STUDIES OF THE BACTERIAL CONTENT OF WASHED MILK CANS

SUMMARY: Colony counts at 30° were greater than 10 times those at 37° in a high proportion of rinses of washed milk cans, the difference being most marked in those containing milk scale, where 58% of the colony counts at 30° exceeded 10⁶/can. A high proportion of the microflora was composed of thermoduric bacteria. Of 895 cultures from the milk scale, 33% were micrococci, 28% corynebacteria, 22% streptococci, and 9% were Gram-negative rods. Though aerobic sporing rods constituted only 5% of the microflora of the milk scale, they were present in large numbers and unsatisfactorily washed cans probably constitute one of the main sources of these organisms in milk.     

The Microflora of Steam Sterilized Milking Equipment

S ummary : The classification of 1,466 bacteria, isolated on Yeastrel-milk agar incubated at 30° from 68 rinses of steam sterilized dairy equipment taken at five farms, showed that micrococci were dominant, with corynebacteria and aerobic sporeforming rods frequent, representatives of these three groups constituting 73% of the isolates. The microflora was characterized by the dominance of organisms which were relatively inactive in milk. Typical milk souring organisms such as lactic streptococci and coli-aerogenes organisms were rarely isolated, but anaerogenic Gram-negative rods resembling Achromobacter, Pseudomonas and Flavobacterium were not uncommon on milking machine clusters and on a cooling unit. The glass recorder jars contained a relatively high proportion of sporeformers.     

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.     

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.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

The relationship between physical and chemical conditions and low microbial diversity in the Blue Lagoon geothermal lake in Iceland

Abstract: The Blue Lagoon in Iceland is a shallow geothermal lake with average temperatures of 37°C, pH 7.5 and about 2.5% salinity. It was formed in 1976 from the effluents of the Svartsengi geothermal power plant and is saturated with silica which constantly precipitates in the lake. It has been colonized by a few types of specialized microorganisms which seem to proliferate in this unusual ecosystem. The average bacterial colony count in the lake was 1.3 × 10⁵ ml⁻¹ on plate count agar made with 50% Blue Lagoon fluid but 2.6 × 10⁶ ml⁻¹ when determined with the MPN method. A total of 99 isolates were purified and characterized by 54 phenotypic tests and then grouped using Numerical Taxonomy. At similarity values of 80%, one major cluster was formed containing 85% of the isolates. Four representative strains from this cluster were further characterized and all shown to be Gram-negative, obligately aerobic, non-motile rods. They were oxidase positive, catalase negative and grew optimally at 45°C and in 3.5% NaCl with doubling time of about 80 min.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

THE NORMAL FLORA OF THE BOVINE RUMEN II. QUANTITATIVE BACTERIOLOGICAL STUDIES

SUMMARY: A simple medium and dilution count technique was evolved for determining the viable count of rumen bacteria. Material from three fistulated cows was examined and the viable population found to vary within the range 10⁷-10¹²/g; the most frequently occurring values were 10⁸-10¹⁰/g (94.0% of 350 counts). Observations were made of the homogeneity of rumen contents, of variations in count with time after sampling and between cows, and of variation from day to day, hour to hour, before and after feeding and on different diets. The variations in the viable count, even with gross changes in diet, were never greater than those occurring in a single animal on a fixed diet. Microscopic examinations showed Gram-positive cocci and rods to be the most frequently occurring types of bacteria, Gram-negative organisms of the coli-aerogenes type being present only in low numbers.     

THE SANITATION OF FISH BOXES. I. THE QUANTITATIVE AND QUALITATIVE BACTERIOLOGY OF COMMERCIAL WOODEN FISH BOXES

SUMMARY: The cleaning efficiency of hosing wooden market fish boxes with cold water, the usual commercial practice, was very low, even when high pressure jets were used. After washing, the internal surfaces could still carry up to 20 × 10⁶ bacteria/cm². Even prolonged steaming failed to sterilize the surfaces. Coryneform organisms accounted for over 50% of the total bacterial flora, although these have usually been reported as present in only low proportions on both fresh and spoiling fish. The major subsidiary groups were species of Achromobacter and Pseudomonas , constituting about 18% and 14% of the population respectively. A few micrococci and flavobacteria were also present.
The conditions of counting, i.e. whether roll tubes or Petri dishes were used, whether incubation was at 0°, 20° or 37°, and whether the media were based on sea or tap water, could markedly influence both the magnitude of the count and the proportions of the different types of bacteria which could be isolated.     

