OPTIMIZATION OF IN VITRO SELECTIVE MEDIA FOR SELECTING ANTHRACNOSIS-RESISTANT FLAX CELLS

Цель исследований – оптимизация селективных сред для проведения отбора in vitro каллусных клеток льна, устойчивых к культуральному фильтрату штаммов возбудителя антракноза и создание in vitro новых генотипов, устойчивых к болезни. В результате исследований уточнен состав культурального фильтрата штаммов антракноза. Выявлено, что токсичность культуральных фильтратов не зависела от вирулентности используемых штаммов – более токсичными оказались культуральные фильтраты штаммов 784 (сильновирулентного) и 780 (средневирулентного) (загнивание и отмирание первичных корешков на 5 сутки наблюдали у 67 – 88% проросших семян), менее токсичны – штаммы 793 (сильновирулентный) и 788 (слабовирулентный) (на 5 сутки загнивание и отмирание первичных корешков отмечено у 9 – 15% проросших семян). Установлено, что морфогенные очаги формировались активнее у генотипов, морфогенный каллус которых переносили на среду с аналогичной или более высокой концентрацией культурального фильтрата. Показано, что на 14 сутки во втором пассаже с большей частотой формировались морфогенные каллусы, почки и побеги при использовании в первом и втором пассажах селективной среды, содержащей культуральный фильтрат в концентрации 40 мл/л, или в первом пассаже – 40 мл/л, а во втором – 44 мл/л. Выделены генотипы, сохраняющие устойчивость к антракнозу в течение трёх поколений на уровне 50 – 60%: НО-78 х Ленок, HJI-103-2 х Ленок, НЛ-40-1 х Ленок, HЭ-38 х Росинка, НЭ-36 х Ленок, НЭ-17 х Ленок, HЭ-16-2 х Росинка. Research objective – optimization of selective media for in vitro selection of flax callus cells resistant to culture filtrate of anthracnose pathogen strains and in vitro creation of new disease-resistant genotypes. As a result of the research, the composition of the culture filtrate of anthracnose strains was clarified. It was revealed that the toxicity of cultural filtrates did not depend on the virulence of the strains used - cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of primary roots on day 5 was observed in 67 - 88% of germinated seeds), less toxic - strains 793 (strongly virulent) and 788 (weakly virulent) (on the 5th day, decay and death of primary roots was noted in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes, the morphogenic callus of which was transferred to a medium with a similar or higher concentration of the culture filtrate. It was shown that on the 14th day in the second passage, morphogenic callus, buds and shoots were formed with a greater frequency when using in the first and second passages a selective medium containing a culture filtrate at a concentration of 40 ml/l, or in the first passage - 40 ml/l, and in the second - 44 ml/l. Genotypes were identified that retain resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, HJI-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.

Sterility Test of Syringes As A Pharmaceutical Preparation That Obtained From Pasar Pramuka

Syringes are one of the pharmaceutical preparations that are in high demand. In healthcare institutions, syringes are used to aid in patient care and examination. Pharmaceutical preparations, such as various syringes, are widely available in the Pasar Pramuka. Syringes must be free from microbes and used syringes should not be reused. Microbiological sterility tests can be performed on a syringe to determine whether it is sterile or not. The purpose of the study is to test the sterility of syringes obtained from the Pasar Pramuka. A random sample of a syringe is being used in the study as an experimental method. The syringes were isolated and incubated for 14 days in Fluid Thioglycollate Medium (FTM) and Trypticase Soy Broth (TSB) at 30-35 oC and 20-25 oC, respectively, with frequent observations. If FTM and TSB media were turbid, then isolated into selective media based on their microbe as controls, namely Clostridium sporogenes ATCC 19404, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633, Salmonella typhi. The results showed that the B syringe was turbid on FTM media and did not contain pathogenic microbes after being identified on selective media, as in controls. The A and C syringes on TSB media were turbid, and after identification on selective media, Candida albicans and Aspergillus brasiliensis were found. In conclusion, the A, B, and C syringes are not steril.

