Antibiotics Assays - Examples of Pharmaceutical Microbial Assays

EXAMPLES OF PHARMACEUTICAL MICROBIAL ASSAYS

The microbial assays have been effectively extended to a plethora of pharmaceutical preparations i.e., the secondary pharmaceutical products. However, this particular section will deal with only the following three types of such products, namely :

(1) Antibiotics,

(2) Vitamins, and

(3) Amino Acids.

Antibiotics Assays

The microbial assays of ‘antibiotics’ are usually carried out by two standard methods as per Indian Pharmacopoea (1996), namely :

Method A i.e., the ‘Cylinder-Plate Method’ as discussed in Section 10.1.3.1 and Section 10.3.1.

Method B i.e., the ‘Turbidimetric Method’ as described in Section 10.1.3.2.

A comprehensive account of the ‘Antibiotic Assays’ shall now be dealt with under the following sub-heads :

Standard Preparation and Units of Activity

Standard preparation may be defined as— ‘the authentic sample of the appropriate antibi-otic for which the potency has been precisely determined with reference to the appropriate inter-national standard’

However, the potency of the standard preparation may be duly expressed either in Interna-tional Units (IU) or in μg.mg^–1 with respect to the ‘pure antibiotic’.

Important Points

(1) Standard Preparation for India are adequately maintained at the Central Drugs Labora-tory, Kolkata. A unit referred to in the ‘official assays’ and ‘tests’ refers to the specific activity con-tained in such an amount of the respective standard preparation as is duly indicated by the Ministry of Health and Family Welfare, Government of India from time to time.

(2) Standard Preparation may be suitably replaced by a ‘working standard’ prepared by any laboratory that must be compared at definite intervals under varying conditions with the ‘standard’.

[A] Media. The media necessarily required for the preparation of ‘test organism inocula’ are duly made from the various ingredients as listed specifically in Table : 10.1. However, one may make minor modifications of the individual ingredients as and when required or ‘reconstituted dehydrated media’ may be employed provided the resulting media have either almost equal or definitely better growth-promoting characteristic features, and ultimately give a similar standard curve-response.

Method : Dissolve the various prescribed ingredients in sufficient distilled water (DW) to pro-duce 1L, and add sufficient 1M sodium hydroxide or 1M hydrochloric acid, as required so that after sterilization the pH must be as stated in Table : 10.1.

TABLE 10.1. Composition of Media : Quantities in g.L^–1

[B] Buffer Solutions : Prepare the buffer solutions by dissolving the quantities (see Table 10.2) of K₂HPO₄ and KH₂PO₄ in sufficient distilled water to produce 1L after adjusting the pH with 8 M . H₃PO₄ or 10 M.KOH. The buffer solutions are duly sterilized after prepares and the final pH specified in each case must be the one that is obtained after sterilization.

Table 10.2 : Buffer Solutions

Preparation of Standard Solution

In order to prepare a ‘Stock Solution’, dissolve a quantity of the Standard Preparation of a given antibiotic, weighed accurately and precisely, and dried previously as duly indicated in Table 10.3 in the solvent specified in the said Table ; and subsequently dilute to the required concentration as indicated specifically. It is advisable to store the ‘Stock Solution’ duly in a refrigerator (+ 1–5°C), and use within the stipulated period indicated.

On the particular day intended for carrying out the assay, prepare from the ‘Stock Solution’ at least five or even more test dilutions whereby the successive solutions increase stepwise in concentra-tion, invariably in the ratio 1 : 1.25 for method A or smaller for method B. Use the final diluent specified and a sequence in a such a manner that the middle or median should have the concentration as specified duly in Table 10.3.

Preparation of Sample Solution

Based on the available information for the ‘drug substance’ under investigation (i.e., the ‘un-known’) assign to it an assumed potency per unit weight or volume, and on this assumption prepare on the day of the assay a ‘Stock Solution’ and test dilution(s) as duly specified for each individual antibi-otic in Table 10.3, taking particular care to use the same final diluent as employed for the Standard Preparation. The assay with 5 levels of the Standard necessarily requires only one level of the ‘un-known’ at a concentration assumed very much equal to the ‘median level’ of the ‘Standard’.

