ASSAY OF ANTIBIOTICS BY TURBIDIMETRIC (OR NEPHELOMETRIC) METHOD

A large number of antibioics, namely : chlortetracycline, doxycyline, gentamicin, neomycin,
streptomycin, tobramycin and the like may be assayed tubidimetrically with fairly good accuracy.
 Assay of Chlorotetracycline
Theory. Inoculate a medium consisting of : peptone : 6 g, beef extract : 1.5 g, yeast extract : 3 g,
sodium chloride : 3.5 g, D-glucose monohydrate : 1.0 g, dipotassium hydrogen orthophosphate : 3.68 g,
potassium hydrogen orthophosphate : 1.32 g and dissolve in sufficient water to produce 1 L with a
known quantity of a suspension of Staphylococcus aureus (NCTC 6571**) so as to obtain a readily
measured opacity after an incubation of about 4 hours. The micro-organisms must exhibit a sensitivity to
the antibiotic under investigation to such an extent that a sufficiently large inhibition of growth takes
place in the prevailing conditions of the test.
In actual practice, it is always advisable that the inoculated medium should be used immediately
after its preparation. Using a phosphate buffer of pH 4.5 (dissolve 13.61 g of KH2 PO4 in about 750 ml
of water, adjusting the pH to 4.5 with 0.1 M NaOH and diluting to 1 L with water), prepare solutions of
the Standard Preparation and the substance under investigation at concentrations presumed to be equal.
To enable the validity of the assay to be examined, it is desirable to use at least three doses of the
Standard Preparation and of the substance being examined. It is also advisable to use doses in logarithmic
progression in a parallel line assay.
Materials Required : Standard chlortertracyline ; sterilized media (as described above) : 1 L ;
authentic and pure strain of microorganism Staphylococcus aureus (NCTC 6571) ; formaldehyde solution
(34–37% w/v) 10 mL ; matched identical test tubes : 20 ;
Procedure : Distribute into identical test-tubes an equal volume of standard tetracycline solution
and the sample to be examined (having presumed equal concentrations) and add to each tube an equal
volume of inoculated nutrient medium (for instance 1 mL of the solution and 9 ml of the medium). Prepare
at the same time two control tubes without the chlorotetracycline, one containing the inoculated medium
and the other identical with it but treated immediately with 0.5 mL of formaldehyde solution. These tubes
are used to set the optical apparatus employed to measure the growth.
Place all the tubes, randomly distributed, in a water-bath or other suitable means of bringing all the
tubes rapidly to 35–37°C i.e., the incubation temperature and maintain them at that temperature for 3 to 4
hours, taking due precautions to ensure uniformity of temperatures and identical incubation times. After
incubation, stop the growth of the microorganisms by adding 0.5 mL of formaldehyde solution, each tube
and subsequently measures the opacity to at least three significant figures using a suitable

optical apparatus. From the results calculate the potency of the substance being examined i.e.,
chlortetracycline by standard statistical methods.*
Note. (a) Rectilinearity** of the dose-response relationship, transformed or untransformed, is often
obtained only over a very limited range. It is this range that must be used in calculating
the activity and it must include at least three consecutive doses in order to permit
rectilinearity to be verified,
(b) Use in each assay the number of replications per dose sufficient to ensure the required
precision. The assay may be repeated and the results combined statistically to obtain the
required precision and to ascertain whether the potency of the antibiotic being examined
is not less than the minimum required.
 Cognate Assays
A few other official antibiotics in BP (1993) may also be assayed by adopting the method stated
above, but using specific micro-organism, definite final pH of the medium, pH of the phosphate buffer,
potency of solution (U per ml) an the incubation temperature. A few typical examples

Assay of Vitamins
As or late the judicious exploitation of various microorganisms as dependable and reliable
‘analystical tools’ in a well organized Quality Assurance Laboratory (QAL) for the precise determination
of a plethora of Vitamins and amino acids.
Merit of Microbial Assays. There are several well-known merits of microbial assays as enumerated
under :

