Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance

The capacity of six basal media to support the growth of thermophilic campylobacters was tested. The most successful was Nutrient Broth No. 2 (Oxoid) solidified with New Zealand agar but it gave at best only a 9% recovery rate. Various blood products, iron compounds, detoxifying agents, reducing agents, growth stimulants and an antimetabolite were added to the selected basal medium and counts of inoculated organisms were compared with counts on basal medium containing 5% lysed horse blood. Of 22 supplements tried only blood, Fildes' peptic digest of blood, heamatin, iron salts, charcoal, sodium metabisulphite and sodium pyruvate greatly improved the basal medium. The ability of these supplements used singly and in combinations to facilitate aerotolerance of campylobacters was investigated. Two aspects of aerotolerance were tested; (a) the ability of the supplements to sustain the viability of campylobacters seeded onto culture plates left on the bench for up to 6 h before microaerobic incubation; and (b) the ability of the supplements to facilitate the growth of campylobacters at increasing oxygen tension (6, 10 and 17% oxygen). A combination of 0.4% charcoal, 0.025% ferrous sulphate and 0.025% sodium pyruvate was found to be as effective as blood in both tests.     

The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide

B olton , F.J. C oates D. & H utchinson , D.N. 1984. The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. Journal of Applied Bacteriology 56 , 151–157.
Nutrient agar plates stored in light and air for 48 h became inhibitory for Campylobacter jejuni, C. coli and nalidixic acid-resistant, therrnophilic campylobacter (NARTC) strains. All five campylobacter test strains showed > 5 log reduction in counts on media which had been stored in light and air. Media stored in the dark and/or in a reduced atmosphere did not become inhibitory and supported the growth of campylobacters. Ferrous sulphate, sodium pyruvate, blood, charcoal or sodium metabisulphite, compounds frequently used as supplements in campylobacter media, were added to nutrient agar prior to storage of media in light and air. All additives except sodium metabisulphite prevented the accumulation of photochemically generated toxic oxygen derivatives and allowed growth of test strains. In qualitative tests to determine the ability of supplements to neutralize hydrogen peroxide, blood was the most active, charcoal and sodium pyruvate slightly less active and ferrous sulphate and sodium metabisulphite the least active. The results of this study confirm that supplements in campylobacter media act as quenching or detoxifying agents and not as enrichment factors.     

Evaluation of medium supplements for in vitro cultivation of Wuchereria bancrofti

Third-stage larvae (L3) of Wuchereria bancrofti molt to the fourth stage in an in vitro culture medium composed of NCTC 135 and Iscove's modified Dulbecco's medium (1:1; v/v) supplemented with 10% human serum and a mixture of anti-bacterial and anti-mycotic agents. In the present investigation this culture medium was used to examine the effects of different concentrations of human serum, medium supplements, and serum replacements on larval growth, development, and molting. Several medium supplements and serum replacements were evaluated including hemin, Nutridoma, and a mixture of soybean lipids, bovine serum albumin, and transferrin. The supplements tested could not support larval growth and development in the absence of serum and they did not have an enhancing effect on larval growth and development in combination with human serum. A medium supplement of 30% human serum resulted in molting of 80-94% of L3s and optimum growth to the mid to late fourth stage. This culture system provides an excellent alternative to experimentally infected animals as a source of larvae undergoing the third molt and fourth-stage larvae for screening potential anti-filarial compounds and for immunologic and biochemical studies.     

The enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis is enhanced under conditions where reactive oxygen species are neutralized

