Effect of culture media and incubation temperature on growth of selected strains of Francisella tularensis.

The rate and amount of growth of 4 field isolates and reference strain ATCC 6223 of Francisella tularensis were evaluated on isolation media with 2 different agar bases and with different supplements and incubated at 25 C, 35 C, and 42 C. Biochemical reactions on conventional differential media with and without cysteine were evaluated. Two of the field isolates and the reference strain were F. tularensis subspecies tularensis (formerly biovar tularensis or Type A), and 2 isolates were subspecies holarctica (formerly subspecies palaearctica or Type B). Bacto cystine heart blood agar supplemented with 1% hemoglobin, glucose cystine heart blood agar, and brain-heart infusion blood agar supported good growth of all 4 field strains, with the most luxuriant growth occurring on Bacto cystine heart blood agar with hemoglobin. Heart infusion blood agar and trypticase soy blood agar supported growth of the field isolates, although growth was diminished and delayed. Strain 6223 was distinctly fastidious and failed to grow on heart infusion or trypticase soy blood agars. Growth of strain 6223 was best on Bacto cystine heart blood agar with hemoglobin. The agar base did not affect growth unless the supplements became limiting, in which case Bacto agar base generally supported growth better than BiTek agar base. Incubation at 35 C was optimum for all 5 strains. Growth at 42 C was slow, with the greatest decrease in the rate and amount of growth occurring with field isolates of F. tularensis subspecies tularensis. Strain 6223 did not grow at 25 C, and the 4 field isolates grew slowly at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the suitability of phase-contrast and immunofluorescence technics in the laboratory diagnosis of porcine dysentery]

Immunofluorescence technique, compared with the method of phase contrast, does not appear to be better for laboratory diagnostics of swine dysentery because neither of these methods can be used for distinguishing between pathogenic and nonpathogenic strains of treponemas. The number of treponemas contained in faeces should still be considered to be the main criterion in laboratory diagnostics. In clinically healthy pigs from stocks which never suffered from dysentery treponemas were found only in few cases and always in small numbers. The numbers of treponemas contained in the faeces of dysenteric pigs were several times higher. Antigenic relationship of one nonpathogenic and three pathogenic strains of Treponema hyodysenteriae was proved by the agglutination and fluoresence methods.     

Effect of transport enrichment medium, transport time, and growth medium on the detection of Campylobacter fetus subsp. venerealis.

The combination of medium and growth conditions, including transport enrichment medium (TEM), transport time, TEM incubation time, and growth medium, that best support Campylobacter fetus subsp. venerealis while inhibiting contaminants was studied. The 3 TEMs evaluated, Weybridge, Cary-Blair, and 0.85% saline solution, were inoculated with preputial smegma spiked with C. fetus subsp. venerealis and held in the laboratory for 4 or 24 hours before inoculation onto growth medium. The effect of overnight incubation at 37 C of the TEM was also evaluated. Median scores of C. fetus subsp. venerealis growth and microbial contaminant inhibition were compared within TEM, transport time, overnight incubation, and growth medium groups using the Mann-Whitney U-test and the Kruskal-Wallis test. The proportion of samples with any growth or contamination within each group was also compared using the chi-square test. Results suggest that the growth of C. fetus subsp. venerealis was influenced by 3 of the 4 criteria evaluated. Weybridge TEM more effectively maintained the organism than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time of 4 hours rather than 24 hours (P < 0.001) and avoiding overnight incubation of TEM at 37 C (P < 0.001) were associated with improved growth. Significant differences were not identified among growth media; however, Skirrow Campylobacter agar appeared to yield slightly better growth than did either blood agar or Greenbriar Plus agar. Contaminant growth was also influenced by 3 of the 4 variables. Weybridge TEM inhibited contaminant growth more effectively than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time was not associated with contaminant growth. Eliminating overnight incubation of TEM reduced contamination (P < 0.01). Skirrow agar was preferred to both blood agar and Greenbriar Plus agar for suppression of contaminants on solid medium (P < 0.001). These results suggest that the detection of C. fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM and are transferred to Skirrow agar the same day they arrive in the laboratory.     

