Microbiology Questions

1.      Describe the toxins produced by Cl. Welchii.          

2.      Represent the mechanism of development of Cl. Tetani

infection                                                                      

3.      Which microorganisms are responsible for enteric fever? Diagrammatically represent the course of disease development. Mention its clinical features and complication?                                                         

4.      Define sterilization. Describe the physical modes of

sterilization.                                                                

5.      Represent the path of disease development of C. diptheriae infection. Mention its clinical features.           

6.      Explain                                                       

a.      Shick’s test

b.      Complement fixation test

c.       Coomb’s test

Widal test.

Complement Fixation Test

Complement Fixation Test

The complement fixation test (CFT) is used to detect the presence of specific antibodies in the patient’s serum. This test is based on the use of complement, a Biologically labile serum factor that causes the immune cytolysis i.e. lysis of antibody coated cells.

Principle of complement fixation test

                 It is the nature of the complement to be activated when there is formation of antigen-antibody complex.

·        The first step is to heat the serum at 56°C to destroy patient’s complement.

·      A measured amount of complement and antigen are then added to the serum. If there is presence of antibody in the serum, the complement is fixed due to the formation of Ag-Ab complex.

·        If no antibody is present then the complement remains free.

·  To determine whether the complement has been fixed, sheep RBCs and antibodies against sheep RBCs are added.

In the positive test : The available complement is fixed by Ag-Ab complex and no hemolysis of sheep RBCs occurs. So the test is positive for presence of antibodies.

In the negative test : No Ag-Ab reaction occurs and the complement is free. This free complement binds to the complex of sheep RBC and it’s antibody to cause hemolysis, causing the development of pink color.

Procedure

The following figure shows the steps involved in the procedure of complement fixation test.

Interpretation

Positive test : The available complement is fixed by Ag-Ab complex and no hemolysis of sheep RBCs occurs. So the test is positive for presence of antibodies.

Negative test : No Ag-Ab reaction occurs and the complement is free. This free complement binds to the complex of sheep RBC and it’s antibody to cause hemolysis, causing the development of pink color.

Coombs test

A Coombs test (also known as Coombs' test, antiglobulin test or AGT) is either of two clinical blood tests used in immuno-hematology and immunology. The two Coombs tests are the direct Coombs test (DCT, also known as direct antiglobulin test or DAT), and the indirect Coombs test (also known as indirect antiglobulin test or IAT)

1. The direct Coombs test is used to detect these antibodies or complement proteins that are bound to the surface of red blood cells; a blood sample is taken and the RBCs are washed (removing the patient's own plasma) and then incubated with anti-human globulin (also known as "Coombs reagent"). If this produces agglutination of RBCs, the direct Coombs test is positive, a visual indication that antibodies (and/or complement proteins) are bound to the surface of red blood cells.
2. The indirect Coombs test is used in prenatal testing of pregnant women and in testing blood prior to a blood transfusion. It detects antibodies against RBCs that are present unbound in the patient's serum. 

Mechanism

The two Coombs tests are based on the fact that anti-human antibodies, which are produced by immunizing non-human species with human serum, will bind to human antibodies, commonly IgG or IgM.

Animal anti-human antibodies will also bind to human antibodies that may be fixed onto antigens on the surface of red blood cells (also referred to as RBCs), and in the appropriate test tube conditions this can lead to agglutination of RBCs.

The phenomenon of agglutination of RBCs is important here, because the resulting clumping of RBCs can be visualised; when clumping is seen the test is positive and when clumping is not seen the test is negative.

Common clinical uses of the Coombs test include the preparation of blood for transfusion in cross-matching, atypical antibodies in the blood plasma of pregnant women as part of antenatal care, and detection of antibodies for the diagnosis of immune-mediated haemolytic anemias.

 

 

 Direct Coombs test

The direct Coombs test (also known as the direct antiglobulin test or DAT) is used to detect if antibodies or complement system factors have bound to RBCs surface antigens in vivo. The DAT is not currently required for pre-transfusion testing but may be included by some laboratories.

Examples of diseases that give a positive direct Coombs test

The direct Coombs test is used clinically when immune-mediated hemolytic anemia (antibody-mediated destruction of RBCs) is suspected. A positive Coombs test indicates that an immune mechanism is attacking the patient's own RBCs. This mechanism could be autoimmunity, alloimmunity or a drug-induced immune-mediated mechanism.

Indirect Coombs test

The indirect Coombs test (also known as the indirect antiglobulin test or IAT) is used to detect in-vitro antibody-antigen reactions. It is used to detect very low concentrations of antibodies present in a patient's plasma/serum prior to a blood transfusion. In antenatal care, the IAT is used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. The IAT can also be used for compatibility testing, antibody identification, RBC pheno-typing, and titration studies.

 

Examples of clinical uses of the indirect Coombs test

Blood transfusion preparation

Main articles: blood transfusion and cross-matching

The indirect Coombs test is used to screen for antibodies in the preparation of blood for blood transfusion. The donor's and recipient's blood must be ABO and Rh D compatible. Donor blood for transfusion is also screened for infections in separate processes.

·         Antibody screening

A blood sample from the recipient and a blood sample from every unit of donor blood are screened for antibodies with the indirect Coombs test. Each sample is incubated against a wide range of RBCs that together exhibit a full range of surface antigens (i.e. blood types).

·         Cross matching

The indirect Coombs test is used to test a sample of the recipient's serum for antibodies against a sample of the blood donor's RBCs. This is sometimes called cross-matching blood.

Courtesy - Wikipedia.

Differnece between Endotoxin and Exotoxin

Differences Between

Exotoxins and Endotoxins

Toxins and enzymes play important role in pathogenecity. Toxins are of two types:

Exotoxins are usually heat labile proteins secreted by certain species of bacteria which diffuse into the surrounding medium.

Endotoxins are heat stable lipopolysaccharide-protein complexes which form structural components of cell wall of Gram Negative Bacteria and liberated only on cell lysis or death of bacteria.

Some of the differences between 
Exotoxins and Endotoxins are as follows:

S.N.	Exotoxins	Endotoxins
1	Excreted by organisms, living cell	Integral part of cell wall
2	Found in both Gram positive and Gram Negative bacteria	Found mostly in Gram Negative Bacteria
3	It is polypeptide	It is lipopolysaccharide complex.
4	Relatively unstable, heat labile (60°C)	Relatively stable, heat tolerant
5	Highly antigenic	Weakly immunogenic
6	Toxoids can be madeby treating with formalin	Toxoids cannot be made
7	Highly toxic, fatal in µg quantities	Moderately toxic
8	Usually binds to specific receptors	Specific receptors not found
9	Not pyrogenic usually, Toxin Specific	Fever by induction of interleukin 1 (IL-1) production, Shock
10	Located on extrachromosomal genes (e.g. plasmids)	Located on chromosomal genes
11	Filterable	Not so
12	It has no enzymatic activity	It has mostly enzymatic activity
13	Its molecular weight is 10KDa	Its molecular weight is 50-1000KDa
14	On boiling it get denatured.	On boiling it cannot be denatured.
15	Detected by many tests (neutralization, precipitation, etc)	Detected by Limulus lysate assay
16	Examples: Toxins produced byStaphylococcus aureus, Bacillus cereus, Streptococcus pyogenes, Bacillus anthrcis(Alpha-toxin, also known as alpha-hemolysin (Hla))	Examples: Toxins produced by E.coli, Salmonella Typhi, Shigella, Vibrio cholera(Cholera toxin- also known as choleragen)
17	Diseases: Tetanus, diphtheria, botulism	Diseases: Meningococcemia, sepsis by gram negative rods