(1) These are as precise and accurate as the ‘chemical methods’.
(2) These are invariably quite simple, convenient, not-so-cumbersome, and above all definitely
inexpensive.
(3) A very small quantum of the ‘sample’ is required for the recommended microbial assay.
(4) They hardly need any elaborated instrumentation.
(5) These microbial assays do require the following essential criteria, such as :
• ascertains continuous checks for consistency of results,
• ensures specificity, and
• prevents any possible interferences.
(6) Automation of microbial assays may essentially overcome any possible limitations, accuracy
of observations, and the sample-handling capacity to a significant extent.
Example : In an ‘automatic photometric assay’ the following activities do take place in a
sequential manner, namely :
􀁑 measures the exact quantum of antibiotic present in a given solution,
􀁑 incorporates requisite quantum of inoculum and nutrient medium,
􀁑 incubates the resulting mixture for 100 minutes,
􀁑 transfers the incubated mixture to photometer cell, and
􀁑 results are adequately read and recorded.
Principle. It has been amply proved and established that there are some specific microbes which
predominantly require vitamin (factor) for their usual normal growth phenomenon ; and, therefore, are
quite sensitive to the extremely small quantities of the much desired ‘factor’. Nevertheless, it is precisely
the critical inherent ability of these particular microorganisms (i.e., the ‘test organisms’) to carry
out the synthesis of the ‘factor’ being determined. This ultimately gives rise to the fundamental basis of
the microbial assay of vitamins. To accomplish the ultimate objective, the ‘test organism’ is duly
inoculated in the highly specialized culture media that are essentially complete in every possible respects
except the presence of the ‘factor’ under investigative study. In reality, it evidently caters for the
‘control’ wherein either little or almost minimal growth of microbes is duly exhibited. Importantly, in
another set of parallel/identical experiments, one may incorporate meticulously the ‘graded quantities
of factor’ the thus the ultimate growth of the test organism (i.e., response) is observed adequately.
However, one may observe invariably that the ‘response’ (i.e., growth of the ‘test organism’) is directly
proportional to the ‘factor’ (i.e.,quantum of the dose) actually incorporated to the culture medium.
Microbial assays of the following three water-soluble vitamins would be discussed individually
in the sections that follows :
(a) Calcium Pantothenate,
(b) Niacin (or Niacinamide), and
(c) Vitamin B12 (or Cyanocobalamin).
Calcium Pantothenate
It refers to one of the B complex vitamins (or vitamin B complex). The various steps involved
for the assay are enumerated under sequentially :
(1) Reagents. The various reagents essentially required for the assay of ‘calcium pantothenate’
are :

(a) Standardized Stock Solution. Each mL of this stock solution consists of 50 mcg of calcium
panthothenate. It may be prepared by carefully dissolving 50 mg of BPCRS* calcium pantothenate
in 500 mL of double-distilled water ; 10 mL of 0.2 M acetic acid, 100 mL of 1.6% (w/v) sodium acetate ;
and volume made upto 1 L with DW.
Note : The resulting solution must be stored under a layer of ‘toluene’ in a refrigerator.
(b) Standard Solution. The standard solution should contain approximately 0.04 mcg of calcium
pantothenate in 1 mL, and is duly prepared by diluting the Standard Stock Solution (a).
(c) Test Solution. The test solution essentially contains nearly the same equivalent amount of
calcium pantothenate as present in the Standard Solution (a) above i.e., 0.4 mcg.mL–1 prepared in
double-distilled water.
(d) Culture Medium. The culture medium is composed of the following solutions and ingredients
:
(i) Casein hydrolysate solution** : 25 mL
(ii) Cysteine-tryptophane solution : 25 mL
(iii) Polysorbate-80 solution*** : 0.25 mL
(iv) Dextrose (anhydrous) : 10 g
(v) Sodium acetate (anhydrous) : 5 g
(vi) Adenine-guanine-uracil solution : 5 mL
(vii) Riboflavin-Thiamine hydrochloride-Biotin Solution : 5 mL
(viii) PABA****-Niacin-Pyridoxine hydrochloride solution : 5 mL
(ix) Calcium pantothenate solution A : 5 mL
(x) Calcium pantothenate solution B : 5 mL
The culture medium is usually prepared by dissolving both anhydrous dextrose and sodium
acetate in previously mixed solutions and the pH is carefully adjusted to 6.8 with 1 M.NaOH solution.
The final volume is duly made upto 250 mL with distilled water and mixed thoroughly.
(2) Stock Culture of Organism : The stock culture of organism may be prepared dissolving 2
g water-soluble yeast extract in 100 mL DW, 500 mg anhydrous dextrose, 500 mg anhydrous sodium
acetate, and 1.5 g agar. The resulting mixture is heated gently so as to dissolve the agar. Now, 10 mL of
hot solution is transferred to test tubes and sterilized at 121°C by keeping in an upright position. The
‘stab culture’***** is now prepared duly in three tubes employing Lactobacillus plantarum, incubated
at 30 to 37°C for 16 to 24 hours, and stored in a refrigerator ultimately.