TABLE 10.3 : Stock Solutions and Test Dilutions of Standard Preparations

For Amphotericin B, further dilute the stock solution with dimethylformamide to give concentrations of 12.8, 16, 20, 25 and 31.2 μg per ml prior to making the test solutions. The test dilution of the sample prepared from the solution of the substance being examined should contain the same amount of dimethylformamide as the test dilutions of the Standard Preparation.

For Bacitracin, each of the standard test dilutions should contain the same amount of hydrochloric acid as the test dilution of the sample.

For Nystatin, further dilute the stock solution with dimenthylformamide to give concentrations of 64.0, 80.0, 100.0, 125.0 and 156.0 μg per ml prior to making the test dilutions. Prepare the standard response line solutions simultaneously with dilutions of the sample being examined. The test dilution of the sample pre-pared from the solution of the substance being examined should contain the same amount of dimethylformamide as the test dilutions of the Standard Preparation. Protect the soultions from light.

When making the stock solution of Polymyxin B, add 2 ml of water for each 5 mg of the weighed Standard Preparation material.

Where indicated, dry about 100 mg of the Standard Preparation before use in an oven at a pressure not exceeding 0.7 kPa at 60° for 3 hours, except in the fine of Bleomycin (dry at 25° for 4 hours), Novobiocin (dry at 100° for 4 hours), Gentamicin (dry at 110° for 3 hours) and Nystatin (dry at 40° for 2 hours).

Where two-level factorial assays are performed use the following test doses per ml; Amphotericin B, 1.0 to 4.0 μg; Bacteracin, 1.0 to 4.0 Units; Kanamycin Sulphate, 5.0 to 20.0 units; Streptomycin, 5.0 to 20.0 μg.

[Adapted From : Indian Pharmacopoea. Vol. II, 1996] 10.6.1.4.

Test Organisms

The various test organisms for each antibiotic is duly listed in Table 10.4, along with its prop-erly documented identification number in the following recognized and approved compendia as :

·        American Type Culture Collection (ATCC)

·        National Collection of Type Cultures (NCTC)

·        National Collection of Industrial Bacteria (NCIB).

Usually maintain a ‘culture’ on the slants of the medium, and under the specified incubation conditions as mentioned duly in Table 10.5, and transfer weekly to fresh slants.

TABLE 10.4 : Test Organisms for Microbiological Assays of Antibiotics

·        American Type Culture Collection, 21301 Park Lawn Drive, Rockville, MD 20852, USA.

·        National Collection of Type Cultures, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, England.

·        National Collection of Industrial Bacteria, Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen 98 DC, Scotland.

[Adapted From : Indian Pharmacopoea, Vol. II, 1996]

TABLE 10.5 : Preparation of Inoculum

Methods of preparation of test organism suspension

1. Maintain the test organism on slants of Medium A and transfer to a fresh slant once a week. Incubate the slants at the temperature indicated above for 24 hours. Using 3 ml of saline solution, wash the organism from the agar slant onto a large agar surface of Medium A such as a Roux bottle containing 250 ml of agar. Incubate for 24 hours at the appropriate temperature. Wash the growth from the nutrient surface using 50 ml of saline solution. Store the test organism under refrigeration. Determine the dilution factor which will give 25% light transmission at about 530 nm. Determine the amount of suspensions to be added to each 100 ml of agar of nutrient broth by use of test plates or test broth. Store the suspension under refrigeration.

2. Proceed as described in Method 1 but incubate the Roux bottle for 5 days. Centrifuge and decant the supernatant liquid. Resuspend the sediment with 50 to 70 ml of saline solution and heat the suspension for 30 minutes at 70°. Wash the spore suspension three times with 50 to 70 ml of saline solution. Resuspend in 50 to 70 ml of saline solution and heat-shock again for 30 minutes. Use test plates to determine the amount of the suspension required for 100 ml of agar. Store the suspension under refrigeration.

