STUDIES WITH H.

PERTUSSIS'
II. MAINTENANCE OF CULTURES IN PHASE I

Received for publication June 28, 1939

Shackell's (1909) method of desiccation from the frozen state

was applied first by Hammer (1911) to the preservation of viable
bacteria. Rogers (1914), with an improved technique, extended
this work on a larger scale to lactic-acid-forming bacilli. Swift
(1921) applied the same principle of desiccation in the maintenance of streptococci and pneumococci in the original state of
virulence, and similarly Elser, Thomas and Steffen (1935) were
able to maintain a wide range of organisms including the meningococcus and gonococcus, in viable state, over a period of many
years.
More recently, by use of the Lyophile apparatus (Flosdorf
and Mudd, 1935), Siler and his associates (1936) at the Army
Medical School have maintained their cultures of S-58 virulent
Eberthella typhosa without dissociation over a period of years.
Freshly-opened containers of Lyophile organisms are used
routinely in preparation of mass cultures for vaccine preparation.
Similarly, Welch, Borman, and Mickle (1939) have used the
Lyophile apparatus to maintain cultures of Klebsiella pneumoniae
in unaltered form.
In the case of Hemophilus pertussis, workers generally rely on
fresh isolations to insure that their cultures are in Phase I (Leslie
and Gardner's terminology, 1931). This is the practice recommended for preparation of vaccines by the Referee on Methods
and Reagents for the Diagnosis and Control of Whooping Cough
1 This work has been aided by a grant from the United States Public Health
Service.
256

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E. W. FLOSDORF AND ANNE C. KIMBALL

Department of Bacteriology, University of Pennsylvania Medical School

256

E. W. FLOSDORF

AND ANNE C. KIMBALL

METHOD

We have used the Cryochem apparatus for carrying out desiccation from the frozen state (Flosdorf and Mudd, 1938). Properly identified cultures isolated from a cough plate are transferred to a test tube slant of Bordet-Gengou medium containing
fresh defibrinated blood in appropriate amount. These are
harvested by scraping into saline or milk. If in saline, the suspension is added to an equal volume of sterile skimmed milk.
This mixture is then distributed in 0.05 ml. amounts into small
vials not carrying rubber stoppers and dried according to a procedure previously described (Flosdorf and Mudd, 1935, 1938).
With these small volumes, it is advisable to freeze the suspension
of bacteria in milk by placing a freezing mixture around the containers after attachment to the manifold of the Cryochem apparatus. By using larger containers, with 0.5 ml. or more of
material in each, the cryochemic degassing and self-freezing procedure may be used. In such case, it is not necessary to place a
greater number of organisms in the containers, but only to use
more milk in order to provide more liquid for evaporation during
self-freezing. The Lyophile apparatus likewise may be used;
the advantages of the Cryochem procedure lie mostly in economy

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for the Standard Methods Committee on Diagnostic Procedures

and Reagents (American Public Health Association Year Book,
1935-1936). In the present report we are able to demonstrate
that cultures of H. pertussis may be preserved in Phase I for
extended periods by desiccation from the frozen state. By this
means several advantages may be gained. In systematic studies
the same strains may be used over a period of years. Although
H. pertussis has been generally considered to be of a single type,
the recent reports of a Para form (Eldering and Kendrick, 1937,
1938), or an atypical type (Bradford and Slavin, 1937), indicate
that such differences must be guarded against. Similarly, differences in hemolytic activity are found. Furthermore, continual
isolation is laborious and at times a sufficient number of cases
of whooping cough in the proper stage forisolationof the organisms
are difficult to find.

