BACTERIOLOGICAL ISOLATION IN THE RESTAURANT OF HAWLER CITY KURDISTAN REGION, IRAQ

Nutrient Agar is a universal medium containing agar, meat extract, yeast extract and peptone. NA media is often made in large quantities, stored under sterile conditions, and then reheated when needed. Repeated heating can reduce the number of bacterial colonies that grow because the components that make up the media become damaged. This study aimed to determine the effect of repeated heating of NA (Nutrient Agar) media 4 times on the Total Plate Count (TPC) test results for Staphylococcus aureus and Escherichia coli bacteria. The number of bacteria that grew on the media with varying amounts of heating was calculated, and the results showed that repeated heating 4 times caused a decrease in the number of bacterial colonies that grew on the NA media. The ANOVA test gave a value of p = 0.000 for the two types of bacteria separately, which showed that there is a significant difference between the number of bacteria in varying amounts of media heating. This research concluded that repeated heating of NA (Nutrient Agar) media affects the TPC test results for Staphylococcus aureus and Escherichia coli.

Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix.Graphical abstract

Aims: To develop a sampling procedure for PCR-based screening of bacterial leaf spot (BLS)-causing xanthomonads without DNA extraction from infected tomato plants. Place and Duration of Study: University of Copenhagen, Denmark and Sokoine University of Agriculture, Morogoro, Tanzania between July 2008 and November 2010. Methodology: Flinders Technology Associates (FTA) plant cards and Chromatography paper or Whatman paper strips (WPS) were spotted with bacterial suspensions from 24h-old cultures from reference strains of BLS-causing xanthomonads, or sap obtained by grinding or hand maceration of plant tissue, were used as templates in PCR reactions or isolation of live bacterial cells on Nutrient agar (NA) media. Samples were tested by PCR with Xan 7 genus/-specific Xanthomonas primers or in multiplex with 26S rRNA primers. Isolation of bacteria was done by streaking aliquots of 75 μl of a suspension from a disc Research Article British Biotechnology Journal, 3(4): 556-574, 2013 557 (2-mm-punch by Harris Micro Punch®) in triplicate, removed from each of the FTA plant card and WPS onto NA media. Results: The FTA plant card spotted with pure cultures of reference strains of xanthomonads and sap from grinding or direct maceration of plant tissue resulted in more clear PCR bands (402 bp) and (594 bp of rRNA gene in multiplex) than the WPS samples. Sensitivity of detection by the FTA paper-based PCR was ≈ 5.0 x 10, while that of the WPS was > 1.0 x 10 CFU/ml. The WPS (but not the FTA) was proved to be useful for saving living bacteria cells for up to one week of storage at ambient temperatures. Conclusion: Both FTA plant card and WPS can be used for PCR detection of BLScausing xanthomonads in tomato. However, the FTA plant card is recommended as it produced clearer PCR products than WPS. WPS is recommended for experiments requiring isolation of live bacterial cells on NA media.

Service activities are carried out as an educational medium for IMMIM Islamic Boarding School students through material presentation and direct practice in making Nutrient Agar (NA) media. So, it can increase knowledge about making good media. This activity was also carried out so that students knew an in-depth explanation of the stages of making, usefulness and how important Nutrient agar media is as a growth medium and observation of the morphology of Eschericia coli bacteria. This counseling activity provided satisfaction for the participants, more than 95% of the participants strongly agreed with this activity. Community service activities have a good impact on the participants. The knowledge and understanding of IMMIM Islamic Boarding School students increased about Nutrient Agar media, the correct stages of making Nutrient Agar, the usefulness of Nutrient Agar media, and the importance of Nutrient Agar media as a growth medium and observation of the morphology of Escheria coli bacteria.

