Replica Plating Technique Research Articles

Estimating the population-level prevalence of antimicrobial-resistant enteric bacteria from latrine samples

BackgroundLogistical and economic barriers hamper community-level surveillance for antimicrobial-resistant bacteria in low-income countries. Latrines are commonly used in these settings and offer a low-cost source of surveillance samples. It is unclear, however, whether antimicrobial resistance prevalence estimates from latrine samples reflect estimates generated from randomly sampled people.MethodsWe compared the prevalence of antimicrobial-resistant enteric bacteria from stool samples of people residing in randomly selected households within Kibera—an informal urban settlement in Kenya—to estimates from latrine samples within the same community. Fecal samples were collected between November 2015 and Jan 2016. Presumptive Escherichia coli isolates were collected from each household stool sample (n = 24) and each latrine sample (n = 48), resulting in 8935 and 8210 isolates, respectively. Isolates were tested for resistance to nine antibiotics using the replica-plating technique. Correlation- and Kolmogorov–Smirnov (K–S) tests were used to compare results.ResultsOverall, the prevalence values obtained from latrine samples closely reflected those from stool samples, particularly for low-prevalence (< 15%) resistance phenotypes. Similarly, the distribution of resistance phenotypes was similar between latrine and household samples (r > 0.6; K–S p-values > 0.05).ConclusionsAlthough latrine samples did not perfectly estimate household antimicrobial resistance prevalence, they were highly correlated and thus could be employed as low-cost samples to monitor trends in antimicrobial resistance, detect the emergence of new resistance phenotypes and assess the impact of community interventions.

E. Coli Bactria Deactivation by Plasma Jet with Replica Plate Technique

In this research, non-thermal plasma system (plasma jet) of argon gas is designed to work at normal atmospheric pressure and suitable for work as well as use in medical and biotechnological applications, also applied this technique in the treatment of The E. Coli bacteria and show the role of the applied voltage on the killing rate of bacteria, and we obtained a 100% killing rate during the time of 5 minutes.

Comparison of Membrane Filtration and Replica Plate Technique to Detect Fecal Coliform

Introduction: Membrane filtration (MF) is one of the widely used technique on a routine basis. On the other hand, replica plate (RP) technique can be used to transfer existing bacterial colonies in two plates which even allows pinpointing the original colony. The aim of this study is to comparatively detect the cfu/100 mL of fecal coliform using membrane filtration and replica plate techniques.Methods: In the study, a total of 25 bottled water were selected from the local market in Kathmandu valley. The total coliform count was detected using MF, while fecal coliform was detected using both MF and RP technique.Results: It was found that the average cfu/100 mL for total coliform, fecal coliform (MF) and fecal coliform (RP) were 143.38, 49.82 and 51.00 respectively. Pearson correlation coefficient calculated between total coliform and fecal coliform (MF), total coliform and fecal coliform (RP), fecal coliform (MF) and fecal coliform (RP) were found to be 0.695, 0.733 and 0.990 respectively; implying a positive correlation Conclusions: It has been demonstrated that intrinsic and extrinsic factors influence colony forming units. Furthermore, RP is a more sensitive method for screening fecal coliforms although both MF and RP can be efficiently used.

Greenbeards in yeast: an undergraduate laboratory exercise to teach the genetics of cooperation

Recent years have seen a dramatic increase in our understanding of the social behaviour of microbes. Here, we take advantage of these developments to present an undergraduate laboratory exercise that uses the cooperative flocculating behaviour of yeast (Saccharomyces sp.) to introduce the concept of inclusive fitness and teach the genetics of cooperation. Students generate their own data using co-cultures of various yeast strains and perform statistical analyses to test whether kin selection or greenbeard effects determine the cooperative flocculating behaviour. The lab has run successfully for two consecutive years in a second year course with some 1, 200 students per year at the University of Toronto, Canada. We discuss the benefits of using microbes to teach social evolution, describe the set-up and learning outcomes of the laboratory exercise, and then outline possible extension and variants of the lab. In addition to providing students with the opportunity to use a model organism to study social behaviour, students are also taught common laboratory skills, such as replica plating and sterile techniques. Ultimately, while the genetics of cooperation has traditionally been taught through computer simulations and evolutionary games, this exercise demonstrates a way to experimentally introduce the topic.

Fitness and competitive growth comparison of methicillin resistant and methicillin susceptible Staphylococcus aureus colonies.

