MICROBIAL ASSAY OF ANTIBIOTICS
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.
Thermostatic control is required at several stages of a microbial assay, when culturing a microorganism 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.
Measuring transmittance within a fairly narrow frequency band requires 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 a 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 x 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 6mm ± 0.1mm 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 cylinder to remove all residues. An occasional acidbath, e.g. with about 2 M 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 x 125 mm or 18 mm x 150 mm that are relatively uniform in length, diameter, and thickness
and substantially free from 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.
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, exclusive 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 antibiotic 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 samples. 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 per cent or more than 150 per cent 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 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.
Carry out the microbiological assay by Method A or Method B.
A. Cylinder-plate or Cup-plate method
Inoculate a previously liquefied medium appropriate to the assay (Tables 1 and 3) with the requisite quantity of suspension of the micro organism, add the suspension to the medium at a temperature between 40º and 50º and immediately pour the inoculated medium into the petri dishes or large rectangular plates to give a depth of 3 to 4 mm (1 to 2mm for nystatin). Ensure that the layers of medium are uniform in thickness, by placing the dishes or plates on a level surface.
Store the prepared dishes or plates 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 2 and 3, prepare solutions of known concentrations of the standard preparation and solutions of the corresponding assumed of concentrations 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 3. Apply the solutions to the surface of the solid medium in sterile cylinders or in cavities prepared in the agar. The volume of solution 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 sterilized 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 under examination 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 4º, as appropriate, as a period of preincubation diffusion to minimise the effects of variation in time between the application of the different solutions. Incubate them for about 18 hours at the temperature indicated in Table 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 monograph. Some of the usual assay designs are as follows.
(a) One-level assay with standard curve
Standard Solution. Dissolve an accurately weighed quantity of the Standard Preparation of the antibiotic, previously dried where necessary, in the solvent specified in Table 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 solution, 5 dilution (solutions S1 to S5) representing 5 test levels of the standard and increasing stepwise in the ratio of 4:5. Use the diluent specified in Table 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 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 accommodate 72 cylinders or cavities. A set of 3 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 solution. For each unknown preparation use a set of 3 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 concentration tested on each sets of three plates, and average also all 36 readings of solution S3. The average of the 36 readings of solution 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:
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 solution. If 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 S3 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.
(b) Two-level factorial assay
Prepare parallel dilutions containing 2 levels of both the standard (S1 and S2) and the unknown (U1and 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 diameters of the zones of inhibition.
Estimation of potency. Sum the diameters of the zones of each dilution and calculate the percentage potency of the sample (in terms of the standard) from the following equation :
Per cent potency = Antilog (2.0 + a log I
where in a may have a positive or negative value and should be used algebraically and
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 per cent or greater that 150 per cent of the standard, the assay is invalid and should be repeated using higher or lower dilutions of the same solution. The potency of the sample may be calculated from the expression.
(c) 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 unknowns.
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 plates assay and care should be taken to ensure freedom from such substances 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 3) and increasing stepwise in the ration 4:5. Select the median concentration (Table 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 3) previously seeded with the appropriate test organism (Table 3).
At the same time prepare three control tubes, one containing the inoculated culture medium (culture control), another identical with it but treated immediately with 0.5 ml of diluteformaldehyde solution (blank) and a third containing uninoculated culture medium.
Place all the tubes, randomly distributed or in a randomized block arrangement, in an incubator or water-bath and maintain them at the specified temperature (Table 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 (2.4.7).
Estimation of potency. Plot the average absorbances for each absorbances on the arithmetic scale and concentrations on the logarithmic 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.
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