Heatley NG, A method for the assay of penicillin. Biochem J.1944; 38(1): 61–65
A Method for the Assay of Penicillin
(received 18 December 1943)
Practical details of the method
i) Pouring the plates:
------- 4 in. Petri dishes are poured to a depth of 3-5mm with
a simple medium such as ---------
ii) Seeding the plates:
Staphylococcus aureus, no.6571 of the National Collection of
Type Cultures, is used as test organism.-------- The plates are
seeded by placing on each a small volume of a 16-24 hr. broth
culture (or a 10-100-fold dilution of such a culture). --------
iii) Preparation of solutions for assay:
The pH of the solution should be within the range 5.0-8.0.
iv) Placing and filling cylinders:
--------- Carefully placed on the surface of the agar. There may
be a very brief sizzling sound (though the cylinder should scarcely
be hot enough to cause this) and a perfectly fluid-tight seal is
made between agar and the cylinder. The cylinders are not
pressed into agar. The solution to be assayed are then placed
in the cylinders, care being taken that there is no air space
between the fluid and the surface of the agar. The exact volume
of fluid seems to make little difference to the assay value, but
in practice the fluid is filled level with the top of the cylinder.
v) Specifications for cylinders:
The cylinders may be made of glass or vitreous porcelain, gazed
or ungazed. 9.6±0.2 mm, by 5.1±0.2 mm. internal diameter and
7.2±0.1mm. external diameter. one end is bevelled internally
and the shape edge is ground perfectly plane; the other end is
colored for easy identification. ------------------
vi) Incubation:
------ 37 incubator for 16-24 hr. Incubation is usually allowed to
proceed overnight but the zones of inhibition are quite distinct
after less than 10 hr. and, if left longer, increase in size only
very slightly. ------------
vii) Measurement of zones of inhibition:
viii) Arranging standard and unknown solutions on the plates:
In prepare ring the plates a number of factors such as batch of
medium, length of autoclaving, drying time of plates, number
of times the incubator is opened during drying, density of
bacterial suspension used for seeding, etc., are not easy to
control in practice.
It has been found that the of the curve relating the diameter or
the zones of inhibition to Concentration of penicillin, as well as
the absolute assay value for any given sample, may vary from
day to day, probably depending on these variables.
Preliminary investigations showed that a quantitative study of
the effects involved would a lengthy and difficult task; the method
is still therefore largely empirical. It is, however, a simple matter
to determine the shape of the curve afresh each day by setting up
with the--------
ix) The penicillin standard:
x) Reproducibility: limit of accuracy
A quantitative idea of the reproducibility of the method is afforded
by a series of 27 assays of a given preparation against the same
standard. Each single potency value was derived from the mean
of four zone measurements, the values being 75, 75, 66, 77, 88.5,
63, 68, 72, ------------ 79, 63.5, 81, 73. The coefficient of variation
of this series (Series A) is 9.0% .
Two further series with ten assays in each were the following
results: Series B: 56, 56, 62, 61, 51.5, 56.5, 57, 55, 56, 60;
coefficient of variation, 5.6%. Series C: 70.5, 62, 72, 68.5,
67, 72, 65, 71.5, 65, 68.5; coefficient of variation, 5.0% .
------------------
The scatter within tests and between successive tests is illustrated
by Table 1, which gives the zone measurements actually recorded
for the standard solutions set up in a consecutive series of assays.
-------- If 9% is taken as the coefficient of variation which may
be expected, then for P=0.01 the limits of accuracy for a single
assay in quadruplicate(i.e. a value derived from measurement
of four zones) will be rise to +- 23.2%. For an assay in Triplicate
the limits will be rise to +- 26.9% and for an assay derived from
two zone measurements only they will be +- 36.7%.
For P=0.05, the limits will be +- 19.5%, +- 22.7% and 27.7%
respectively. In series B and C the degree of reproductively
was considerably greater than this, though the reason is not
known.
xi) Possible modifications and applications of the method:
Substitutes for cylinders:
------ instead of cylinders, circular disks of filter paper which
are merely placed on the surface of a seeded plate and impregnated
with the solution to be tested.
----------
Application to substance other than penicillin:
Great caution must be exercised ------- when applied to other
antibiotics. If the inhibitor is not diffusible, the method is obviously
of no use. The composition of the agar may need spcial attention(e.g.
the presence of peptones wpuld presumably interface with sulphonamides
; penicillin B or notatin gives large zones of inhibition in the presence
of glucose, but not in its absebce). --------
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Any questions: write to Keiji Hagiwara, MD keijihagiwara@gmail.com
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