TELLURITE-EGG AGAR, A SELECTIVE AND DIFFERENTIAL MEDIUM FOR THE ISOLATION OF COAGULASE POSITIVE STAPHYLOCOCCI

SUMMARY: The preparation and use of tellurite-egg agar (TEA), a medium for the isolation of coagulase positive staphylococci, are described. The medium, based on that of Ludlam (1949), was non-inhibitory to coagulase positive staphylococci and colonies on it of these organisms could easily be distinguished from contaminating types. Comparisons were made between the medium and three other media in common use for the isolation of coagulase positive staphylococci.     

Application of a chromogenic medium and the PCR method for the rapid confirmation of L. monocytogenes in foodstuffs

Detection of Listeria monocytogenes in foodstuffs by conventional cultivation methods carried out according to EN ISO guidelines is rather time-consuming. Therefore, two alternative methods were applied for rapid confirmation of L. monocytogenes in foodstuffs. Inoculum from liquid selective broth was plated on PALCAM and OXFORD agar and on chromogenic agar medium RAPID L. mono. Suspect colonies from PALCAM were confirmed according to EN ISO standards and by the multiplex PCR method. In total, 990 samples of foodstuffs were investigated and 63 strains of L. monocytogenes were isolated. The chromogenic medium RAPID L. mono provided results comparable to PCR, it is easier to handle and provides considerable financial savings.     

Limitations of Fibrinogen-Polymyxin Medium in Detecting Coagulase-Positive Staphylococci in Raw Milk

A fibrinogen-polymyxin medium and Staphylococcus Medium 110 were used in the isolation of coagulase-positive staphylococci in raw milk. Results indicated that both media allow the growth of some rods and of many coagulase-negative cocci. A significantly greater number of coagulase-positive staphylococci were identified by the tube test than were revealed by halo formation on fibrinogen-polymyxin medium.     

Staphylococci in Competition: V. Effect of Eggs, Eggs Plus Carbohydrates, and Lipids on Staphylococcal Growth

As is generally known, custards have been frequently involved in staphylococcal food poisonings and are regarded by some as an ideal culture medium. Previous studies showed that high carbohydrate concentrations repressed the growth of competing saprophytic species, allowing the growth of staphylococci in a mixture rather than stimulating the growth of the staphylococci. In an extension of those studies, the influence of the egg constituent of custard was investigated to determine its role in affecting the growth of pathogenic staphylococci in a competing mixture of psychrotrophic saprophytes. The normal competitive effect of the saprophytes on the growth of staphylococci was very slightly affected by the addition of 25% whole egg to the growth media. Approximately 9% egg yolk alone added to the medium resulted in a slight increase in the length of the lag period of the psychrotrophs and a slight increase in the number of staphylococci which grew. Addition of 25% whole egg plus 14.5% sucrose resulted in repression of saprophyte growth similar to that seen in high sucrose concentrations. Staphylococcal growth was more extensive in the presence of both whole egg and sucrose than in the presence of either ingredient alone. Incorporation of 4% corn oil in media was effective in repressing growth of the saprophytes at 37 C only. This allowed the staphylococci to dominate the population. At lower temperatures, staphylococci were unable to compete effectively. Buffering media of high carbohydrate content resulted in lengthened lag periods for the psychrotrophs and the appearance of very large staphylococcal populations.     

Isolation of motile aeromonads from foods of animal origin

The purpose of this work was to select an available medium for Aeromonads isolation as well as to point out the Aeromonads presence in different foodstuffs of animal origin. Out of 15 tested media for Aeromonas isolation, two media, were selected: DFS as control medium and AGOS medium, both giving superposable results. 404 samples of foodstuffs of animal origin were examined; Aeromonads testing was performed qualitatively and quantitatively. 142 samples with a positivity percent of 51.40% were qualitatively tested. 262 samples with a positivity percent of 48.85% were quantitatively tested.     

The plaque-forming cell response of human blood lymphocytes. I. PFC response of lymphocytes to formalin-treated staphylocci.

