THE DETERMINATION OF COLI-AEROGENES ORGANISMS IN RAW MILK BY SURFACE SMEARS ON DRIED EOSIN METHYLENE BLUE AGAR PLATES

SUMMARY: The determination of the coli-aerogenes content of raw milk by smearing 0·1 or 0·2 ml of suitable dilutions on dried Levine's eosin methylene blue agar plates and incubating at 37° or 30°, was found too unselective as a routine technique. Only about two-thirds of the colonies showing the characteristic appearance of coli-aerogenes bacteria were confirmed as such by the formation of acid and gas in MacConkey's broth. Appreciable proportions of the numerous rose coloured colonies and of the very small dark colonies with metallic sheen, which are not considered to be coliaerogenes bacteria, formed acid and gas in MacConkey's broth.     

THE BACTERIAL CONTENT OF FARM DAIRY EQUIPMENT

SUMMARY: The results obtained during the examination of 2,310 advisory rinses of farm dairy equipment by means of colony count on Yeastrel milk agar at 30°, coli-aerogenes test at 30° and milk spoilage organisms (MSO) test at 22°, are discussed in relation to attainment of proposed satisfactory colony count levels. A much higher proportion of rinses of milking machine clusters gave unsatisfactory results than those of metal equipment. The MSO test (3 days, 22°) was a more sensitive indicator of the presence of milk spoilage organisms than the coli-aerogenes test, and is recommended for routine use. Results for milking machine clusters sterilized with steam were much better than those for clusters claimed to have been cleansed by chemical methods.     

COLI-AEROGENES BACTERIA FROM FARM DAIRY EQUIPMENT AND RAW MILK

SUMMARY: A series of 1,040 cultures of coli-aerogenes bacteria isolated at 30° from farm dairy equipment and raw milk was classified according to the recommendations of the Coliform Sub-Committee of the Society for Applied Bacteriology (Report, 1949). The distribution of species and types isolated from milk did not differ materially from that for dairy equipment, Klebsiella cloacae, K. aerogenes and Citrobacter freundii being the dominant species, while the incidence of Escherichia coli was relatively low. There was no marked seasonal incidence of any type, though E. coli I formed a slightly higher proportion of the coli-aerogenes microflora in winter, whereas K. aerogenes I was slightly higher in summer. Many of the cultures of the dominant types, K. cloacae, K. aerogenes I and Cit. freundii I, did not form acid and gas from lactose in two days at 37°. Consequently 37° negative, anaerogenic and paracolon strains constituted a relatively high proporton (49%) of the coli-aerogenes microflora.     

A COMPARISON OF 30° AND 37° AS INCUBATION TEMPERATURES IN THE PRESUMPTIVE COLIAEROGENES TEST FOR RAW AND PASTEURIZED MILK

SUMMARY: Incubation at 30° and 37° for the presumptive coli-aerogenes test for raw and pasterurized milk has been investigated. There were more positives at the lower temperature and it is suggested that in this test, incubation at 30° might provide a much better guide to the hygienic quality of both raw and pasterurized milk. The ability of the coli-aerogenes bacteria studied to ferment lactose in MacConkey's broth at 30° but not at 37° was found to be a stable factor which was unchanged by prolonged storage on agar slopes at room temperature or on continued incubation in MacConkey's broth at temperatures above the optimum for lactose fermentation.     

THE DETERMINATION OF THE COLI-AEROGENES CONTENT OF MILK AND DAIRY EQUIPMENT BY PLATING ON VIOLET RED BILE AGAR INCUBATED AT 30°

SUMMARY: Violet red bile agar (VRB) incubated at 30° for 20–24 hr was as good an indicator of coli-aerogenes bacteria in milk as MacConkey's broth. A high proportion (82%) of the large, deep red colonies considered to be formed by coli-aerogenes bacteria were confirmed as such. A British brand of dehydrated VRB agar was as suitable as an American brand of this medium for determining the coli-aerogenes content of milk. All the strains of typical coli-aerogenes bacteria tested formed red colonies. In a small proportion of cases the diameter was less than 0·5 mm. The only other milk bacteria which formed colonies resembling those of coli-aerogenes organisms were some acid forming strains of Gram-negative rods. Coli-aerogenes bacteria, determined on VRB agar at 30°, generally constituted only a small proportion of the microflora of fresh raw milk and of farm dairy equipment.     

