Page 379 - The Veterinary Laboratory and Field Manual 3rd Edition

348 Susan C. Cork, Willy Schauwers and Roy Halliwell

Table 7.5 Interpretation of the bLank dILuEnt rEaGEnt
bilirubin test. Potassium sodium tartrate 9.0 g
Potassium iodide 5.0 g
Green or blue colour Time Bilirubin
(min) (mg/100 ml)* Sodium hydroxide 0.2 M to 1000 ml.
-No green/blue colour 5 <0.2
+ slight colour 5 0.2–0.5 To prepare 0.85% saline solution, dissolve 8.5 g
++ moderate colour 5 0.5–0.8 NaCl in 1 l of distilled water. Prepare the blank
change** reagent in a similar manner to that outlined for
+++ Strong colour 5 (1) 0.8–1.4 the Biuret reagent.
change
++++ Strong*** 1 1.4–2.0 MEtHod
• Pipette reagents out as outlined in Table 7.6.
+++++ Intense 1 > 2.0
Mix the contents of each of the tubes and
Notes: *Values may also be expressed in µmol/l (x 17.1) see place in a water bath at 37°C for 15 min.
Tables 7.2 and 7.3. **A reaction of ++ or +++ would require • Using a colorimeter set at a wavelength
further investigation. ***Results in the range of (++++/+++++)
would be considered high. of 530 or 565 nm, measure the OD of the
samples against a reagent blank. The blank
is used to ‘zero’ the machine before sample
Serum total protein by the Biuret ODs are read.
method • Calculation – The protein concentration:

PrIncIPLE OD value of the sample × conc. of the stan-
A violet colour complex is formed by the reac- dard used / OD value of the standard (that
tion of serum proteins and peptides with a is, 50 g/l)
copper sulphate (Biuret) reagent. Other non-
protein nitrogen complexes such as creatinine
and urea do not react. IntErPrEtatIon
See Table 7.3 for normal serum protein values.
bIurEt rEaGEnt
Potassium sodium tartrate 45.0 g (159.48 Haemoglobin determination

mmol)
There are a number of methods available to deter-
Cupric sulphate 0.5 H O 15.0 g (60.08 mine haemoglobin levels in blood samples but most
2
mmol) are based on the principle of comparing a colour
reaction in the test sample against that of a control
Potassium iodide 5.0 g (30.12 mmol)
reagent which has a known haemoglobin level (see
Add sodium hydroxide 0.2 M (200 mmol/l) Chapter 5). To a large extent the reliability of the
to make 1000 ml. methods depends on the stability of the coloured
samples being measured. Cyanmethaemoglobin,
Dissolve the tartrate in approximately 400 ml used in the Drabkin method, is more stable but has
of 0.2 M NaOH; Add, while stirring, the cupric the disadvantage of being a toxic chemical. There
sulphate: continue to stir until in solution. Add are also a number of kit tests now available for Hb
the potassium iodide and when dissolved make determination (see also Chapter 2). The alkaline
up to 1000 ml (1 l) with 0.2 M NaOH. haematin method is outlined below.

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