Exponential developments of both Methicillin resistant Staphylococcus aureus (MRSA) 3R ve 36R and methicillin susceptible Staphylococcus aureus (MSSA) 27S were evaluated in the presence and absence of oxacillin. The strains were isolated from the specimens collected in microbiology department. It was also determined the transfer of mecA gene from 3R to 27S strain by using the replica plate technique. It was observed that the presence of antibiotics in the preliminary culture had a positive impact on the growth of the secondary culture of MRSA isolates. Comparison results of Rt bacteria in three different mixed cultures, assessed with Tukey's HSD test, showed a significant statistical difference among the groups. The values were as following; on the first day; Df: 2, F: 60.90, P: 0.0001, second day; Df:2, F:90.56, P: 0.0000, and third day; Df:2, F:4.86, P:0.0557. As a result of the study, we can suggest that the gene expression levels of the transferred antibiotic resistance genes could help us in both controlling hospital originated sickness and developing new strategies to prevent the spread of resistant bacteria.

Utilization of Dimethoate by Wild Type Pseudomonas putida from Polluted Sites in Iran

Pseudomonas putida was isolated from organophosphate contaminated soil by enrichment method. Dimethoate optimally was degraded at 0.8 g L(-1) in mineral solution and showed the maximum tolerance at 4 g L(-1) in nutrient broth. Dimethoate was degraded two folds more than orthophosphate. Total protein was increased to 287 mg L(-1) in single culture. Anti-acetyl choline esterase (AChE) Activity was proportionally displayed of dimethoate level (0.2-2 g L(-1)). Dimethoate (2 g L(-1)) was remedied 50 and 100% by p putida after 48 and 96 h, correspondingly. It means that its anti-AChE effect diminished to 70-95%. The strains were lost the dimethoate degrading plasmids by 200 mg L(-1) of acridine orange and converted to dimethoate-sensitive strains. This plasmids were transferred to sensitive strains that were consecutively confirmed by replica plating technique in mineral medium containing dimethoate. We are supposed to simply applying organophosphate degrading plasmids for cleaning the anti-nerve agents.

Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation.

Mesenchymal stem cells give rise to osteoprogenitors that proliferate and differentiate into identifiable preosteoblasts, osteoblasts, bone lining cells and osteocytes. To identify and establish a molecular profile for the more primitive and uncharacterized cells in the lineage, relatively rare (<1%) osteoprogenitors present in primary cultures of fetal rat calvaria cell populations were identified by a replica plating technique. Since the cell number was limited in each colony sampled, we used global amplification PCR to analyze the repertoire of genes expressed in osteoprogenitors. We established a molecular fingerprint and a developmental sequence based on simultaneous expression patterns for both known osteoblast-associated markers (collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, PTH1R and osteocalcin) and potential regulatory molecules (i.e. FGFR1, PDGF-Ralpha and PTHrP). By analysis of 99 osteoprogenitor and osteoblast colonies captured by replica plating at different developmental stages, we found: (1) a recognizable cohort of cells considered more primitive than committed osteoprogenitors; (2) a cohort of early progenitors transiently expressing bone sialoprotein; and (3) that mRNAs for FGF-R1, PDGF-Ralpha and PTH1R were expressed earlier than other markers and tended to increase and decrease in relative concert with the osteoblast-specific markers. The observations suggest that within the osteoblast differentiation sequence both discrete stages and continua of changing marker expression levels occur with variation in expression for any given marker. This combined approach of replica plating and global amplification PCR allows molecular fingerprinting of definitive primitive osteoprogenitors and will aid in identifying novel developmental stages and novel differentiation stage-specific genes as these cells progress through their differentiation sequence.

An analysis of the spontaneous mutation rate measurement in filamentous fungi

Mutations related to gene methG1 of Aspergillus nidulans were analyzed, in order to study a mathematical model for the determination of the mutation rate per nucleus per generation, in filamentous fungi. A replica plating technique was used to inoculate, in a single operation, 26 colonies of the strain, into Petri dishes containing culture medium, and the nine central colonies were analyzed for size and number of conidia in each colony. Using this technique, several central colonies were analyzed with regard to the appearance of mutation, and the number and type of reversions were determined for each colony. The frequencies obtained for each reversion were analyzed, in order to verify if their distribution was in accordance with that of Greenwood and Yule. The data obtained allowed us to conclude that, using the mathematical model studied, it is possible to determine the mutation rate per nucleus, per generation, in filamentous fungi.

Development of a New Most-probable-number Method for Enumerating Methanotrophs, Using 48-well Microtiter Plates.

