Helicobacter pylori in Liquid Culture: Evaluation of Growth Rates and Ultrastructure

This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (0.1% wt/vol), agar (0.3% wt/vol), or blood agar blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140–200 nm) were seen in the cytoplasms. Incubation of cultures on rotary shakers at 120 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol) or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and the cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells of H. pylori showed on electron micrographs clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at −80°C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and Brucella broth without cryopreservative (in descending order). Received: 10 November 1997 / Accepted: 29 January 1998     

Electron microscopic observation of Branhamella catarrhalis

The hemagglutination (HA) test was done on 85 strains of Branhamella catarrhalis, isolated from sputum of patients with respiratory infections; 53% were HA positive strains. Three HA positive and three HA negative strains were selected and were observed under the electron microscope. The bacterial cell wall appeared to be lobulated and its total thickness was about 38 nm. The nuclear region consisted of whorls or fibrils and dense bodies. Five strains were fimbriated and one strain was nonfimbriated. The size of fimbriae was about 68 nm in length and 4.5 nm in width. The fimbriae of Branhamella catarrhalis were densely arranged and peritrichous in distribution. There was no change of fimbriation between broth and agar cultures.     

Physicochemical properties of Shiga toxigenic Escherichia coli

AIMS: To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture. METHODS AND RESULTS: Cellular surface charge was determined using zeta potential measurements. Hydrophobicity of the isolates was determined using bacterial adhesion to hydrocarbons assay, hydrophobic interaction chromatography and contact angle measurements. Microbial adhesion to solvents was used to determine the electron donor/acceptor characteristics. No differences of surface charge measurements were found between broth and agar grown cultures. Isolates belonging to serogroup O157 and serotypes O26:H11 and O111:H- were significantly (P < 0.05) less negatively charged than other STEC serotypes tested. All strains were hydrophilic with most methods and demonstrated a lower hydrophobicity in agar culture compared with broth culture. All strains demonstrated a strong microbial adhesion to chloroform indicating that STEC possess an electron donor and basic character. A relationship between serogroup O157 and other STEC serotypes was apparent using principal-component analysis (PCA). CONCLUSIONS: Combining the results for physicochemical properties using PCA differentiated between strains belonging to the O157 serogroup and other STEC/non-STEC strains. PCA found similar results for broth and agar grown cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Particular serotypes of STEC possess similar physicochemical properties which may play a role in their pathogenicity or potential attachment to various surfaces.     

Pseudomonas putrefaciens Isolates from Clinical Specimens

A total of 109 cultures of Pseudomonas putrefaciens isolated from clinical specimens were studied. The cultures were separated into two groups. The majority of the group 1 isolates, comprising 31 cultures, were characterized by (i) growth in plain nutrient broth, but no growth in broth supplemented with NaCl at concentrations of 7% and above, (ii) no growth on Salmonella-Shigella (SS) agar, and (iii) production of acid from the carbohydrates, sucrose, maltose, arabinose, and dextrin. Most group 2 isolates, comprising 78 cultures, were (i) unable to grow in plain nutrient broth, but grew well in broth supplemented with NaCl at a concentration of 7 to 10%, (ii) able to grow on SS agar, and (iii) unable to produce detectable amounts of acid from any of the carbohydrates tested except for variable results with glucose and fructose.     

Occurrence of a Lysogenic Streptomyces sp. on the Nodule Surface of Black Gram (Vigna mungo (L.) Hepper)

A lysogenic Streptomyces sp., strain NS.A4, which was isolated from the nodule surface of black gram (Vigna mungo (L.) Hepper), was found to inhibit rhizobia of fast-and slow-growing strains of cowpeas and soybeans. It exhibited plaques when there was a change in cultural conditions. Repeated culturing of the organism in nutrient agar and broth confirmed the infection of Streptomyces sp. strain NS.A4 by an actinophage. Addition of the culture filtrate of Streptomyces sp. strain NS.A4 to shaken broth cultures of three other Streptomyces spp. resulted in phage infection.     

Influence of Incubation Temperature and Sodium Heptadecyl Sulfate (Tergitol No. 7) on the Isolation of Salmonellae from Pork Sausage

Cultures of 68 samples of fresh pork sausage purchased locally were incubated at 37 and 43 C, with and without Tergitol No. 7 (sodium heptadecyl sulfate) added to the tetrathionate-Brilliant Green enrichment broth. The results indicated an advantage in incubating the tetrathionate broth at 43 C rather than 37 C in attempting to isolate salmonellae from pork sausage. Without Tergitol, more samples were positive at 43 C than at 37 C, but with Tergitol there was no difference. The higher temperature suppressed the competing gram-negative bacteria and permitted Salmonella to grow in relatively pure culture, thus providing an advantage for isolating and identifying the organisms. Tergitol dispersed and emulsified the fat which improved the isolation of Salmonella when the cultures were incubated at 37 C but not at 43 C. Brilliant Green-sulfadiazine agar was superior to bismuth sulfite agar for isolating salmonellae from tetrathionate broth cultures of fresh pork sausage.     

