THE AMERICAN

JOURNAL OF HYGIENE
VOL. 27

MAY, 1938

No. 3

BY

L. J. REED AND H. MUENCH

(Received for publication October 26th, 1937)

An endpoint in biological procedures such as titration of sera or

viruses is usually taken as the dilution at which a certain proportion
of test animals react or die. The advantages of using the dilution by
which half the animals are affected have been set forth by Gaddum (1)
on the basis of pharmacological tests. This endpoint is less affected by
small chance variations than is any other; the worst in this respect
being the 100 per cent point so frequently used.
The best method of determining the endpoint is the use of large
numbers of animals at dilutions near the value for 50 per cent reaction,
and the interpolation of the correct value by the procedures described
by Gaddum. But this is scarcely practical in a great deal of immunological work, where the titer may vary between wide limits in any given
test; while highly refined statistical procedures are hardly justified in
view of the uncontrolled variables involved.
To cover a wide range of possible variations in titer, it is generally
necessary to use a large number of small groups of test animals at
different dilutions. The following method utilizes, at least in part,
the large, total number involved. It is essentially the one given by
Reed (2), somewhat simplified and adapted to rapid use. The effect is
that of using, at the two critical dilutions between which the endpoint
lies, larger groups of animals than were actually included at these
1
This method has been used in the estimation of endpoints at the Yellow Fever
Laboratories of the International Health Division of The Rockefeller Foundation,
New York City.
2
From the Department of Biostatistics, The John Hopkins University School of
Hygiene and Public Health, and the International Health Division of The Rockefeller Foundation.
493

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A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT

ENDPOINTS l ' 2

494

L. J . REED AND H. MUENCH

dilutions. By inclining to equalize chance variations, the method

tends to define the point more nearly than would be possible if it were
simply interpolated between the two bracketing results.
For purposes of illustration the results of a hypothetical titration of
a protective serum are given in table 1. Six mice are tested at each

Total
Dilution
a

Alive
6

Dead
c

1 : 1
2
4
8
16
32
64
128
256

6
6
5
6
4
2
2
0
1

0
0
1
0
2
4
4
6
5

Per cent
mortality

Alive
d

Dead
e

32
26
20
15
9
5
3
1
1

0
0
1
1
3
7
11
17
22

0
0
5
6
25
58
79
94
96

dilution and the results, in numbers surviving and dying, are entered
in columns "b" and "c." It is assumed that a mouse surviving at a
given dilution of serum would have survived at a lower dilution.
Column "b" is therefore added from the bottom, and the subtotal
for each dilution is entered in column "d" as the accumulated number
of mice surviving at this plus higher dilutions. Conversely, a mouse
dead at a given dilution would have died at any higher one. Column
"c" is added from the top, and the subtotals in column "e" give the
cumulative numbers of mice dying at given dilutions plus those lower.
Percentage mortality is calculated from columns "d" and "e" and is
entered in column "/." For example, of a total of twelve mice treated
with a dilution of 1 : 16, nine survived at this or higher dilutions while
three died at this or lower ones. The calculated mortality rate is
therefore 25. per cent.
Figure 1 illustrates the method. The two lines represent columns
"d" and "e" and meet at the assumed point of 50 per cent mortality,
in this case, about three-quarters of the distance from dilution 1 : 16
to 1 : 32. It is obvious that the position of the endpoint would not
be changed by an indefinite number of deaths at higher dilutions, or
of survivals at lower. The "accidental" occurrence of survivals at

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TABLE 1

Titration of a hypothetical protective serum

METHOD OF ESTIMATING FIFTY PEE CENT ENDPOINTS

495

Mice surviving
(Col. "d", table l)

10

16

128

32

1:256

Dilutions
FIGURE 1.

Accumulation of deaths and survivals.

high dilutions, or of deaths at low, would shift the point if one or the
other predominated. This shift is less for the 50 per cent point than
for any other, but it should be eliminated as far as possible.
Since highly susceptible or refractory animals are occasionally encountered in most types of test, it is to be expected that these "accidental" survivals and deaths will occur, at times, at practically any
dilution. They should, in general, tend to cancel each other if an
equal number of dilutions is taken on each side of the endpoint. This
may be accomplished by abridging the original results, as has been
done in table 2.
TABLE 2
Abridged tabulation of the results of table 1
Total
Dilution
a

Alive
b

Dead
c

1 :8
16
32
64
128

6
4
2
2
0

0
2
4
4
6

Per cent
mortality

Alive
d

Dead
e

14
8
4
2
0

0
2
6
10
16

0
20
60
83
100

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Mice dyin
(Col. "e", table 1

496

L. J. REED AND H. MUENCH

Since, in table 1, 1 : 32 was the dilution nearest the endpoint, this is

taken as the central value of the new table, the two above and two
below being retained. Three or four dilutions on each side could be
used as well and would be preferable if mortality increased slowly with
dilution. Columns "d," "e," and " / " are constructed exactly as
before, but are now based on the smaller number of results.
The 50 per cent point evidently lies between 1 : 16 and 1 : 32, but
nearer the latter. It is assumed that, since the mortality at 1 : 32 is
40 per cent above that at 1 : 16, while the 50 per cent point is 30
per cent above, the endpoint is 30 per cent/40 per cent or three100

50

.2

.6

.8

1.0

FIGURE 2. Graphical interpolation of final titer.

quarters of the distance from 1 : 16 to 1 : 32. The formula for the

proportionate distance of the endpoint above the dilution giving next
below 50 per cent mortality is:
50 per cent (mortality at dilution next below)
(mortality next above) (mortality next below)

Proportional distance

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Calculation of results

METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS

497

Since dilutions are increasing on a logarithmic scale, it is necessary

to obtain the final reading as follows:
Logarithm of 16 (lower dilution)
0.75 (propor. dist.) X log 2 (dilution factor)

1.4299

which makes the endpoint at a dilution of 1 : 26.9 or approximately

1 :27.
This part of the calculations can more quickly be carried out
graphically, as shown in figure 2. The lower axis of a sheet of "arithlog" paper is scaled from zero to one, representing the total distance
between dilutions. A line is drawn from the lower left corner to the
point on the right representing the dilution factor (in this case two).
With dividers, the distance from the axis to this line is measured off at
the proportionate distance (here 0.75). The dividers are then moved
to the vertical axis with the lower point on the line corresponding to
the lower dilution, here sixteen. The upper point falls on the scale
reading for the endpoint, in this case just below 27.
If the titer to be found is that of an infective agent instead of a
protective serum, "deaths" and "survivals" are merely reversed in
direction, the method otherwise remaining the same.
BIBLIOGRAPHY
1. GADDUM, J. H.

1933 Reports of Biological Standards, III. Methods of Biological Assay

Depending on a Quantal Response. Med. Res. Council, London;
Special Report Series, No. 183.
2. DUGGAR (ED.), B. J.

1936 Biological Effects of Radiation. McGraw-Hill, New York City; Chap.

II, L. J. Reed: Statistical Treatment of Biological Problems in
Irradiation.

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Sum Gog. of endpoint)

1.2041
0.2258