AIM: To investigate the effect of neutralization of reactive oxygen species (ROS-neutralized conditions) on the enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis using selective and nonselective media. METHODS: Pure cultures of E. coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute sodium hypochlorite, at free chlorine levels of 0.6 and 0.9 microg ml(-1), respectively, and then enumerated at 37 degrees C by surface plate counts on nonselective nutrient (N) agar and on several selective media, either under (i) standard aerobic conditions; (ii) aerobic conditions using growth medium, supplemented with 0.05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions designed to neutralize ROS, using a combination of 0.05%-w/v sodium pyruvate in the growth medium, together with incubation in an anaerobic jar. RESULTS: The counts obtained on the nonselective medium were lowest under aerobic conditions in unsupplemented medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and highest for ROS-neutralized conditions. Counts for the selective media were often lower than those for nonselective N (nutrient) agar, with enhancement under peroxide-neutralized conditions and a further increase in counts under ROS-neutralized conditions. Broadly similar observations were made for three other strains of each organism. CONCLUSIONS: Chlorine-injured E. coli and Ent. faecalis become sensitive to ROS, giving higher counts under ROS-neutralized enumeration conditions than under conventional aerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement in counts observed under ROS-neutralized conditions indicate that the addition of pyruvate to the growth medium may not fully counteract the effects of sublethal injury under aerobic conditions, which is a novel observation. Thus, ROS-neutralized conditions may be required for optimal enumeration of faecal indicator bacteria. Furthermore, the lower counts, obtained using selective media indicate that the sensitivity of chlorine-injured bacteria to selective agents is not necessarily reversed under ROS-neutralized conditions.     

Antibody production and growth of mouse hybridoma cells in Nutridoma media supplements

Traditionally, cell culturists have relied upon the addition of serum to culture medium for the growth and maintenance of cell lines. However, many aspects of the use of serum in tissue culture are problematic. Cell culture supplements that circumvent the need for serum are readily available and provide a consistent protein composition. This defined environment allows the antibody to be more easily purified from culture supernatants. Nutridoma media supplements were formulated to support the growth of lymphoblastoid cells in a defined culture environment. In this study, Nutridoma media supplements were tested in parallel with serum-containing cultures to determine if Nutridoma supplemented medium is effective in supporting hybridoma cell growth and antibody production in three hybridoma cell lines. Data, based on cell growth and antibody production, show the importance of basal media selection when serum is replaced with Nutridoma media supplements. SDS-PAGE results show that cell supernatants from Nutridoma supplemented cultures contain very few contaminating proteins.     

Selective Culture Medium to Survey the Incidence of Haemophilus Species

A culture medium for the selective isolation of Haemophilus species is described. Bacitracin and nutritional supplements were incorporated in a rich basal agar medium to which rabbit blood was added to distinguish hemolytic species. Colony counts of seven typed strains of H. influenzae on this medium were within practical limits of counts on other media tested for clinical use. The bacitracin medium was as reliable as hemoglobin-agar for detecting H. influenzae and more sensitive for detecting other Haemophilus species in a clinical survey with the advantage of selectivity.     

Culture of Ethiopian Strains of Borrelia recurrentis

A number of standard bacteriological media with supplements were tested for their ability to support in vitro growth of Ethiopian strains of Borrelia recurrentis. Propagation of 18 out of 21 strains occurred in Trypticase soy yeast broth to which bovine albumin (fraction V), N-acetyl glucosamine, and sodium pyruvate had been added. This medium supported a population of 10(7) organisms per ml and yielded a harvest of four to five times the original inoculum during the logarithmic phase of growth. Maximal yield varied from 1.4 x 10(7) to 3.4 x 10(7) organisms per ml. Generation time in optimal media was 11.3 h. Lesser multiplication of organisms occurred in other media tested. Strains from primary cultures were infective for the green monkey. Recovery of viable organisms from subculture has not been successful.     

Influence of phytohormones,carbohydrates, aminoacids,growth supplements and antibiotics on somatic embryogenesis and plant differentiation in finger millet

Cultured caryopses of finger millet (Eleusine coracana GAERTN) produced callus from shoot apices or mesocotyls depending upon the concentration of picloram and combination of cytokinins in MS basal medium. On subsequent subcultures, numerous somatic embryos differentiated from the callus on MS medium supplemented with picloram and kinetin. The embryos germinated into complete plants on medium devoid of phytohormones. When different carbohydrates were tested, basal medium containing glucose and sucrose produced the highest frequency of germinating somatic embryos. Supplementation of MS basal medium with a variety of aminoacids, osmotic agents and growth supplements had an adverse effect on the germination of embryos. Incorporation of different antibiotics such as carbenicillin, cefotaxime and streptomycin sulfate enhanced plant differentiation from somatic embryos. Cytological analysis of regenerated plants showed normal diploid chromosome number in their root tips.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - BA benzyl adenine - 2,iP 6---dimethylallylamino purine - Kn kinetin - Z zeatin     