3. Maintain the test organism on 10 ml agar slants of Medium G. Incubate at 32° to 35° for 24 hours. Inoculate 100 ml of nutrient broth. Incubate for 16 to 18 hours at 37° and proceed as described in Method 1.

4. Proceed as described in Method 1 but wash the growth from the nutrient surface using 50 ml of Medium 1 (prepared without agar) in place of saline solution.

[Adapted From : Indian Pharmacopoea, Vol. II, 1996]

Preparation of Inoculum

The method of preparation of the microbial suspensions for preparing the inoculum for the assay of various antibiotics is clearly stated in Table 10.5. In an event when the suspensions are duly prepared by these methods, one may accomplish and observe that the growth characteristic features are fairly uniform in order that the inoculum could be determined by carrying out the following trials.

For Method A. After the suspension is prepared, as given under Table 10.5, add different volumes of it to each of several different flasks containing 100 ml of the medium specified in Table 10.4 (the volume of suspension suggested in Table 10.4 may be used as a guide). Using these inocula, prepare inoculated plates as described for the specific antibiotic assay. While conducting cylin-der-plate assays, double layer plates may be prepared by pouring a seed layer (inoculated with the desired micro-organism) over a solidified uninoculated base layer. For each Petri dish, 21 ml of the base layer and 4 ml of the seed layer may be generally suitable. Fill each cylinder with the median concentra-tion of the antibiotic (Table 10.4) and then incubate the plates. After incubation, examine and measure the zones of inhibition. The volume of suspension that produces the optimum zones of inhibition with respect to both clarity and diameter determines the inoculum to be used for the assay.

For Method B. Proceed as descirbed for Method A and, using the several inocula, carry out the procedure as described for the specific antibiotic assay running only the high and low con-centrations of the standard response curve. After incubation, read the absorbances of the appropriate tubes. Determine which inoculum produces the best response between the low and high antibiotic con-centrations and use this inoculum for the assay.

Apparatus. All equipment is to be thoroughly cleaned before and after each use. Glassware for holding and transferring test organisms is sterilised by dry heat or by steam.

Temperature Control

Thermostatic control is required in several stages of a microbial assay, when culturing a micro-organisms and preparing its inoculum and during incubation in a plate assay. Closer control of the temperature is imperative during incubation in a tube assay which may be achieved by either circulated air or water, the greater heat capacity of water lending it some advantage over circulating air.

Spectrophotometer

Measuring transmittance within a fairly narrow frequency band requiers a suitable spectrophotometer in which the wavelength of the light source can be varied or restricted by the use of a 580 nm filter for preparing inocula of the required density, or with a 530 nm filter for reading the absorbance in a tube assay. For the latter purpose, the instrument may be arranged to accept the tube in which incubation takes place, to accept a modified cell fitted with a drain that facilitates rapid change of contents, or preferably fixed with a flow-through cell for a continuous flow-through analysis. Set the instrument at zero absorbance with clear, uninoculated broth prepared as specified for the particular antibiotic, including the same amount of test solution and formaldehyde as found in each sample.

Cylinder-Plate Assay Receptacles

Use rectangular glass trays or glass or plastic Petri dishes (approximately 20 × 100 mm) having covers of suitable material and assay cylinders made of glass, porcelain, aluminium or stainless steel with outside diameter 8 mm ± 0.1 mm, inside diameter 6 mm ± 0.1 mm and length 10 mm ± 0.1 mm. Instead of cylinders, holes 5 to 8 mm in diameter may be bored in the medium with a sterile borer, or paper discs of suitable quality paper may be used. Carefully clean the cylinders to remove all residues. An occasional acid-bath, e.g., with about 2M nitric acid or with chromic acid solution is needed.

Turbidimetric Assay Receptacles

For assay tubes, use glass or plastic test-tubes, e.g., 16 mm × 125 mm or 18 mm × 150 mm that are relatively uniform in length, diameter, and thickness and substantially free form surface blemishes and scratches. Cleanse thoroughly to remove all antibiotic residues and traces of cleaning solution and sterilise tubes that have been used previously before subsequent use.