(3) Preparation of Inoculum. The cells consequently obtained from the stock culture, (a) above,
organism are duly transferred to a sterile tube containing 10 mL of the culture emdium (d). Finally, it is
incubated at 30 to 37°C for a duration of 16–24 hours.
(4) Methodology. The various steps involved are as stated below :
(i) Standard Solution (b) is added to five test tubes in varying amounts viz., 1, 2, 3, 4 and 5
mL in duplicate.
(ii) To each of the five above test tubes plus another four similar tubes without any standard
solution is added 5 mL of culture medium, and the final volume made upto 10 mL with
DW.
(iii) Now, volumes of test solution (c) corresponding to either three or more of the levels as
taken above, are incorporated carefully to similar test tubes, in duplicate.
(iv) To each test tube 5 mL of the medium solution, and volume is made upto 10 mL with
DW. Thus, we may have two separate racks :
First Rack : Having complete set of standard plus assay tubes ; and
Second Rack : Having duplicate set only.
(v) Tubes of both the series are duly heated in an autoclave at 121°C for 5 minutes only ;

Note. (1) For the assay of niacin, it is cultured in the assay tubes by actually transferring to the
ensuing liquid culture medium comprising of the basic medium having an optimized quantum
of added niacin.
(2) To obtain a measurable response the amount of niacin present in each tube may range
between 0.05 to 0.5 mcg.
(1) Reagents. The various reagents used for the microbial assay of niacin are as enumerated
under :
(a) Standard Stock Solution of Niacin (I). It essentially contains 100 mcg.mL–1 of niacin
USPCRS*.
(b) Standard Stock Solution of Niacin (II). It consists of 10 mcg.mL–1 of niacin USPCRS;
and is prepared by dilution of solution (I) in the ratio 1 : 10, i.e., 1 mL of solution (I) is made
up to 10 mL in DW.
(c) Standard Niacin Solution. It critically contains niacin ranging between 10-40 ng
(i.e.,nanogram). mL–1, and may be prepared from Solution II by an appropriate dilution
with DW.
(d) Basal Culture Medium Stock Solution. The basal culture medium stock solution may be
prepared by the following requisite proportion of various ingredients and solutions as enumerated
under :
(i) Casein hydrolysate solution** : 25 mL
(ii) Cystine-tryptophane solution : 25 mL
(iii) Anhydrous dextrose : 10 g
(iv) Anhydrous sodium acetate : 5 g
(v) Adenine-guanine-uracil solution : 5 mL
(vi) Riboflavin-Thiamine hydrochloride-Biotin Solution : 5 mL
(vii) PABA-Calcium patothenate-Pyridoxine
hydrochloride solution : 5 mL
(viii) Niacin solution A : 5 mL
(ix) Niacin solution B : 5 mL
The culture medium is duly perpared by carefully dissolving anhydrous dextrose and anhydrous
sodium acetate into the previously mixed solutions, and adjusting the pH precisely to 6.8 by the dropwise
addition of 1 M.NaOH. The final volume was made up to 250 mL with DW.
(e) Culture Medium. Into a series of labeled ‘test tubes’ containing 5 mL of the Basal Culture
Medium Stock Solution [(d) above] 5 mL of water containing exactly 1 mcg of niacin are
incorporated carefully. The sterilization of all these ‘test tube’ are carried out by first plugging
each of them with cotton, and subsequently autoclaving them at 121°C for 15 minutes.
(2) Preparation of Inoculum. Transfer from the stock culture of Lactobacillus plantarum
cells aseptically into a sterilie test tube containing 10 mL of culture medium [(e) above]. The resulting
culture is duly incubated at a temperature ranging between 30–37°C for a duration of 16–24 hours. The
cell suspension of the said organism is termed as the inoculum.

