Lecithin agar for detection of microbial phospholipases.

Lecithin agar was developed on which phospholipase C produced turbid zones and phospholipase A produced clear zones. Reactions on lecithin agar agreed 74% of the time with reactions in egg yolk broth. On lecithin agar, interpretation was easier, phospholipase A was detectable, and opaque zones were visible 1 or 2 days earlier than on egg yolk agar. All constituents of the medium can be autoclaved.     

高产磷酯酶C菌株筛选及其抗血小板功能的研究

在湖北省、湖南省、江苏省、山东省、广东省156个点采土样1268份,利用卵黄琼脂的LB培养基初筛获得产生乳白色晕圈的菌株648株;再用卵黄琼脂滤纸圆片检测获得产生乳白色晕圈和透明圈菌株266株;这些菌株用卵黄琼脂杯蝶法筛选出产生大于13．0mm乳白色晕圈及少量透明圈共73株,经过菌生物量、卵黄杯碟法和抗兔血小板聚集率的测定,从73株菌中筛选出15株,菌号为：183,198,247,424,587,596,692,744,754—1,791—2,779,970,998,1107,1182;将此15菌株经菌生物量、卵黄琼脂杯碟法、NPPC法和抗人血小板聚集率的测定,从中选出754—1,779,970,1107四株菌进行分类鉴定和已知菌CW—W—90—3菌对照比较的研究。     

A microassay for the quantitative measurement of egg yolk-reactive enzyme activity produced byPseudomonas cepacia

A newly developed microassay offers a sensitive method for quantitating egg yolk reactivity in culture supernatants and samples prepared during enzyme purification. Equal volumes of supernatant, saline, egg yolk suspended in saline, and buffer were incubated in microtiter wells at 37°C, and the resulting turbidity was measured quantitatively with an ELISA reader at 410 nm. The microassay was used to screen culture supernatants from nine clinical isolates ofPseudomonas cepacia, and the results were compared with those obtained when the isolates were screened on egg yolk agar. The microassay was also used to detect egg yolk reactivity in ammonium sulfate-precipitated fractions of the culture supernatant of one strain, Pc224c, and to determine which fraction of egg yolk contained the substrate for the activity.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.     

The Egg Yolk Reaction Produced by Several Species of Bacteria

S ummary . A study of the egg yolk reaction produced by various species of bacteria has shown that it is caused by the hydrolysis of phosphatidylcholine. Staphylococcus aureus and Serratia marcescens degrade phosphatidylcholine by a series of hydrolytic reactions which are initiated by phosphatidylcholine: cholesterol o -acyltransferase. The degradation of phosphatidylcholine results in the formation of a sub-surface, opaque homogeneous zone around colonies growing on egg yolk agar and a collar of lipid material floating on the surface of egg yolk broth. This reaction may not be shown by all species growing in egg yolk media but it can be demonstrated if enzyme extracts are used.     

Enumeration of Food-Borne Clostridium perfringens in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

The SFP (Shahidi-Ferguson perfringens), TSC (tryptose-sulfite-cycloserine), EY (egg yolk)-free TSC, and OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agars have been tested for their suitability to enumerate Clostridium perfringens in naturally contaminated foods. Complete recoveries of C. perfringens were obtained in each of the four media, but only the TSC and EY-free TSC agars were sufficiently selective to ensure subsequent confirmatory tests without interference from facultative anaerobes. Because of some disadvantages associated with the use of egg yolk, EY-free TSC agar is recommended for enumeration of C. perfringens in foods. Several conditions for convenient shipment of foods and C. perfringens isolates with minimum loss of viability have been tested. The highest viable counts were preserved when foods were mixed 1:1 (wt/vol) with 20% glycerol and kept in a container with dry ice. Isolated C. perfringens strains remained viable for at least 2 weeks at ambient temperatures on blood agar slopes with a 2% agar overlay in screw-cap culture tubes.     

The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar

The potassium tellurite concentration, 0.01% w/v, in Baird-Parker agar has been recommended for plasma coagulase media such as pig or rabbit fibrinogen agar. Comparative tests have shown that with some strains of Staphylococcus aureus this level of potassium tellurite is too inhibitory and it should be reduced four-fold to 0.0025% w/v to maximize the isolation rate. It is postulated that egg yolk in Baird-Parker agar has a protective effect on staphylococci against the inhibitory action of tellurite.     

The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar

The potassium tellurite concentration, 0.01% w/v, in Baird-Parker agar has been recommended for plasma coagulase media such as pig or rabbit fibrinogen agar. Comparative tests have shown that with some strains of Staphylococcus aureus this level of potassium tellurite is too inhibitory and it should be reduced four-fold to 0.0025% w/v to maximize the isolation rate. It is postulated that egg yolk in Baird-Parker agar has a protective effect on staphylococci against the inhibitory action of tellurite.     

The in vitro development from egg to prepupa of Campoletis sonorensis (Hymenoptera: Ichneumonidae) in an artificial medium: importance of physical factors

A further attempt to improve the in vitro rearing of the solitary larval endoparasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae) was made after third instars were obtained in previous studies. Respiration was an important factor for the parasitoid's continued development: both small diet volumes that exposed the larval cuticle to air and O(2)greatly improved growth and development of C. sonorensis. Fifth instars were obtained when the parasitoid larvae were reared in medium volumes of 0.25ml and 0.1ml per well. Increasing the O(2) tension of the air resulted in larger larvae and prepupae. The agar layer under the nutritional medium was designed to slowly remove water from the diet. The results indicated that a drier environment was important for the parasitoid at the terminal stage. One percent and 0.8% agar gels, which removed water from the diet more rapidly, provided a drier environment and resulted in improved larval growth compared to the 0.6% agar gel. The detrimental effect of higher concentration of chicken egg yolk mixed in the diet was eliminated by using a dry egg yolk layer covered by an agar layer. This sandwich technique resulted in more parasitoid larvae molting to the final larval instar (5th) and the formation of many prepupae. However, the development of prepupae was premature and none of the fifth instar larvae successfully formed a normal cocoon.     