STUDIES WITH H. PERTUSSIS

257

TESTING FOR PHASE I CHARACTERISTICS

The cultures have been found to have all the original characteristics of Phase I with respect to colonyform, hemolytic activity, morphology, toxicity, and agglutination. We have tested
these properties of cultures grown from organisms maintained in
the Cryochem-dried form for over two years. The serological

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and convenience of operation. Similarly, large glass vacuum

desiccators may be used for the Cryochem procedure with the
containers attached by glass tubes passing through a rubber
stopper in the lid (Flosdorf, Boerner, Lukens and Ambler, 1940).
However, the limited capacity and the inconvenience and danger
of such apparatus makes questionable the advisability of its use
routinely, particularly on a large scale.
The desiccation is allowed to continue overnight and then the
containers are hermetically sealed by fusion of the glass with a
flame. Although we have always evacuated the containers prior
to sealing, this may be unnecessary. In the case of guinea-pig
complement, hermetically sealing the product in a container
filled with air has proven to be quite satisfactory (Flosdorf,
Boerner, Lukens and Ambler, 1940). Furthermore, the work
of Swift (1921) would indicate that air in contact with at least
certain organisms yields satisfactory results. With certain types
of equipment, however, sealing in vacuo makes it simpler to maintain sterility.
The sealed containers have been stored at 5 to 80C. When a
fresh culture is desired, a container of the desiccated organisms is
opened. After scratching the glass with a file, the container, for
breaking, is wrapped under a cloth impregnated with antiseptic
to prevent the spreading of dry organisms as air rushes in. Excess antiseptic should be squeezed out of the cloth, however, to
avoid sucking the liquid into the container when the vacuum is
broken. Sterile water is then added. With a small loop,
Bordet-Gengou medium in a petri dish is plated in the usual
fashion. In this first culture generation 72 to 96 hours may be
required to obtain satisfactory colonies. Colonies may then be
picked from the plate and transfers made in the usual fashion.

258

E. W. FLOSDORF AND ANNE C. KIMBALL

TABLE 1
Agglutination with once-absorbed Phase I immune rabbit serum
AGGLUTINATION TEST-FINAL SERUM DILUTIONS
ABSORBING

TEST ANTIGEN

ANTIGEN

1:1600 1:3200 1:6400

1:100

1:200

1:400

1:800

tr.

Phase IV

iso- 4
lated Phase I
tr.
Phase IV

None

Freshly

tr.

"Cryochem
Phase I"
"Cryochem
Phase I"
Phase IV

None

Freshly

isolated Phase I
Phase IV

Freshly

isolated Phase I
Phase IV

effect of the incubation, if any, on the titer of the serum. The

effect was negligible.
If the Cryochem-dried organisms had become degraded and
had lost their Phase I antigenic structure as a result of long storage
in dry form, it should have been made evident by lack of absorption of the agglutinins from the known Phase I antiserum.
Included in Table I are recorded the results of a control test in
which a known degraded-Phase strain which grows luxuriantly
on plain or glycerol-agar was used in an attempt to absorb the
Phase I antibody. The agglutination titer against Phase I
organisms was not thereby reduced, indicating complete specifi-

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properties have been tested by using the organisms cultured from

Cryochem-preparations to absorb antibodies from serum of
rabbits immunized with freshly isolated organisms and then
testing the absorbed serum in agglutination with freshly isolated
strains as the test antigen. For absorption of agglutinins, 7 ml.
of a 1:10 dilution of the Phase I immune serum were incubated 30
minutes at 370C. and overnight at 6-8 with 1.0 ml. of packed
organisms suspended in 2 ml. saline. In table 1 are recorded the
results of a typical experiment. As a control experiment, the
serum was treated identically as in absorption to determine the

STUDIES WITH H. PERTUSSIS

259

DISCUSSION

The fundamental importance in antibacterial immunity of the

combination of agglutinins with surface antigens of bacteria is
well established. With non-flagellated organisms the use of such
surface reactions as agglutination or phagocytosis therefore provides distinguishing methods of assay for effective surface reactants in either serum or antigen. Complement fixation, and
precipitin testing with soluble antigens, are of diagnostic value
but do not distinguish surface antigen-antibody combination
from phenomena involving other antigens. Consequently we
believe the demonstration by agglutinin-absorption of the presence of the Phase I antigenic cellular surface in Cryochem-dried
organisms to be the crucial test for the maintenance of pertinent
Phase I serological characteristics.
Since Cryochem-dried H. pertussis may be maintained without
essential alteration of serological and other properties, use of
such a drying procedure, as in the case of other organisms,
would appear to be justified. We are using this method in our
current studies on the antigenic surface-structure of H. pertussis
(Flosdorf, Kimball, Chambers, 1939) and feel confident that over
a period of years we shall be dealing with an organism of essentially constant properties.
2Since going to press, Miller, J. J., and Silverberg, R. J., in an excellent
paper, "Agglutinative Reaction in Relation to Pertussis and to Prophylactic Vaccination against Pertussis with Description of a New Technic," J.

