1. Results of viable count

2. Count the number of colonies on each plate ( both circular and spindle shaped) Find the average count for each dilution (3 plates for each dilution) Record your results in the table For calculation, select the petri plates ( the dilution) containing between 30 and 300 colonies. Calculate the number of bacteria (CFU) per milliliter of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added (here=1ml) If more than one dilution give colony count in the range 30- 300, calculate the viable count using both dilutions, then calculate the average CFU/ml.

3. Colony count 10 -3 10 -2 10 -1 Plate # 1 2 3 Average Number of CFU per ml= Average colony count /dil factor x amount plated (1 ml)

4. 10 -1 10 -2 10 -3 Example: If count in 1 st dilutuion plates (average) 10 -1 = 330 colonies Count in 2 nd dilution plates (average) 10 -2 = 35 Count in 3 rd dilution plates ( average) 10 -3 = 2 Viable count= 35/ 10 -2 = 35oo CFU/ml

5. Blood film Blood is composed of a liquid called blood plasma (containing proteins, minerals) and blood cells suspended within the plasma. The blood cells present in blood are red blood cells (also called RBCs or erythrocytes), white blood cells (leukocyte) and platelets.blood plasmablood cellsred blood cellswhite blood cellsplatelets White blood cells are of two types: Granulocytes (granulated cytoplasm) Agranulocytes ( non granular cytoplasm)

6. Granulocytes: Neutrophiles. Eosinophiles, Basophiles Agranulocytes are: Monocytes and lymphocytes Blood film is stained with leishman stain

7. Blood film showing erythrocytes ( gray) and leucocytes (pink)

8. Serology In vitro Antigen- Antibody reactions Antigen- Antibody reactions are classified according to the physical state of antigen into: -Agglutination reactions: antigens are cells or particles -Precipitation reactions: antigens are soluble -Flocculation reactions: antigens are suspended -Complement fixation: indicated by positive or cell lysis

9. Blood grouping (hemagglutination reactions) Hemagglutination reactions are used in typing of blood According to the presence of 2 surface antigens (A, B) on red blood cells, there are 4 combinations of these antigens giving 4 different blood groups: A, B, AB,O Another surface antigen exist on human RBCS : Rh factor or D antigen. Accordingly, individuals are either Rh-positive or Rh- negative

10. Procedure A person’ s blood group is determined by mixing 3 separate drops of blood on a slide with anti A serum, antiB serum, antiD serum in each of 3 squares of the marked slide After mixing each square with separate toothpicks, look for agglutination

11. Interpretation The plasma contains antibody against the absent antigens. For example people with blood group A have antibodies to B in their plasma If agglutination occur with A antiserum, the blood group is A, If it occurs with B antiserum, the blood group is B, if it occurs with both antisera, Blood group is AB. The absence of agglutination with both antisera indicates that Blood group is O If agglutination occurs with anti- D antiserum, the person is Rh – positive, if not, the person is Rh - negative

13. Type of serological reaction: hemagglutination reaction Blood group is ……..

14. Complement fixation test (CF tests) Can be used for both antigen or antibody detection. Example for screening of syphilis Complement proteins are usually found in an inactive form. Once activated, they become involved in a chemical cascade It is the cell- lysing ability of activated complement components that is important in CF tests

15. Principle The complement fixation tests depend upon 2 distinct systems: 1- test system: involves Ag and Ab (one of which is known, the other is unknown)+ complement: If Ag and Ab are specific for one another, they will combine and fix the added complement 2- Indicator system: sensitized sheep RBCs is used as an indicator system to test for the presence of free complement. If the complement has been fixed by Ag-Ab complex, none will be available for lysis of sensitized sheep RBCs. If Ag and Ab are not specific for each other, the complement remains free to attach to sensitized sheep RBCs and lyse them Therefore: a positive complement fixation test gives no hemolysis, a negative test gives hemolysis

16. Positive complement fixationNegative complement fixation

17. Procedure 1- The patient serum is heated in a water bath at 56 C for 30 min to inactivate any complement present in serum 2- the patient serum is then serially diluted in wasserman tubes 2- A kn amount of antigen is added to each serum dilution 3- Fixed amount of complement is added to each tube Mixture is incubated for 1 hr at 37 C The indicator system is then added, incubated at 37C for 30 min Read for hemolysis Control tube is included in the experiment with no serially diluted antibody : control tube must show hemolysis

18. Results Positive complement fixation: all tubes show no hemolysis, except control tube Positive complement fixation with titer: results show non hemolysed tubes, then hemolysed tubes. Control tube is hemolysed Negative complement fixation: all tubes show hemolysis including control tube Anti complementary reaction: control tube shows no hemolysis, indicating that something wrong is present in the system, preventing complement from lysis the indicator system

19. Titer: reciprocal of the highest dilution (or least concentration) of antibody which give a detectable antigen antibody reaction For example: if the dilutions of ½, ¼, 1/8, 1/16 show no hemolysis, then dilution of 1/32 show hemolysis The titer will be 16