苜蓿黄萎病菌中国菌株生物学特性研究

为了解我国苜蓿黄萎病菌的生物学特性,于2007-2008年间对苜蓿黄萎病菌进行了分离、鉴定及生物学特性研究。采用常规组织分离法从新疆伊犁地区新源县苜蓿黄萎病标样中分离到一种真菌(VA001),依据Koch′s法则,并结合形态学、培养特性及ITS序列分析,鉴定为黑白轮枝菌(Verticillium albo-atrum Reinke et Berthold)。适合该菌生长的培养基有甜瓜培养基、马铃薯蔗糖培养基、马铃薯培养基,适合产孢的培养基有马铃薯葡萄糖培养基、甜瓜培养基、梅干培养基。在碳源和氮源中,麦芽糖(Maltose)、乳糖(Lactose)、甘露醇(Mannitolum)、赖氨酸(Lysine)、牛肉膏(Beef extract)、氨基乙酸(Aminoacetic acid)、组氨酸(Histidine)有利于病菌的生长;蔗糖(Sugar)、果糖(Fructose)、乳糖(Lactose)、硝酸钠(Sodium nitrate)、丙氨酸(Alanine)有利于病菌的产孢。苜蓿黄萎菌生长和产孢的适宜pH值为7.0-9.5,温度为25℃。     

Growth of Mycobacterium paratuberculosis in radiometric, Middlebrook and egg-based media

The ability of BACTEC radiometric 7H12 broth, Middlebrook 7H10 Tween broth, Middlebrook 7H10 agar, and Herrold's egg-yolk medium to provide early detection of Mycobacterium paratuberculosis was evaluated. The minimum detection times in days for the various media were: 7H12, 9; 7H10 agar, 23 (plate), 28 (slant); 7H10 Tween broth; 27; and Herrold's egg-yolk medium, 43 (plate), 49 (slant). The radiometric broths provided the earliest detection of M. paratuberculosis, and 3625 organisms ml-1 were required to produce a positive, radiometric growth-index reading. Of the non-radiometric plate and slant media evaluated, microscope examination of the translucent 7H10 agar plate resulted in the earliest detection and highest mean colony counts (387) as compared with Herrold's egg-yolk agar plate (208). Similar results were noted for 7H10 and Herrold's egg-yolk agar slants; however, accurate colony counts could not be determined because of confluent growth. All media were supplemented with 2 micrograms ml-1 of mycobactin J and excess amounts of this supplement inhibited the growth of M. paratuberculosis in radiometric 7H12 media.     

Adherence of Bordetella avium to turkey tracheal mucosa: effects of culture conditions

Adherence of Bordetella avium to the tracheal mucosa of turkeys was evaluated, using bacteria grown under different culture conditions. Several solid and liquid media were used at incubation times of 12, 24, 36, or 48 hours with incubation temperatures of 18, 26.5, or 35 C. Adherence of B avium was greatest when the bacteria were grown on solid media at 35 C. Use of Bordet-Gengou or brain-heart infusion agar was associated with significantly greater (P less than 0.05) adherence compared with adherence of bacteria grown on other media. Adherence was greatest, using cultures in the stationary phase of growth; however, with some media, adherence diminished when incubation was extended beyond 36 hours. Adherence of B avium was reduced but not completely prevented when cultures were incubated at 18 C.     

Comparison of media used for the primary isolation of Mycobacteriurm bovis by veterinary and medical diagnostic laboratories

Veterinary and medical laboratories engaged in the cultural diagnosis of bovine or human tuberculosis were requested to supply samples of the media that they routinely use for the primary isolation of M. bovis. Fourteen laboratories supplied 7 basic media types; these were Lowenstein-Jensen, Stonebrink's, modified Middlebrook 7H11 agar, tuberculosis bovine blood agar, egg yolk agar, Gerloff's egg and Herrold's egg yolk. Two strains of M. bovis were used to test the media, strain AN5, a glycerol-tolerant laboratory strain and M86/90 a glycerol-sensitive wildtype strain. AN5 grew well on all media with the exception of Herrold's and strain M86/90 did not grow on media containing glycerol and grew poorly on Herrold's medium. It is recommended that Lowenstein-Jensen with pyruvate (but without glycerol), Stonebrink's, modified Middlebrook 7H11 and tuberculosis bovine blood agar should be considered the media of choice for the primary isolation of M. bovis. Egg yolk agar also proved adequate for this purpose in the trial. This medium may be suitable for routine use but to date experience with its use is limited.     

