4.3 Experiment A: Determination of urinary sugars using Benedict's solution, CLINISTIX™ and CLINITEST™ tablets

Procedure (label all tubes with a marker pen to avoid confusion):

1. First, you will set up a series of six standard glucose solutions of known concentration to mimic the test that can be done with CLINITEST™ tablets. Do NOT use the tablets at this stage (they are too expensive to "waste" on a set of standards). Using the 2% glucose solution and distilled water, set up a series of 6 test tubes containing respectively 2%, 1%, 0.75%. 0.5%. 0.25% and 0% glucose, each in a final volume of 200 µl.
2. Add 400µl Benedict's solution to each tube, mix, and place in a boiling water bath for exactly 90 seconds.
3. Remove and compare the colours in these tubes to the chart that comes with the CLINITEST™ tablets. How do your test and the commercial test compare for sensitivity?.
4. Now you will use the CLINITEST™ tablets to measure the reducing sugar content of the five urine samples. Place 5 drops of each of the five urines in the bottom of five separate wide glass test tubes using the dropper provided with the tablets (make sure you rinse the dropper between each urine sample to avoid cross-contamination). Next, add 10 drops of water to each tube.
5. Add one CLINITEST™ tablet to each tube and watch while the complete boiling reaction takes place. Do NOT shake the tubes during boiling or during the 15 seconds after boiling has stopped. Once 15 seconds have elapsed after boiling has finished, shake the tubes and compare the final colour to the colours on the chart provided with the tablets. Record the % reducing sugar in the Table below..
6. Finally, test fresh samples of each of the urines with the CLINISTIX™ strips. Dip the reagent end of the strip in a urine sample, remove, wait EXACTLY 10 seconds, then compare the colour to the chart on the side of the reagent strip bottle and record the result in the Table below.

1. Using 32 mM lithium acetoacetate and distilled water, set up a series of six test tubes containing respectively 32, 16, 8, 3.2, 1.6 and 0 mM acetoacetate, each in a final volume of 200 µl.
2. Add 200 µl colour reagent to each tube, mix and leave for 10 minutes.
3. Compare the colours in these tubes to the chart that comes with the KETOSTIX™. How do your test and the commercial test compare for sensitivity? (Remember that your acetoacetate concentrations will have been halved by mixing with an equal volume of colour reagent). This simple experiment illustrates the basis of KETOSTIX™.
4. Finally, measure the apparent ketone content of the five urine samples using the KETOSTIX™ reagent strips. Dip the reagent end of the strip in a urine sample, remove, wait EXACTLY 15 seconds, then compare the colour to the chart on the side of the reagent strip bottle and record the result in the Table below.

Procedure (label all tubes with a marker pen to avoid confusion):

1. Using 4% albumin and distilled water, set up a series of six test tubes containing respectively 4.0, 0.6, 0.2, 0.06, 0.02 and 0% albumin, each in a final volume of 200 µl. (Hint: to make the 0.06% and 0.02%, first make 200 µl of a 1 in 10 dilution of the 4% (becomes 0.4%) and use this, rather than the 4% albumin).
2. Add 200 µl tetrabromophenol blue solution to each tube and mix.
3. Compare the colours in these tubes to the chart that comes with the ALBUSTIX™. How do your test and the commercial test compare for sensitivity? (Remember that your albumin concentrations will have been halved by mixing with an equal volume of dye solution). This simple experiment illustrates the basis of ALBUSTIX™.
4. Following the instruction sheet enclosed with the ALBUSTIX™, measure the protein content of the urine samples. Note that you must wait 60 sec before reading the colour on the strip.

On the basis of the results of experiments A, B and C, which urine sample corresponds to which case?

This part of the practical will be described and demonstrated to you in groups.

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