Rothera’s test – Definition, Principle, Procedure, Result

What is Rothera’s test?

  • Rothera’s test is a specialized laboratory procedure designed for the qualitative identification of ketone bodies in urine. Ketone bodies are by-products of fat metabolism, comprising primarily of beta-hydroxybutyrate (78%), acetoacetic acid (20%), and acetone (2%).
  • Under typical physiological conditions, fats undergo complete metabolism, resulting in the production of water and carbon dioxide. Consequently, only a negligible amount of ketone bodies (approximately 1mg/24 hrs) are excreted in the urine.
  • However, certain conditions can disrupt this balance. For instance, during periods of starvation or in cases of uncontrolled diabetes, the liver synthesizes ketone bodies as an alternative energy source.
  • When the production rate of these ketone bodies surpasses the body’s ability to utilize or excrete them, the excess is eliminated through the urine, a condition termed as ketonuria. Notably, acetone, being volatile, can also be excreted via the breath.
  • Ketonuria is commonly associated with conditions such as starvation, diabetes mellitus, prolonged vomiting, severe diarrhea, and diets that are high in fats but low in carbohydrates. If left unchecked, an elevated concentration of ketone bodies can lead to metabolic acidosis, particularly in diabetic individuals.
  • For the detection of ketones in urine, several methods have been developed, including Gerhardt’s test, Lang’s test, Lindeman’s test, Han’s test, and the Tablet test. However, Rothera’s test stands out as a primary method. All these tests, in essence, are grounded on the principles of the Rothera’s nitroprusside test.
  • In summary, Rothera’s test serves as a crucial diagnostic tool in the identification of ketone bodies in urine, providing valuable insights into underlying metabolic and physiological disturbances.

Definition of Rothera’s test

Rothera’s test is a diagnostic laboratory procedure used for the qualitative detection of ketone bodies in urine, commonly employed to identify conditions like uncontrolled diabetes or starvation where increased fat metabolism leads to elevated ketone production.

Aim of Rothera’s Test

  • To detect the presence of Ketone bodies within the supplied urine sample.

Rothera’s Test Principle

Rothera’s Test Principle is grounded in the chemical reaction between acetoacetic acid and acetone with an alkaline solution of sodium nitroprusside. This interaction culminates in the formation of a distinct purple-colored complex. The test is sensitive enough to detect concentrations above 1-5 mg/dl for acetoacetic acid and 10-20 mg/dl for acetone. Notably, this method does not identify the presence of beta-hydroxybutyrate.


Requirement for Rothera’s Test

For the execution of Rothera’s Test, specific requirements are essential to ensure accurate results. These include:

  1. Urine Specimen: A sample of urine is the primary material tested to detect the presence of ketone bodies.
  2. Test Tubes: These are used to hold the urine sample and facilitate the reaction with the reagents.
  3. Rothera’s Powder: A specialized blend comprising:
    • Sodium Nitroprusside: Weighing 0.75 grams, it acts as a reactive agent in the test.
    • Ammonium Sulphate: Weighing 20 grams, it aids in the formation of the characteristic purple complex. The components are thoroughly mixed and pulverized to achieve a uniform consistency.
  4. Liquor Ammonia (Ammonium Hydroxide): This alkaline solution is pivotal in the reaction process, facilitating the interaction between the urine sample and Rothera’s powder.

It is imperative to have these components in the specified quantities and conditions to ensure the reliability and accuracy of the Rothera’s Test.


Procedure of Rothera’s Test

The Rothera’s Test is a systematic procedure designed to detect the presence of ketone bodies in urine. Here’s a detailed step-by-step procedure based on the provided content:

  1. Preparation: Begin by ensuring that the test tube is clean and sterile to avoid any contamination.
  2. Urine Sample: Carefully pour approximately 5ml of the urine sample into the prepared test tube.
  3. Addition of Rothera’s Powder: Introduce 1 gram of Rothera’s powder to the test tube containing the urine. It’s crucial to ensure that the powder is uniformly mixed with the urine to facilitate an accurate reaction.
  4. Incorporation of Ammonium Hydroxide: Subsequently, add 1 to 2 ml of concentrated ammonium hydroxide to the mixture. This will form a distinct layer over the urine sample.
  5. Observation: Pay close attention to the interface between the urine and the ammonium hydroxide layer. The appearance of a pink-purple ring at this juncture indicates the presence of ketone bodies in the urine.

This procedure, when followed meticulously, provides a reliable indication of ketonuria, aiding in the diagnosis of conditions like uncontrolled diabetes or starvation.


Results of Rothera’s Test

The results of Rothera’s Test are determined based on the appearance or absence of a specific colored ring at the interface of the urine and ammonium hydroxide layers. Here’s a comprehensive interpretation of the test outcomes:

  1. Positive Result:
    • Indicator: An immediate emergence of a purple permanganate-colored ring at the interface.
    • Interpretation: This denotes the presence of ketone bodies in the urine sample, confirming a positive result.
    • Grading: The intensity of the colored ring can be further graded to quantify the concentration of ketone bodies. The gradation is as follows:
      • Trace: Minimal presence of ketone bodies.
      • + (Plus): Low concentration.
      • ++ (Double Plus): Moderate concentration.
      • +++ (Triple Plus): High concentration.
      • ++++ (Quadruple Plus): Very high concentration.
  2. Negative Result:
    • Indicator: Absence of the purple permanganate-colored ring at the interface.
    • Interpretation: This signifies that the urine sample does not contain ketone bodies, leading to a negative result.

