On the antibacterial action of cultures of Penicillium with special by alicejenny


									On the antibacterial           action of cultures of
a Penicillium,         with special reference to
their use in the isolation of
B. injluenzae

            1929   l   Alexander   Fleming

                                       Fleming, Alexander. 1929. On the Antibacterial
                                       Action of Cultures of a Penicillium, with Special
                                       Reference to Their Use in the Isolation of B.
                                       jnflrunzae. British ]ournal of Experimental Path-
                                       ology, Vol. 10, pages 22fS236.

WHILE   WORKING WITH STAPHYLOCOCCUS          formed      in the mould      culture   and
variants a number     of culture-plates      which had diffused into the surround-
were set aside on the laboratory bench       ing medium. It was found that broth
and examined from time to time. In           in which the mould had been grown
the examinations these plates were nec-      at room temperature       for one or two
essarily exposed to the air and they         weeks had acquired marked inhibitory,
became contaminated    with various mi-      bactericidal   and bacteriolytic    proper-
cro-organisms.   It was noticed       that   ties to many of the more common
around a large colony of a contam-           pathogenic bacteria.
inating    mould   the staphylococcus
colonies became transparent     and were
                                                   CHARACTERS    OF   THE   MOULD
obviously undergoing     lysis.
   Subcultures   of this mould       were       The   colony    appears   as a white
made and experiments conducted with          fluffy mass Lvhich rapidly      increases
a view to ascertaining     something    of   in s&e and after a few davs sporulates,
the properties of the bacteriolytic   sub-   the centre becoming      dark green and
stance    which   had evidently      been    later in old cultures darkens to almost
186                                                                   CHEMOTHERAPY

