = K

Flagella = H

 

From this, bacteria can be classified by according to their antigenic structure, identify antigenic composition, and aid in disease diagnosis.

 

Procedure:

Follow the given handout

A. Phagocytosis

In this lab we will create or obtain periotoneal exudates cells (PEC) and then add Bacillus cereus.  After incubation, a gram stain was preformed and results were observed.

B. Bacterial Agglutination

Draw two ovals on the slide, one on each side.  Add 1 drop of Salmonella typhimurium O antiserum to each oval.  Add S. Typhimurium to one, and Bacillus cereus to the other.  Observe results.

 

Observations:

Magnification at Oculuar: 10X; Objective Lens: 40X Total magnification: 400X

 

1. Phagocytosis: Three types of cells/objects seen

1. Purple: Bacillus bacteria, rod shaped (like a pill)

2. Pink: Phagocyte

3. Phagocytosis of the bacteria: a phagocyte (pink/reddish bubble) with a dark             purple center (bacteria).

 

 

 

 

2. Bacterial Agglutination:

Salmonella typhimurium – had agglutination

Bacillus cereus – Had no agglutination, just hazy white. 

 

 

 

 

Discussion:

 

In this lab we investigated the properties of phagocytes and bacterial agglutination. In the first part PEC (peritoneal exudates cells) were obtained and placed with Bacillus cereus, and then a gram stain was applied.  It was then observed as to what would happen.  The gram stain stained the phagocytes purple and the Bacillus cereus pink.  Under a microscope, both could be seen separately.  It was also observed that the phagocytes had phagocytosized the bacteria and there are two colors.  In the center, where the bacteria is located is, is pink, and surrounding it (phagocyte) is purple.

 

In the second part of lab, the properties of bacterial agglutination were studied.   Bacteria is agglutinated by specific antisera directed against a mosaic of antigens making up the bacterial cell surface, capsule, or flagella.  For this lab, the detection of antibodies against O antigens in an immune system was performed.  If the sample agglutinated, then the O antigen is present in the sample.  The Salmonella typhimurium when added to the Salmonella typhimurium O antiserum agglutinated indicating an antibody-antigen cross-linking (agglutination) reaction. Salmonella typhimurium reacted with the Salmonella typhimurium O antiserum because it is the antigen that the Salmonella typhimurium  can bind to (i.e. Salmonella typhimurium O antiserum is the antibody, while Salmonella typhimurium is the antigen).  When Bacillus cereus was added to Salmonella typhimurium there was no reaction because Bacillus cereus is not the corresponding antigen for Salmonella typhimurium that will cause it to cross-react.