scholarly journals Effects of Growth Medium, Inoculum Size, and Incubation Time on Culturability and Isolation of Soil Bacteria

2005 ◽  
Vol 71 (2) ◽  
pp. 826-834 ◽  
Author(s):  
Kathryn E. R. Davis ◽  
Shayne J. Joseph ◽  
Peter H. Janssen
Keyword(s):  
New Media ◽  
Growth Medium ◽  
Incubation Time ◽  
Low Frequency ◽  
Viable Count ◽  
Inoculum Size ◽  
Bacterial Cells ◽  
Phylogenetic Groups ◽  
Solid Media ◽  
Pure Cultures

ABSTRACT Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes (including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.

scholarly journals Occurrence of Bacterial Stem Rot, Caused by Erwinia chrysanthemi, on Field-Grown Tomato in Florida

Plant Disease ◽  
1998 ◽  
Vol 82 (7) ◽  
pp. 831-831 ◽  
Author(s):  
D. O. Chellemi ◽  
H. A. Dankers ◽  
K. Hill ◽  
R. E. Cullen ◽  
G. W. Simone ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Methyl Esters ◽  
Sole Source ◽  
Erwinia Chrysanthemi ◽  
Identification System ◽  
Bacterial Cells ◽  
Sterile Water ◽  
Microbial Identification ◽  
Pure Cultures ◽  
Tomato Isolate

In September 1997, wilted 4-week-old tomato (Lycopersicon esculentum Mill.) plants were observed in a commercial production field in St. Lucie County, FL. Closer inspection of affected plants revealed hollow stems and petioles with dark, water-soaked lesions. Diseased tissue was macerated and streaked onto nutrient agar (NA) and crystal violet pectate (CVP) agar. After incubation for 2 days at 30°C, isolates produced pits on the CVP agar. Isolates were transferred onto NA and the incubation and transfer procedure was performed two additional times to obtain pure cultures. Suspensions of bacterial cells were injected into tomato and tobacco leaves to test for a hypersensitive or pathogenic reaction. Isolates produced collapsed necrotic tissue on tomato while no reaction was observed on tobacco. Tests for differentiating species and subspecies in the ‘carotovora’ group of Erwinia were conducted following the protocol of Dickey and Kelman (1). With known cultures of E. carotovora subsp. carotovora and E. chrysanthemi as controls, the isolate from tomato was determined to function as a facultative anaerobe, utilize asparagine as a sole source of carbon and nitrogen, and give positive reactions for pectate degradation, phosphatase, and growth at 37°C. Known cultures of E. carotovora subsp. carotovora, E. chrysanthemi, and the tomato isolate were grown on trypticase soy broth agar for 24 h at 28°C and their cellular fatty acids derivatized to fatty acid methyl esters (FAMEs). Statistical analyses of FAME profile data (MIDI Microbial Identification System, Newark, DE, version 3.60) identified the tomato isolate as Erwinia chrysanthemi. Pathogenicity was determined by inoculating 50-day-old tomato plants (cv. SunPride) with a suspension of E. chrysanthemi obtained from nutrient broth plates incubated at 24°C for 60 h. Three plants each were inoculated with the E. chrysanthemi identified from tomato, sterile water, and known cultures of E. chrysanthemi and E. carotovora subsp. carotovora by placing a drop at the junction of the petiole and stem and passing a sterile needle through the drop into the stem. Plants were maintained in a greenhouse. Dark, water-soaked cankers were observed on the stems of plants inoculated with E. chrysanthemi, including the tomato isolate and E. carotovora subsp. carotovora, after 7 days. No symptoms were observed on plants inoculated with sterile water. Reisolation of the pathogen and identification was performed with tissue from one of the symptomatic inoculated plants. Analyses of FAMEs confirmed E. chrysanthemi as the causal agent. This is the first report of E. chrysanthemi causing a vascular disease of field-grown tomato in Florida. Reference: (1) R. S. Dickey and A. Kelman. 1988. Pages 44–59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. American Phytopathological Society, St. Paul, MN.