black. In four or five days a bright yel-        In the rest of this article allusion
low colour is produced which diffuses         will constantly be made to experiments
into the medium. In certain condi-            with filtrates of a broth culture of this
tions a reddish colour can be observed        mould, so for convenience         and to
in the growth.                                avoid the repetition of the rather cum-
   In broth the mould grows on the            bersome phrase “mould broth filtrate,”
surface    as a white      fluffy growth      the name “penicillin”     will be used.
changing     in a few days to a dark          This will denote the filtrate of a broth
green felted mass. The broth becomes          culture of the particular     penicillium
bright yellow and this yellow pigment         with which we are concerned.
is not extracted by CHC&. The reac-
tion of the broth becomes markedly
                                              METHODSOF EXAMINING CULTURES FOR
alkaline, the PH varying from 8.5 to 9.
Acid is produced       m 3 or 4 days in
glucose and saccharose broth. There              The simplest method of examining
is no acid production        in 7 days in     for inhibitory power is to cut a fur-
lactose, mannite or dulcite broth.            row in an agar plate (or a plate of
   Growth is slow at 37’C. and is most        other suitable culture material),     and
rapid about 2O’C. No growth is ob-            fill this in with a mixture of equal parts
served under anaerobic conditions.            of agar and the broth in which the
   In its morphology    this organism is a    mould has grown. When this has solid-
penicillium and in all its characters it      ified, cultures of various microbes can
most closely     resembles     P. rubrum.     be streaked at right angles from the
Biourge (1923) states that he has never       furrow to the edge of the plate. The
found P. rubrum in nature and that it         inhibitory substance diffuses very rap-
is an “animal de laboratoire.”        This    idly in the agar, so that in the few
penicillium is not uncommon         in the    hours before the microbes show visible
air of the laboratory.                        growth it has spread out for a centi-
                                              metre or more in sufficient concentra-
                                              tion to inhibit growth of a sensitive
                                              microbe. On further incubation it will
                                              be seen that the proximal portion of
   A number of other moulds were              the culture for perhaps one centimetre
grown in broth at room temperature            becomes transparent, and on examina-
and the culture fluids were tested for        tion of this portion of the culture it
antibacterial substances at various in-       is found that practically all the mi-
tervals up to one month. The species          crobes are dissolved, indicating that
examined were: Eidamia viridiscens,           the anti-bacterial substance has con-
Botrytis cineria, Aspergillus fumigatus,      tinued to diffuse into the agar in suffi-
Sporotricbum, Cladosporium, Penicil-          cient concentration to induce dissolu-
hum, 8 strains. Of these it was found         tion of the bacteria. This simple
that only one strain of penicillium           method therefore suffices to demon-
produced      any inhibitory     substance,   strate the bacteria-inhibitory      and
and that one had exactly the same cul-        bacteriolytic properties of the mould
tural characters     as the original one      culture, and also by the extent of the
from the contaminated      plate.             area of inhibition gives some measure
   It is clear, therefore,  that the pro-     of the sensitiveness of the particular
duction of this antibacterial substance       microbe tested. Figure 2 shows the
is not common to all moulds or to all         degree of inhibition    obtained  with
types of penicillium.                         various microbes tested in this way.
Fleming   l    The antibacterial action of cultures of a Penicillium           187
   l%e inhibitory power can
be accurately       titrated  by
making serial dilutions of
penicillin in fresh nutrient
broth, and then implanting all
the tubes with the same vol-
ume of a bacterial suspension
and incubating them. The in-
hibition can then readily be
seen by noting the opacity of
the broth.
   For the estimation of the
antibacterial power of a mould
culture it is unnecessary to
filter as the mould grows only
slowly at 37’C., and in 24
hours, when the results are
read, no growth of mould is
perceptible. Staphylococcus is
a very suitable microbe on
which to test the broth as it is
hard , lives well in culture, grows         water and weak saline solutions. My
rapi J ly, and is very sensitive to pen-    colleague, Mr. Ridley, has found that
icillin.                                    if penicillin is evaporated at a low
   The bactericidal power can be            temperature to a sticky mass the active
tested in the same way except that at       principle can be completely extracted
intervals measured quantities are ex-       by absolute alcohol. It is insoluble in
planted so that the number of sur-          ether or chloroform.
viving microbes can be estimated.              Rate of development of inhibitory
                                            substance in culture. A 500 cc. Erlen-
              PROPERTIES   OF   THE         meyer flask containing ZOO cc. of
      ANTIBACTERIAL        SUBSTANCE        broth was planted with mould spores
                                            and incubated at room temperature
   Ejfect of heat. Heating for 1 hour       (IO’ to 2O’C.). The inhibitory power
at 56’ or 8O’C. has no effect on the        of the broth to staphylococcus was
antibacterial power of penicillin. Boil-    tested at intervals,
ing for a few minutes hardly affects it.
Boiling for 1 hour reduces it to less         After         Complete inhibition in
than one quarter its previous strength        5 days         I in 20 dilution.
if the fluid is alkaline, but if it is        6 ”            1 in 40       ”
neutral or very slightly acid then the        7 99           1 in 200      ”
reduction is much less. Autoclaving           8 9,           1 in 500     ”
for 20 minutes at IlS’C. practically           Grown at 2O’C. the development
destroys it,                                of the active principle is more rapid
  Effect of filtration. Passage through     and a good sample will completely in-
a Seitz filter does not diminish the        hibit staphylococci in a I in 500 or 1
antibacterial power. This is the best       in 800 dilution in 6 or 7 days. As the
method of obtaining sterile active          culture ages the antibacterial power
mould broth,                                falls and may in 14 days at 2O’C. have
   Volubility. It is freely soluble in      almost disappeared.
188                                                                  CHEhtOTHERAPY