cooled to ambient temperature, added 1 drop of inoculum (3) to each tube except two of
the four tubes that specifically has no ‘standard solution’ (i.e., the uninoculated tubes),
and mixed thoroughly. The tubes are adequately incubated at 121°C at 30–37°C for 16–
24 hours.
(vi) Transmittance of the various tubes is measured with a spectrophotometer at wavelength
ranging between 540–660 nm.
(5) Calculation. First of all, a standard concentration response curve is plotted between the
transmittance Vs log mL (volume) of the standard solution in each tube. In this way, the response is
duly calculated by summing up the two transmittances for each level of the test solution.
Finally, the exact concentration of the calcium pantothenate in the ‘test sample’ is determined
accurately with the aid of the standard concentration-response curve obtained.
 Niacin (or Niacinamide)
Preamble. In this particular assay the most appropriate organism should be such that must be
able to fully use up there five vital and important components, namely : niacin, nicotinuric acid,
miacinamide, niacinamide, nucleoside, and coenzymase (an enzyme). This organism that may critically
satisfy the aforesaid requirements happens to be Lactobacillus plantarum. Interestingly, this acid
forming organism is found to be quite incapable to afford the synthesis of niacin for its on-going metabolic
processes. A few other equally important criteria of this organism are as given under :
• Non-pathogenic in nature
• Easy to culture
• Least affected by various stimulatory or inhibitory constituents usually present in ‘pharmaceutical
formulations’ containing niacin.
• Conveniently grown upon a rather simple stab culture comprising of gelatin, yeast extract,
and glucose.

(3) Methodology. The various steps that are involved in the microbial assay of niacin are described
as under in a sequential manner :
(i) First and foremost the ‘spectrophotometer’ is duly calibrated according to the procedural
details mentioned in the ‘official compendia’*.
(ii) Standard Niacin Solution is added in duplicate into various Standard Niacin Tubes
in varying quantities viz., 0, 0.5, 1.0, 1.5, 2.0, 2.5 ...... 5.0 mL respectively. To each of
these tubes add 5.0 mL of the Basal Culture Medium Stock Solution [(d) above] plus
sufficient distilled water to make 10 mL.
(iii) Test Solution Tubes containing varying amounts of niacin are carefully prepared by
making in duplicate 1, 2, 3, 4, and 5 mL respectively of the ‘test solution’. To these
tubes are added 5 mL of the Basal Culture Medium Stock Solution [(d) above], and
followed by water to make upto 10 mL.
(iv) All the tubes obtained in (iii) above are duly plugged with cotton, and adequately sterilized
in an ‘autoclave’ (for 15 minutes at 121°C).
(v) After having brought down the hot tubes to the ambient temperature, they are carefully
inoculated asepticlly with one drop of inoculum [(2) above], and subsequently between
30–37°C for a duration of 16 to 24 hours.
(vi) Having set the percentage transmittance at 1 for the ‘uninoculated blank’, the various
transmittance of the inoculated tubes is duly noted, and recorded.
(4) Calculation : First of all a ‘Standard Curve’ is plotted for niacin between :
• standard transmittances for each level of Standard Niacin Solution, and
• exact quantum of niacin (in mcg) present duly in the respective tubes.
Thus, from the ‘Standard Curve’, one may easily obtain the niacin precisely present in the ‘test
solution’ of each tube by interpolation.
Finally, the exact niacin content of the ‘test material’ may be calculated from the ‘average
values’ duly obtained from at least six tubes which should not vary by more than ± 10% with respect to
the average values.
 Vitamin B12 [or Cynocobalamin]
It is pertinent to state here that the ‘basic culture medium’ employed for the assay of vitamin
B12 is found to be extremely complex in nature, and essentially comprises of a large number of varying
constituents in the form of a mixture in solution.
Various steps are as follows :
(1) First set of tubes contains solely the measured quantum of a Standard Cyanocobalamin
Solution.
(2) Second set of tubes essentially comprise of the graded volumes of the ‘test sample’ (i.e.,
unknown).
(3) All the ‘tubes’ (i.e., first set + second set) are carefully inoculated with a small quantity of
the culture of Lactobacillus leichmanni, and subsequently incubated duly.

(4) The precise extent of growth is assayed by measuring the percentage transmittance by the
help of a standardized (calibrated) spectrophotometer.
(5) The concentration-response curve is now prepared mediculously by plotting the following
two observed parameters :
• Transmittance values (i.e., response), and
• Different concentrations (i.e.,dose) of Standard cyanocobalamin solution.
(6) Ultimately, the exact quantum of vitamin B12 duly present in the given ‘test sample’ (i.e.,
unknown) is calculated based on the ‘Standard Curve’ by the interpolation.10.7.4. Assay of Amino Acids
As discussed earlier the critical and specific requirements of a microorganism for an ‘amino
acid’ may be employed categorically to assay the exact quantum of the amino acid duly present in a
plethora of pharmaceutical formulations or even food products by allowing the particular organism
to grow optimally in a medium containing all the ‘essential requirements’, and thus the measured
doses of the ‘substance’ called be assayed accurately.