Lactic acid bacteria are beneficial bacteria for the health of the body by improving the balance of the intestinal microflora. Lactic acid bacteria can generally be isolated from fermented foods, fruits, vegetables and meat. Inasua is a traditional fermented fish product originating from Central Maluku (Teon, Nila and Serua islands) wherein the fermentation process is carried out at room temperature for a certain time. The purpose of this study was isolation and identification based on the morphological characteristics of lactic acid bacteria in inasua. Isolation of Lactic Acid Bacteria was carried out using the pour plate method and the streak method on the media de Man, Rogosa and Sharpe Agar (MRSA) and Nutrient Agar media. The culture was incubated at 370C for 48 hours. The growing colonies were observed for morphological characteristics and GRAM staining of bacteria was carried out. The results of isolation of BAL from inasua in the media de Man, Ragosa and Sharpe Agar (MRSA) + CaCO3 obtained 4 bacterial isolates, namely INS-A1, INS-A2, INS-A3 and INS-A4 and only 2 isolates that have the characteristics of positive GRAM with Bacillus forms, namely INS-A2 and INS-A4. It is suspected that the 2 isolates were isolates of Lactic Acid Bacteria. The results of isolation on Nutrient Agar (NA) media contained 5 isolates namely INS-B1, INS-B2, INS B3, INS-B4, INS-B5 which have characteristics as negative GRAM shaped Bacillus

Ginseng (Panax ginseng) is an economically valuable medicinal herb mainly planted in Jilin Province, China. In September 2013, during harvest, suspected bacterial rots were observed on ginseng roots with about 10% incidence in Fusong County, Jilin Province, China (127°29.48' N, 42°11.12' E). Rotted roots completely lost their economic value. Symptoms on roots began as water-soaked lesions, and developed rapidly into a soft, watery, decayed mass within 3 to 5 days. Three diseased root tissues were surface-sterilized in 70% ethanol for 30 s, rinsed 3 times in sterilized water and cut into small pieces (2 to 3 mm). Tissues were then macerated for 5 min in sterilized water, streaked onto nutrient agar (NA) medium, and incubated at 28°C for 2 days. Representative colonies were selected from each plate and further purified by sub-culturing onto NA medium. Five strains of the bacteria were gram-negative, short straight rods, 0.5 to 1.0 × 1.5 to 3.0 μm with a single, polar flagellum. Colonies were round, smooth, translucent, and yellowish green on NA medium. The bacteria were identified based on physiological and biochemical tests as follows (3): They were levan and potato rot negative, oxidase, aerobic, and arginine dihydrolase positive, converted nitrate to N2, hydrolyzed gelatin, produced nitrites from nitrates, produced pyocyanin, and grew at 41°C. Bacterial identity was further confirmed by amplifying the 16S rRNA (1,461 bp), gyrB (1,134 bp), and 16S-23S ITS genes (523 bp) with 27F/1492R, UP1/UP2, and L1/L2 primer sets, respectively. The 16S rRNA gene sequence (NCBI Accession No. KJ156527), gyrB gene sequence (KJ748373), and 16S-23S ITS gene sequence (KJ748374) had 99% identity to that of Pseudomons aeruginosa strain BS01 (JQ229778), ATCC25011 (FJ652721), and ATCC15522 (AB547908), respectively. The strains were also identified by using BD Phoenix-100 Automated Microbiology System (BD Ltd., New Jersey) as P. aeruginosa with 99% confidence. A pathogenicity test was conducted by spraying a suspension of five strains individually (108 CFU/ml) onto 4-year-old ginseng roots (cv. Damaya) wounded with a sterilized needle. Five ginseng roots were inoculated with each strain and five ginseng roots were inoculated with sterilized water as controls. All inoculated plants were maintained at 28°C with 80 to 85% relative humidity. Soft, watery tissue rot symptoms developed 3 to 5 days after inoculation, and were similar to those observed on the diseased plants under natural conditions. In contrast, no symptoms developed on control plants. The bacteria were readily re-isolated from inoculated plants and identified as P. aeruginosa using bacterial colony morphology, physiological and biochemical tests, as well as sequence analysis of the 16S rRNA gene, fulfiling Koch's postulates. The bacterium was not isolated from control plants. P. aeruginosa has been reported to cause diseases in a variety of plants including onion (1,2), arabidopsis, and sweet basil (4). To our knowledge, this is the first report of P. aeruginosa causing ginseng root rot in China.