    The antibacterial power of penicil-      and non-pathogenic, are inhibited by
 lin falls when it is kept at room tem-     penicillin. The first table shows the in-
 perature.                                  hibition by the agar plate method and
    If the reaction of penicillin is al-    the second shows the inhibitory power
 tered from its original pH of 9 to a       when diluted in nutrient broth.
pH of 6.8 it is much more stable.              Certain interesting facts emerge
    The small drops of bright       ellow   from these Tables. It is clear that pen-
fluid which collect on the sur Y   ace of   icillin contains a bacteria-inhibitor
the mould may have a high antibac-          substance which is very active towar Bs
terial titre. One specimen of such          some microbes, while not affecting
fluid completely inhibited the growth       others. The members of the coli-
of staphylococci in a dilution of 1 in      typhoid group are unaffected as are
 20,000 while the broth in which the        other intestinal bacilli such as I3.
 mould was growing, tested at the same      pyocyaneus    [Pseudomonas],      B. p-o-
time, inhibited staphylococcal growth       teus   [Proteus]   and V.        cholerae
 in 1 in 800.                                 [Vibrio]. Other bacteria which are in-
    If the mould is grown on solid           sensitive to penicillin are the enteroc-
 medium and the felted mass picked           occus, some of the Gram-negative
 off and extracted in normal salt solu-      cocci of the mouth, Friedlander’s
tion for 24 hours it is found that the       pneumobacillus [ Klebsiella], and B.
extract has bacteriolytic properties.       infiuenzae (Pfeiff er) [ Hemophilus 1,
    If this extract is mixed with a thick   while the action on B. dysenteriae
suspension of staphylococcus suspen-         (Flexner) [ Shigella], and B. pseudo-
sion and incubated for 2 hours at            tuberculosis   rodentium is almost neg-
45’C. it will be found that the opacity     ligible. The anthrax bacillus is com-
of the suspension has markedly dimin-       pletely inhibited in a 1 in IO dilution
ished and after 24 hours the previously     but in this case the inhibitory influence
opaque suspension will have become          is trifling when compared with the
almost clear.                               effect on the pyogenic cocci.
   Influence of the medium on the anti-         It is on the pyogenic cocci and on
bacterial titre of the mould culture.       bacilli of the diphtheria group that
So far as has been ascertained nutrient     the action is most manifest.
broth is the most suitable medium for           Staphylococci are very sensitive, and
the production of penicillin. The ad-       the inhibitory effect is practically the
dition of glucose or saccharose, which      same on all strains, whatever the colour
are fermented by the mould with the         or type of the staphylococcus.
production of a&d, delays or prevents          Streptococcus     pyogenes is also very
the appearance of the antibacterial         sensitive. There were small differences
substance. Dilution of the broth with       in the titre with different strains, but it
water delays the formation of the anti-     may be said generally that it is slightly
bacterial s&stance and diminishes the       more sensitive than staphylococcus.
concentration    which is ultimately           Pneumococci are equally sensitive
reached.                                    with Streptococcus      pyogenes.
                                               The green streptococci vary very
                                            considerably, a few strains being al-
                                            most unaffected while others are as
                                            sensitive as S. pyogenes. Gonococci,
   Tables II and III show the extent        meningococci, and some of the Gram-
to which various microbes, pathogenic       negative cocci found in nasal catar-
Flewzing - The antibacterial action of cultures of a Penicillium                       189

             TALW IL-Znbibitory        Power of Penicillin on Various Microbes
                                      (Agar Plate Method)

                                                                Extent of inhibition in mm‘
                                                                 from penicillin embedded
                                                                   in agar, serum agar, or
                  Type   of microbe                                   blood agar plates

Experiment   1:

  S~apbylococcus pyogenes                                                  23
  Smeptococcus pyogenes                                                    17
  Streptococcus viridans (mouth)                                           17
  Diphtheroid bacillus                                                     27
  Sarcina                                                                  10
  Micrococcus lysodiekhus                                                   6
  Micrococcus from air (1)                                                 20
  Micrococcus from air (2)                                                  4
  B. anhacis ~Bacillusl                                                     0
  B. ty@9orz0 [Salmonellal                                                  0
  Enterococcus [Streptococcus?   1                                          0

Experiment   2:

  Stapl9ylococcus pyogenes                                                 24
  Streptococcus pyogenes                                                   30
  Sweptococcus viridans (mouth)                                            25
  Pneumococcus                                                             30
  Diphtheroid bacillus                                                     35
  B. pyocyaneus IPseudomonas                                                0
  B. pneumoniae (Friedlander)   [Klebsiellal                                0
  B. coli [Escherichial                                                     0
  B. paratyphosus A LSalmonella1                                            0

Experiment   3:

  Staphylococcus pyogenes                                                  16
  Gonococcus [Neisserial                                                   16
  Meningococcus [Neisserial                                                17

Experiment   4:

  Staphylococcus pyogenes                                                  17
  Staphylococcus epidermidis                                               18
  Streptococcus pyogenes                                                   15
  Streptococcus viridans (faeces)                                           5
  B. dipbtheriae (2 strains) [Corynebacteriuml                             14
  Diphtheroid bacillus                                                     10
  Gram-negative coccus from the mouth (1)                                  12
  Gram-negative coccus from the mouth (2)                                   0
  B. cofi [Escherichial                                                     0
  B. influenzae (Pfeiffer) 6 strains [Hemophilusl                           0
                                                           TABLE III.-MGbitory                 Power of Penicillin on Difierent                Bacteria.’
                                                                                    Dilution      of penicillin      in broth
                                                      l/f.             l/lo.       1/20.         1/46.         1/80.       l/lOO. 1/200. 1/40
    Staphylococcus       aweus                        0                0           0             0             0           0      0      0
            ,,           epidemidis                   0                0           0             0             0           0      0      0
    Pneumococcus                                      0                0           0             0             0           0      0      0
    Streptococcus        (haemolytic)                 0                0           0             0             0           0      0      0
                         viridam (mouth)              0                0           0             0             0           0
                        faecalis                      ++               ++          ++            ++            ++
    B.   antbracis [Bacillus1                         0                0                         +                         E
    B.   pseudo-tuberculosis rodentium                                              :+                         E
    B.   pullorum [Salmonella]                        :                :            ++            z                        E
    B.   dysenteriae [Shigella]                                                                   ++                       ++
    B.   coli [Ekcherichia]                           :+               z            z
    B.   typhosus [Salmonella]
    B.   pyocyaneus [Pseudomonas]                     ::               z            z
    B.   proteus [Proteus]
g   v.    cbolerae   [Vibrio]                         z                z           :z

                                                                                                               1/60.      1/120.          1/300.     1/600
         B. dipbthriae     (3 strains) [Corynebacterium]                                                       0           *              ++
         Streptococcus    pyogenes (13 strains)                                                                0          0               0          z
               ,9                                                                                              0          0               2          ++
                          fae&s         (i:   ZZ)                                                              ++
               9,                                                                                              0
                          viridans at random        from      faeces       (1 strain)
               t                *,             9,            ??
                                                                           (2 strains)                         0
                ,.              9,           99          $9                (1 strain)                          0
                ,,              $9           9,          M                 (I strain)
                ,,              9,           9,         9,                 (1 strain)                          :+
                9.              ,,                                                                             0
                                      at random     from mouth             (1 strain)
                9,              ,,           ,*         ,,                 (2 strains)                         0
                ,.              9.           *,         ,,                 (1 strain)                          0

