PREPARATION OF ANTIBIOTIC STOCK SOLUTION

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Anantibiotic stock solutionis defined as a concentrated solution of an antimicrobial preparation that will be diluted to some lower concentrated solution for actual use. They are usually used to save preparation time, conserve materials, reduce storage space, and improve the accuracy with which working lower concentration solutions are prepared in the course of an experiment. Antibiotic stock solutions are concentrated solutions from which a working concentration (that is more diluted in form than the stock solution) can be obtained. The antibiotic stock solution of a particular antimicrobial agent is more concentrated than the actual working concentration of the same antibiotic. An antibiotic stock solution is prepared by weighing out an appropriate portion of a pure antibiotic powder by measuring out an appropriate volume of a pure liquid or diluent and diluting the drug to a known volume.

The antibiotic is usually ordered from the manufacturer as salt or powder; and it is this form that is used to get the stock solution of the drug through dilution. The dilution to be carried out usually depends on the required concentration unit of the antibiotic. For example, to prepare a solution with a desired molarity, weigh out an appropriate mass of the antibiotic, and dissolve it in a portion of its unique solvent; and then bring the preparation to the desired volume. Distilled water (dH2O) is commonly used for the dissolution of antibiotics especially when other solvents are not available (Table 1). Antibiotic stock solutions can be prepared by dilution methods – in which the antibiotic solutions are often prepared by diluting a more concentrated stock solution. In this technique, a known volume of the antibiotic stock solution is transferred to a new container or bottle and brought to a new volume at room temperature. Since the total amount of solute is the same before and after the dilution, it is known analytically that:

Ca × Va = Cb × Vb

Ca = is the stock solution’s concentration

Va = is the volume of stock solution being diluted

Cb = is the dilute solution’s concentration

Vb = is the volume of the dilute solution.

Note: The type of glassware used to measure Va and Vb depends on how exact the antibiotic solution’s concentration must be known.

Antibiotic powders must be accurately weighed and dissolved in the appropriate diluents to yield the required concentration, using sterile glassware such as conical flasks, beakers and test tubes (Table 1). The antimicrobial activity of the prepared antibiotic stock solution should be evaluated using standard strains of stock microbial cultures such as Escherichia coli ATCC 25922 as recommended by the American Type Culture Collection (ATCC).

Table 1: Stock solution and working concentration of different antibiotics

AntibioticStock solution (mg/ml)SolventWorking concentration (µg/ml)Dilution (µl/ml)
     
Ampicillin50*H20501.0
Carbenicillin50*H20501.0
Chloramphenicol35*EtOH or MetOH351.0
D-cycloserine100.1M *NaPO4 (pH 8.0)101.0
Erythromycin20EtOH201.0
Gentamicin10H2O10    1.0
Kanamycin50H2O501.0
Nalidixic acid30NaOH301.0
Neomycin10H2O800   80
Rifampicin50MetOH1002.0
Streptomycin100H2O1001.0
Tetracycline15EtOH151.0
* H2O = water; EtOH = ethyl alcohol; MetOH = methyl alcohol; NaPO4= sodium phosphate;

Antibiotic stock solutions found to be satisfactory after the evaluation can be aliquoted in 5 ml volumes and frozen or stored at -20ºC or at -60ºC in the dark. Expired antibiotic powders should not be used to make antibiotic stock solutions because such antibiotic powders may give inconsistent results. The antibiotic stock solutions to be prepared should be made to a final concentration of 10 mg/ml or 10 times the highest concentration to be tested and then diluted to an appropriate concentration in a broth medium. The manufacturer’s instruction or guideline should always be adhered to when preparing antibiotic stock solutions of antimicrobial agents or antibiotics. As aforementioned, distilled water or sterile water is generally used to prepare antibiotic stock solutions. However, some antibiotics including but not limited to streptomycin, erythromycin, nalidixic acid and chloramphenicol require some special solvents for preparation because they are insoluble in water. For antibiotics that are insoluble in water, minimal amount of the solvent (e.g. ethyl alcohol) is added to the antibiotic powder in a drop-wise manner until the antibiotic dissolves. Then, the final volume of the antibiotic solution is obtained using sterile water. To get the actual amount or concentration of the antibiotic required for the final (desired) volume of the antibiotic solution, the following formular is used:

Example 1. What is the amount of antibiotic powder required to prepare 3 ml of 10 mg/ml solution of drug “A” with 99.4 % potency (994 µg/mg)? The following solution applies:

A = 3 x 10000 / 994 30.2 mg

Thus, 30.2 mg of the antibiotic powder is required for the preparation of the antibiotic stock solution. In order words, weigh out 30.2 mg of the powder and dissolve it in 3 ml of sterile distilled water. An analytical weighing balance is best suitable for weighing out the antibiotic powder.

Antibiotic stock solutions are prepared using the following formula:

Where:

P = potency of the antibiotic base,

V = volume in ml required,

C = final concentration of solution and

W = weight of the antimicrobial to be dissolved in V.

FURTHER READING

Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India. 

Block S.S (2001). Disinfection, sterilization and preservation. 5th edition. Lippincott Williams & Wilkins, Philadelphia and London.

Courvalin P, Leclercq R and Rice L.B (2010). Antibiogram. ESKA Publishing, ASM Press, Canada.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Hugo & Russell’s Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA. Pp.152-172.

Ejikeugwu Chika, Iroha Ifeanyichukwu, Adikwu Michael and Esimone Charles (2013). Susceptibility and Detection of Extended Spectrum β-Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29.

Finch R.G, Greenwood D, Norrby R and Whitley R (2002). Antibiotic and chemotherapy, 8th edition. Churchill Livingstone, London and Edinburg.

Russell A.D and Chopra I (1996). Understanding antibacterial action and resistance. 2nd edition. Ellis Horwood Publishers, New York, USA.

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