Growth of Helicobacter pylori in various liquid and plating media

The objectives of this research were to compare commonly used liquid and plating media to elucidate whether one medium provided superior growth of Helicobacter pylori in vitro. The liquid media compared were Mueller-Hinton broth, brain heart infusion broth and H. pylori special peptone broth, formulated in this laboratory. No significant differences in growth rates were noted and shaking during the incubation of broths was not essential for good growth. The plating media compared included Columbia agar, Mueller-Hinton agar, modified Glupczynski's Brussels campylobacter charcoal agar, Johnson-Murano agar and H. pylori special peptone agar (HPSPA). None of the non-specific plating media that have been used historically to culture H. pylori exhibited any particular advantage. However, HPSPA provided an obvious advantage in colony size. Helicobacter pylori special peptone agar enhances the cultivation of H. pylori and could improve the recovery of the bacterium from clinical samples in vitro.     

THE RESISTANCE OF TWO STRAINS OF BACTERIUM COLI TYPE I TO DRYING UNDER ATMOSPHERIC CONDITIONS

SUMMARY: Suspensions of two strains of Bacterium coli type I were dried as thin films under atmospheric conditions and the numbers of organisms determined before and after drying. Three methods were used to grow the culture; in two the culture was grown in broth and in the other on agar slopes.
Strain 28.D.10 was less sensitive than strain NCTC 5934. After culturing in broth NCTC 5934 showed irregular daily fluctuations in sensitivity to drying; 28.D.10 became more sensitive for the first 2–3 weeks and thereafter less sensitive. Suspensions prepared from 18 hr plate cultures were more sensitive than from 24 hr plate cultures; a change from peptone water to Lemco broth for daily culturing slightly decreased the sensitivity to drying. With both strains suspensions prepared from broth cultures were more sensitive than those from agar slopes.
Continuous daily culturing of 28.D.10 on a solid medium as compared with broth decreased the sensitivity of suspensions and over a long period the culture appeared to be more stable. When strain NCTC 5934 was grown on a solid medium the suspension was as sensitive to drying as that obtained when broth was used but the daily fluctuation of results appeared to be less.
A decrease in the number of cells in the 24 hr broth culture of 28.D.10 coincided in time with an increase in the sensitivity to drying of the suspension prepared from the same culture.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

A NOTE ON THE USE OF ROGOSA AND MODIFIED ROGOSA MEDIA FOR THE ENUMERATION AND CULTURE OF LACTOBACILLI

SUMMARY: The modified Rogosa media (acetate broth and acetate agar) developed by Mabbitt & Zielinska (1956) have been compared with tomato juice media for the growth of representative Lactobacillus species.
Most of the species tested grew markedly better in acetate broth than in tomato dextrose broth, but some cultures did not grow well in the acetate broth although they grew normally in tomato dextrose broth. The acetate agar was satisfactory for counting those lactobacilli cultured at 30° but was inferior to tomato dextrose agar for counting those grown at 37°.     

Comparison of microbiological culture methods in screening allograft tissue. Swab versus nutrient broth

In order to reduce the risk of transmission of infectious diseases through transplantation of tissue allografts, one should examine tissues for the presence of microorganisms. However, there are no detailed tissue banking guidelines describing the culture method or incubation time to be used. Therefore, we compared two culture methods--blood agar plate versus Wilkins Chalgren broth--and three incubation times--2, 7 or 14 days for their performance. The ultimate aim is to use the optimal setting as standard operating procedure (SOP) for tissue allograft cultures. From 70 consecutive donors, 919 tissue samples were taken. All 919 tissue samples were incubated on blood agar as well as in Wilkins Chalgren broth for 7 days. 567 of these 919 tissue samples were left to incubate up to 14 days. Wilkins Chalgren broth yielded 24.5% (139/567) positive cultures after 14 days of incubation. This was slightly more than the growth on blood agar after 14 days (22.9%--130/567) (p=n.s.) and significantly more than the growth in Wilkins Chalgren broth after 7 days of incubation (21.9%--124/567) (p<0.05). Based on these results, Wilkins Chalgren broth has been implemented as the SOP. Since the yield of positive cultures increased from 2 to 7 days of incubation in broth (1.8 times) and the variability of species cultured from 7 to 14 days of incubation shifted towards mostly microorganisms known to be common contaminants, we established the cut-off at 7 days of incubation in Wilkins Chalgren broth.     

Improved method for detection of Vibrio parahaemolyticus in seafood.

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction,electron microscopy and evaluation of bactericidal properties

The evolution of nanotechnology and the production of nanomedicine from various sources had proven to be of intense value in the field of biomedicine. The smaller size of nanoparticles is gaining importance in research for the treatment of various diseases. Moreover the production of nanoparticles is eco-friendly and cost effective. In the present study silver nanoparticles were synthesized from Carica papaya leaf extract (CPL) and characterized for their size and shape using scanning electron microscopy and transmission electron microscopy, respectively. Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDS/EDX) and X-ray diffraction spectroscopy (XRD) were conducted to determine the concentration of metal ions, the shape of molecules. The bactericidal activity was evaluated using Luria Bertani broth cultures and the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using turbidimetry. The data analysis showed size of 50–250 nm spherical shaped nanoparticles. The turbidimetry analysis showed MIC and MBC was >25 μg/mL against both Gram positive and Gram negative bacteria in Luria Bertani broth cultures. In summary the synthesized silver nanoparticles from CPL showed acceptable size and shape of nanoparticles and effective bactericidal activity.     