         .LFxEND:    0 z no growth;           k = trace    of growth;          + z poor        growth;    ++        = normal    growth.
Fleming   l   The antibacterial action of cultures of a Penicikm                   191
rhal conditions are about as sensitive is necessary to inhibit completely the
as are staphylococci. Many of the culture in broth. In the weaker con-
Gram-negative cocci found in the centrations it will be seen that at first
mouth and throat are, however, quite there is growth of the staphylococci
insensitive.                                and only after some hours are the
   B. dipbtberiae [ Corynebacterium]        cocci killed off. The same thing can
is less affected than staphylococcus but be seen if a series of dilutions of pen-
is yet completely inhibited by a lYO icillin in broth are heavily infected
dilution of a fair sample of penicillin.    with staphylococcus and incubated. If
    It may be noted here that penicillin,   the cultures are examined after 4 hours
which is strongly inhibitory to many        it may be seen that growth has taken
bacteria, does not inhibit the growth       place apparently equally in all the
of the original penicillium which was tubes but when examined after being
used in its preparation.                    incubated overnight, the tubes con-
                                            taining penicillin in concentrations
         THE RATE OF KILLING OF             greater than 1 in 300 or 1 in 400 are
       STAPHYLOCOCCI    BY PENICILLIN       perfectly clear while the control tube
                                            shows a heavy growth. This is a clear
    Some bactericidal agents like the
                                            illustration of the bacteriolytic action
hypochlorites are extremely rapid in
                                            of penicillin.
their action, others like flavine or no-
varsenobillon are slow. Experiments
                                                      TOXICITY OF PENICILLIN
were made to find into which category
penicillin fell.                               The toxicity to animals of power-
    To 1 cc. volumes of dilutions in fully antibacterial mould broth filtrates
 broth of penicillin were added 10 appears to be very low. Twenty cc.
 c.mm. volumes of a I in 1000 dilution      injected intravenously into a rabbit
of a staphylococcus broth culture. The      were not more toxic than the same
 tubes were then incubated at 37’C. quantity of broth. Half a cc. injected
 and at intervals 10 c.mm. volumes intraperitoneally into a mouse weigh-
 were removed and plated with the ing about 20 gm. induced no toxic
 following result:                          symptoms. Constant irrigation of large
                                            infected surfaces in man was not ac-
                Number of colonies devel- companied by any toxic symptoms,
                  oping after sojourn in    while irrigation of the human con-
                pemcillin in concentrations
                         as under:          junctiva every hour for a day had no
                                            irritant effect.
                Control I/80 1/40 1/20 l/l0     In &TO pencillin which completely
                                            inhibits the growth of staphylococci
 Before              27 27 27 27 27         in a dilution of 1 in 600 does not inter-
After 2 hours       116 73 Sl 48 23         fere with leucocytic function to a
After 4% hours       a    13    1    2    5 greater extent than does ordinary
 After 8 hours       w     0    0    0 0    broth.
 After 12hours       m     0    0    0 0
                                            USE   OF PENICILLIN     IN THE   ISOLATION
  It appears, therefore, that penicillin
                                             OF B.   lNFLu#?Ni?AE        (PFEIFFER)
belongs to the group of slow acting
                                                     AND OTHER      ORGANISMS
antiseptics, and the staphylococci are
only completely killed after an inter-        It sometimes happens that in the
val of over 4% hours even in a con-         human body a pathogenic microbe
centration 30 or 40 times stronger than     may be difficult to isolate because it
192                                                                           CHEMOTHERAPY