Assay Designs

Microbial assays gain markedly in precision by the segregation of relatively large sources of potential error and bias through suitable experimental designs. In a cylinder-plate assay, the essential comparisons are restricted to relationships between zone diameter measurements within plates, exclu-sive of the variation between plates in their preparation and subsequent handling. To conduct a turbidimetric assay so that the difference in observed turbidity will reflect the differences in the antibi-otic concentration requires both greater uniformity in the environment created for the tubes through closer thermostatic control of the incubator and the avoidance of systematic bias by a random placement of replicate tubes in separate tube racks, each rack containing one complete set of treatments. The essential comparisons are then restricted to relationships between the observed turbidities within racks.

Within these restrictions, two alternative designs are recommended; i.e., a 3-level (or 2-level) factorial assay, or a 1-level assay with a standard curve. For a factorial assay, prepare solutions of 3 or 2 corresponding test dilutions for both the standard and the unknowns on the day of the assay, as described under Preparation of the Standard and Preparation of the Sample. For a 1-level assay with a standard curve, prepare instead solutions of five test dilutions of the standard and a solution of a single median test level of the unknown as described in the same sections. Consider an assay as preliminary if its computed potency with either design is less than 60% or more than 150% of that assumed in preparing the stock solution of the unknown. In such a case, adjust its assumed potency accordingly and repeat the assay.

Microbial determinations of potency are subject to inter-assay variables as well as intra-assay variables, so that two or more independent assays are required for a reliable estimate of the potency of a given assay preparation or unknown. Starting with separately prepared stock solutions and test dilutions of both the standard and the unknown, repeat the assay of a given unknown on a different day. If the estimated potency of the second assay differs significantly, as indicated by the calculated standard error, from that of the first, conduct one or more additional assays. The combined result of a series of smaller, independent assays spread over a number of days is a more reliable estimate of potency than that from a single large assay with the same total number of plates or tubes.

Methods.

The microbiological assay of antibiotics may be carried out by Method A or Method B.

[A] Cylinder-Plate or Cup-Plate Method

Inoculate a previously liquefied medium appropriate to the assay (Tables 10.1 and 10.3) with the requisite quantity of suspension of the micro-organisms, add the suspension to the medium at a tempera-ture between 40° and 50° and immediately pour the inoculated medium into Petri dishes or large rectan-gular plates to give a depth of 3 to 4 mm (1 to 2 mm for nystatin). Ensure that the layers of medium are uniform in thickness, by placing the dishes or plates on a level surface.

The prepared dishes or plates must be stored in a manner so as to ensure that no significant growth or death of the test organism occurs before the dishes or plates are used and that the surface of the agar layer is dry at the time of use.

Using the appropriate buffer solutions indicated in Tables 10.2 and 10.3, prepare solutions of known concentration of the Standard Preparation and solutions of the corresponding assumed concen-trations of the antibiotic to be examined. Where directions have been given in the individual monograph for preparing the solutions, these should be followed and further dilutions made with buffer solution as indicated in Table 10.3. Apply the solutions to the surface of the solid medium in sterile cylinders or in cavities prepared in the agar. The volume of soluiton added to each cylinder or cavity must be uniform and sufficient almost to fill the holes when these are used. When paper discs are used these should be sterilised by exposure of both sides under a sterilising lamp and then impregnated with the standard solutions or the test solutions and placed on the surface of the medium. When Petri dishes are used, arrange the solutions of the Standard Preparation and the antibiotic to be examined on each dish so that they alternate around the dish and so that the highest concentrations of standard and test preparations are not adjacent. When plates are used, place the solutions in a Latin square design, if the plate is a square, or if it is not, in a randomised block design. The same random design should not be used repeatedly.

Leave the dishes or plates standing for 1 to 4 hours at room temperature or at 4°, as appropriate, as a period of pre-incubation diffusion to minimise the effects of variation in time between the applica-tion of the different solutions. Incubate them for about 18 hours at the temperature indicated in Table 10.3. Accurately measure the diameters or areas of the circular inhibition zones and calculate the results.