THE MULTIPLICATION OF COLI-AEROGENES BACTERIA IN MILK STORED AT 3–5°

SUMMARY: When 108 samples of individual farm milk supplies were held at 3—5° for 72 hr, 35·2% showed increases in coli-aerogenes organisms of over one-hundredfold and 10·2% increases of more than one-thousandfold. The coli-aerogenes microflora after refrigeration was dominated by Klebsiella cloacae and K. aerogenes I. While some strains of all the coli-aerogenes types isolated showed growth, though sometimes scanty, on yeast-dextrose agar in 5 days at 3—5°, the majority of the strains of K. cloacae showed luxuriant growth under these conditions and can be considered as typical facultative psychrophiles of milk.     

COLI-AEROGENES BACTERIA IN FARM WATER SUPPLIES

SUMMARY: A series of 825 cultures of coli-aerogenes bacteria isolated at 30° and a series of 735 cultures isolated at 37° from 645 samples of farm water supplies were classified according to the recommendations of the Coliform Sub-Committee of the Society for Applied Bacteriology (Report, 1949). Klebsiella constituted 50% of the cultures isolated at 30°, whereas Escherichia coli I was the dominant type, forming 57%, among the cultures isolated at 37°. It would thus appear that isolation at 30° is as selective for Klebsiella as isolation at 37° is for Escherichia . Coli-aerogenes organisms, mainly 37° negative strains of Citrobacter freundii I and K. cloacae , were found in waters of high sanitary quality derived from protected springs and wells; but the coli-aerogenes microflora of polluted water was dominated by E. coli I, which formed 43% of the isolates at 30° and 76% of those at 37°. The results for a series of fortnightly samples from 11 farm water supplies showed a marked seasonal variation in the incidence of different types isolated at 30°; E. coli I formed a higher proportion in summer than in winter, while 37° negative strains of Klebsiella and Citrobacter formed a higher proportion in winter than in summer.     

ADVISORY MICROBIOLOGICAL STANDARDS AND TOLERANCES FOR RAW MILK

SUMMARY: To study the value of recent modifications of microbiological tests used for advisory purposes, samples of tuberculin tested milk, taken at weekly intervals over a 5 year period from the Trawscoed Experimental Husbandry Farm and selected at random during a 12 months period from farms in Wales, were examined by a temperature compensated keeping quality test at 22°, colony count on Yeastrel milk agar in 3 days at 30° and coli-aerogenes colony count on violet red bile agar in 20–24 hr at 30°.
The results show that milk produced and handled under hygienic conditions can be expected to have colony counts of less than 2 × 10⁴/ml and coli-aerogenes colony counts less than 10/ml when examined within 18 hr of milking.     

FURTHER OBSERVATIONS ON THE BACTERIAL CONTENT OF WATER IN WATERCRESS BEDS

SUMMARY: Observations on two watercress beds in Kent have confirmed previous findings (Jones & Baker, 1955) that even in comparatively well-protected beds the coli-aerogenes content of the water may be high in summer. They have also shown that in spite of circumstances which would lead one to expect pollution, as in the case of one bed, the coli-aerogenes content can be low in winter. The season appears to influence the content in the water of coli-aerogenes bacteria, pectate liquefying bacteria and those capable of growing at 37°, but in the case of those growing at 22° other factors can play a more important part, as higher counts were not uncommon in winter. Seasonal effects were less marked with inlet samples; they were less influenced by atmospheric temperature, and little extraneous contamination occurred. Numbers increased as the sampling point moved from inlet to outlet.     

THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.     