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 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
different volumes of it to each of several different flasks containing 100 ml of the medium specified in
(the volume of suspension suggested  may be used as a guide). Using these
inocula, prepare inoculated plates as described for the specific antibiotic assay. While conducting cylinder-
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 concentration
of the antibiotic  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 concentrations
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 concentrations
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 microorganisms
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, 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 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 temperature
between 40° and 50° and immediately pour the inoculated medium into Petri dishes or large rectangular
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  prepare solutions of
known concentration of the Standard Preparation and solutions of the corresponding assumed concentrations
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 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 application
of the different solutions. Incubate them for about 18 hours at the temperature indicated
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.

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 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 accommodate
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 concentration
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 =
3 2
5
a + b + c − e
; H =
3 2
5
e + d + c − a
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 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.
[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 diameters
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 = 1 2 1 2
1 2 1 2
(U + U ) – (S + S )
(U + U ) + (S – S )
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 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-plate 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 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 organism
(Table 10.3).
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 dilute formaldehyde 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
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 logarithmic
scale. Construct the best straight response line through the points either by inspection or by means
of the following expressions :
L =
3 2
5
a + b + c − e
; H =
3 2
5
e + d + c − a
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.*

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 :
(a) Antibiotics, (b) Vitamins, and (c) 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 antibiotic
for which the potency has been precisely determined with reference to the appropriate international
standard’
However, the potency of the standard preparation may be duly expressed either in International
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 Laboratory,
Kolkata. A unit referred to in the ‘official assays’ and ‘tests’ refers to the specific activity contained
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. 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 produce
1L, and add sufficient 1M sodium hydroxide or 1M hydrochloric acid, as required so that after
sterilization the pH must be as stated

[B] Buffer Solutions : Prepare the buffer solutions by dissolving the quantities
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.

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 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 concentration,
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
 Preparation of Sample Solution
Based on the available information for the ‘drug substance’ under investigation (i.e., 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’ and test dilution(s) as duly specified for each individual antibiotic
 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 ‘unknown’
at a concentration assumed very much equal to the ‘median level’ of the ‘Standard’.

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 prepared
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.

Test Organisms
The various test organisms for each antibiotic is duly listed in Table 10.4, along with its properly
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.

1. Use Medium A containing 300 mg of manganese sulphate per litre.
2. For Pseudomonas aeruginosa in the assay of Carbenicillin, use the dilution yielding 25% light transmission,
rather than the stock suspension, for preparing the inoculum suspension.
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

ANALYTICAL METHODS FOR MICROBIAL ASSAYS

There are several sophisticated analytical methods that are used most abundantly for the precise
quantitative methods microbial assays, such as :
(a) High Performance Liquid Chromatography (HPLC),
(b) Reverse-Phase Chromatography (RPC), and
(c) Ion–Pair (or Paired-Ion) Chromatography,
These three chromatographic techniques shall now be discussed briefly in the sections that follows
:
 High Performance Liquid Chromatography [HPLC]
Preamble : Giddings* (1964) rightly predicted that the careful and meticulous application of
relatively ‘small particulate matter’ under the influence of excessively enhanced flow pressure could
definitely improve upon the performance of ‘Liquid Chromatography’ significantly ; and ultimately
one could easily, accomplish an appreciably high number of ‘theoretical plate numbers’. Towards the
later half of 1960s world’s two eminent scientists, Horvath and Lipsky at Yale University (USA), came
forward with the first ever HPLC, and named it as ‘high pressure liquid chromatography’. Nevertheless,
the early 1970s the world witnessed the ever glorious technological supremacy by producing and

using very small silanized silica particles that gainfully permitted the usage of small-volume longer
columns absolutely urgent and necessary to yield the much desired high-resolution performance. In
fact, the latest HPLC is, therefore, commonly known as the ‘high-performance liquid chromatography’
across the globe.
Principles : The particle size of the stationary phase material predominantly plays an extremely
vital and crucial role in HPLC. In actual practice, high-efficiency-stationary phase materials
have been duly researched and developed exclusively for HPLC with progressively smaller partricle
size invariably known as ‘microparticulate column packings’. These silica particles are mostly uniform,
porous, with spherical or irregular shape, and with diameter ranging betwene 3.5 to 10 μm.*
The bonded-phase supports normally overcome a good number of cumbersome and nagging
serious problems that are invariably encountered with the adsorbed-liquid phases. Thus, the molecules
containing the stationary phase i.e., the surfaces of the silica particles are covalently bonded upon a
silica-based support particle.
Example : Siloxanes are duly formed by heating the silica particles in diluted acid for 24–48 hrs.
in order to give rise to the formation of the reactive silonal moiety as depicted below :