Selection of the assay design should be based on the requirements stated in the individual mono-graph. Some of the usual assay designs are as follows.

[A.1] One-Level Assay with Standard Curve

Standard solution. Dissolve an accurately weighted quantity of the Standard Preparation of the antibiotic, previously dried where necessary, in the solvent specified in Table 10.3, and then dilute to the required concentration, as indicated, to give the stock solution. Store in a refrigerator and use within the period indicated. On the day of the assay, prepare from the stock solutions, 5 dilutions (solutions S₁ to S₅) representing five test levels of the standard and increasing stepwise in the ratio of 4 : 5. Use the dilution specified in Table 10.3 and a sequence such that the middle or median has the concentration given in the table.

Sample solution. From the information available for the antibiotic preparation which is being examined (the “unknown”) assign to it an assumed potency per unit weight or volume and on this assumption prepare on the day of the assay a stock solution with the same solvent as used for the standard. Prepare from this stock solution a dilution to a concentration equal to the median level of the standard to give the sample solution.

Method. For preparing the standard curve, use a total of 12 Petri dishes or plates to accommo-date 72 cylinders or cavities. A set of three plates (18 cylinders or cavities) is used for each dilution. On each of the three plates of a set fill alternate cylinders or cavities with solution S₃ (representing the median concentration of the standard solution) and each of the remaining 9 cylinders or cavities with one of the other 4 dilutions of the standard solution. Repeat the process for the other 3 dilutions of the standard solutions. For each unknown preparation use a set of three plates (18 cylinders or cavities) and fill alternate cylinders or cavities with the sample solution and each of the remaining 9 cylinders of cavities with solution S₃.

Incubate the plates for about 18 hours at the specified temperature and measure the diameters or the zones of inhibition.

Estimation of potency. Average the readings of solution S₃ and the readings of the concentra-tion tested on each set of three plates, and average also all 36 readings of solution S₃. The average of the 36 readings of soluiton S₃ is the correction point for the curve. Correct the average value obtained for each concentration (S₁, S₂, S₄ and S₅) to the figure it would be if the readings for solution S₃ for that set of three plates were the same as the correction point. Thus, in correcting the value obtained with any concentration, say S₁, if the average of 36 readings of S₃ is, for example, 18.0 mm and the average of the S₃ concentrations on one set of three plates is 17.8 mm, the correction is + 0.2 mm. If the average reading of S₁ is 16.0 mm, the corrected reading of S₁ is 16.2 mm. Plot these corrected values including the average of the 36 readings for solutions S₃ on two-cycle semilog paper, using the concentrations in Units or μg per ml (as the ordinate logarithmic scale) and the diameter of the zones of inhibition as the abscissa. Draw the straight response line either through these points by inspection or through the points plotted for highest and lowest zone diameters obtained by means of the following expressions :

L ₌ [ 3a + 2b + c – e] / 5 _{; H =} [ 3e + 2d + c – a] /5

where

L = the calculated zone diameter for the lowest concentration of the standard curve response line.

H = the calculated zone diameter for the highest concentration of the standard curve response line.

c = average zone diameter of 36 readings of the reference point standard solution.

a, b, d, e = corrected average values for the other standard solutions, lowest to highest concentrations, respectively.

Average the zone diameters for the sample solution and for solutions S₃ on the plates used for the sample soluiton. If the sample gives a large average zone size than the average of the standard (solution S₃), add the difference between them to the zone size of solution S ₃ of the standard response line. If the average sample zone size is smaller than the standard values, subtract the difference between them from the zone size of solution S₃ of the standard response line. From the response line read the concentration corresponding to these corrected values of zone sizes. From the dilution factors the potency of the sample may be calculated.

[A.2] Two-Level Factorial Assay

Prepare parallel dilutions containing 2 levels of both the standard (S₁ and S₂) and the unkown (U₁ and U₂). On each of four or more plates, fill each of its four cylinders or cavities with a different test dilution, alternating standard and unknown. Keep the plates at room temperature and measure the diam-eters of the zones of inhibition.