Lactic acid bacteria isolated from fish gut produce conjugated linoleic acid without the addition of exogenous substrate

The production of conjugated linoleic acid (CLA) by four strains of lactic acid bacteria isolated from fish, i.e., Leuconostoc mesenteroides H20, Leuconostoc mesenteroides H22, Leuconostoc lactis H24 and Lactobacillus pentosus H16, was evaluated in MRS broth and on MRS agar. The bioconversion and production of CLA by resting cells were also assessed. Linoleic acid was detected in cultures grown on agar at percentages of up to 18.3% (w/w) of total fatty acid, and conjugated isomers were found in the fatty acid profiles of Lactobacillus pentosus H16. The percentage of CLA relative to total fatty acid increased from 5.68 ± 1.65% to 23.69 ± 0.79% when resting cells were removed from agar plates and incubated without the addition of exogenous linoleic acid as a substrate. When Lactobacillus pentosus H16 cells were incubated with linoleic acid, cyclization and changes in monounsaturated fatty acid percentages were observed instead of conjugation. These results show that growth on a solid support is required for CLA production. More significantly, an increase in the CLA content could be achieved by incubating resting cells without exogenous substrate.     

Conventional laboratory agar media provide an iron-limited environment for bacterial growth

Abstract The outer membrane proteins of Escherichia coli and Pseudomonas aeruginosa grown in a number of conventional laboratory media were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) High-molecular-weight proteins similar to those produced by these strains in an iron-limited chemically defined medium were detected in cells grown on the surface of various agar media. In contrast, these proteins were not produced or were only poorly expressed by the corresponding broth cultures or by cells grown an agar supplemented with iron. A catecholic substance could be detected in DST agar extracts subsequent to bacterial growth which was produced to a lesser extent in IST agar and in broth cultures.     

OBSERVATIONS ON THE ISOLATION OF SALMONELLAE FROM SELENITE BROTH

SUMMARY: Studies of growth curves of enterobacteria in selenite broth containing different carbohydrates showed that whereas mannitol and lactose brought about a steep fall in coli-aerogenes bacteria, mannitol improved the growth of Salm. typhi-murium . With mixed cultures of Cit. freundii I and Salm. typhi-murium the presence of lactose, utilizable by the former, adversely affected the viable count of the latter.
Comparative studies with routine faeces specimens showed that selenite broth was an efficient selective medium with MacConkey's agar; but much better results were obtained when it was combined with deoxycholate-citrate agar. Gentian violet introduced into selenite broth improved its selectivity for most Salmonella types when MacConkey's agar was used for final isolation.     

Chromogenic detection of antigen in bacteriophage plaques: a microplaque method applicable to large-scale screening

We have developed a simple and rapid (24 h) enzyme-linked immuno-detection method to screen for rare antigen-positive phage among large numbers of antigen-negative ones. Horse-radish peroxidase-antibody conjugate, incorporated into the soft agar layer of a plaque assay system, is precipitated locally by antigen produced during plaque formation, and is detected by standard chromogenic methods. The method has been used to screen plaques of bacteriophage beta tox+ for the presence of diphtheria toxin and related cross-reacting material. When phage were plated on very dense bacterial lawns, they formed minute plaques (microplaques). Because of the high local concentration of antigen generated by lysis of the dense lawn, the microplaques gave more intense chromogenic signals than larger plaques formed on less dense Corynebacterium diphtheriae lawns. Thus, antigen-positive microplaques could be easily recognized even in the presence of very large numbers of antigen-negatives. In a reconstruction experiment, small numbers of antigen-positive phage were detected with high efficiency (greater than 75%) against a background of 3.8 X 10(4) antigen-negatives/cm2 of agar surface (equivalent to 2.4 X 10(6) plaques/9 cm petri plate). This screening method should facilitate isolation of phage mutants affecting production of given antigens and may be of particular value in detecting specific genes cloned into phage vectors.     

Sub-lethal damage of Listeria monocytogenes after long-term chilled storage at 4 degrees C

The possibility that long term in vitro chilled storage may result in sub-lethal damage to Listeria monocytogenes cells was investigated by comparing growth of chill-stored (starvation at 4 degrees C) and fresh cultures on selective and non-selective media. Growth of freshly grown cells was minimally (3-8%) affected by selective LSAMM agar compared with non-selective Brain Heart Infusion agar. In contrast, numbers of chill-stored strains were reduced by greater than 99% after direct plating on the same selective and non-selective media. Furthermore, chill-stored strains were able to grow in standard selective broth (Listeria Selective broth and Fraser broth) only if undiluted inocula (approximately 10(5)-10(6) cfu ml-1) were used, whereas they were capable of growth in Brain Heart Infusion broth even when the lowest dilutions were used (approximately 10(1) cfu ml-1). The potential public health consequences of this finding for the isolation of Listeria monocytogenes from foods is considered.