麋鹿产气荚膜梭菌分离鉴定方法的建立与初步应用

探索了产气荚膜梭菌在营养琼脂、甘露醇卵黄琼脂和血琼脂平板上的菌落形态和培养特点,优化了产气荚膜梭菌的分离方法,并对分离菌株进行生化鉴定;在此基础上对北京南海子麋鹿苑和江苏大丰麋鹿国家级自然保护区的麋鹿自然感染情况进行了调查。结果发现,粪样中的产气荚膜梭菌在营养琼脂上生长呈半透明边缘不整的白色菌落,接种甘露醇卵黄琼脂和血琼脂平板,分别出现伴有卵磷脂酶乳光浑浊带的粉红色火山口状菌落和伴有双溶血环的灰绿色勋章样菌落。生化试验结果确认这些分离株均为产气荚膜梭菌。对北京南海子麋鹿苑和江苏大丰麋鹿国家级自然保护区麋鹿粪样检测发现,阳性率分别为13.04%(3/23)和19.51%(8/41)。说明本研究建立的麋鹿产气荚膜梭菌分离鉴定方法简便快速,确实可行;麋鹿产气荚膜梭菌自然感染率较高,应加强麋鹿产气荚膜梭菌病的防控。     

Effects of source and washing of erythrocytes on growth of bacterial pathogens from the bovine mammary gland

Effects of source and washing of RBC on quantitative growth and hemolytic zone sizes of common bacterial pathogens of the bovine mammary gland were evaluated. Blood samples used to prepare the blood agar media were obtained from 10 adult dairy cows, 10 dairy calves, and 10 sheep. Hemolytic zone sizes produced by Staphylococcus aureus were significantly (P less than 0.01) larger on blood agar prepared with washed RBC than on blood agar prepared with nonwashed RBC, regardless of RBC source. With the exception of Corynebacterium bovis, growth of all bacteria was equivalent or significantly higher on medium prepared with washed RBC, compared with that on medium prepared with nonwashed RBC, regardless of RBC source. Significantly higher numbers of C bovis (P less than 0.01) and Streptococcus agalactiae (P less than 0.01) were isolated on medium prepared with washed cow RBC. Significantly higher numbers of Str uberis (P less than 0.01) and S aureus (P less than 0.05) were isolated on medium prepared with washed sheep RBC and washed calf RBC, respectively. Growth of Escherichia coli was not affected by the RBC source. Seemingly, RBC used in the preparation of medium should be washed. The source of RBC, as well as inter-animal variation, also should be considered in the quality control of medium.     

Comparison of six commercially available transport media for maintenance of Serpulina (Treponema) hyodysenteriae.

Two anaerobic (A1 and A2), 1 selective (S1), and 3 conventional (C1, C2, and C3) transport media formulations were compared for their capacity to maintain the viability of Serpulina (Treponema) hyodysenteriae. Initial experiments compared the recovery of S. hyodysenteriae from pure cultures held in each transport medium for 0.5, 1, 2, 3, 5, and 7 days at -40 C, 4 C, 25 C, and 36 C. Subsequent experiments compared each transport medium for maintenance of S. hyodysenteriae in fecal specimens obtained from experimentally infected pigs after holding for up to 7 days at 25 C. In each experiment, the viability of S. hyodysenteriae in each transport medium incubated at each temperature and for each period was determined by inoculating the transport medium onto either trypticase soy agar with 5% sheep blood or selective BJ agar and incubating at 42 C anaerobically. Viability and fecal flora contamination were evaluated blindly after 2-, 4-, and 6-day incubation periods. At -40 C, recovery of viable S. hyodysenteriae from pure culture did not differ among the transport media from 0.5 to 7 days, and all of the transport media consistently maintained the viability of the spirochetes for 7 days. At 4 C, the anaerobic and selective transport media maintained the viability of pure cultures of S. hyodysenteriae significantly better than did the conventional transport media group at day 7 (P = 0.019). At the same temperature, the anaerobic media maintained viability better than did the conventional media at 5 days (P less than 0.042).(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological variants developing in L-form cultures of two strains of Actinomyces spp. of canine origin