Estimation of potency. Sum the diameters of the zones of each dilution and calculate the % potency of the sample (in terms of the standard) from the following equation :

% potency = Antilog (2.0 + a log I)

wherein a may have a positive or negative value and should be used algebracially and

where a = (U₁ + U₂ ) – (S₁ + S₂ ) / (U₁ + U₂) + (S₁ – S₂)

U₁ and U₂ are the sums of the zone diameters with solutions of the unknown of high and low levels.

S₁ and S₂ are the sums of the zone diameters with solutions of the standard of high and low levels.

I = ratio of dilutions.

If the potency of the sample is lower than 60% or greater than 150% of the standard, the assay is invalid and should be repeated using higher or lower dilutions of the same solutions. The potency of the sample may be calculated from the expression.

[ % potency × assumed potency of the sample ] / 100

[A.3] Other Designs

(1) Factorial assay containing parallel dilution of three test levels of standard and the unknown.

(2) Factorial assay using two test levels of standard and two test levels of two different un- knowns.

[B] Turbidimetric or Tube Assay Method

The method has the advantage of a shorter incubation period for the growth of the test organism (usually 3 to 4 hours) but the presence of solvent residues or other inhibitory substances affects this assay more than the cylinder-plate assay and care should be taken to ensure freedom from such sub-stances in the final test solutions. This method is not recommended for cloudy or turbid preparations.

Prepare five different concentrations of the standard solution for preparing the standard curve by diluting the stock solution of the Standard Preparation of the antibiotic (Table 10.3) and increasing stepwise in the ratio 4 : 5. Select the median concentration (Table 10.3) and dilute the solution of the substance being examined (unknown) to obtain approximately this concentration. Place 1 mL of each concentration of the standard solution and of the sample solution in each of the tubes in duplicate. To each tube add 9 ml of nutrient medium (Table 10.3) previously seeded with the appropriate test organ-ism (Table 10.3).

At the same time prepare three control tubes, one containing the inoculated culture medium (cul-ture control), another identical with it but treated immediately with 0.5 mL of dilute formaldehyde solu-tion (blank) and a third containing uninoculated culture medium.

Place all the tubes, randomly distributed or in a randomized block arrangement, in an incubator or a water-bath and maintain them at the specified temperature (Table 10.3) for 3 to 4 hours. After incubation add 0.5 mL of dilute formaldehyde solution to each tube. Measure the growth of the test organism by determining the absorbance at about 530 nm of each of the solutions in the tubes against the blank.

Estimation of potency. Plot the average absorbances for each concentration of the standard on semi-logarithmic paper with the absorbances on the arithmetic scale and concentrations on the logarith-mic scale. Construct the best straight response line through the points either by inspection or by means of the following expressions :

where

L = the calculated absorbance for the lowest concentration of the standard response line.

H = the calculated absorbance for the highest concentration of the standard response line.

a, b, c, d, e = average absorbance values for each concentration of the standard response line lowest to highest respectively.

Plot the values obtained for L and H and connect the points. Average the absorbances for the sample and read the antibiotic concentration from the standard response line. Multiply the concentration by the appropriate dilution factors to obtain the antibiotic content of the sample.

Precision of Microbiological Assays

The fiducial limits of error of the estimated potency should be not less than 95% and not more than 105% of the estimated potency unless otherwise stated in the individual monograph. This degree of precision is the minimum acceptable for determining that the final product complies with the official requirements and may be inadequate for those deciding, for example, the potency which should be stated on the label or used as the basis for calculating the quantity of an antibiotic to be incorporated in a preparation. In such circumstances, assays of greater precision may be desirable with, for instance, fiducial limits of error of the order of 98% to 102%. With this degree of precision, the lower fiducial limit lies close to the estimated potency. By using this limit, instead of the estimated potency, to assign a potency to the antibiotic either for labelling or for calculating the quantity to be included in a prepara tion, there is less likelihood of the final preparation subsequently failing to comply with the official requirements for potency.