When such microparticulate-bonded-phases are compactly packed into a column, the tiny size
of these particles affords a substantial resistance to the ensuing solvent flow ; and, therefore, the mobile
phase has got to be pumped via the column at a flow rate ranging between 1 to 5 cm3 . min– 1.
Advantages of HPLC : The advantages of HPLC are as stated below :
(1) Highly efficient, selective, and broad applicability.
(2) Only small quantum of sample required.
(3) Ordinarily non-destructive of sample.

(4) Rapidly amineable and adaptable to ‘Quantitative Analyses’.
(5) Invariably provide accurate, precise, and reproducible results.
HPLC-Equipments : Modern HPLC essentially comprises of seven vital components, namely :
(a) solvent reservoir and degassing system, (b) pressure, flow, and temperature, (c) pumps and sample
injection system, (d) columns, (e) detectors, (f) strip-chart recorder, and (g) data-handling device and PCbased
control.
Fig. 10.5 represents the HPLC chromatogram of peritoneal (PT) fluid from a subject having an
impaired renal function to whom ‘Cefotaxime’, an antibiotic has been administered intraperitoneally.
Cefotaxime (CTX) gets metabolized to microbioligically ‘active’ and ‘inactive’ metabolites.
PT Fluid : Peritoneal Fluid
DACM : Desacetyl Cefotaxime (Active)
CTX : Cefotaxime
UP1 and UP2 : Two microbiologically inactive metabolites

Reverse-Phase Chromatography [RPC]
The Reverse-Phase Chromatography (RPC) or Reversed-Phase HPLC (RP-HPLC) is
invariably employed for the separation of organic compounds.
In RPC, specifically a relatively nonpolar stationary phase is employed along with such polar
mobile phase as :
􀁏 methanol, acetonitrile, tetrahydrofuran, water, or
􀁏 mixture of organic solvents and water.
Organic Solvent—the organic solvent is usally termed as the ‘modifier’ e.g., acetonitrile.
Water—Water content is mostly varied according to the required polarity.
Methanol—It is used for acidic compounds.
Acetonitrile—It is employed for basic compounds.
Tetrahydrofuran (THF)—It is usually used for those compounds having large dipoles comparatively.
In fact, most of these solvents do have low viscosity and are UV-transparent.
Bonded Phases—The abundantly used bonded phases are :
􀁑 n-Octyldecyl (i.e., C-18 chain),
􀁑 n-Decyl (i.e., C-8 chain), and
􀁑 Phenyl Moieties
Polar-Reversed Phase Columns— The polar-reversed phase columns essentially are
polyethylene glycol (PEG) which contain either moieties that interact with polar analytes e.g., phenolic
compounds, multiaromatic ring systems, and hydroxyl-containing compounds.
 Ion-Pair (or Paired-Ion) Chromatography
Importantly, perhaps the most valuable of the secondary equilibria variants usually encountered
in the ‘pharmaceutical analysis’ being the ion-pair formation, that may be adequately expressed
for a reversed-phase LLC-System

It has been duly observed that the ion-pair AB thus formed is capable of partitioning very much
into the ensuing stationary phase. However, in many instances the ions A+ and B– fail to do so by virtue
of the fact that their ultimate polarity gain entry into the stationary-phase gradually thereby the evolved
chromatographic resolution is controlled exclusively by the so called ion-pairing phenomenon.
It is, however, pertinent to state here that one may invariably come across a host of ‘drug substances’
that are either acidic or basic in character ; and, therefore, they may be duly rendered into ionic
by carefully regulating the pH of the ensuing mobile phase. In short, ion-pair chromatography possesses
an enormous applicability in the separation of drug substances.
Examples : A few-typical examples pertaining to the ion-pair chromatography are as described
under :
(1) Separation of Niacin, Niacinamide, Pyridoxine, Thiamine and Riboflamin. The admixture
of five vitamins can be separated effectively by making use of the
sodium hexanesulphonate as the ion-pairing agent, on a C—18 column
i.e., ODS-column
(2) Antihistamines and decongestants may be separated efficaciously on a phenyl column.