Survival of Bacteria in “Soul Foods” at 10-Centigrade

1981 ◽  
Vol 44 (4) ◽  
pp. 271-274
Author(s):  
ADELLE W. STEWART
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Salmonella Typhimurium ◽  
Food Samples ◽  
Viable Count ◽  
Inoculum Size ◽  
Farm Families ◽  
Farm Family ◽  
Collard Greens ◽  
Naturally Occurring

The fate of naturally occurring and added bacterial pathogens was determined in “soul foods” purchased at local supermarkets and farm families while the foods were stored under conditions simulating those used for retail distribution, home storage, and preparation before use. Viable count determinations for 10 samples at the end of a 5-day period at 10 C showed considerable decreases in comparison to the inoculum size, indicating that growth was not promoted. Escherichia coli survived in all the food samples but the populations decreased by 1 to 9 log cycles/g of food. Salmonella typhimurium survived in 59% of the food samples. Except for farm family collard greens and sausage (encased), Staphylococcus aureus remained viable in all of the foods tested an d was the only survivor in cracklings (cooked) obtained from both sources. Clostridium perfringens was detected in farm family sweet peas and 23% of the pig offal samples.

Effect of Four Presumptive Coliform Test Media, Incubation Time and Product Inoculum Size on Recovery of Coliforms from Dairy Products

1985 ◽  
Vol 48 (5) ◽  
pp. 388-392 ◽  
Author(s):  
B. C. COOKE ◽  
M. A. JORGENSEN ◽  
A. B. MacDONALD
Keyword(s):  
Bile Salt ◽  
Media Studies ◽  
Incubation Time ◽  
Dairy Products ◽  
Limited Range ◽  
Inoculum Size ◽  
Media Type ◽  
Defined Media ◽  
Media Comparison ◽  
Better Than

The need to consider the interrelationship of relevant procedural parameters in media comparison studies has been shown. In this study, the influence of media-type on recovery of coliforms from dairy products was found to be of less importance than the effects of incubation time and inoculum size. Statistically significant media-type effects were found, however, and these contrasted with the observations of other workers in that bile-salt-based media performed better than chemically-defined media. These findings indicate that media studies too specifically designed may yield conclusions with limited range of application.

scholarly journals Fast and reliable procedure developed to generate soft rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) Tn5 mutants resistant to bacteriophage infection

2020 ◽  
Vol 159 (1) ◽  
pp. 227-231
Author(s):  
Robert Czajkowski
Keyword(s):  
Growth Medium ◽  
Aminolevulinic Acid ◽  
Soft Rot ◽  
Lytic Bacteriophage ◽  
Bacterial Cells ◽  
Bacteriophage Infection ◽  
Genes Encoding ◽  
Tn5 Mutants ◽  
Bacterial Mutants ◽  
Fast Procedure

AbstractA simple and fast procedure has been developed to generate soft rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.) Tn5 mutants in genes encoding receptors used by bacteriophages to interact with their hosts, for the follow-up studies. The procedure is inexpensive and does not require any specialized tools and/or dedicated technical support. The neomycin-resistant SRP Tn5 mutants are generated via conjugation with a transposon donor Escherichia coli ST18 strain (requiring 5-aminolevulinic acid (5-ALA) to survive) carrying pFAJ1819-mini-Tn5-neoR. The conjugation is done on solid medium supplemented with 5-ALA. After conjugation bacterial cells are collected, suspended in liquid bacterial medium, added to the suspension containing lytic bacteriophages and incubated for the additional 30 min with shaking (120 rpm). During this stage, the transposon recipients (Pectobacterium spp. and/or Dickeya spp. Tn5 mutants), susceptible to bacteriophage infection are lysed. Likewise, due to the lack of 5-ALA in the growth medium, E. coli ST18 (transposon donor) cells die at this stage. Finally, after incubation, the bacterial mutants with the Tn5 insertions, resistant to phage infection are selected on solid growth medium supplemented with neomycin. The Tn5 insertion sites are sequenced to acquire knowledge about the Tn5-distrupted genes and their putative function in phage-host interactions. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes in a short time (ca. 48 h) that can be later characterized for various other phenotypic features. In this study, as a proof-of-concept, this method has been used to generate Dickeya solani IPO2222 Tn5 mutants resistant to infection caused by the lytic bacteriophage ɸD5.