The plaque-forming cell and proliferative responses of human peripheral blood lymphocytes induced by formalin-treated Staphylococcus aureus of the Cowan strain were studied in vitro. Human blood mononuclear cells were incubated for 6 days with staphylococci in culture medium RPMI 1640 supplemented with 10% human AB serum. The number of anti-sheep erythrocyte plaque-forming cells was determined by the Jerne technique. Lymphocyte proliferation was measured by [³H]thymidine incorporation. Individual lymphocyte donors could be classified as high or low responders to staphylococci. Lymphocyte proliferation appeared necessary for the generation of plaque-forming cells. The plaque-forming cell response was greatly influenced by the source of the human AB serum used in the culture medium. The addition of hydrocortisone to the culture medium augmented the plaque-forming cell response. Human B lymphocytes prepared by passage through a column containing Sepharose 4B conjugated to anti-human F(ab)₂ generated plaque-forming cells when incubated with staphylococci. However, the addition of T lymphocytes to these B-lymphocyte preparations augmented the plaque-forming cell response to staphylococci.     

Application of current methods for isolation and identification of staphylococci in raw bovine milk

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.     

Design and performance of a new medium for the quantitative recovery of Staphylococcus aureus from recreational waters

Several reports have suggested that staphylococci, and especially Staphylococcus aureus, are useful indicators of pollution of recreational waters. The lack of a selective, accurate and reliable recovery system for the quantification of Staph. aureus from water has been the principal obstacle to the evaluation of their use as indicators. In this study, several inhibitory substances and different nutrient sources have been evaluated for the formulation of a new selective medium. The medium designed, BFR-0 agar, recovers more than 75% of staphylococci and allows Staph. aureus to be identified.     

Design and performance of a new medium for the quantitative recovery of Staphylococcus aureus from recreational waters

Several reports have suggested that staphylococci, and especially Staphylococcus aureus , are useful indicators of pollution of recreational waters. The lack of a selective, accurate and reliable recovery system for the quantification of Staph. aureus from water has been the principal obstacle to the evaluation of their use as indicators. In this study, several inhibitory substances and different nutrient sources have been evaluated for the formulation of a new selective medium. The medium designed, BFR-0 agar, recovers more than 75% of staphylococci and allows Staph. aureus to be identified.     

Some Microbiological Aspects of Staphylococcal Mastitis

(1) Mannitol fermentation is a reasonably reliable method for the detection of coagulase positive staphylococci in milk. This reliability can be improved if mannitol fermentation is carried out under anaerobic conditions.(2) Among hemolytic strains of staphylococci isolated from milk, beta hemolytic staphylococci predominate. Bovine and sheep blood agar plates give similar hemolytic patterns, but the hemolysis is more pronounced on sheep blood agar.(3) Gelatin liquefaction cannot be relied upon for the selection of coagulase positive staphylococci in milk.(4) Urease production is a feature of the majority of coagulase positive staphylococci isolated from milk.(5) Tellurite Glycine agar medium is not satisfactory for the selection of coagulase positive staphylococci in milk.     

Effect of lantibiotic gallidermin against biogenic amine-producing faecal staphylococci from ostriches and pheasants

In ostriches and pheasants, there is still limited information relating to staphylococci and their properties. Biogenic amines (BAs) are nitrogenous low-molecular-weight substances with biological functions in animals, plants and microorganisms. In this study, we focused on BA production by targeted faecal staphylococci from ostriches and pheasants and their sensitivity to lantibiotic bacteriocin gallidermin. Gallidermin belongs in a group of polycyclic proteinaceous antimicrobial substances. Thirty-six faecal staphylococci (24 strains from 140 ostriches, 12 from 60 pheasants) comprising different species were tested. Staphylococci from ostriches and pheasants did not produce tryptamine-TRYP, putrescine–PUT, cadaverine–CAD or histamine–HIS. Production of tyramine-TYM, phenylethylamine–PEA was high or very high (100–1000 mg/L). Production of spermine–SPM and spermidine–SPD by staphylococci was very low or low although in the case of staphylococci from pheasants medium production of SPM was found. Because of the risk posed by BAs for consumers, the control of BA-producing bacteria is important from the points of view not only of safety assessment of food-producing animals but also of human health safety. The sensitivity to gallidermin in biogenic amine-producing staphylococci from ostriches and pheasants detected here is the most promising indication for further application of gallidermin for veterinary purposes. The novelty of our study lies in testing the ability of faecal staphylococci from ostriches and pheasants to produce BAs and in their treatment with gallidermin which has so far not been tested in this way.     