Development of a protocol for genetic transformation of Malus spp

A protocol to produce transgenic shoots of Malus X domestica cv Greensleaves was optimized using two gene constructs previously used to create parthenocarpic tomato, Ino-IaaM and DefH9-IaaM. The aim was to obtain sufficient nº of transgenic shoots for in vitro multiplication, transfer to soil, grafting and testing for parthenocarpy in the next years. We investigated the effects of two modifications of a previous published protocol: 1) co-transformation with an Agrobacterium containing “VIP” genes in the gene construct and 2) two different hormones or hormone combinations. More shoot regeneration was obtained with a combination of three hormones (BA:NAA:TDZ) during co-cultivation instead of IBA and no co-transformation was performed using the VIP gene. For the DefH9-IaaM transgene, 21.04% regeneration was achieved for this treatment instead of 8.95% achieved with “IBA treatment” and 4.42% with the Agrobacterium co-transformation treatment. More shoot regeneration occurred with the combination of three hormones (BA:NAA:TDZ) instead of with only IBA and no co-transformation was performed using VIP gene. Experiments using Ino-IaaM confirmed the results shown for the DefH9-IaaM transgene. The regenerated shoots were multiplied in selective media containing kanamycin and roots were obtained.

Evaluation of Three Culture Media for Isolation of Burkholderia cepacia Complex from Respiratory Samples of Patients with Cystic Fibrosis

Burkholderia cepacia complex (BCC) is a significant pathogen causing respiratory disease in individuals with cystic fibrosis (CF). Diagnosis is typically achieved by isolation of BCC on selective culture media following culture of sputum or other respiratory samples. The aim of this study was to compare the efficacy of three commercially available selective media for the isolation of BCC. The three media comprised Burkholderia cepacia selective agar (BCSA; bioMérieux), BD Cepacia medium (BD: Becton–Dickinson) and MAST Cepacia medium (MAST laboratories). Each medium was challenged with 270 respiratory samples from individuals with CF as well as an international collection of BCC (n = 26) and 14 other isolates of Burkholderia species at a range of inocula. The international collection was also used to artificially “spike” 26 respiratory samples. From a total of 34 respiratory samples containing BCC, 97% were recovered on BD and 94% were detected on MAST and BCSA. All three media were effective for isolation of BCC. BCSA was much more selective than the other two media (p < 0.0001) meaning that fewer isolates required processing to exclude the presence of BCC.

Mutagenesis and Identification of Sugarcane Mutants Using Survival on Polyethylene Glycol and Leaf Damage under Managed Water Stress

Drought causes severe damage to sugarcane, reducing its product yield. Given Thailand’s weather conditions and topography, a breeding program to develop new sugarcane genotypes with a high tolerance for water stress is important to the sugarcane industry. This study created new water stress tolerant sugarcane genotypes using ethyl methanesulfonate (EMS) mutagenesis in the sugarcane cultivar Khon Kaen 3. Using 16 mM of EMS for 4 h induced callus mutagenesis (survival rate, 57.5%). The survival rates of calli treated with 10 mM of EMS for 2 and 4 h in selective media with 15% PEG were higher than that of non-EMS-treated calli. The selected calli survived and grew on selective media with 20% PEG, while non-EMS-treated calli did not grow. The mutant plantlets developed from EMS-treated calli on selective media with 20% PEG for 4 weeks had varying survival rates: 72.25% (10 mM of EMS for 2 h), 75.85% (10 mM of EMS for 4 h), and 60.61% (16 mM of EMS for 4 h). Both healthy mutant sugarcane plants (2,086) and non-mutant plants (234) were cultured on the media with 20% PEG for 16 weeks. Of these, 462 mutant sugarcane plantlets survived and developed on the media, but all the non-mutant sugarcane plantlets died during the selection process. Mutagenesis induced using treatment 4 produced the highest frequency of mutant sugarcane plantlets with water-stress tolerance (45.5%). In total, 136 selected mutant sugarcane plants were transplanted to a greenhouse for evaluation under managed water stress. Fourteen mutant sugarcane plants stayed green after the third cycle of water stress, but the KK3 sugarcane cultivar showed damage on 50% of the leaves. Thus, EMS mutagenesis and evaluation using in vitro and greenhouse methods were successful in developing new sugarcane clones with high water-stress tolerance, which is important for sugarcane breeding programs.