Volatiles produced by Bacillus mojavensis RRC101 act as plant growth modulators and are strongly culture-dependent

A disease survey was carried out in Tamil Nadu, India during 2021 to assess the prevalence of diseases in major rice-growing areas and characterise the pathogens associated with infected plants. During the survey, rice plants at the seedling to grain-filling stage showing water-soaked, elongated, light brown lesions in the leaf margins and tips (Figure 1) were collected along with grains in a polythene bag, labelled and sealed. Leaves showing yellowing and drying from tips and margins were cut into small pieces, surface-sterilised in 1% sodium hypochlorite for one minute, rinsed three times with sterile distilled water and then allowed to air dry. After drying, the tissues were soaked for 30 minutes at room temperature in 0.5 ml of sterile distilled water and then a loop full of suspension was streaked onto nutrient agar (NA) medium. For the isolation of pathogen from infected seed, discoloured grain samples were surface sterilised in 3% sodium hypochlorite for one minute, rinsed thrice with sterile distilled water and allowed to air dry. Surface-sterilised grains were placed in a Petri plate containing NA medium. These plates were incubated at 28°C for one to two days. Upon incubation, straw to yellow-coloured colonies emerged. The colonies were further purified through single colony isolation on NA. The purified colonies were yellow in colour, translucent and undulate. (Figure 2.) The colonies exhibited umbonate surfaces with slightly concave centres (Al-Kidsawey et al., 2020; Delétoile et al., 2009). The pathogenicity of the isolates was demonstrated by inoculation of isolates on rice seedlings (cv. Co 39). For this, bacterial isolates were incubated in nutrient broth for 48 hours at 27°C. The bacterial suspension was adjusted to a concentration of 109 cfu/ml by adding sterile water. The bacterium was inoculated by cutting the tip (about 3 to 4 cm) of the fully expanded uppermost leaf with scissors and dipping in the bacterial suspension. Controls used sterile distilled water instead of the bacterial suspension. The inoculated plants were covered with polythene sheet overnight and incubated under laboratory conditions. The inoculated leaves developed progressive necrosis with colour shifts from pale green to brown within 3 to 14 days post inoculation, whereas the control plants exhibited no symptoms. Koch's postulates were fulfilled by isolating the bacterium on NA which produced yellow, flat to convex translucent colonies after 48 hours incubation. The bacterium was further subjected to biochemical tests using the KB002 HiAssorted Biochemical Test Kit (Himedia, Germany). The bacterium showed a positive reaction for citrate utilisation, phenylalanine deamination, nitrate reduction, glucose and arabinose utilization. It showed a negative reaction to lysine utilisation, ornithine utilization, urease, H2S production, adonitol, lactose and sorbitol utilization. Based on the morphological characters and biochemical tests, the bacterium was identified as a Pantoea sp. To further characterise the pathogen, DNA extraction was done, and a PCR assay was performed using universal primers 27F and 1492R targeting the 16S rRNA gene. Sequencing of the amplicon revealed that the bacterial isolates had 93.7% identity with Pantoea agglomerans type strain SGAir0210 (GenBank Accession No. CP028033.1). The partial 16S rRNA gene sequences of two strains of P. agglomerans were deposited in GenBank (OP784578 and OP784743). Pantoea agglomerans causing bacterial leaf blight and glume discolouration has been reported from Brazil, South Korea, Turkey and Venezuela (Aksoy & Boluk, 2019; Carrer Filho et al., 2018; González et al., 2015; Lee et al., 2010). Pantoea agglomerans also causes infection in humans (Al-Kidsawey et al., 2020; Delétoile et al., 2009)). Bacterial leaf blight in rice caused by P. stewartii subsp. indologenes has been reported from Tamil Nadu, India (Vinodhini et al., 2017). However, to our knowledge, this is the first report of P. agglomerans causing rice leaf blight and grain discolouration in India. Further studies are needed to ascertain the prevalence of P. agglomerans in commercial rice cultivars in other parts of India. We gratefully acknowledge the facilities provided by DST-FIST Lab, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.