occurs in association with others which              favourable   for the growth      of B. in-
grow more profusely and which mask                   fluenzae must be used, e.g. boiled
it. If in such a case the first microbe              blood agar, as by the repression of the
is insensitive to penicillin and the ob-             pneumococci      and the staphylococci
scuring microbes are sensitive, then by              the symbiotic     effect of these, so fa-
the use of this substance these latter               miliar in cultures of sputum on blood
can be inhibited while the former are                agar, is lost and if blood agar alone
allowed to develop normally. Such an                 is used the colonies of B. influenzae
example occurs in the body, certainly                may be so minute as to be easily
with I?. in@enzue        (Pfeiffer) and prob-        missed.
ably with Bordet’s whooping-cough                       From    a number        of observations
bacillus and other organisms. Pfeiffer’s             which have been made on sputum
bacillus, occurring         as it does in the        postnasal and throat swabs it seems
respiratory     tract, is usually associated         likely that by the use of penicillin,
with streptococci,         pneumococci,       sta-   organisms of the B. influence         group
phylococci       and Gram-negative         cocci.    will be isolated from a great variety
All of these, with the exception                of   of pathological     conditions  as well as
some of the Gram-negative             cocci, are     from individuals      who are apparently
highly sensitive to penicillin and by                healthy.
the addition of some of this to the
medium the           can be completely         in-                  DISCUSSION
hibited whi Y B. influenzae is unaf-
fected. A definite quantity of the pen-                 It   has been demonstrated       that a
icillin may be incorporated            with the      species of penicillium produces in cul-
molten      culture     medium      before    the    ture a very powerful antibacterial sub-
plates are made, but an easier and very              stance which affects different bacteria
satisfactory      method is to spread the            in different degrees. Speaking gener-
infected material, sputum, nasal mucus,              ally it may be said that the least sensi-
and so forth, on the plate in the usual              tive bacteria    are the Gram-negative
way and then over one half of the                    bacilli, and the most susceptible       are
plate spread 2 to 6 drops (according                 the pyogenic      cocci. Inhibitory    sub-
to potency)         of the penicillin.      This     stances have been described         in old
small amount of fluid soaks into the                 cultures of many organisms; generally
agar and after cultivation for 24 hours              the inhibition is more or less specific
it will be found that the half of the                to the microbe which has been used
plate without the penicillin will show               for the culture,      and the inhibitory
the normal growth while on the peni-                 substances are seldom strong enough
cillin treated half there will be nothing            to withstand even slight dilution with
but B. influenzae with Gram-negative                 fresh nutrient material. Penicillin is not
cocci and occasionally             some other        inhibitory   to the original penicillium
microbe. This makes it infinitely easier             used in its preparation.
to isolate these penicillin-insensitive                 Emmerich     and other workers have
organisms, and repeatedly            B. influen-     shown that old cultures       of B. pyo-
zae has been isolated in this way when               cyanem acquire a marked bacteriolytic
they have not been seen in films of                  power. The bacteriolytic      agent, pvo-
sputum      and when it has not been                 cyanase,    possesses properties    similar
possible to detect them in plates not                to penicillin in that its heat resistance
treated with penicillin.          Of course if       is the same and it exists in the filtrate
this method is adopted then a medium                 of a fluid culture, It resembles peni-
Fleming   * The antibacterial action of cultures of a Penicillium                  193
cillin also in that it acts only on certain   in this paper, and to our mycologist,
microbes. It differs however in being         Mr. la Touche, for his suggestions as
relatively extremely weak in its action       to the identity of the penicillium.
and in acting on quite different types
of bacteria. The bacilli of anthrax,                         SUMMARY
diphtheria, cholera and typhoid are
those most sensitive to pyocyanase,                1. A certain   type of penicillium
while the pyogenic cocci are unaf-            produces in culture a powerful anti-
fected, but the percentages of pyo-           bacterial substance. The antibacterial
cyaneus filtrate necessary for the in-        power of the culture reaches its maxi-
hibition of these organisms was 40, 33,       mum in about 7 days at 2O’C. and
40 and 60 respectively            (Bocchia,   after 10 days diminishes until it has
 1909). This degree of inhibition is          almost disappeared in 4 weeks.
hardly comparable with 0.2% or                    2. The best medium found for the
less of penicillin which is necessary to      production of the antibacterial sub-
completely inhibit the pyogenic cocci         stance has been ordinary nutrient
or the 1o/O  necessary for B. dipbtheriae.    broth.
   Penicillin, in regard to infections             3. The active agent is readily fil-
with sensitive microbes, appears to           terable and the name “penicillin” has
have some advantages over the well-           been given to filtrates of broth cul-
known chemical antiseptics. A good            tures of the mould.
sample will completely inhibit sta-               4. Penicillin loses most of its power
phylococci, Streptococcus pyogenes            after 10 to 14 days at room tempera-
and pneumococcus in a dilution of 1           ture but can be preserved longer by
in 800. It is therefore a more powerful       neutralization.
inhibitory agent than is carbolic acid            5. The active agent is not de-
and it can be applied to an infected          stroved by boiling for a few minutes
surface undiluted as it is non-irritant       but ‘in alkaline solution boiling for 1
and non-toxic. If applied, therefore, on      hour markedly reduces the po\ver.
a dressing, it will still be effective even   Autoclaving for 20 minutes at 115’C.
when diluted 800 times which is more          practically destrois it. It is sohtblc in
than can be said of the chemical an-          alcohol but insoluble in ether or chlo-
tiseptics in use. Experiments in con-         roform.
nection with its value in the treatment           6. The action is very marked on
of pyogenic infections are in progress.       the pyogenic cocci and the diphtheria
   In addition to its possible use in the     group of bacilli. Many bacteria are
treatment of bacterial infections pen-        quite insensitive, e.g. the coli-tvphoid
icillin is certainly useful to the bac-       group, the influenza-bacillus -group,
teriologist for its power of inhibiting       and the cnterococcus.
unwanted microbes in bacterial cul-               7. Penicillin is non-toxic to animals
tures so that penicillin insensitive          in enormous doses and is non-irritant.
bacteria can readily be isolated. A           It doses (sic) not interfere with leu-
notable instance of this is the very          cocytic function to a greater degree
easy isolation of Pfeiffer’s bacillus of      than does ordinary broth.
influenza when penicillin is used.                8. It is suggested that it mav be an
   In conclusion my thanks are due to         efficient antiseptic for application to,
my colleagues, Mr. Ridley and Mr.             or injection into, areas infected with
Craddock, for their help in carrying          penicillin-sensitive microbes.
out some of the experiments described             9. The use of penicillin on culture
194                                                                   CHEMOTHERAPY
plates renders obvious many bacterial           IO. Its value as an aid to the isola-
inhibitions which are not very evident       tion of B. influenzae has been demon-
in ordinary cultures.                        strated.