Irish kefir-like grains: their structure, microbial composition and fermentation kinetics

The structure of six Irish kefir samples was studied in the electron microscope, and the microbial composition and fermentation kinetics during growth in 10% reconstituted skim milk at 21°C. The microbial composition of the six samples was similar; at the end of the fermentation the counts of lactococci, leuconostocs, lactobacilli, acetic acid bacteria and yeasts were 10⁹, 10⁸, 5 × 10⁶, 10⁵ and 10⁶ ml⁻¹ respectively; the levels of acetic acid bacteria and lactobacilli showed some intersample differences. Lactate was the major metabolite followed in order by ethanol, acetate and acetoin. The final concentrations of L-lactate produced (66–90 mmol kg⁻¹) were 10-fold higher than those of D-lactate. Acetate and ethanol concentrations varied from 4 to 14 and 2 to 40 mmol kg^−'1 respectively. The rates of citrate utilization and concentration of acetoin produced during growth differed between samples. Scanning electron microscopy showed not only variation between the interior and exterior of the sample but also large variation between different sections of the interiors and exteriors of the same sample. Long and short, and straight and curved rods and yeasts were seen in all samples, the curved rods observed in the interior, but lactococci were seen on the surface of only one sample. There were no gross differences in structure between samples.     

Rapid Counting of Bacteria in Rinses of Milking Equipment by a Membrane Filtration - Epifluorescent Microscopy Technique

Membrane filtration and epifluorescent microscopy were used for the rapid enumeration of bacteria in rinses of milking equipment. Rinse solution (10 ml) was filtered through a 0.6 µm pore size Nuclepore membrane filter and the bacteria retained stained with acridine orange. The clump count of orange fluorescing bacteria on the membrane correlated well with the corresponding plate count ( r =0.83). The technique is rapid, taking approximately 10 min, and is sufficiently sensitive to detect 1000 bacteria ml rinse (equivalent to 5 × 10³ bacteria/m² of milking equipment surfaces by the method currently in use).     

The Microbiology of Built Up Poultry Litter

The numbers of viable bacteria in built up poultry litter were found to be 10¹⁰-10¹¹/g fresh weight and appeared to be little affected by factors such as age, temperature, moisture content and pH. Counts for unused litter and poultry droppings were lower. In built up litter of high alkalinity coryneform bacteria were predominant; micrococci occurred sporadically and small numbers of nocardias, streptomycetes, aerobic spore formers and streptococci were encountered. A variety of Gram negative bacteria also occurred, the numbers of which appeared to be controlled by alkalinity; they were less abundant in litters where the pH and buffering capacity were high. Strongly alkaline conditions also tended to lower the fungal counts but had no effect on the count of enterococci.     

THERMODURIC ORGANISMS IN MILK. PART V. A SIMPLIFIED TESTING TECHNIQUE

SUMMARY: Thermoduric colony counts at 30° of laboratory pasteurized milk determined by the roll tube or agar strip methods were lower than those obtained by the standard Petri plate method. The differences in colony count were not of such magnitude, however, as to be likely to result in many errors in grading if a thermoduric bacterial content of greater than 10⁴/ml is accepted as an index of unsatisfactory cleansing of dairy equipment.
The three methods examined were simpler and more economical than the Petri plate technique, but the agar strip method, as described, using the standard loop, was the simplest and gave a sufficiently reliable estimate of the thermoduric colony count for advisory purposes.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Semi-automated counting of bacteria and somatic cells in milk using epifluorescence microscopy and television image analysis

An epifluorescent microscope fitted with a 'Chalnicon' closed circuit television camera linked to an Optomax System III image analyser was used to count bacteria and somatic cells on membrane filters prepared from milk by the direct epifluorescent filter technique (DEFT). For both bacteria and somatic cells, the semi-automated DEFT count of low count milk exceeded the visual DEFT count but this difference became proportionately less as the count increased. There was close agreement between the semi-automated and visual DEFT counts over the range 10⁵-5 times 10⁶/ml for bacteria and 3 times 10⁵-5 times 10⁶/ml for somatic cells. The semi-automated DEFT count of bacteria and somatic cells correlated well with the plate count and Coulter count respectively, with correlation coefficients of 0.83 and 0.81.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

Isolation and characterization of Azotobacter chroococcum from the roots of Zea mays

Abstract Bacteria showing rapid growth on a nitrogenfree medium and acetylene-reducing activity were isolated from maize roots collected from agricultural soils in Spain. The isolates were Gram-negative motile rods and were identified as Azotobacter chroococcum . Acetylene-reducing activity and microbial counts were determined on root segments from 7- and 30-day-old plants. Rates obtained were in the range of 0.0053–0.848 nmol C₂H₂· g⁻¹· h⁻¹. Root populations were 1.4–6.0 × 10⁴ micro-organisms · g⁻¹. These results showed that there was an association between A. chroococcum strains and roots of maize planted in some Spanish soils.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

THE OCCURRENCE OF A FLAVOBACTERIUM SPECIES IN CHILLED WATER

during the examination of chilled water from a churn immersion cooler it was observed that when the water was not changed at frequent intervals the colonies on standard nutrient agar incubated at 22° became entirely composed of a Flavobacterium sp. Colony counts greater than 10⁶/ml. were obtained at 3–5° and 22°. Although the dominant organism produced no visible change in litmus milk yet 0.1 ml. of chilled water clotted the milk within 48 hr. at 22°.