THE BACTERIOLOGY OF FARM WATER SUPPLIES: A STUDY OF THE COLONY COUNT IN 72 HOURS AT 22°

SUMMARY: The colony count at 22° of farm water supplies from springs and wells was mainly composed of biochemically inactive, non-pigmented, Gram-negative rods. Water from a stream polluted with farmyard sewage showed a similar dominance of Gram-negative rods, but orange or yellow pigmented colonies were more abundant. There were few 37° positive coli-aerogenes bacteria in either the farm water supplies or the sewage polluted stream, and Bact. coli type I was rare.
A high proportion of the bacteria from farm water supplies fermented milk in 3 days at 22°; a third developed acid, 15% proteolysis and 6.4% ropiness.
Contamination of pure spring water with surface soil from a heavily grazed pasture resulted in a hundredfold increase in colony count with aerobic sporing rods replacing Gram-negative rods as the dominant organisms, but coli-aerogenes bacteria were absent.     

COLD-TOLERANT FERMENTATIVE GRAM-NEGATIVE ORGANISMS FROM MEAT AND OTHER SOURCES

SUMMARY: From various chilled meats, twenty-eight strains of coli-aerogenes bacteria and one Aeromonas were isolated which grew well at +1±5° and some at −1±5°. The optimum growth temperature for most of these strains was nearer 37° than 30°. Nine strains (including the Aeromonas ) fermented lactose rapidly, the remainder slowly or not at all. All the strains which fermented lactose rapidly with the production of gas gave positive presumptive coli-aerogenes tests in MacConkey's broth at 30°, but only five were positive at 37°; none was positive at 44°. Because such organisms can attain populations of millions/cm², they could confuse the interpretation of presumptive coli-aerogenes tests made on chilled meat.     

The Microflora of Hand Washed Milk Bottles

S ummary . The examination of a series of 713 milk bottles cleansed by hand washing at producer-retailer farm dairies showed that though nearly 60% attained satisfactory bacteriological standards, about 30% gave high colony counts (>600/bottle), while coli-aerogenes organisms were found in 17% and milk spoilage organisms in 25% of them. The microflora of efficiently cleansed bottles, with colony counts of <200/bottle, was dominated by micrococci and aerobic sporeforming rods. Only 3.7% of the 259 cultures from these bottles gave acid reactions in litmus milk in 72 h at 22°. Inefficiently cleansed bottles, with colony counts of >600/bottle, had quite a different type of microflora which was usually dominated by Gram negative rods (achromobacteria, nonfluorescent pseudomonads and flavobacteria). A much higher proportion (19%) of the 393 cultures from these bottles gave acid reactions in litmus milk.     

Coli-aerogenes Bacteria on Plants

S ummary . Specimens of plant foliage and flowers collected in the region of Attica from various situations and geographical regions were examined over a period of 13 months for the presence of coli-aerogenes bacteria able to grow at 37°. Twenty-five (12.2%) of 204 specimens carried coli-aerogenes bacteria of which Escherichia coli type I was isolated from 12 (5.8%). Coli-aerogenes bacteria were seldom isolated from plants grown in uninhabited areas, and they occured more frequently during summer and autumn.
Vegetables and fruits sold in the central market of Athens are frequently contaminated with coli-aerogenes bacteria. Of 76 specimens of vegetables examined 66 (86.8%) were positive for coli-aerogenes bacteria, and 17 (22.3%) for E. coli type I. The figures from 41 specimens of fruits were 26 (65.8%) and 4 (9.7%), respectively.     

Summer quiescent behaviour of the millipede Ommatoiulus moreleti (Julida: Julidae)

The millipede Ommatoiulus moreleti (Lucas), (Julidae) originates in the south-west Iberian Peninsula, and has successfully invaded parts of southern Australia with hot, dry summers. The species is inactive in the field during summer but is active in autumn, particularly following rains, and in spring. There is some activity in winter. In this paper, the temperature and humidity determinants of seasonal behaviour are analysed, in particular the conditions causing summer quiescence.
Experiments using a millipede treadmill apparatus at different temperatures at 95% RH showed an almost linear response of locomotor activity with temperature between 3 and 21°C, but when the temperature was increased to 28°C, the millipedes became quiescent. Activity could he restored by reducing the temperature.
When the millipedes were exposed to 45% RH there was comparatively little activity at any temperature. Field evidence indicates ii rapid breaking of quiescence with surface moisture
The success of O. moreleti in invading the surface-litter habitat in South Australia can he explained by its ability to avoid desiccation; its adaptations include its quiescent behaviour during summer and its ability to moult from a winter (reproductive) to B summer (non-reproductive) form.     