Effect of penicillin on the intensity of formation of penicillin-resistant staphylococcal populations in the suppurative foci of patients]

The dynamics of sensitivity to penicillin of staphylococcal populations in purulent inflammatory foci of patients treated and not treated with antibiotics was estimated according to 4 indices. No reliable differences in the dynamics of sensitivity to penicillin were found in 2 groups of the patients, when estimation was performed with respect to the frequency of the penicillin resistant or penicillin sensitive staphylococci and detection of the penicillin resistant staphylococci by direct inoculation of the focal excretion to the medium with penicillin. A reliable increase in the percentage of the penicillin resistant staphylococci in the microbial population was observed only in the patients treated with penicillin.     

Drug Resistance of Staphylococci VI. Genetic Determinant for Chloramphenicol Resistance

Naturally occurring strains of staphylococci which are resistant to chloramphenicol (CM) inactivate this antibiotic. One of the inactivation products of CM showed the chromatographic behavior of 3-acetoxychloramphenicol. Induction of resistance occurred after prior exposure to subinhibitory concentrations of the antibiotic. The resistance of induced populations, as well as CM-inactivation ability, was decreased when they were grown in CM-free medium. The CM-inactivation property was transduced together with CM resistance. Transductional analysis and CM-resistance elimination experiments indicated that CM resistance in naturally occurring strains of staphylococci is mainly accounted for by inactivation of the drug.     

Use of Shake Cultures in a Semisolid Thioglycolate Medium for Differentiating Staphylococci from Micrococci

The standard diagnostic test for differentiating staphylococci from micrococci is based on the ability of the former to produce acid anaerobically in a glucose-containing growth medium. This test has been modified to provide greater convenience, easier interpretation of results, and better correlation with deoxyribonucleic acid (DNA) base composition. In the modified test, shake cultures in Brewer's fluid thioglycolate medium with 0.3% agar added are observed for growth in the anaerobic zone of the tubes. This test was applied to 125 strains of staphylococci and micrococci, and all except two strains gave results that were consistent with other criteria. Of particular interest were eight strains of Micrococcus saprophyticus and three strains of M. lactis that have a DNA composition of 30 to 37% guanine plus cytosine (GC). All 11 of these cultures produced anaerobic growth and thus would be classified as staphylococci. Strains of M. lactis that have a high GC content in their DNA grew only aerobically. Some cultures of staphylococci produced characteristic band patterns of anaerobic growth and other cultures produced only a few anaerobic colonies from an inoculum of 10(6) to 10(7) cells. These observations suggest some interesting genetic and metabolic capabilities in such cultures.     

Ultrastructure,in vitro and in vivo,of staphylococci exposed to antibiotics

The ultrastructure of staphylococci from the peritoneal fluid of mice treated with oxacillin and from sputum of a patient treated with ampicillin was comparable to the structure of staphylococci grown on filter membranes exposed to oxacillin, but was different from the same organisms grown in broth with oxacillin. The selection of a solid phase support growth medium, such as a filter membrane for in vitro studies of drug induced morphology, appears necessary if such studies are to reflect bacterial ultrastructure in vivo.     

Selective nutrient medium for the isolation of Bacillus cereus

A selective dried medium for the isolation of B. cereus from clinical material and foodstuffs has been developed. The medium has high selective properties which ensure the isolation of B. cereus from microbial association in pure culture, thus making it possible to accelerate further identification of the microorganisms.     

