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.     

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.     

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.     

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.     

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.     

STUDIES OF THE DETERMINATION OF THE BACTERIUM COLI TYPE I CONTENT OF FARM WATER SUPPLIES

SUMMARY: A – comparison of the suitability of brilliant green bile broth and MacConkey's broth at 44° for the detection of Bacterium coli type I in farm water supplies, showed that 83.1% of the samples had no difference in the number of positive tubes at 44°, and only 5 samples (1.7%) had a significantly higher number of positive tubes in MacConkey's broth.
Of 707 strains of coli-aerogenes bacteria isolated from 44° positive tubes of both media, 94.5% were Bact. coli type I. Strains of Bact. coli type II and Bact. aerogenes type I which were 44° positive constituted 3.7% and 0.4% respectively, all of which were indole negative at 44°. In addition there were 10 strains (1.4%) of 44° positive Intermediate type II, 9 of which were indole positive at 44°.
An appreciable number (6.6%) of Bact. coli type I strains failed to give a positive indole reaction in 24 hr at 44°.     

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.     

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.     

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.     

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 VALUE OF THE ASSOCIATION ESCHERICHIEAE-GROUP D STREPTOCOCCI IN THE DIAGNOSIS OF CONTAMINATION IN FOODS

SUMMARY: The tests for faecal contamination in foods, based on 'indicator species', should be reconsidered for the following reasons. First, in Europe, Escherichia coli is regarded as a specific index of such contamination; but, apparently through the use of antibiotics, the proportion of human faeces containing Klebsiella has risen from 5.2% in 1947 to 48.4% in 1956–7. Moreover, E. coli and other coli-aerogenes organisms may be absent from the gut of certain animals, notably the pig. Second, while the usual methods of counting coli-aerogenes bacteria do not permit the isolation of strains which ferment lactose slowly or not at all (i.e. paracolons), these occur in 35.6% of samples of human faeces. Further, the recommended methods of detecting such strains are not wholly satisfactory. Third, the origin of the coli-aerogenes bacteria is uncertain.
It is thus necessary to consider other organisms which normally inhabit the intestinal tract of humans and other animals. As to Clostridium perfringens , its source is doubtful, for it may come from faeces or from soil, and many foods contain only soil strains. The group D streptococci, on the other hand, are excellent indicators of faecal contamination. They are constant or frequent in the intestines of man and animals, and often more numerous there than coli-aerogenes bacteria. Their specificity as an index of faecal contamination is high and their investigation is easy with the selective media now available. Associated with the coli-aerogenes bacteria, they justify the diagnosis of faecal contamination in a foodstuff.     

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.     

THE RAPID IDENTIFICATION OF ESCHERICHIA COLI I BY THE PRODUCTION AT 44° OF BOTH INDOLE AND GAS FROM LACTOSE

SUMMARY: The modification of the 44° test proposed by Mackenzie, Taylor & Gilbert (1948) is useful for the rapid identification of Escherichia coli I in water and foods. False positive tests caused by other coli-aerogenes bacteria, or by their association with other organisms, can be considered rare. Only a few E. coli I fail to produce indole or ferment lactose at 44°, and further confirmation for routine purposes is necessary only when the results at 44° are discordant, e.g. indole positive-lactose negative or indole negative-lactose positive.     

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.     

CAUSES OF GREENING OF UNEVISCERATED POULTRY CARCASES DURING STORAGE

SUMMARY: The greening of uneviscerated poultry carcases stored at 15° is due to the production of hydrogen sulphide following multiplication of bacteria in the gut. The main groups present in the gut after death are faecal streptococci, coli-aerogenes organisms, lactobacilli and clostridia, but their relative importance in causing greening has not yet been established. The hydrogen sulphide diffuses from the gut into the muscle tissue and there reacts with the haem pigments of blood and muscle to form derivatives which microspectroscopically are indistinguishable from sulphaemoglobin.
The delay of greening at lower storage temperatures is consistent with this view; and it is suggested that mechanically plucked birds green more rapidly over the ribs than hand plucked ones because the shaking distributes bacteria from the caecum along the gut.     

THE EFFECT OF VARIOUS WATER BACTERIA ON THE GROWTH OF ESCHERICHIA COLI I IN MACCONKEY'S BROTH AT 37° AND 42°

SUMMARY: The growth of a streptomycin resistant strain of Escherichia coli I in MacConkey's broth at 37° or 42° was not inhibited by water organisms of the genera Micrococcus, Proteus, Achromobacter and Pseudomonas , but the presence of large numbers of other members of the coli-aerogenes group resulted in retardation or inhibition of its growth.     

THE ASSOCIATED GROWTH OF STREPTOCOCCUS LACTIS AND ESCHERICHIA COLI

SUMMARY: The possibility that contamination of farmhouse starters by coli-aerogenes bacteria may be a factor in producing the subtle flavour of farmhouse Cheddar cheese has been discussed. The associated growth of Strep. lactis and E. coli I at 30° and 37° resulted in the rapid disappearance of E. coli from the mixtures, even though it had been the dominant organism in some of them originally. Mixtures containing Strep. lactis and an anaerogenic strain of E. coli still contained this variant at the end of a month, although in no definite ratio and in a very much reduced proportion. It is concluded that the components of coli-lactic starters to be used in the manufacture of cheese should be combined together in the vats.     

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 OBSERVATIONS ON THE VIABILITY OF SEWAGE BACTERIA IN RELATION TO SELF-PURIFICATION OF MUSSELS

SUMMARY: Autoclaved sea water and water removed from the shell cavity of mussels supported the growth of coli-aerogenes bacteria. Attempts to reproduce conditions which had favored multiplication of coli-aerogenes bacteria in purified mussels failed.
'Enrichment' of sea water by repeated re-use for storage of successive lots of mussels, in conjunction with sand filtration and aeration under conditions equivalent to 60 weeks of approved purification, did not promote the growth of coli-aerogenes bacteria.
A culture of Salmonella typhi and Salm. paratyphi B survived for a few days only in sea water favourable to the slow multiplication of Bacterium coli type I.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.