SUMMARY:Recently isolated virulent strains of Haemophilus pertussis grow well in a partially defined medium, but are more exacting in their growth requirements and conditions of growth than avirulent strains. The optimal pH for growth is 7-6-7.8. Mechanical agitation by stirring, spinning or aeration with either nitrogen or air, inhibits growth. Gaseous conditions, however, are critical in that there is an optimal relationship of surface area to volume of medium. Mechanical agitation and aeration stimulate the growth of avirulent strains.Virulent strains require starch for growth, and there is a close relationship between virulence, specific agglutinability and starch requirement. Avirulent strains with low specific agglutinability grow readily in the absence of starch.When starch in a standard medium is replaced by amylose, there is an increase in the final amount of growth. Amylopectin, glycogen and dextrans are about onethird as effective as starch; other carbohydrates, gums and inorganic adsorbents support little growth. Charcoal can replace starch, but has only c. 70% of its effect.The amino-acid requirements of H . pertussis are satisfied by acid-hydrolysate of casein, the optimal concentration being 7 yo. All strains utilize aspartic and glutamic acids, serine, threonine, glycine, alanine and proline, but the rate of utilization is greater with virulent than with avirulent strains. For good growth yeast extract is needed but can be replaced by nicotinamide, nicotinic acid or cozymase, provided sulphur-containing amino-acid is also present. Virulent strains of H.pertussis require a sulphur-containing acid for growth, which may be supplied by yeast extract. Cysteine, cystine or glutathione, but not methionine, act as sources of essential sulphur.For many years the optimal medium for the cultivation of Haenwphilus pertussis has been the solid medium originally recommended by Bordet & Gengou (1906). Strains grown on this medium maintain their biological properties unaltered for varying lengths of time. As the preparation of this medium necessitates the addition of either human or animal blood, Hornibrook attempted to replace it by a fluid medium (Hornibrook, 1939). Cohen & Wheeler (1945) modified Hornibrook's medium by adding copper and ferrous sulphates and increased the amounts of potassium phosphate, magnesium chloride and soluble starch. We used Cohen & Wheeler's modification as a basal medium, having first confirmed that the organism grew well in it.Our investigation was primarily intended to establish whether liquid partially defined culture media would permit growth of organisms suitable for vaccine production, that is to say, of bacilli that maintained the antigenic properties characteristic of a freshly isolated strain. It was considered probable that the physical and nutritional requirements of the organism would need careful control under the particular conditions of growth in the medium. The purpose of this communication is to report a detailed study of these requirements and of 346J . Ungar and...

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SUMMARY: A bacteriological and histological study was made of the sequence of events in unprotected and protected mice, after intracranial challenge with Bordetella pertussis. No difference in the results was obtained whether mice were injected through the parietal bone or through the foramen magnum. Injected material was distributed throughout the subarachnoid space and ventricles. In unprotected mice organisms multiplied continuously until death. Bacterial multiplication was limited to the cranial cavity, in which it was practically confined to the ciliated layer over the ependyma. There was no invasion of the brain substance. The histological changes in the brain included purulent meningitis, choroiditis, polymorphonuclear infiltration of perivascular spaces and cerebral substance, haemorrhages, hydrocephalus and necrosis of nerve cells. In protected mice there was a brief initial multiplication of organisms and then their rapid elimination. Histologically the brains of immune mice after challenge were characterized by an intense mononuclear-cell response, which persisted for 2-3 weeks, subsiding slowly during the following 10 weeks.

SUMMARY: Of 46 strains of Haemophilus pertussis, 34 recently isolated strains were all agglutinated by Phase 1 antiserum. Of 12 laboratory strains, 5 had lost their agglutinability. The agglutinable strains were precipitated by aluminium phosphate, and lysed by sodium hydroxide or 10% sodium desoxycholate and were virulent to mice. The virulent strains, which were precipitable by aluminium phosphate, produced toxic substances in a fluid culture. Precipitation by aluminium phosphate provides a quick method for distinguishing virulent and avirulent strains of H. pertussis.Suspensions of strains of Haemophilus pertussis that are agglutinated by a Phase 1 antiserum have been found also to be completely precipitated with aluminium phosphate, whereas strains that become non-agglutinable by the Phase 1 antiserum also lose this precipitability (Ungar & Muggleton, 1948). The object of the present communication is to show the relationship between precipitability, agglutinability and certain other biological properties of different strains of H . pertussis, either freshly isolated from recent cases of pertussis or maintained in the laboratory for some time.It has long been known that strains of H . pertussis maintained in artificial culture can pass through a series of serologically differentiable phases. In our study no attempt was made to classify the strains used into phases, except that we assigned to Phase 1 all strains that were agglutinated to a high titre (over 1/8000) with a rabbit antiserum prepared by inoculation with a freshly isolated strain.Altogether we tested 46 strains, of which 34 were received, immediately after isolation, from the Central Public Health Laboratory of the Public Health Laboratory Service or from the North Western Group Laboratory of the L.C.C., and one from Prof. G. A. H. Buttle of the School of Pharmacy, University of London. The remainder were stock strains maintained in this laboratory for various periods; of these, six had been repeatedly subcultured on BordetGengou medium and were no longer agglutinable with the Phase 1 antiserum and the rest had been freeze-dried and stored. All the strains were kept freezedried during the investigation and frequent culture from the freeze-dried state was made to ensure that the strains used in the tests remained constant.Aluminium phosphate precipitation of the strains A standard method of testing the precipitability of the strains was used throughout, The suspensions from 48 hr. Bordet-Gengou cultures were washed twice, resuspended in saline containing 0.2% formaldehyde, to give a concentration of c. lolo organisms/ml., and held at room temperature overnight.

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