Daily and seasonal activity patterns of the muskrat Ondatra zibethicus as revealed by radiotelemetry

Activity patterns of the muskrat Ondatra zibethicus L. were examined during summer and winter in Delta Marsh, Manitoba, Canada (50°11'N, 98°23'W). Animals exhibited intermittent activity throughout the day with a mean periodicity close to 6 h in both seasons. The 24-h activity pattern in summer was typically bimodal with major peaks occurring between sunset and sunrise. Muskrats were more diurnal in winter when maximal levels of activity occurred in late afternoon and early evening (1500–2000). Pronounced inter- and intra-individual variability in daily activity ensured continuous, or nearly continuous occupation of winter shelters by a variable number of muskrats, and thus enchanced microclimate stability. Daily variation in activity and weather were weakly correlated both summer and winter. Of the environmental variables tested, only photoperiod and wind speed in summer, and photoperiod and air temperature in winter correlated significantly with total time spent in lodge or burrow per day.     

COLI-AEROGENES BACTERIA OF PASTEURIZED MILKS, WITH SPECIAL REFERENCE TO PATHOGENIC SEROTYPES OF ESCHERICHIA COLI

SUMMARY: A total of 375 samples of retail pasteurized milk in bottles was examined from January to the end of August 1955, for coli-aerogenes bacteria and especially for pathogenic serotypes of Escherichia coli. These serotypes were not found, though 101 of the samples contained bacteria of the coli-aerogenes group. During the summer months, June-August, the percentage of contaminated samples was about 40% and 10% of the samples contained E. coli I or II. The epidemiological significance of these findings is discussed.     

SOME BIOCHEMICAL CHARACTERISTICS OF FRESHLY ISOLATED STRAINS OF THE GENUS SERRATIA

SUMMARY: Many of 110 strains of Serratia , isolated from soil, water, milk and dairy equipment, were biochemically closely related to the coli-aerogenes bacteria. Acid and gas was formed from glucose in 14 days at 30° by 53% and from lactose and MacConkey's broth by about 40%. All except one strain gave——++ IMViC reactions.
An inverse relationship was observed between depth of pigmentation and carbohydrate fermentation. Complete loss of pigment in mutant strains was not uncommon, and was associated with loss of proteolytic properties and increase of saccharolytic activity.
The majority of the strains had psychrophilic characteristics: 75% grew at 3–5°. Most strains showed moderate growth at 37°, but only 7 formed red pigment at that temperature.
All strains resembled Serratia marcescens in morphology, containing minute coccoid rods smaller than those of coli-aerogenes bacteria.     

The water and temperature relationships of Ommatoiulus moreletii (Diplopoda: Iulidae)

During summer, Ommatoiulus moreletii aggregates in cool, moist sites. O. moreletii prefers high relative humidities and temperatures of 20 to 25°C throughout the year. These preferences explain the summer aggregations.
O. moreletii develops a strong ability to survive exposure to low humidity and high temperature during summer. This may explain why summer mortality of the millipede has only been demonstrated during an extremely hot and dry summer. The percentage water contents of O. moreletii are higher during spring and summer than during autumn and winter. They are also higher in small animals than in large. The ecological and physiological significances of these facts are discussed.     

A COMPARISON OF SOME MILK REJECTION TESTS

SUMMARY: Examination of 202 samples of raw milk showed that the combination of a pH paper strip test with a second sorting test (80 or 76% alcohol or rapid acidity) provided a basis to select milk which required a more time-consuming test before grading accurately.
Samples of pasteurized milk at the yellow-green stage of fluorescence when examined in ultraviolet light did not clot on boiling and possessed long methylene blue reduction times, whereas at the purple stage of fluorescence the samples clotted on boiling and reduced methylene blue almost instantaneously.