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Methods for Standardization of Antibiotics, Vitamins and Amino Acids

A prescription antibiotic consists of a stable dose of a chemical compound, which is produced either naturally or artificially.

Methods of Standardization of Antibiotics

A prescription antibiotic consists of a stable dose of a chemical compound, which is produced either naturally (by a microorganism) or artificially (by a semi-synthetic process), and which has the ability to suppress (bactericidal) or destroy (bacteriostasis) microorganisms in relatively dilute solutions.

The three main points for standardizing antibiotics are as follows:
  • Regulations governing all aspects of antibiotic testing are in detail and modified periodically by the FDA (Food and Drug Administration).
  • To determine the methods for determining the safety and effectiveness of specific antibiotics, FDA regulations need to be consulted.
  • The actual and apparent measurement of the potency of antibiotics is based on the ability of the substance to inhibit growth of a suitable strain of microorganisms, or the ability to prevent their multiplication.
To measure turbidity, two methods are used: the cup-plate method and the cylinder plate method. By using a vertical cylinder, the antibiotic diffuses through a solid layer of agar in the Petri dish. Adding the microorganism to a cylinder containing an antibiotic solution completely prevents it from growing. In the turbidimetric method, a microbial culture is inhibited from growing in an antibiotic solution in fluid medium that would otherwise be conducive to its rapid growth without the antibiotic.

Methods of preparation

Insufficient water must be used to dissolve all ingredients and 1 M Sodium Hydroxide or 1 M Hydrochloride acid must be added to achieve a pH of between 6.5 and 7.5 after sterilization.

Buffer solution preparation

In a 1000 ml solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, 8 M phosphoric acid or 10 M potassium hydroxide is added to adjust the pH to neutral to achieve a neutral solution.

Standard preparation

As standard preparation, we use an authentic sample of an antibiotic that has been examined against the relevant international standard in order to determine its potency. As standard preparation, the potency is expressed as g per mg of a pure antibiotic international unit.

In a stock solution, the standard preparation of an antibiotic is dissolved in an amount that matches the amount of weighed quantity.

Preparation of the sample

In the preparation of diluted samples, the sample or unknown samples are diluted according to ranges of diluted standards.

Methods of standardization of vitamins

A microbiological vitamin assay uses microorganisms to conduct the test. Growth of microorganisms depends on the presence of vitamins and amino acids. Under proper nutritional conditions, the aim of this essay is to measure the ability of an organism to utilize a substance. The following table provides some examples of microorganisms used as bioassays for vitamins

Material required

Inoculum media, stock solutions, and assay media are needed. We then generate standard curves.

Assay of vitamin B12

Vitamin B12 is also known as cyanocobalamin. Water-soluble vitamins are essential for healthy living. Water-soluble vitamins are essential for healthy living. Macrolytic anemia and Pernicious anemia are caused by B12 deficiency.


Lactobacillus liechmannii is selected as the test organism because it can use free cyanocobalamin. Assays are conducted either by titrimetry or turbidimetry.

Preparation of standard stock solution

A precise weight of Cyanocobalamin reference standard should be weighed and the amount of cyanocobalamin added to enough 25% ethanol (resulting in a solution containing 1.0 g of cyanocobalamin per mL) needs to be added before the solution is stored in a refrigerator. In addition to this stock solution, you can dilute it by adding 1 ml to 99 ml purified water (1 ml is equal to 10 ng) and another 1 ml to 199 ml purified water (1 ml is equal to 0.05 ng).

Test solution

To measure vitamins, an accurate amount of each is dissolved in water. Using diluted HCl or NaOH, the pH is adjusted to 6.0, followed by adding water to reach the desired pH.

Inoculum preparation

Put 10 ml of sterile culture medium in each of two tubes with a loop of Lactobacillus liechmannii derived from a recent sub-culture.

Composition of culture media

For 18 to 24 hours, the culture is incubated at 37 degrees Celsius. A centrifuge is used to separate the supernatant fluid from the incubated culture under aseptic conditions. To suspend the cultured cells in Basal medium stock solution for 10 ml, centrifuge and decant off the supernatant fluid, then centrifuge again. One ml of the uniform cell suspension is transferred to sterile medium and mixed aseptically with 10 ml of the medium. Inoculums are prepared by taking this resulting cell suspension.

Assay procedure - The following ten test tubes should be cleaned, and cyanocobalamin solutions added in 0, 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5 ml. Water is added to the 5 ml of Basal medium solution in each tube to make the volume equal to 10 ml. Four more test tubes should be filled with 1, 2, 3, 4 ml of the test solution to be tested. For each of these, add 5 ml of Basal medium stock solution, add 10 ml of water, and mix. For all test tubes, an autoclave at 121 °C for 15 minutes is used to sterilize them, and then the tubes are cooled at room temperature. The test tubes were incubated for 64 to 72 hours at 30 to 37° C with an inoculum prepared with Lactobacillus liechmannii. Using bromothymol blue as an indicator, titrated each test tube with 0.05 N NaOH until a green color appeared. Take the readings and calculate them.

0.05 N NaOH titration values (in ml) of cyanocobalamin standard solution are used to construct a graph plotting the average titration values against the concentration of standard solution at each level. To calculate the concentration of vitamin B12 as activity per ml, the standard curve is interpolated to produce a line graph. A graph can provide information about cyanocobalamin concentrations.

Methods of standardization of amino acids

  • Standard conditions are required for a microbiological assay to determine the degree of activity of a compound based on the amount required to develop the predicted effect.
  • Microbiological analyses take a lot of time and are not applicable to all amino acids.
  • Some microorganisms need amino acids to grow and reproduce.
  • These microbes rely on specific amino acids in many strains.
  • The growth of such microorganisms will be limited if a small amount of an amino acid is supplied, as measured by turbidimetry, or if only a small amount of pH levels are increased by microtitration.
  • Guithrie and Susi developed a modified version of the microbiological test using diffusion in gels to screen blood samples for the presence of elevated phenylalanine levels in clinical biochemistry laboratories.
  • It is named after its inventors as the Guthrie test, and it is one of the most commonly used microbiological methods to evaluate amino acids.
  • In the assay, phenylalanine is evaluated by comparing its ability to inhibit Bacillus subtilis growth against the chemical competitor, β-2- theienylalanine.
  • This test makes use of an agar plate, onto which suspensions of B.subtilis spores are added, along with growth nutrients and an additional amount of β-2 thienylalanine for supplementation.
  • In addition to the filter paper soaked in blood, phenylalanine standards are also soaked in blood and placed on the agar surface. They are then incubated overnight at 37°C.
  • When phenylalanine concentrations in blood discs are high enough, β-2 thienylalanine can't prevent bacterial growth.
  • The discs themselves show growth zones around them.
  • We measure the circumference of each zone of growth the next day, and correlate this with phenylalanine levels.
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Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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