A large number of antibiotics, namely : chlortetracycline, doxycyline, gentamicin, neomycin, strepto-mycin, tobramycin and the like may be assayed tubidimetrically with fairly good accuracy.
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.
Standard chlortertracyline ; sterilized media (as described above) : 1 L ; authen-tic and pure strain of microorganism Staphylococcus aureus (NCTC 6571) ; formaldehyde solution (34-37% w/v) 10 ml ; matched identical test tubes : 20 ;
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 chlortetracycline, 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 measure 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.
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) and the incubation temperature. A few typical examples are given in Table 20.1 below:
In order to obtain the required rectilinearity it may be necessary to select from a large number three consecutive doses, using corresponding doses of the standard preparation and of the substance being examined. (BP, 1993, Appendix XIV A, p, 167 and 168).