We developed a new most-probable-number (MPN) method for enumeration of methanotrophs, using 48-well microtiter plates. With this method, a microtiter plate which has been inoculated with serial dilutions of a sample is placed in a plastic desiccator containing methane and shaken. The proposed method gave estimates of population densities of a pure culture of Methylocystis sp. strain M and of methanotrophs in a paddy soil sample similar to those obtained by a conventional MPN method using serum bottles. The new microtiter plate MPN method with an additional replica-plating technique made it possible to enumerate small populations of methanotrophs in cornfield and forest soils. It also requires less equipment, labor, and time than the conventional serum bottle MPN method.

The frequency of common progenitors for adipocytes and osteoblasts and of committed and restricted adipocyte and osteoblast progenitors in fetal rat calvaria cell populations.

In fetal rat calvaria (RC) cell populations, adipocyte differentiation is stimulated by both dexamethasone (Dex) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], whereas osteoblast differentiation is stimulated by Dex but inhibited by 1,25(OH)2D3. We examined whether the osteoblastic and adipocytic colonies were derived from a common progenitor, from committed and restricted adipocyte and osteoblast progenitors, or from both and whether the adipocyte progenitors stimulated by 1,25(OH)2D3 constitute a population of progenitors that is different from that stimulated by Dex. RC cells were isolated by sequential enzyme digestion yielding five populations designated I-V. In population I the effect of Dex on adipocyte formation was greater than that of 1,25(OH)2D3, whereas the effect of 1,25(OH)2D3 was greater than that of Dex in populations III-V. We next applied replica plating techniques to further investigate the response characteristics of individual osteoprogenitors and adipocyte progenitors by looking at the fate of duplicate colonies derived from the same progenitor under different culture conditions. RC cells were plated at 1,000-1,500 cells/100 mm culture dish and a 17-microm mesh polyester membrane overlaid onto master dishes on day 4 or day 5 and removed on day 11 or day 12. Then, replicas and master dishes were cultured separately in medium containing either Dex, 1,25(OH)2D3, or Dex plus 1,25(OH)2D3 for a further 17-21 days and then fixed and stained with both Sudan IV and the von Kossa technique. Nine hundred twenty-seven matched colonies present on both master dishes and replica membranes were screened and colonies were classified as either adipocytic, osteoblastic (bone or osteoid), or fibroblastic. Results show convincingly that most of the osteoprogenitors present in fetal RC cells are committed and restricted to the osteoblastic cell lineage (95.29%); that the 1,25(OH)2D3-responsive adipocyte progenitors are different from the Dex-responsive adipocyte progenitors, but both are restricted to form adipocytes and finally; and that a common osteoblastladipocyte progenitor is present in a low frequency (4.71% of osteoprogenitors).

DETERMINAÇÃO DA ATIVIDADE ANTIMICROBIANA "IN VITRO" DE QUATRO ÓLEOS ESSENCIAIS DE CONDIMENTOS E ESPECIARIAS

Determinou-se a atividade antimicrobiana "in vitro" de quatro óleos essenciais de condimentos e especiarias (canela, cravo, gengibre e menta), em três diferentes concentrações (10,0; 1,0 e 0,1%) sobre vinte e um microrganismos (sete leveduras e quatorze bactérias). Para tanto, foram utilizados inóculos padronizados destas leveduras e bactérias, previamente crescidos em caldo nutriente e semeados pela técnica de plaqueamento em réplica, em placas de Petri contendo ágar nutriente + tween 20, sem tratamento (controle) e com as diferentes concentrações dos óleos essenciais estudados. Os resultados, obtidos após incubação a 30 °C por 30 dias, demonstraram que, dentro dos parâmetros utilizados no experimento, os óleos essenciais com maior atividade antimicrobiana foram os de canela e menta, nas concentrações de 10,0 e 1,0%. O óleo de cravo também inibiu completamente o crescimento de todos os microrganismos testados na concentração de 10,0%.

Rapid screening for endo-β-1,4-glucanase and endo-β-1,4-mannanase activities and specific measurement using soluble dye-labelled substrates

Soluble dye-labelled substrates were prepared for the specific and rapid screening and the measurement of endo-β-1,4-glucanase and endo-β-1,4-mannanase activities. Soluble carboxy-methylcellulose and locust bean galactomannan were dyed with Remazol Brilliant Blue R. A specific screening procedure was developed to isolate genes coding for cellulolytic and hemicellulolytic enzymes from a Cellulomonas sp. Ce1B7 library. The assay is advantageous for the simple, rapid and direct detection of cellulase and hemicellulase activities in a large number of clones as found in gene libraries without replica plating or blotting techniques. The prepared dye-labelled substrates can also be used for the quantitative measurement of specific endo activities of cellulolytic and hemicellulolytic enzymes because the undigested substrate can be precipitated and the amount of soluble dye, determined spectrophotometrically, is proportional to enzyme activity.

