ALTHOUGH bacterial invasion of the developing ovum is known to occur (Haines, 1939), it is fairly clear that the microbial decomposition of eggs encountered in commercial handling is due, in the main, to the invasion of the egg by spoilage-producing micro-organisms after laying. This in turn depends on (a) the inherent porosity of the shell, and (b) the treatment the egg receives, e.g. washing. The present work is an attempt to gain information on these two factors.THE POROSITY OF THE SHELLThe porosity of the shell may be studied (1) by histological methods and (2) by measuring the rate of movement of liquids or gases through the shell under a given pressure gradient. Difficulties have been met in applying both these techniques. The first has given a useful picture not, however, amenable to quantitative interpretation, whilst the second has shown that porosity is a relative term, varying at different points on the shell and in successive eggs from the same hen.(1) Histology P1. IX, fig. 1 is a transverse section ( x 120), normal to the surface, of the eggshell. Four layers can be distinguished. These are: first, the cuticle, composed mainly of fibres of mucin (Moran & Hale, 1936); secondly, the spongy layer consisting of crystals of calcite more or less normal to the cuticle; thirdly, the mammillary layer, also consisting of crystals of calcite, which however do not appear to be definitely orientated; and fourthly, the inner shell membrane.P1. IX, fig. 2 shows a section of a shell, again normal to the surface ( x 100), in which pores or V-shaped openings can be seen stretching from the outside (cuticle not present) to the mammillary layer. It is evident that these pores do not pass right through the shell as has been claimed by Marshall & Cruickshank (1938). The diameter of one of these openings was 13 ,u at the top, 6 , at the bottom. P1. IX, fig. 3 is a section ( x 75), cut parallel with the surface of the shell, in the third or mammillary layer, near the inner shell membrane. The extreme irregularity of the structure is shown, with spaces of various sizes between the calcite crystals which possibly form canals or pores leading to the interior of the egg.

1. Examination of several hundred eggs suggests that a high proportion (98%) of the whites of fresh eggs, and a slightly smaller proportion of the yolks (93%), are sterile.2. The shell of the egg is heavily infected with a heterogeneous flora, includingProteusandPseudomonasbacteria capable of producing rotting.3. The rots found in imported New Zealand and Australian eggs, and in English stored eggs, may be grouped into black rot, red rot, green rot, and a miscellaneous group.4. Black rot is brought about chiefly by strains ofProteus, but some species ofPseudomonascause some blackening. Red and green rots are due to infection with particular strains ofPseudomonas.5. A “fishy” odour is developed during the multiplication of certain atypical coliform organisms in the white, and a strong “cabbage-water” smell is often found after the growth ofPseudomonasspecies.6. Washing eggs under clean conditions has no effect on immediate bacterial penetration. Washing removes a protective coating so that if the eggs are subsequently soaked in a bacterial suspension, much more penetration of bacteria occurs than with untreated controls.7. Detailed descriptions of the coliform andProteusorganisms isolated are given. It is shown that the strains ofProteusfrom the eggs here investigated are antigenically not related toP. melanovogenesfound by Miles and Hainan to be the cause of black rot in South African eggs.8. It does not at present seem possible to assign specific names to the organisms isolated. The utilization of carbon sources by the species ofPseudomonasobtained from eggs, and by certain stock strains, and the possibility of a grouping on that basis, is discussed.

Data are given showing the concentration of pure ozone required to inhibit the growth of, and to destroy, various micro-organisms when growing on agar, in nutrient broth, a synthetic medium, or simply suspended in water. The following is shown:1. Different organisms vary in their susceptibility towards this gas. Achromobacter and Pseudomonas strains, such as occur on chilled meat, are the most resistant. On the whole, mould fungi are about as susceptible as bacteria.2. Very much higher concentrations are required to arrest established growth than can be used if inoculation and admission of inhibitor are coincident.3. Lower concentrations are inhibitory at lower temperatures. These results are ascribed to dissipation of the ozone by combination with products of bacterial metabolism. It seems that any factor which diminishes growth will augment the effectiveness of ozone.4. Comparatively small concentrations (less than 10 p.p.m.) suffice to destroy bacteria suspended in water. Somewhat larger concentrations inhibit growth in a synthetic medium.5. Still larger quantities (several hundred p.p.m.) are required in nutrient broth.6. Nutrient broth treated with ozone will support little or no bacterial growth, due to change of pH, growth taking place slowly if the pH is restored to its original value. Inhibition of growth in such a medium is therefore a complex process depending in part upon the secondary effects of decomposition of the medium.7. To kill them, bacteria require still more ozone (in some cases several thousand p.p.m.) if growing on agar. These results are ascribed to combination of ozone with the supporting medium.8. When applied to organisms growing on food, combination with ozone not only spoils the food, but it makes it difficult to interpret the effect on the microbes in terms of the ozone applied.9. The inhibitory concentrations are higher than humans can tolerate.10. Ozone destroys the dehydrogenating enzymes of the cell, and it is suggested that its germicidal action may be partly due to interference with cellular respiration.The work described in this paper was carried out as part of the programme of the Food Investigation Organization of the Department of Scientific and Industrial Research.

1. Examination of the “slime” obtained on lean beef stored at temperatures just above zero Centigrade has shown it to be composed chiefly of organisms of the Achromobacter group.2. A study of about 120 strains of these organisms has been carried out and the characteristics of typical strains are given in detail.3. Data of the rate of growth of these organisms on meat and in broth are given.4. The dependence of the “storage life” of the meat on the initial bacterial load is shown.

IN a previous communication [Haines, 1931] it was shown that bacterial proteases are produced by organisms growing in simple synthetic media made up from carefully purified substances, and the influence of salts of calcium and magnesium on growth and protease formation was discussed. The methods used to estimate protease formation, namely, liquefaction of gelatin as evidenced by inability to set in ice-water and lack of coagulation of caseinogen on addition of acid, were however too crude to yield quantitative data as to the relative efficiency of the media in stimulating or inhibiting enzyme formation. In particular, it is to be expected that small amounts of gelatinase will be formed on certain media, insufficient in quantity to cause entire liquefaction, which will escape detection by such a method. More sensitive ways of measurement were therefore sought, and changes in viscosity of a mixture of gelatin and enzyme seemed to offer a ready means of following gelatinase action.Manning [1924] showed that the viscosity of 5 % gelatin could be measured in the ordinary way in an Ostwald viscometer provided that the determinations were carried out at 350 or above. Below 350 the gelatin is in gel form and possesses appreciable rigidity, but at 35°and above it is a sol. The shape of the viscosity-time curve also depends on the previous treatment of the gelatin, but is reproducible if the gelatin be made up under standard conditions. EXPERIMENTAL.The organisms used were freshly isolated from dung. In the previous work [1931] the strains of Proteus obtained would not grow in synthetic media. One strain has subsequently been isolated, however, which grew well, and the experiments described in this paper have been conducted with it and a strain of Pseudomonas. The characteristics of these organisms are listed in Appendix I. In general the organisms were grown on nutrient agar, PH 7 4, overnight at 200, the surface growth was scraped off, and washed and centrifuged in sterile saline three times. 1 cc. of the final suspension was used for inoculation purposes. The routine technique was the same as described in the previous paper, all substances being purified in a similar manner and carefully cleaned