The purpose of this research was to describe the characteristics and number of microbes that grow in the gastrointestinal tract of catfish and to determine the potential of microbes as probiotics. The type of this research was observational conducted on 30 September-15 October 2018 at the Microbiology Laboratory, Faculty of Science and Technology, Universitas Airlangga, Surabaya. Microbial isolation using NA (Nutrient Agar), MRSA (de Man, Rogosa and Sharpe Agar) and PDA (Potato Dextrose Agar) media. The fish that used is catfish that are bred in ponds at Desa Tlasih, Kecamatan Tulangan, Kabupaten Sidoarjo, East Java. The identification includes microscopic and macroscopic characteristics. Based on the research that has been done, it can be concluded that there are 8 kinds of bacterial colonies in NA media, on PDA media there are 8 kinds of fungi, and on MRSA media there are 2 bacterial colonies and each has different macroscopic and microscopic characteristics. The total number of bacteria growing in NA and MRSA media respectively were 8.7 x 104 CFU / gram and 1.2 x 105 CFU / gram. It is suspected that there are potential bacteria as beneficial probiotics for catfish which still need further research.
 
 
 Keywords: catfish, gastrointenstinal tract, microbes, bacteria, fungi

Background: Reducing microbial load in the oral cavity will provide an additional rationale for the prevention of dental diseases. Antibacterial properties of topical fluorides and chlorhexidine (CHX) are well documented with certain limitations. However, effects of topical applications of 2% sodium fl uoride solution (NaF soln), 1% CHX gel, and 2.26% NaF varnish on plaque microfl ora and following its termination are less explored. Hence, there is a need to study the comparative effects on these agents in vivo. Aim: The aim was to assess quantitative and qualitative changes in the plaque microflora following topical application of 2% NaF soln, 1% CHX gel, and 2.26% NaF varnish. Materials and Methods: This was a double-blind, randomized, parallel-group clinical trial. A total of willing sixty schoolchildren of Nellore city with apparently good health aged 9–13 years were randomly allocated to three interventional groups as follows: Group A (NaF soln, 20), Group B (CHX gel, 20), and Group C (NaF varnish, 20). NaF soln and CHX gel were applied four times on a weekly interval and NaF varnish was applied only once. Once patients enrolled to participate in the clinical trial, an identification number was sequentially assigned, allowing maintenance of a double-blind, randomized study design. Plaque samples were collected at baseline (P0) and at seven different time intervals (P1: 1st week, P2: 2nd week, P3: 3rd week, P4: 4th week, P5: 8th week, P6: 12th week, and P7: 16th week) and cultured on nutrient agar medium. The number of microbial colony-forming units (CFUs) was calculated using a digital colony counter. Statistical significance within and between groups was calculated using Chi-square test, paired t-test, ANOVA, Dunnett's multiple comparison test, and Tukey's multiple comparison test. Results: There was reduction in the CFU in all the three groups at P2, P3, and P4 when compared to P0 (P 0.05). Single application of NaF varnish was equally potent as four times application of NaF soln and CHX gel, but there was no statistical significance in-between groups in terms of CFU reduction (P > 0.05). P7 sample estimation showed Lactobacilli species with 100% resistance followed by other organisms. Conclusion: All the three agents used demonstrated significant reduction of microbial load in plaque but allowed recolonization following termination of intervention.

Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections

The study was conducted aiming at the isolation and identification of Salmonella and Escherichia coli (E. coli) from different brands of poultry feeds sold in Savar, Dhaka, Bangladesh. Seven different poultry feeds were subjected to microbiological analysis. All these samples were analyzed by culturing in different media such as nutrient broth (NB), nutrient Agar (NA), SS Agar (Salmonella-Shigella Agar), BGA (brilliant-green Agar), Mac Conkey, DHL and EMB (eosin methylene blue) media. Total bacterial colonies of all the samples were counted separately on the nutrient Agar media. Hence, bacteria were counted as 9.5×105 in the feed sample C (Layer) which was found to be the highest in number among the poultry feeds. Total viable count (TVC) of Salmonella and E. coli in the feed samples were as 0 to 6.75×104 and 0 to 3.05×104 respectively. Both organisms were found in 71.43% and 57.14% of the analyzed feed samples, respectively. The highest number of Salmonella was found in sample C (Layer) feeds and that of E. coli was found in sample B (Grower) feeds. The widespread occurrence of Salmonella and E. coli in poultry feeds reinforces the need for effective control measures, hygiene in processing and handling of feeds. Keywords: Salmonella; Escherichia coli; Poultry feeds; Total viable count; Contamination; Hygiene. © 2011 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi:10.3329/jsr.v3i2.7128 J. Sci. Res. 3 (2), 403-411 (2011)