Effects of oxygen tension and supplements to the culture medium on activation and development of bovine follicles in vitro

Our laboratory developed a method for culturing small pieces of bovine and baboon ovarian cortex, rich in primordial follicles, that supports the initiation of follicle growth and development to the primary stage. However, only a few follicles progressed to the secondary stage. The purpose of the current experiments was to determine if changes in culture conditions, specifically oxygen concentration and supplements to the culture medium, would facilitate the primary to secondary follicle transition. In Experiment 1, ovarian cortical pieces from late-gestation bovine fetuses were cultured with 2, 5, 20, or 60% oxygen in Waymouth's medium plus ITS+ (insulin, transferrin, selenium plus linoleic acid and BSA). Although the three lower concentrations of oxygen were generally equivalent in promoting follicle activation and growth, the highest concentration (60%) had deleterious effects on follicle survival after 7 days in culture, reducing the number of healthy follicles to about 35% of the number observed with 20% oxygen (P<0.05). In Experiment 2, bovine ovarian cortical pieces were cultured in the standard gas mixture (5% CO(2) in air) with graded doses of fetal bovine serum (FBS, 2.5, 5, or 10%) in the presence or absence of 0.5 or 1x ITS+. All concentrations of FBS alone were much less effective at maintaining follicular health and supporting the initiation and progression of follicular growth than was ITS+. However, 5 and 10% FBS alone increased the percentage of healthy primordial and primary follicles by about twofold (P<0.05) in the absence of ITS+ and in the presence of 0.5x ITS+, they enhanced the primary to secondary follicle transition by 10- and 9-fold, respectively. Thus, of the culture conditions evaluated, 20% oxygen and medium containing 0.5x ITS+ plus 5% or 10% FBS were the most effective for promoting follicular health and development.     

Comparison of supplements* to enhance recovery of heat‐injured Salmonella from egg albumen

Aims: The purpose of this study was to determine the proficiency of supplements to enhance the recovery of Salmonella from heat‐treated liquid egg albumen on solid agar media. Methods and Results: Salmonella‐inoculated albumen, heated at 53·3°C for 4 min, was plated on 39 combinations of solid media with or without the addition of 12 supplements. Greater numbers of Salmonella (P < 0·05) recovered with the addition of 1·0 g l^?1 ferrous sulfate (FeSO₄) than with any other supplements, except for 0·5 or 1·0 g l^?1 3′3′‐thiodipropionic acid (TDP), which recovered equivalent populations. Addition of 1·0 g l^?1 sodium pyruvate or 6·0 g l^?1 yeast extract plus 1·0 g l^?1 sodium pyruvate supported greater resuscitation than unsupplemented tryptic soy agar (TSA) or supplementing with 0·01 or 0·1 g l^?1 N‐propyl gallate, 10 g l^?1 activated charcoal, 0·1 g l^?1 KMnO₄ or 50 mg l^?1 ethoxyquin. The remaining supplements supported recovery of equivalent numbers of Salmonella, which were fewer cells than recovered with 1·0 g l^?1 FeSO₄, yet greater populations than recovered with 50 mg l^?1 ethoxyquin. Conclusion: Supplementation of plating media with FeSO₄, TDP or sodium pyruvate enhanced recovery of sublethally injured Salmonella from albumen. Significance and Impact of the Study: Pasteurizing albumen impedes recovery of pathogens. These results suggest that the addition of supplements to plating media may assist resuscitation and colony development of heat‐injured salmonellae.     