Studies in intermicrobial symbiosis. Saccharomyces cerevisiae and Lactobacillus casei

1972 ◽  
Vol 18 (11) ◽  
pp. 1733-1742 ◽  
Author(s):  
R. D. Megee III ◽  
J. F. Drake ◽  
A. G. Fredrickson ◽  
H. M. Tsuchiya
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Continuous Culture ◽  
Mixed Culture ◽  
Growth Medium ◽  
Lactobacillus Casei ◽  
Mixed Cultures ◽  
Genetic Characteristics ◽  
Culture Techniques ◽  
Pure Cultures

Saccharomyces cerevisiae and a riboflavin assay strain of Lactobacillus casei have been propagated anaerobically in mixed culture. Both batch and continuous culture techniques were used. By varying the concentrations of glucose and riboflavin in the growth medium, it was possible to produce symbioses of commensalism + competition, competition, and mutualism + competition. In short, the interaction prevailing is determined by the medium as well as by the genetic characteristics of the organisms. The behavior of the mixed cultures in these situations was predicted from data taken on pure cultures of the organisms.

Colony formation by Bact. lactis aerogenes on solid media containing antibacterial agents

1952 ◽  
Vol 215 (1123) ◽  
pp. 565-565
Keyword(s):  
Inoculum Size ◽  
Survival Times ◽  
Fluctuation Test ◽  
Solid Media ◽  
Statistical Variation ◽  
Toxic Concentration ◽  
Solid Agar ◽  
Agar Media ◽  
Different Cultures ◽  
The Given

A study has been made of the formation of colonies of Bact. lactis aerogenes on solid agar media containing antibacterial substances (brilliant green, 1-phenyl semicarbazide, phenyl mercuric nitrate, phenol, thymol and chloramphenicol) at such concentrations that a small fraction only of the inoculated cells develop. The pattern of behaviour varies from drug to drug and sometimes from culture to culture with a given drug. As the toxic concentration increases, colonies diminish in number, in size or in both. Anomalous dependence in some cases upon inoculum size, and the appearance in others of satellites to the main colonies, indicate the operation of co-operative effects probably depending upon diffusion of metabolites or antagonists. The statistical variation in the number of developing colonies is greater for different cultures than for samples of a given culture (as in the well-known fluctuation test for mutations), but the behaviour of a culture may depend upon the aeration, and upon the precise conditions of the test. The variances show no apparent relation to the ease of production of resistance to the given drug. Nor does the scatter of the survival times in liquid media containing phenol (no resistance developable) differ much from that in chloramphenicol (resistant forms readily produced). Consideration of the factors determining the successful formation of a colony on a drug plate suggests that the fluctuation test for the demonstration of mutations must be applied with great reserve.

Acoustic Band Gap Extension in One-Dimensional Solid/Fluid Phononic Crystal Heterostructure

2014 ◽  
Vol 22 (04) ◽  
pp. 1450010 ◽  
Author(s):  
Xu Yang Xiao ◽  
Run Ping Chen
Keyword(s):  
Band Gap ◽  
Phononic Crystal ◽  
Low Frequency ◽  
Wide Band ◽  
Normal Incidence ◽  
Wide Band Gap ◽  
Longitudinal Waves ◽  
One Dimensional ◽  
Solid Media ◽  
Fluid Media

The propagation of elastic longitudinal waves in one-dimensional (1D) phononic crystals (PNCs) consisting of alternating solid and fluid media is comprehensively analyzed in theory. We demonstrate the acoustic band gap (ABG) structure determined by the dispersion relation for longitudinal waves at normal incidence. According to the band structure, we design a sub-PNC by setting a reasonable thickness ratio of fluid and solid media, and then form a phononic heterostructure by merging this PNC and other PNC designed in advance. We have shown that the wide band gap exists in such a phononic heterostructure for elastic longitudinal waves at normal incidence. For oblique incidence, the wide band gap shifts towards high frequency regions, meanwhile a low-frequency band gap is split.