The present study has shown that a catalase-positive Actinomyces-like organism, UCD 19-480, failed to grom from its clinical fluid origin as normal colonies on three types of L-form media, although all three media were later shown to support it from a blood agar plate source. The growth of 19-480 from a fresh sulfur granule into a droplet of modified Barile Yaguchi Eveland (BYE) L-form broth was studied in a sealed slide incubated at 25–28°C for 11 weeks. Within 7 days, very long, bizarre branched filaments were detected near the crushed granule mass. Microcolonies were present at 5 weeks with radial filaments extending 0.5 cm from a core. The core consisted of pleomorphic vesicles arranged in a ring. Crystals appeared within and around the granule, but not elsewhere. After repeated passage on blood agar plates, 19-480 and a second, non-granule strain of recent origin, 339-180, were inoculated into BYE L-form broths as well as broths of new design. Unique membranous structures exhibiting actinomycete morphology were detected at 3 weeks in the 19-480 trypticase soya cultures containing putrescine and at 9 weeks in BYE L-form broths. They were restricted to the deep portions of certain broth types at predictable times and were not present in incubated controls. They fluoresced intensely with acridine orange and were weakly Gram-positive. ‘Pseudocolony’-like formations were detected at 8 weeks in the 3% NaCl + 5% sucrose BYE broth culture of UCD 19-480. It is suggested that the absence of normal colonies of these agents of L-form plate cultures of clinical fluids is due to growth of spreading variants unique to the actinomycete group.     

The role of hyaluronic acid capsular material of Streptococcus equi subsp. zooepidemicus in mediating adherence to HeLa cells and in resisting phagocytosis.

Hyaluronic acid is thought to be one of the critical virulence factors of Streptococcus equi subsp. zooepidemicus. The present study was designed to study the role of hyaluronic acid capsular material in mediating adherence and to resist the phagocytosis of the host's immune defence. The studies were performed with two encapsulated S. equi subsp. zooepidemicus and two unencapsulated phase variants. The bacteria had been previously isolated from diseased pigs and monkeys in Indonesia. The presence of capsular material was determined using the hyaluronic acid decapsulation test and by electron microscopic studies. Both encapsulated bacteria showed mucoid colonies after cultivation on blood agar, grew with diffuse colonies in soft agar media and reacted negatively in the salt aggregation test. The unencapsulated bacteria grew with small colonies on blood agar, formed compact colonies in soft agar media and reacted positively in the salt aggregation test. Adherence and phagocytosis studies revealed that the encapsulated bacteria adhered significantly more to HeLa cells and were less phagocytosed by murine macrophages compared to unencapsulated bacteria. Pretreatment of the HeLa cells using hyaluronic acid or pretreatment of the bacteria by hyaluronidase decreased the adherence value of encapsulated bacteria. Pretreatment of bacteria with pronase had no effect. The presented results strongly indicate that the hyaluronic acid capsular material contributes to adherence properties of S. equi subsp. zooepidemicus and might help the bacteria to resist phagocytosis by macrophages.     

Factors neutralizing the bacteriocidal effect of lysolecithin

The investigations indicate that a variety of non-dialyzable proteins and peptides, including hemoglobins, blood serum proteins, casein, soy protein and hydrolyzed proteins (peptones) are able to neutralize the bacteriocidal effect of lysolecithin.A number of lysolecithin-resistant bacteria are shown to produce lysolecithin-inhibiting metabolites that also promote growth of sensitive organisms in lysolecithin-containing media. On lysolecithin-con- taining agar this can result in a characteristic satellite growth of sensitive organisms around resistant “mother colonies”. Stable resistant mutants were easily selected from a wild type of Staphylococcus aureus after heavy inoculation on lysolecithin-containing nutrient agar.The bacterial lysolecithin-neutralizing factors examined are not considered to be of enzymatic nature. The factors in culture filtrate of Escherichia coli were separated into two active fractions by gel filtration. Due to extremely small amounts of the substances responsible for the neutralizing activity, chemical analyses of these fractions proved problematic, and only a few amino acids could be demonstrated.The neutralizing activity of the bacterial factors, and some of the proteins and peptides, resisted 100° C, or more, for several min.Some aspects of the lysolecithin-inhibitor-interaction are discussed.     

A synthetic, selective culture medium for Pseudomonas aeruginosa

Selective and differentiating media consisting of simple chemical components have been developed, both in solid and in liquid form, for culturing Pseudomonas aeruginosa from foodstuffs. These media work on the basic principle that Gram-negative bacteria can utilize ammonia as inorganic nitrogen source. This principle has already been utilized for developing a culture medium for coliform bacteria (Szita and Biró, 1986; Szita et al., 1988). P. aeruginosa can produce the ammonia needed for its growth by decomposing acetamide. The liquid synthetic medium was compared with the nitrofurantoin broth and the solid one with cetrimide agar by parallel inoculation of 60 raw milk samples and 20 P. aeruginosa pure cultures. The main advantages of the synthetic media are their high selectivity, high sensitivity and rapidity. Owing to their advantageous properties, the new media can be recommended for replacing cetrimide agar and nitrofurantoin broth.     