Isolation of mammalian cell mutants that are X-ray sensitive, impaired in DNA double-strand break repair and defective for V(D)J recombination

The Chinese hamster lung V79-4 cell line was infected with a Moloney murine leukemia retrovirus and the infected cells were subsequently screened for mutants that were sensitive to X-rays using a toothpicking/96-well replica plating technique. Four independent mutants that were sensitive to X-irradiation (sxi-1 to sxi-4) were isolated from 9000 retrovirally infected colonies. A pulse-field gel electrophoresis (PFGE) assay demonstrated that all of the sxi mutants were impaired in DNA double-strand break (DSB) repair, thus providing a molecular explanation for the observed X-ray sensitivity. Interestingly, additional PFGE experiments demonstrated that for any given X-ray dose all of the mutants incurred more DNA DSBs than the parental V79-4 cell line indicating there may be some inherent fragility to sxi chromosomes. Cross-sensitivity to other DNA-damaging agents including bleomycin, mitomycin C and methyl methanesulfonate indicated that sxi-2, sxi-3 and sxi-4 appear to be specifically hypersensitive to genotoxic agents that cause DNA DSBs, whereas sxi-1 appeared to be hypersensitive to multiple types of DNA lesions. Lastly, in preliminary experiments all of the sxi mutants demonstrated an inability to carry out V(D)J recombination, a somatic DNA rearrangement process required for the assembly of lymphoid antigen receptor genes. Thus, the sxi cell lines have interesting phenotypes which should make them valuable tools for unraveling the mechanism(s) of DNA DSB repair and recombination in mammalian cells.

A test of the directed mutation hypothesis in Escherichia coli MCS2 using replica plating

Excision of phage Mu from Escherichia coli MCS2 was originally put forth as a clear example of directed mutation. This claim was considerably weakened, however, by subsequent evidence that Mu excision occurs during starvation regardless of fitness consequences. Here, I use the classical replica-plating technique to examine Mu excision during starvation of MCS2. The results support the conclusion that Mu excision is not directed but is, instead, nonspecifically induced by starvation.

Binding of human high-molecular-weight salivary mucins (MG1) to Hemophilus parainfluenzae.

In human saliva, two different mucin populations can be distinguished, viz., high-molecular-weight mucins (MG1, mol. wt > 1 x 10(6)) and low-molecular-weight mucins (MG2, mol. wt approximately 125 kD). The carbohydrate moiety of MG1 displays a wide spectrum of oligosaccharide structures, varying in composition, length, branching, and acidity. The biological significance of the heterogeneity in carbohydrate structures of mucins is unclear. The present investigation focused on the question whether MG1, because of its diverse carbohydrate side-chain population, can bind to a large variety of oral micro-organisms. A replica plate technique, in combination with immunochemical detection with monoclonal antibodies against MG1, was used to screen in vivo human oral microflora for the presence of micro-organisms which could bind the high-molecular-weight salivary mucin MG1. Binding to purified MG1 was established for Hemophilus (para)influenzae species, whereas other species, including Streptococcus and Staphylococcus, were negative. MG1 binding to Hemophilus parainfluenzae could be abolished by protease treatment of MG1. In contrast, periodate acid treatment, partial deglycosylation, or addition of monosaccharides did not affect MG1 binding to H. parainfluenzae, indicating that MG1 carbohydrate side-chains were not directly involved in the binding. The binding was pH-dependent, showing an increase when the pH was lowered from 8.0 to 4.0. These data indicate that MG1 can be bound in a selective manner by Hemophilus spp. and suggest that the 'naked' unglycosylated polypeptide moiety of MG1 is involved in its binding to Hemophilus parainfluenzae.