Growth Inhibition of Staphylococci by Sodium Thiosulphate

The addition of sodium thiosulphate to a medium as neutralizer of an iodine antiseptic resulted in unexpected growth inhibition of various strains of staphylococci and micrococci. The minimum growth inhibiting concentration varied with different strains. The inhibitory effect of sodium thiosulphate was more pronounced in media with low pH values than in those with high pH values, and was diminished by the addition of Tween 80. The action was also found to depend on the concentration of l -cystine in the medium. It is suggested that the use of sodium thiosulphate be avoided in growth media designed to neutralize iodine in disinfection efficiency tests when staphylococci or micrococci are used as test organisms.     

Properties of Corynebacterium diphtheriae cultivated in the medium prepared from inedible raw materials

A nutrient medium for the cultivation of C. diphtheriae toxigenic strain was developed on the basis of raw materials unsuitable for use as foodstuffs and its main physico-chemical and cultivation properties were studied. The morphological, cultural, biochemical and toxigenic properties of C. diphtheriae cultivated in the experimental medium with horse serum were evaluated. As revealed in this study, C. diphtheriae retained their properties after prolonged cultivation and storage in the newly developed medium, both liquid and with agar added. The medium has a number of advantages: it is economical, raw materials for its production are readily available, the medium is free of ballast substances.     

Staphylococci in Competition: IV. Effect of Starch and Kind and Concentration of Sugar on Staphylococcal Growth in Mixed Populations

Foods containing large amounts of carbohydrate have frequently been involved in staphylococcal food poisoning. Custard has been considered to be a highly favorable culture medium for staphylococci; however, it may be a selective medium rather than an ideal one. The influence of dextrose, lactose, and sucrose in varying amounts from 0.25 to 18%, and of starch, on the growth of staphylococci in mixed populations with saprophytes was determined. The inhibitory effect of the sugars was much greater on the saprophyte population than on the staphylococci. Of the three sugars, sucrose was most inhibitory to the saprophytes. It greatly decreased their lag periods as the concentration of sugar increased. Dextrose was the least inhibitory; in fact, 0.5% dextrose gave considerable stimulus to saprophyte growth. This sharply repressed staphylococcal growth. Lactose occupied an intermediate position. Rapid onset of the death phase of the staphylococci was observed in all increased sugar concentrations and seemed to be a pH effect rather than a result of competition. Sucrose exerted an inhibitory effect on the growth of saprophytes at and above room temperature. In the presence of 2.5% corn starch, staphylococcal growth in mixed cultures was slightly inhibited, while the death phase was sharply accelerated. Thus, carbohydrates exert their influence on staphylococcal growth in mixed cultures through their effect on the saprophytes by decreasing or increasing competition.     

Polymyxin-Coagulase-Mannitol-Agar: I. A Selective Isolation Medium for Coagulase-Positive Staphylococci1

A selective, differential plating medium was developed for the isolation and identification of coagulase-positive and mannitol-fermenting staphylococci. Coagulase produced by growing Staphylococcus aureus caused an opaque zone of fibrin to form around each colony. Several strains of S. aureus produced a visible coagulase reaction by 8 hr, and all strains gave a positive reaction before 12 hr. Mannitol fermentation was usually observed between 12 and 36 hr. Rabbit serum was filtered through Sephadex G-100 to obtain plasmin- and plasminogen-free coagulase-reacting factor (CRF). False-negative reactions, caused by staphylokinase and staphylococcal Müller factor action on plasminogen, were eliminated when this CRF was used. False-positive reactions by lipolytic, coagulase-negative staphylococci were reduced, since gel filtration removed the serum lipoprotein which served as a primary source of opacity. The addition of 75 mug of polymyxin B per ml selectively retarded the growth of S. epidermidis and minimized false-positive reactions caused by citrate-utilizing gram-negative rods. The preparation, characteristics, and use of the medium are presented.