scholarly journals When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Pravin Malla Shrestha ◽  
Kelly P. Nevin ◽  
Minita Shrestha ◽  
Derek R. Lovley
Keyword(s):  
Genome Sequencing ◽  
Rare Variants ◽  
Low Frequency ◽  
Geobacter Sulfurreducens ◽  
Specific Gene ◽  
Microbial Culture ◽  
Content Type ◽  
Potential Electrode ◽  
Common Strategy ◽  
Pure Cultures

ABSTRACTGeobacter sulfurreducensstrain KN400 was recovered in previous studies in which a culture of the DL1 strain ofG. sulfurreducensserved as the inoculum in investigations of microbial current production at low anode potentials (−400 mV versus Ag/AgCl). Differences in the genome sequences of KN400 and DL1 were too great to have arisen from adaptive evolution during growth on the anode. Previous deep sequencing (80-fold coverage) of the DL1 culture failed to detect sequences specific to KN400, suggesting that KN400 was an external contaminant inadvertently introduced into the anode culturing system. In order to evaluate this further, a portion of the gene for OmcS, ac-type cytochrome that both KN400 and DL1 possess, was amplified from the DL1 culture. HiSeq-2000 Illumina sequencing of the PCR product detected the KN400 sequence, which differs from the DL1 sequence at 14 bp, at a frequency of ca. 1 in 105copies of the DL1 sequence. A similar low frequency of KN400 was detected with quantitative PCR of a KN400-specific gene. KN400 persisted at this frequency after intensive restreaking of isolated colonies from the DL1 culture. However, a culture in which KN400 could no longer be detected was obtained by serial dilution to extinction in liquid medium. The KN400-free culture could not grow on an anode poised at −400 mV. Thus, KN400 cryptically persisted in the culture dominated by DL1 for more than a decade, undetected by even deep whole-genome sequencing, and was only fortuitously uncovered by the unnatural selection pressure of growth on a low-potential electrode.IMPORTANCERepeated streaking of isolated colonies on solidified medium remains a common strategy for obtaining pure cultures, especially of difficult-to-cultivate microorganisms such as strict anaerobes. The results presented here demonstrate that verifying the purity of cultures obtained in this manner may be difficult because extremely rare variants can persist, undetectable with even deep genomic DNA sequencing. The only way to ensure that a culture is pure is to cultivate it from an initial single cell, which may be technically difficult for many environmentally significant microbes.

COMPARATIVE STUDIES ON THE GROWTH OF MYXOBACTERIA ON BACTERIAL SUSPENSIONS

1963 ◽  
Vol 9 (4) ◽  
pp. 577-584 ◽  
Author(s):  
B. Kletter ◽  
Y. Henis
Keyword(s):  
Growth Medium ◽  
Test Organism ◽  
Growth Media ◽  
Multiplication Rate ◽  
Bacterial Cells ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Negative Organisms ◽  
Myxococcus Virescens ◽  
Number Of Bacteria

The growth of Myxococcus fulvus and Myxococcus virescens on a number of bacteria was followed in a liquid medium. Multiplication of the myxobacteria was accompanied by their adsorption on the bacterial cells, by their coagglutination, and by their adsorption on the glass surface of the culture flask. Lysis of the agglutinated bacterial cells and release of their proteins to the growth medium took place prior to an increase in the lytic activity of the growth medium towards the tested bacteria. Soluble proteins reached a higher level in media containing Gram-negative than in those containing the Gram-positive organisms. No difference was observed in the multiplication rate, sporulation, or pigmentation of the myxobacteria tested, when grown on either Gram-positive or Gram-negative organisms. Using Staphylococcus aureus as a test organism, no antibiotic activity in any of the growth media could be detected.

Sign in / Sign up

Export Citation Format

Share Document