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city of the Phase I agglutinin absorption. Even two additional

absorptions with fresh portions of degraded-Phase organisms,
incubating with each additional portion exactly as previously, did
not reduce the agglutination titer against Phase I organisms.
The cross agglutination titer of the Phase I serum against the
degraded-Phase organisms, however, was virtually eliminated in
a single absorption with degraded-Phase organisms, and was
completely removed in the three absorptions. Absorption of the
cross agglutination against degraded-Phase organisms was not
quite complete in a single absorption with the Phase I organisms
but was complete after two absorptions.

260

E. W. FLOSDORF AND ANNE C. KIMBATL

The authors wish to express their appreciation to Dr. Stuart

Mudd for his helpful suggestions.
SUMMARY

REFERENCES

American Public Health Association Year Book. 1935-1936 Tentative methods

for the Bacteriological Diagnosis and Control of Whooping Cough.
Kendrick, P., Referee, Miller, J. J. and Lawson, G. MacL. Assoc.
Referee, 200-206.
BRADFORD, W. L., AND SLAVIN, B. 1937 An organism resembling Hemophilus
pertussis. Am. J. Pub. Health, 27, 1277-1282.
ELDERING, G., AND KENDRICK, P. 1937 A group of cultures resembling both
Bacillus pertussis and Bacillus Bronchisepticus but identical with
neither. J. Bact., 33, 71.
ELDERING, G., AND KENDICK, P. 1938 Bacillus para-pertussis. A species
resembling both Bacillus pertussis and Bacillus bronchisepticus but
identical with neither. J. Bact., 35, 561-572.
ELSER, W. J., THOMAS, R. A., AND STEFFEN, G. I. 1935 Desiccation of sera and
other biological products (including microorganisms) in frozen state
with preservation of original qualities of products so treated. J.
Immunol., 28, 433-473.
FLOSDORF, E. W., BOERNER, F., LUKENS, M., AND AMBLER, T. 1940 Cryochempreserved complement of guinea pig serum. Am. J. Clin. Path., in
press.
FLOSDORF, E. W., KIMBALL, A. C., AND CHAMBERS, L. A. 1939 Studies on H.
pertussis. I. Liberation by sonic vibration of a soluble component
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FLOSDORF, E. W., AND MUDD, S. 1935 Procedure and apparatus for preservation
in "lyophile" form of serum and other biological substances. J.
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FLOSDORF, E. W., AND MUIDD, S. 1938 An improved procedure and apparatus
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HAMMER, B. W. 1911 A note on the vacuum desiccation of bacteria. J. Med.
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LESLIE, P. H., AND GARDNER, A. D. 1931 The phases of Haemophilus pertussis.
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Immunol., 1939, 37, 207-222, have mentioned that they have used the Lyophile
apparatus for keeping their strains. Their data in support of maintenance of
Phase I characteristics, however, were not included.

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It has been demonstrated that desiccation from the frozen

state may be used to preserve the characteristics of Hemophilus
pertussis in Phase I. Antigenic properties have been tested by
agglutinin absorption and found to be characteristic of Phase I.

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ROGERS, L. A. 1914 The preparation of dried cultures. J. Infectious Diseases,

14, 100-123.
SHACKELL, L. F. 1909 An improved method of desiccation, with some applications to biological problems. Am. J. Physiol., 24, 325-340.
SILER, J. F. and the Laboratory staff of the Army Medical School. 1936 Typhoid vaccine studies. Investigation of virulence and antigenic
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SwirT, H. F. 1921 Preservation of stock cultures of bacteria by freezing and
drying. J. Exptl. Med., 33, 69-75.
WELCH, H., BORMAN, E. K., AND MICKLE, F. L. 1939 Preparation and analysis
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