Jute (Corchorus olitorius L.) is the second most important fiber crop after cotton in terms of global production (3). In November 2011, symptoms suggestive of bacterial infection were observed on a seed crop of jute at the CRIJAF research farm, Barrackpore, West Bengal, India. The disease appeared as small, brown, circular spots, usually less than 5 mm in diameter on the leaves and some of the spots were surrounded by a yellow halo. The lesions on the stems were elongated and in some cases were found to girdle the stem. In the later stages of disease, brown sunken spots were found on the green capsules. Disease incidence varied from about 20% to 90% of the total plants in different affected fields at the CRIJAF research farm. Bacterial leaf spot of jute with similar symptoms was reported in 1957 from Sudan (4). Five symptomatic and three asymptomatic leaf samples were collected from different jute fields. Bacterial colonies isolated on nutrient agar medium from infected young leaves were Xanthomonas-like and pale yellow cream in color. Total DNA was extracted from symptomatic as well as asymptomatic leaf samples by using an improved salt concentration and simple sodium acetate CTAB method (2). Single bacterial colonies were transferred to nutrient agar (NA) medium plates and incubated at 28°C for 48 h. Pure colonies from plates were used directly for DNA extraction using the QIAGEN DNeasy Blood and Tissue kit. PCR was carried out with Xanthomonas campestris specific primers NZ8F3/NZ85R3 (1), which generated an amplicon of 530 bp from all the symptomatic leaf samples as well as pure cultures of the isolated bacteria. No amplification was obtained from asymptomatic leaves. The amplicons from the five symptomatic samples collected from the field were sequenced and showed 100% identity with one another, and one sequence (strain JB-CO-13) was deposited in GenBank (Accession No. KC342185). The BLASTn analysis revealed that bacterial strain JB-CO-13 had 100% identity with X. campestris pv. olitorii (EU285213). Nucleotide span and ORF finder (NCBI) analysis indicated the 530-bp PCR amplicon coded part of a gyrase B gene that had 100% identity with a translated gene product (Protein ID: ABX84334). Three leaves of five 1-month-old jute plants (cv. JRO 204) in pot culture were infiltrated each with a separate bacterial strain using suspensions (1 × 105 CFU/ml) in distilled water. The negative control consisted of leaves infiltrated with sterile distilled water. The plants were kept in a greenhouse with mean maximum and minimum temperatures of 28.96 and 21.8°C, respectively. The plants were covered with plastic bags to maintain high relative humidity (>80%). Typical bacterial lesions were recorded on all the inoculated plants after 1 week. No lesions were seen on the negative control. To the best of our knowledge, this is the first report of bacterial leaf spot on C. olitorius caused by X. campestris pv. olitorii from India.

Coffee is a socioeconomic important plant all over the world due to its exportation and how it provides income to the farmers and the country. However, potato taste defect (PTD) affects the Rwandan coffee quality. The smell is reported to be caused by some bacteria that are responsible for the off‐flavor and may also be related to the infestation of Antestia pest which are in its elimination process. The aim of this study was to isolate, biochemically characterize, and identify bacteria producing potato flavor from Rwandan coffee. Five samples were obtained from different regions (Nyamasheke and Nyakizu) of Rwanda. Bacteria were isolated and enumerated in the nutrient agar media followed by culture on nutrient and tryptic soy broth media. Bacteria were also cultured to several carbon sources such as glucose, fructose, sucrose, starch, pectin, and galactose to smell the odor produced by those bacteria. DNA extraction of isolates was done, and the resulting DNA strands were undergone three steps of PCR to be amplified using the forward primer and reverse primer. The identification of bacteria producing potato flavor from Rwandan coffee beans was done through 16S rDNA method followed by sequence analysis using FinchTV software and BLAST. Earthy odor was the mostly produced one for nutrient agar and tryptic soy agar media, and for carbon sources such as sucrose, glucose, pectin, fructose, and galactose. The potato odor was recorded mostly from damaged floaters and hand‐sorted damaged coffee beans. However, other odors such as fruity and ferment were found to be produced by bacteria in coffee beans. The study came up by concluding the presence of different kinds of bacteria including Enterobacteriaceae and Pantoea, which are responsible for the formation of 2‐isopropyl‐3‐methoxypyrazine (IMP) in coffee beans and cause the production of potato flavor.