Stimulation of the growth of cutaneous strains of Peptococcus saccharolyticus by iron, haematin and blood

The growth of nine strains of Peptococcus saccharolyticus was assessed quantitatively by culture Trypticase Soy/yeast extract/Tween 80 agar (TSY-TW) with and without supplementation with iron or haematin and on blood agar, in aerobic, reduced 02 (3% O2 with 8% CO2, 8% H2 and 81% N2) and anaerobic atmospheres. All strains grew better anaerobically and under reduced O2 conditions than aerobically on supplemented or unsupplemented TSY-TW.Supplementation of TSY-TW with iron or haematin resulted in an average 4.4-fold increase in bacterial count in a reduced O2 atmosphere and an average 4.2-fold increase under anaerobic conditions. Under aerobic conditions the increase in count ranged from O to greater than 5000-fold, as some strains failed to grow on unsupplemented TSY-TW but responded well to the supplements of iron or haematin. The highest bacterial counts were obtained on Columbia blood agar incubated anaerobically. However, P. saccharolyticus failed to grow aerobically on plain or heated Columbia blood agar with or without supplements. TSY-TW blood agar supported the growth of the one strain tested under all three atmospheric conditions. The type strain (ATCC 14953) differed from all others in its failure to grow aerobically or in a reduced O2 atmosphere on supplement or unsupplemented media. Colony size varied greatly on different media, in different atmospheres and from strain to strain, being greatest in a reduced O2 atmosphere on Columbia blood agar. There was no correlation between the viable bacterial count and colony size.     

Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples

Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics. Fifty-two strains of campylobacters were isolated from total of 1668 cultures. The various broth/medium combinations did not affect the dominance of C. jejuni over C. coli (total 49 C. jejuni and three C. coli). The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium. Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants. Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate. Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants. Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants. However, these membranes retain campylobacters and must be cultured to avoid underestimation. From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium.     

Isolation and characterization of a novel catalase-negative, urease-positive Campylobacter from cattle faeces

Forty-four strains of a phenotypically unique Campylobacter were isolated from the faeces of 26 of 45 cows in a single herd. Isolation involved enrichment and membrane filtration onto blood agar or plating onto cefoperazone amphotericin teicoplanin agar. The strains exhibited phenotypic characteristics typical for Campylobacter species. However, they were unusual in that they produced urease and copious H₂S in triple sugar iron (TSI) medium, but did not produce catalase. They did not grow aerobically. None of the strains grew on modified cefoperazone charcoal deoxycholate agar (mCCDA). Macrorestriction profiles of chromosomal DNA were prepared for 15 strains using pulsed-field gel electrophoresis (PFGE). Twelve of 15 profiles were identical and all appeared to be closely related. These catalase-negative, urease-positive campylobacters (CNUPC) represent a group not previously reported. Their sensitivity to antibiotics normally used in selective media for campylobacters might explain why they have not previously been encountered. Their ecological significance and importance with respect to human and animal disease remain to be assessed.     

Improved Recoverability of Microbial Colonies from Marine Sponge Samples

Abstract The growth of microorganisms from marine sponge samples was studied on various low to high nutrient solid media using media supplements. The supplements utilized were catalase, sodium pyruvate, and a combination of the two. Medium composition was found to influence the growth response on the supplemented media. Microorganisms on low nutrient media responded more favorably to the media additions than on high nutrient media. Thirty-five percent of the supplemented media demonstrated colony forming unit (CFU) recoveries that were 50% or greater than those of the unamended control plates. Twenty-one percent showed recoveries of more than 100% of the control values, with sodium pyruvate additions providing for the greatest overall increase in recovery, whether alone or in conjunction with catalase. These findings suggest that addition of catalase or sodium pyruvate to solid growth and isolation media may improve recoverability of microorganisms from natural samples. Received: 24 January 2000; Accepted: 9 June 2000; Online Publication: 28 August 2000     

Role of media, protein and energy supplements on maintenance of morphology and DNA-synthesis of small preantral domestic cat follicles during short-term culture

Small preantral follicles (40 to 90 microns in diameter) from domestic cats were cultured for 10 d using different media (M199 and Dulbecco's MEM) and protein (FCS and BSA) supplements. Culture efficacy was determined by Hoechst 33258 staining and estimation of Brom-desoxyuridine (BrdU)-incorporation into oocytes and granulosa cells. Culture in M199 + FCS and in DMEM + FCS resulted in 21.6% and 38.1%, respectively, of morphologically intact preantral follicles. Adding BSA increased the rate of normal follicles to 51.7% in M199 and to 58.6% in DMEM. Oocytes were found in 40% of the follicles, when DMEM and/or BSA supplementation was used, while M199 with FCS induced acute loss of oocytes in 85% of the follicles. About 10% of the oocytes contained degenerating chromatin. Measurement of BrdU-incorporation during culture allows for quick and effective assessment of follicle viability in vitro. Comparison of M199 and Dulbecco's MEM, both with FCS or BSA and DMEM with or without pyruvate/lactate, indicated that Dulbecco's MEM + BSA without pyruvate and lactate is the best medium for culture of cat follicles. However, further research of suitable medium supplements is needed.     

Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements

Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.     

Differential characteristics of catalase-positive campylobacters correlated with DNA homology groups

Eighty-four strains of catalase-positive campylobacters could be placed into seven distinct DNA homology groups (species), corresponding to Campylobacter fetus, "C. hyointestinalis," C. jejuni, C. coli, "C. laridis," "C. fecalis," and aerotolerant campylobacters. The biochemical and physiological characteristics of the strains were examined for their correlation with the homology groups. The characterization tests that provided the most reliable differentiation at the species and subspecies level were growth at 25 and 42 degrees C, sensitivity to cephalothin and nalidixic acid, growth in semisolid media containing 1% glycine and 3.5% NaCl, growth on plates containing 1.5% NaCl, growth in a semisolid minimal medium, anaerobic growth in the presence of 0.1% trimethylamine-N-oxide, hydrogen sulfide production in SIM medium and triple-sugar iron agar, hippurate hydrolysis, nitrite reduction, and growth on plates under an air atmosphere.     

Enumeration of sublethally heated staphylococci in some dried foods.

The effect of 45 substances to restore the salt tolerance of sublethally heat-injured Staphylococcus aureus was tested. Sodium pyruvate, yeast extract, L-histine, casitone (Difco), adenosine triphosphate, and acetylphosphate were effective. For enumeration a repair medium was first used, containing sodium pyruvate and penicillin in 1% skim milk. This step was followed by counting on Baird-Parker agar with penicillinase. This method was selective; fewer than 100 staphylococci/g food could be enumerated and it gave counts about 8 times higher than the method of Giolitti and Cantoni used as a five-tube most probable number technique. Heat injury sensitized S. aureus to polymyxin.     

Culture medium pH is influenced by basal medium,carbohydrate source,gelling agent,activated charcoal,and medium storage method

Summary When four carbohydrates were tested against six commonly cited inorganic basal media, post-autoclave pH was highest for carbohydrate-free and sucrose containing media, and progressively lower for maltoseglucose and fructose-containing media, respectively. Post-autoclave pH for these media without carbohydrates was related to medium buffering capacity. Addition of gelling agents (10 of 11 tested) increased the postautoclave pH of MS medium containing sucrose. Neutralized and acid-washed activated charcoal also increased the post-autoclave pH of liquid and agarsolidified MS medium, and the pH changed further during 8 weeks of storage. Changes in medium pH caused by gelling agents, but not charcoal, could be alleviated by adjusting the pH after their addition but prior to autoclaving.     

Influence of medium composition on the growth and antigen expression of Helicobacter pylori

An evaluation of the ability of various solid and liquid media to support both growth and antigen expression, particularly lipopolysaccharide (LPS) expression, by Helicobacter pylori culture collection strains and clinical isolates was performed. Liquid-based basal media (brain heart infusion, Brucella broth, Mueller–Hinton broth and tryptone soya broth) supported the growth of strains, whereas solid basal media of the same formulation did not support growth. Optimal growth of all strains was obtained on solid and in liquid media containing blood. Supplemented solid media containing supplements other than blood supported growth but only to a small extent. In liquid media excluding blood, serum supplements enhanced growth and horse serum was found to be superior to fetal calf serum. In general, β-cyclodextrin did not increase growth. Mueller–Hinton broth or tryptone soya broth containing horse serum and a nitrogen source such as yeast extract or proteose peptone no. 3 were found to give optimal growth of H. pylori in a blood-free environment. Strains after cultivation in liquid media, irrespective of composition, maintained production of high-molecular weight (mol. wt) LPS with an O side chain independent of medium composition, whereas subculturing on solid media resulted in production of low-mol. wt LPS. Expression of proteins differed in liquid and on solid media, particularly proteins of 57 and 60 kDa, but qualitatively no differences were observed upon supplementation of basal media.