   The history of the development       of   of penicillin or less, while current com-
penicillin is now quite well known. This     mercial production       gives yields of at
paper presents the first scientific obser-   least 5000 pg./ml. But because of the high
vations of this substance.                   activity of penicillin against certain bac-
   Many earlier workers had noted the        teria, he was able to study the effects of
effects of various microorganisms       on   these crude broths. He showed that not
pathogenic bacteria. Fleming was the first   all bacteria were affected equally by pen-
to actually develop a clear conception of    icillin, indicating a high degree of spe-
what this action was due to. It is obvi-     cificity in its action. He further described
ous that he considers penicillin to be a     the bactericidal and bacteriolytic action
substance, and although he does not say      of the antibiotic and showed that a period
this in so many words, he considered it      of growth was needed before either ac-
to be a chemical compound with a defi-       tion was manifest. We know now that
nite structure.   He presented sufficient    this is because penicillin specifically in-
basic information so that later workers      hibits the synthesis of the cell walls of
could develop penicillin into a practical    sensitive bacteria. This means that as they
substance.                                   continue to grow without synthesizing
    He noted that this substance was pro-    cell walls, they are killed or lysed be-
duced only by a specific species of mold.    cause they become osmotically fragile.
This observation is important because it        Fleming perceived only dimly the
showed that the action of the mold broth     practical applications of penicillin. He
filtrate was not due to some nonspecific     obviously was aware that such a sub-
effect, such as a change of PH during        stance would be highly useful if it would
growth.                                      kill pathogenic bacteria in humans, but
   He devised a crude assay for deter-       such a hope seemed much in the future
mining the potency of the substance.         in view of the low broth potencies he
With this assay he was able to study the     was obtaining. It took the development
stability and certain chemical properties    of the sulfa drugs (see page 195) and
of penicillin. He was able to follow its     the impetus of World War II before
production in culture. By current stand-     the tremendous technological and medi-
ards of penicillin production, Fleming’s     cal problems could be adequately solved
material was of very low potency. His        which led to the development of peni-
broths probably had around IO pg./ml.        cillin as a practical chemotherapeutant.

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