PREPARATION OF ANTIGEN

Heat killed / formalinized whole cell bacterin

1. Plate out the bacterial culture from appropriate stock culture onto a BHI agar plate,or other suitable medium. Incubate overnight at 37°C (±2°C).

2.Bacterial Growth is transferred to normal saline solution. Thoroughly mixed .

3.Expose to heat by heating the broth culture for 30 min at 100°C (Free Steam). OR Add Formalin (40% w/v) was added to the broth culture at a final concentration of 0.5% (V/V) and left 48 hrs at room temperature.

4.Centrifuge the killed bacterial suspension and resuspend the sediment in a small volume of normal saline. Repeat the step twice or thrice.

5.Adjust the opacity of suspension to the tube No.4 of Mac Farlands nephlelometer by adding required amount of normal saline.

6. The antigen thus prepared can be used for immunization  or testing antibodies against specific antigen by agglutination tests.

 Preparation of somatic antigen Salmonella typhi antigen-Widal Test

i) Plate out the Salmonella culture from the appropriate stock culture onto a BHI agar plate,or other suitable medium, for single colony growth. Incubate overnight at 37°C (±2°C).

ii) Using a Pasteur pipette, wash off the culture, preferably inside a safety cabinet, with approximately 2 ml of absolute alcohol, and transfer into a sterile universal container.

iii) Leave the antigen for 4–6 hours at room temperature to enable the alcohol to kill the bacteria and detach flagellae.

iv) Spin the universal container in a bench-top centrifuge for 5 minutes at 1000 g. Pour off the liquid and add enough phenol saline to make the antigen up to an opacity equivalent to               suspension with approximately 108 colony-forming units/ml.

v) Carry out standard titration with known serum to ensure that the antigen is positive for the required factor.

vi) Store in a refrigerator at 4°C until required.

 Preparation of sheep RBC suspension for raising haemolysin

  1. Collect 5 ml of blood using Alsever’s solution (Equal proportion) in a centrifuge tube.
  2. Centrifuge at 2000rpm for 6 minutes.
  3. Discard the supernatant plasma and buffy coat without disturbing the RBC.
  4. Add 5ml of Phosphate buffer saline solution to the RBC pack and centrifuge for 2 minutes at 1000rpm.
  5. Repeat step 4 for 2-3 times.
  6. Finally collect the packed cell volume of RBC and prepare the RBC suspensions as required 1%, 10% and 20% for immunization

(For preparing 10% RBC suspension : 1 ml of Packed volume of RBC should be added to 9 ml of Normal saline)

Preparation of Reagents and Standards:

McFarland’s standard

McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range to standardize microbial testing.  An example of such testing is antibiotic susceptibility testing by measurement of minimum inhibitory concentration which is routinely used in medical microbiology and research. If a suspension used is too heavy or too dilute, an erroneous result (either falsely resistant or falsely susceptible) for any given anti microbial agent could occur.

Original McFarland standards were mixing specified amounts of barium chloride and sulfuric acid together. Mixing the two compounds forms a barium sulfate precipitate, which causes turbidity in the solution. A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4).

 The standard can be compared visually to a suspension of bacteria in sterile saline or nutrient broth. If the bacterial suspension is too turbid, it can be diluted with more diluent. If the suspension is not turbid enough, more bacteria can be added.

 McFarland Nephelometer Standards

McFarland Standard No. 0.5 1 2 3 4
1.0% Barium chloride (ml) 0.05 0.1 0.2 0.3 0.4
1.0% Sulfuric acid (ml) 9.95 9.9 9.8 9.7 9.6
Approx. cell density (1X10^8 CFU/mL) 1.5 3.0 6.0 9.0 12.0
 % Transmittance* 74.3 55.6 35.6 26.4 21.5
Absorbance* 0.08 to 0.1 0.257 0.451 0.582 0.669

*at wavelength of 600 nm

 Preparation of Alsever’s Solution

Alsever’s solution is an isotonic, balanced salt solution. It is routinely used as an anticoagulant/ blood preservative, which permits the storage of whole blood at refrigerator temperatures for approximately 10 weeks.

For usage, an equal volume of blood is collected into a container of this product and gently, but thoroughly, mixed. The solution is placed in a refrigerator at 2-8 °C for 2 weeks. The container can then be removed and the plasma layer removed for use. 

Reagents

  • Citric acid C(OH)(COOH)(CH2.COOH)2.H2O
         0.055g
  • Sodium Citrate Na3C6H5O7.2H2O
         0.8g
  • D-Glucose C6H12O6
         2.05g
  • Sodium chloride NaCl
         0.42g
  • Distilled water to make up to 100 mL

 Method

  1. Weigh out reagents into a conical flask.
  2. Dissolve of distilled water and make up to100 mL.
  3. 3. Dispense into sterile 10 mL bottles.
  4. Sterilize by autoclaving at 116°C for 10 minutes. Use slow exhaust.
  5. Allow to cool, then tighten the lids and label the bottles.
  6. Store in the refrigerator. 

Anticoagulant

Acid Citrate Dextrose (ACD)

Reagents

  • Citric Acid C(OH)(COOH)(CH2.COOH)2.H2O
          4.0g
  • Sodium Citrate Na3C6H5O7.2H2O
         11.3g
  • D-Glucose C6H12O6
         11.0g

Method

  1. Weigh out reagents into a conical flask.
    2. Dissolve in 300 mL of distilled water.
    3. Make up to 500 mL with distilled water.
    4. Dispense into 100 mL bottles and put on lids. Do not tighten.
    5. Sterilize by autoclaving at 116°C for 10 minutes. Use a slow exhaust
    6. Allow to cool, then tighten the lids and label the bottles.
    7. Store in the refrigerator. 

Normal saline  0.9%  NaCl

Add 9g  NaCl  to 1000  ml  D/W.  Autoclave  at  15  lbs  for  15  minutes.

Phosphate-buffered saline (PBS)

Reagent Amount to add (for 1× solution) Final concentration (1×)
NaCl 8 g 137 mM
KCl 0.2 g 2.7 mM
Na2HPO4 1.44 g 10 mM
KH2PO4 0.24 g 1.8 mM

 To prepare 1 L of 1× PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.

 Exercise:

  1. Explain:    a. Antigen, b. Bacterin
  2. Enlist Bacterial Antigens.
  3. Name the bacterial antigen products in veterinary use.

References

Joseph Mcfarland,M.D. Jama. (1907).The nephelometer: An instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines.; XLIX(14):1176-1178.

Alsever, J.B., and Ainslie, R.(1941).N.Y. State J. Med.,41, 126.

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