Pelargonium spp., commonly known as geraniums, are among the economically important bedding and pot plants worldwide. In May 2020, irregular and water-soaked lesions were observed on the leaves of Pelargonium × hortorum (L.H. Bailey) in a commercial greenhouse (100 m2) in Rasht County, Gilan Province, with an incidence of 5%. Four diseased plants were sampled for pathogen isolation. Small pieces from the infection zone of symptomatic leaves were surface disinfected in 70% (v/v) ethanol for 2 min, blotted dry and macerated in 5 ml of sterile distilled water (SDW). Droplets of sample suspension were streaked on nutrient agar containing 1% sucrose (SNA) and the plates were incubated at 30°C for 72 h (Basavand et al. 2022). Four single colonies of the dominant bacterium with creamy-like, convex and round appearance on SNA were purified and characterized morphologically and biochemically (Schaad et al. 2001). The isolates were Gram-negative, facultatively anaerobic, catalase positive and positive in pectolytic activity and growth at 37°C. They were negative in production of fluorescent pigment on King's B medium, oxidase and arginine dihydrolase. The strains produced acid from glucose, sucrose, cellobiose, mannitol and raffinose and produced a water-soluble pink pigment on nutrient agar (NA) medium. The colony's color transitioned from white or creamy to pink as the culture aged due to production of carotenoid compounds. Two representative isolates (PL21 and PL22) were further identified by PCR amplification of the partial sequences of 16S rRNA, gyrB and atpD using FD1/RD1 (Weisburg et al. 1991), UP1/UP2r (Yamamoto and Harayama 1995), and 03-F/04-R (Brady et al. 2008) primers, respectively. The amplification products were sequenced and submitted to GenBank: 16S rRNA, PP599026 and PP599031; gyrB, PP619832 to PP619833, atpD, PV067134 to PV067135. BLASTn analyses of the obtained sequences revealed the following identities and query coverages with Erwinia persicina sequences of strains B64 and SR15 deposited in GenBank: 100.0% of 16S rRNA region (900/1200 bp), 99.84-100.0% of gyrB (747/800 bp) and 99.84-100.0% for atpD (629/640 bp). The phylogenetic tree based on the concatenated sequences of the gyrB and atpD genes using the neighbor-joining method with MEGA7 software showed that PL21 and PL22 isolates clustered solely with E. persicina. The cell suspensions of the isolates PL21 and PL22 in SDW were prepared from 24 h old culture grown on NA medium and adjusted turbidimetrically at 600 nm with spectrophotometer to contain 1 × 107 CFU/ml. The suspensions were sprayed on the fully expanded leaves of Pelargonium plants using a refillable mini atomizer. After inoculation, plants were incubated in a greenhouse at 29 ± 2°C and 50 ± 2% relative air humidity. Experiments were repeated twice, each time with three leaf replicates per isolate. SDW was sprayed on negative control leaves. Symptoms were observed on inoculated plants 5 to 6 days post-inoculation, while no symptoms appeared on the plants sprayed with SDW. Bacteria were reisolated only from symptomatic leaves and again identified as E. persicina by 16S rRNA sequencing. To our knowledge, this is the first report of E. persicina causing leaf blight disease of geranium in Iran. This work will enhance awareness among geranium growers, emphasizing the importance of recognizing leaf blight disease caused by E. persicina and implementing effective control measures to minimize or prevent significant economic losses.