Effects of media ingredient substitution and comparison of growth of Flavobacterium psychrophilum among four media

The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.     

Advantages of a new agar medium in the primary isolation of Mycobacterium bovis

Suspect tuberculous lesions from 116 cattle were examined histologically and cultured for Mycobacterium bovis using 5 different media. The media used were: B83, an agar medium incorporating bovine blood and sodium pyruvate; Middlebrook's agar; 2 variations of Stonebrink's medium; L?wenstein-Jensen medium. The B83 medium and a modification of Stonebrink's medium which had a lowered concentration of malachite green were most successful, detecting 95.2% of tuberculous animals when used together. The B83 medium detected isolates approximately 1 week earlier and had more colonies than the Stonebrink's modification. A combination of 2 slopes of B83 and 2 slopes of modified Stonebrink's medium is recommended for routine culture of samples.     

Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis

An unstable L-form of Staphylococcus aureus was identified in milk samples from 3 quarters of 2 cows after treatment with cloxacillin. Milk samples incubated on standard 5% blood agar plates were culture-negative for 7 to 30 days after treatment, but S aureus was reisolated in 80% of 66 samples by additional culturing on enriched L-form media when incubated in 10% CO2 at 37 C. The organism was identified at various phases of reversion of L-form agar and was confirmed on transfer to blood agar plates.     

The effect of culture media on bacteriocin production in various strains of bacteria]

Some staphylococcus and enterococcus strains were used to investigate the effect of culture medium on bacteriocin production. Staphylococcus cohnii SC7, Staphylococcus sp. ZTJ 151, S. saprophyticus SS 877, Enterococcus faecium EF1 and E. faecalis EFG2 were isolated from the rumen wall and contents of lambs, calves and fallow deer, Enterococcus gallinarum EG10 and E. avium EA12 were isolated from the caecum of Japanese quail. The tested bacteria belong to producers with a wide antimicrobial effectiveness spectrum, they have low to medium adherence and urease activity (Tab. III). These culture media were used to test the effect of culture medium on bacteriocin production: nutrient agar no. 2 and VL agar enriched with 2% of glucose and lactose (ZAG, ZAL, VLG, VLL), agar for isolation of faecal streptococci (SA) and the base for blood agar no. 4 and no. 2 (KA4, KA2). The strains Streptococcus bovis AO 24/85 and Staphylococcus aureus Oxford 209 P were used as indicator bacteria. Tables I and II show the results of these tests. The tested strains produced the widest inhibition zones (6 mm) with both indicators on SA medium, and this indicates massive bacteriocin production. On ZAG medium, the zones of enterococci with the AO 24/85 strain were larger size than those of staphylococci, but the zones were dim. All strains with the 209P indicator produced dim zones of the 2mm size. The larger inhibition zones (2-5mm) in comparison with staphylococci were observed in enterococci on the ZAL medium with the AO24/85 strain. The production of tested strains was balanced on VLG agar with respect to the use of both indicators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological properties and classification of strains of Treponema sp. isolated from pigs in poland

Fifty-one treponemas were isolated from pigs. Twenty-three isolates with typical morphology and growth characteristic were beta hemolytic, enteropathogenic, produced indole and with exception of three strains did not ferment fructose. These strains were classified as typical T. hyodysenteriae and were usually isolated from pigs with symptoms of mucohemorrhagic diarrhoea. The seventeen other isolates were weakly beta hemolytic after 48 h incubation, enteropathogenic, 12 out of 17 produced indole, 10 out 17 fermented fructose. These strains were usually isolated from pigs with symptoms of gray-green diarrhoea and classified as T. hyodysenteriae 2 biotype or intermediate type. They may be compared with Treponema sp. isolated by Taylor et al. Eleven non enteropathogenic strains showed typical characteristic for T. innocens. Gas chromatography analysis of the fatty acids production from glucose, showed that all isolated treponemas produced acetate and butyrate. Typical T. hyodysenteriae produced additionally propionate. Strains of T. hyodysenteriae biotype 2 produced propionate or isobutyrate as well.