Veterinary Clinical Medicine-I (General and Systemic)

MICROBIAL TESTS

Protozoa

Density

• ++++ Vigorous
• +++ Abundant - More than 30 Protozoa/low power field (100 X)
• ++ Moderate - 10-30 Protozoa /L.P.F.
• + Few - 1-10 Protozoa / L.P.F.
• - None -
• More the density of protozoa, more active is the rumen fluid.
• Simple inactivity of flora and faune -/+
• Rumen decomposition/alkalosis -/+
• Acute rumen acidosis -

Proportion of large, medium and small infusoria

• Large and medium ciliates in
• Large numbers - Active rumen fluid
• Only small ciliates - Forestomach indigestion

Proportion of dead to live protozoa

• Severe digestive disorders like
  • Rumen acidosis (pH less than 5) - Entire microfauna dead
  • Moderate digestive disorders - Proportion of dead to live increase.

lodophilic acitivity

• Add a drop of Lugol’s lodine to 2-3 drops of rumen fluid on a glass slide and place a cover slip on it and examine under the microscope. Lodophilic activity of protozoa can be recognized by black colouration of starch contents in the protozoa. It can be graded as 0, +, ++, +++ depending on quantity of starch contained. The quantum of starch content of protozoa reflect the degree of digestive disease.

Total protozoal count

• 5 ml of strained rumen liquor (S.R.L.) is diluted to 20 ml with 10% formal Saline. From this, 10 ml of the mixed rumen liquor is taken and 10 drops of 2% Eosin is added to colour the protozoa. The diluted rumen liquor is charged in a Haemocytometer with Neubar ruling. 8 sq.m.m. is counted and is its average is multiplied by 50,000. The result is expressed as total counts per ml (n X 10⁵).

Normal counts

• Mixed ration - 1 X 10⁵ / ml.
• Concentrate ration - 1 X 10⁶ /ml.
• In - Decreased or Nil
• Bloat - Decreased total count, disappearance of Holotrichs
• Acidosis - Nil; if present, only few Entodinia sp.

N. Bacteria

• An air dried smear of rumen fluid is stained by Gram’s method and observed under the microscope.
• Gram positive organism - Violet colour
• Gram negative organism - Red or pink Colour
• Interpretation
  • Basic criteria for interpretation are
    • Presence or absence of morphologically distinguishable bacterial species characteristic of a normal rumen flora - the so called leading bacteria.
    • Multiplicity or uniformity of forms.
    • The ratio of gram positive to gram negative bacteria.
    • Comparison of the smear from the sick animal with that of the healthy animal receiving the same ration.
  • Normal: In the normal pH of rumen fluid – Gram negative bacteria is dominant.
  • Rumen lactacidosis - Proliferation of gram positive cocci and rods at the
  • Expense of gram negative bacteria.
  • Hydrochloric acidosis - Gram positive less than Gram negative bacteria.
• Total bacterial counts
  • Add 5 ml of strained rumen liquor (SRL) and dilute to 25 ml with 10% formal saline. Take 1 ml of this fluid and is again diluted 100 times with 0.85% saline. 0.01 ml of this sample is spread over an area of one square centimeter making use of the guide plate. After the smear has dried, it is stained with Newman’s stain. Atleast 30 fields should be counted in each sear and the average count is to e multiplied y the dilution factor and microscopic factor. The result is expressed as total counts per ml (X x 10⁸).
  • Significance: Normal 10⁷ to 10¹²/ml.

Diagnosis of toxicity by rumen fluid examination nitrie/nitrate toxicity

• Modified diphenylamine test
  • A drop of the test solution (0.5g of diphenylamine, 20 ml distilled water and concentrated sulphuric acid sufficient to make 100 ml) is mixed with a drop of rumen fluid. An intense blue colour within 10 seconds indicates a concentration of greater than 1% nitrate which is more than the permissible level.

Hydrocyanic acid toxicity

• Reagent papers are prepared by mixing 0.5g of picric acid, and 5g sodium carbonate in 100 ml water. Filter papers are dipped in the reagent and allowed to dry in a dark place. A drop of rumen fluid is placed on the test paper. A red discolouration is a positive reaction. The test is designed only to detect free hydrocyanic acid. It may not be positive when cyanides are present in different forms.