A Fourth Complementation Group among Ionizing Radiation-Sensitive Chinese Hamster Cell Mutants Defective in DNA Double-Strand Break Repair

The XR-V9B mutant of Chinese hamster V79 cells which exhibits hypersensitivity to ionizing radiation was isolated by the replica plating technique. The increased sensitivity of XR-V9B cells to X rays (approximately 4-fold, as judged by the D10) was accompanied by increased sensitivity to other DNA-damaging agents such as bleomycin (approximately 17-fold), VP16 (approximately 6-fold), and adriamycin (approximately 5-fold). Only a slightly increased sensitivity was observed after exposure to UV radiation, MMS, or mitomycin C (1.4-, 1.7-, and 2-fold, respectively). As measured by neutral elution after exposure to X rays, XR-V9B cells showed a defect in the rejoining of double-strand breaks (DSBs); after 4 h of repair more than 50% of DSBs remained in comparison to 5% in wild-type cells. No difference was observed in the kinetics of single-strand break rejoining between XR-V9B and wild-type cells, as measured by alkaline elution. To determine whether XR-V9B represents a new complementation group among ionizing radiation-sensitive Chinese hamster cell mutants defective in DSB repair, XR-V9B cells were fused with XR-V15B, XR-1, and V-3 cells, which have impaired DSB rejoining and belong to three different complementation groups. In all cases, the derived hybrids regained the sensitivity of wild-type cells when exposed to X rays, indicating that the XR-V9B mutant represents a new fourth complementation group among X-ray-sensitive Chinese hamster cell mutants defective in DSB repair.

A novel method for in situ screening of yeast colonies with the ?-glucuronidase reporter gene

Expression of the beta-galactosidase gene in yeast has served as a screening marker for many purposes. Here it is shown that in two yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, the beta-glucuronidase (GUS) gene can be used as an alternative marker. Since the histochemical substrate can not be taken up by yeast cells, direct colony screening of plates was found to be impossible. However, by a replica plating technique, GUS expression became visibly detectable within 10 min when the GUS gene was strongly expressed. The staining method could still be performed for expression at a 100-fold lower level, but incubation times of several hours were needed. Furthermore, specific GUS expression levels of yeast protein extracts could be quantified by a fluorometric assay which is both very simple to perform and highly sensitive. Since the GUS gene can also tolerate large N-terminal fusions, this method should be particularly attractive for studying such diverse problems as transcriptional and translational regulation or subcellular localization in yeast.

A comparison of two plating techniques to estimate plasmid stability of a prolonged chemostat culture

A comparison of two plating techniques to estimate the segregational stability ofEscherichia coli RR1 harboring plasmid pBR322 in a chemostat was studied. No significant differences were observed between the spread and replica plating techniques in the beginning of the experiments. However, a noticeable discrepancy between these two methods appeared after approximately 100 hours. This inconsistency can be shown to be statistically significant.

Variation in the ability of Pseudomonas sp. strain B13 cultures to utilize meta-chlorobenzoate is associated with tandem amplification and deamplification of DNA.

Single-colony isolates of Pseudomonas sp. strain B13 were examined for their ability to utilize benzoate (Ben) and meta-chlorobenzoate (3CB) as the sole carbon source. Scoring of B13 cultures by the replica-plating technique revealed that under nonselective conditions, B13 spontaneously formed four different types of colonies: 3CB+ Ben+, 3CB+ Ben-, 3CB- Ben-, 3CB- Ben+. Successive testing of each of the four colony types showed that each produced the same four different types of single-colony isolates. Colonies of each class had characteristic phenotypic properties with respect to the accumulation of Ben or 3CB pathway intermediate products. The physical abundance of a 4.3-kilobase DNA encoding the first three enzymes of the chlorocatechol pathway correlated with the 3CB+ phenotype. Increased abundance of the 4.3-kilobase DNA fragment was the result of tandem amplification.

Isolation and Characterization of Heat-Sensitive, Thermotolerant Defective Mutants of CHO Cells

A replica plating technique was utilized to isolate stable CHO cell mutants that are heat-sensitive and have altered capacities to develop thermotolerance. From a mutagen (EMS) treated population of CHO cells, two strains were isolated. One (HS-36) shows a greatly reduced ability to develop thermotolerance following an initial 45.0 degrees C heat shock. The other (HS-23) also shows a greatly reduced thermotolerance development following a short 45.0 degrees C induction dose, but a greater thermotolerance development following longer 45.0 degrees C induction doses. The dose-survival response following single-dose 45.0 degrees C heating of HS-23 cells suggests the presence of a resistant subpopulation which is not due to contamination from, or reversion to, wild-type cells. Both strains have unique morphological characteristics. Spheroids develop in the central portion of HS-36 colonies, though cells in monolayers are indistinguishable from wild-type parental cells. HS-23 cells grow in firmly attached monolayers, but more than 95% maintain a "rounded" morphology. The remainder show a "flattened" morphology typical of CHO cells. Both strains have parental CHO characteristics as determined by chromosome number, population doubling times, and survival responses to UV light and to gamma rays. Each has maintained its heat-sensitive and altered thermotolerance phenotype for a period of over 6 months in continuous log-phase culture.