Dimethyl sulfoxide (DMSO) stimulates heme synthesis in quail embryonic yolk sac cells

Dissociated yolk sac cells from quail embryos at the definitive primitive streak stage were reaggregated, using a gyratory shaker with or without dimethyl sulfoxide (DMSO). After 24 h of incubation in the shaker, the aggregates were transferred onto a whole egg agar medium containing ⁵⁹Fe, and incubation was continued for an additional 48 h. It was clearly shown that DMSO-treated yolk sac aggregates showed a higher incorporation of radioactive iron into heme than the control culture without DMSO. The maximal stimulatory effect was observed at around 0.75% DMSO.     

Effect of egg yolk concentration on cryopreserving Spanish ibex (Capra pyrenaica) epididymal spermatozoa

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex (Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats (Capra hircus). In Experiment 1, a Tris-citric acid-glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 degrees C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher (P<0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Separation and Formation of Egg Yolk Oil by Solubilizing the Lipoproteins of Spray-dried Egg Yolk into Polypeptides

Egg yolk oil was formed from a hen’s egg without using the traditional charring method and organic solvents. By treating a spray-dried egg yolk suspension with a commercial crude enzyme preparation having protease and lipase activities, the lipids (egg yolk oil) could be easily separated, and a transparent solution of soluble polypeptides was obtained. When the enzyme preparation (4 mg) was added to a 5% spray-dried egg yolk (100 mg) suspension, the reaction mixture became transparent, and the egg yolk oil floated to the surface of the reaction mixture within 3 h. In the case of a 10% spray-dried egg yolk suspension, a transparent solution and egg yolk oil could be successfully obtained within 4 to 5 h by using a larger reaction vessel and a 0.2M lactate buffer. About 87% of the total polypeptides in the initial reaction mixture was recovered from the transparent solution, while about 83% of the total phospholipids was recovered from the floating egg yolk oil.     

Agar Concentration in Counting Clostridium Colonies

Decreasing the agar concentration of a counting medium from the usual 1.5% resulted in larger colonies with less interference from gas in Clostridium botulinum 115B and C. sporogenes PA 3679. Optimal agar concentration was 0.65% for C. botulinum with 24-hr incubation and 0.50% for C. sporogenes with 48-hr incubation. Lower concentrations yielded growth too diffuse for counting. Motility was considered the explanation for increased colony size in softer agar. The greater the degree of motility, the greater would be the diffusibility expected, and thus the higher the agar concentration required to insure discrete colonies. For quantitating motility, evaluations were made by use of microscopic examination of liquid cultures and rate of diffusion in a semisolid medium. With both criteria, the degree of motility of C. botulinum 115B clearly exceeded that of C. sporogenes PA 3679. Small-colony variants of C. botulinum in 0.65% agar yielded only small colonies on subculture, with a corresponding decrease in degree of motility of the cells by both criteria. Colony size of the nonmotile C. perfringens ATCC 3624 was unaffected by lowered agar concentrations.     

Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Cryopreservation of Spanish ibex (Capra pyrenaica) sperm obtained by electroejaculation outside the rutting season

The effectiveness of electroejaculation for obtaining Spanish ibex sperm samples for freeze preserving outside the rutting season was evaluated—the aim being to optimise biological resources for the establishment of germplasm banks. The effect of different egg yolk concentrations (6% or 12%, v/v) in diluents of different buffer composition (Tris-citric acid buffer or Tes-Tris buffer) on frozen-thawed samples of the above also investigated. Experiments were undertaken with six ibex males in February-May, and involved four different semen samples from each animal with four combination of extender, respectively: Tes-Tris-glucose (TTG)-6% egg yolk, TTG-12% egg yolk, Tris-citric acid-glucose (TCG)-12% egg yolk, TCG-6% egg yolk. The results show that electroejaculation is a useful way of obtaining sperm samples from Spanish ibex outside the rutting season (i.e., at a time coinciding with plasma testosterone levels of <0.4 ng/ml). According to the results of the eosin-nigrosin staining and the hypo-osmotic swelling test, the freezing-thawing process significantly reduced the viability and membrane integrity of the spermatozoa extended with TTG-6% egg yolk, TTG-12% egg yolk, and TCG-12% egg yolk, but did not affect these variables in spermatozoa extended with TCG-6% egg yolk. Therefore, the use of Tris-citric acid-based extenders containing low concentrations of egg yolk is recommended for cryopreserving Spanish ibex spermatozoa obtained by electroejaculation outside the rutting season.     

An improved selective and diagnostic medium for isolation and counting of Listeria spp. in heavily contaminated foods

Columbia agar base containing 0·001% acriflavine, 1·5% lithium chloride, 0·25% phenyl ethanol, 0·05% aesculin. 0·05% ferrous salt, 1% mannitol, 2·5% egg yolk emulsion and 0·008% phenol red (ALPAMY), recovered Listeria monocytogenes and some strains of Listeria seeligeri quantitatively, but suppressed Listeria ivanovii and virtually all other bacteria common in fresh foods. When used with foods processed for safety, repair on non-selective buffered glucose tryptone soya peptone yeast extract catalase agar must precede the use of ALPAMY.     

Immunodiffusion method for detection of type A Clostridium botulinum

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.