In essence, Rothera’s Test provides a clear and precise indication of the presence or absence of ketone bodies in urine, aiding in the diagnosis and monitoring of conditions like diabetes and starvation.

Rothera’s Test
Rothera’s Test

Safety precautions when performing Rothera Test

When conducting the Rothera Test, it is imperative to adhere to specific safety precautions to ensure the accuracy of results and prevent potential hazards. Here’s a detailed overview of the safety measures based on the provided content:

  1. Apparatus Sterilization: Prior to the test, ensure that all apparatus, especially those requiring sterility, are thoroughly cleaned and sterilized. This step is crucial to prevent contamination which might interfere with the test results.
  2. Chemical Handling: All chemicals, including reagents used in the test, should be handled with utmost care. Proper techniques should be employed to prevent spillage, breakage, or any other accidents that might compromise the safety of the experiment.
  3. Avoid Direct Contact with Urine: It is essential to avoid direct skin contact with the urine sample. Utilize appropriate tools and wear protective gloves when handling urine specimens to prevent potential contamination and ensure personal safety.
  4. Use of Test Tube Holders: When manipulating test tubes, especially those containing specimens or reactive chemicals, always use test tube holders. Direct handling with bare hands can pose risks, especially if the test tube is hot or contains hazardous substances.
  5. Proper Storage of Apparatus: After the completion of the test, all apparatus should be cleaned and stored in their designated places. This not only ensures the longevity of the equipment but also minimizes the risk of accidents in the laboratory.

In summary, meticulous adherence to these safety precautions is paramount to ensure the integrity of the Rothera Test and safeguard the well-being of those conducting it.



What is the primary purpose of Rothera’s Test?
a) To detect glucose in urine
b) To detect proteins in urine
c) To detect ketone bodies in urine
d) To detect blood in urine

Which of the following is NOT detected by Rothera’s Test?
a) Acetone
b) Acetoacetic acid
c) Beta-hydroxybutyrate
d) Glucose


What color indicates a positive Rothera’s Test?
a) Blue
b) Green
c) Pink-purple
d) Yellow

Which chemical is used in Rothera’s Test to form a colored complex with ketone bodies?
a) Sodium chloride
b) Sodium nitroprusside
c) Sodium bicarbonate
d) Sodium hydroxide

In which condition is ketonuria commonly observed?
a) Hypertension
b) Uncontrolled diabetes
c) Anemia
d) Dehydration

Which layer is formed on top during the Rothera’s Test procedure?
a) Urine layer
b) Acetone layer
c) Ammonium hydroxide layer
d) Water layer

What is the significance of a negative Rothera’s Test?
a) Presence of ketone bodies in urine
b) Absence of ketone bodies in urine
c) Presence of glucose in urine
d) Presence of proteins in urine

Which of the following is a component of Rothera’s powder?
a) Ammonium chloride
b) Ammonium sulphate
c) Ammonium carbonate
d) Ammonium nitrate

In which layer does the pink-purple ring appear in a positive Rothera’s Test?
a) At the bottom
b) In the middle
c) At the interface
d) Throughout the test tube

Which of the following conditions can lead to increased production of ketone bodies?
a) Overhydration
b) High carbohydrate diet
c) Starvation
d) High protein diet


What is Rothera’s Test used for?

Rothera’s Test is a qualitative laboratory test used to detect the presence of ketone bodies in urine.

Why is it important to detect ketone bodies in urine?

Detecting ketone bodies in urine can indicate conditions like uncontrolled diabetes or starvation, where the body starts breaking down fats for energy, leading to ketone production.

How is Rothera’s Test performed?

The test involves adding a specific amount of Rothera’s powder to a urine sample in a test tube, followed by layering with ammonium hydroxide. A positive result is indicated by the formation of a pink-purple ring at the interface.

What does a positive Rothera’s Test indicate?

A positive result, indicated by a pink-purple ring, means that ketone bodies are present in the urine.

Can Rothera’s Test detect all types of ketone bodies?

No, Rothera’s Test primarily detects acetoacetic acid and acetone. It does not detect beta-hydroxybutyrate.

Is Rothera’s Test specific to diabetes?

While the test is commonly used to detect ketonuria in diabetic patients, it can also indicate ketonuria due to other conditions like starvation or prolonged vomiting.

How accurate is Rothera’s Test?

Rothera’s Test is a qualitative test, meaning it can confirm the presence or absence of ketone bodies but does not quantify their concentration. It is considered reliable for detecting ketonuria.

Are there any other tests to detect ketone bodies in urine?

Yes, there are other tests like Gerhardt’s test, Lang’s test, and tablet tests. However, many of these are based on the principle of Rothera’s nitroprusside test.

What precautions should be taken while performing Rothera’s Test?

It’s essential to handle chemicals with care, avoid direct contact with urine samples, use sterilized apparatus, and always use test tube holders when handling test tubes.

Can I perform Rothera’s Test at home?

While there are home-based urine ketone test strips available, Rothera’s Test is typically performed in a laboratory setting due to the chemicals involved and the need for accurate interpretation of results.

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