715. Evaluation of Four Chromogenic Selective Media for the Isolation of Candida auris

Background Candida auris is an emerging healthcare-associated multi-drug resistant pathogen able to cause severe invasive infections. Public health agencies have emphasized the need for active surveillance of C. auris to enable prompt infection control measures in healthcare institutions. Four commercially available chromogenic selective media were evaluated for the isolation of C. auris in this study. Methods Four chromogenic selective media (Brilliance Candida (BC), CHROMagar Candida (CC), CHROMagar Candida Plus (CCP) and chromID Candida (CAN2)) were evaluated with 52 fungal and 20 bacterial strains. Isolates were identified with Vitek matrix-assisted laser desorption/ionization mass spectrometry (MS). 1 µL spots of broth subcultures of each organism were inoculated onto chromogenic media and incubated as per manufacturer’s instructions. Plates were read at 24, 36 and 48 hours. Sabouraud Dextrose agar (fungi) or blood agar (bacteria) were inoculated to obtain viable counts. All reads were categorized into color groups. Sensitivity and specificity were determined based on phenotypic characteristics after 48h of incubation. Additionally, spots of serially diluted C. auris type strain CDC B11903 were assessed for expression of the expected C. auris phenotype at 36, 48 and 72 h. Probit regression model (R version 3.6.3) was used to estimate limit of detection (LOD) for each medium at each time-point. Results A minimum of 36h incubation (optimally &gt; 48 h) was required for observation of distinct color and colony morphology. CCP performed best overall, with 100% sensitivity and 96% specificity. Sensitivity was 100% across all 4 media, but specificity was reduced (BC – 66%, CAN2 – 83%, CC – 81%). The lowest LOD was observed with CCP at 4 colony-forming units (CFU). See figure 1 for details. Figure 1. Comparison of limit of detection for C. auris by type of chromogenic selective media across various time-points. Conclusion Rapid isolation and accurate identification of C. auris from samples, including those from non-sterile sites, are crucial for the management of C. auris infections and in curtailing nosocomial transmission. Chromogenic media are simple to use, inexpensive and scalable; ideal for routine diagnostics and high volume screening samples. Of the 4 media evaluated, CCP is best suited to screen for C. auris, with subsequent confirmation of identification using MS or genotypic methods. Disclosures Lynette Phee, MD PhD, Creative Life Science Co. Ltd (Creative Media Plate) (Other Financial or Material Support, Provided some of the CHROMagar Candida and CHROMagar Candida Plus plates free-of-charge for evaluation purposes.)Fort Richard Laboratories (Other Financial or Material Support, Provided some of the CHROMagar Candida Plus plates free-of-charge for evaluation purposes.)

Molecular Detection and Antimicrobial Susceptibility of Salmonella Species in Chickens

The study was intended for molecular detection of Salmonella Spp isolated from chicken. A total of 160 samples comprising of 40 liver, 40 spleen, 40 lungs and 40 intestines were collected directly into buffered peptone in universal bottles at the poultry slaughter houses of the four districts in Jos South Local Government area of Plateau State. The samples were enriched in 10 ml of Rappaport-Vassiliades broth and cultured onto Xylems Lysine Deoxycholate (XLD) agar for the isolation of bacteria. The isolated bacteria were identified by studying staining characteristics, cultural properties on different selective media, biochemical tests, catalase and coagulase test, and finally by PCR. Out of 160 samples, 65 (41%) samples were found positive for Salmonella Spp on XLD and 24 (37%) positive by biochemical analysis. Two(2) Salmonella isolates were amplified by 942 bp gene based PCR. Antimicrobial sensitivity test was carried out to ascertain the susceptibility of the organism to various antibiotics. Its results showed that the Salmonella isolates were resistant to amoxycillin (100%) and erythromycin (100%), gentamicin (100%), Clindamycin (100%) streptomycin (100%), tetracycline (100%), sulphamethoxazole/trimethoprim (100%) but sensitive to Ceftiofur (100%).

Selecting for and Checking Cells with HGPRT Deficiency for Hybridoma Production

For drug-selective media to work for hybridoma selection, myeloma cells expressing a mutation abrogating the function of their HGPRT gene (and subsequently unable to produce purines for DNA biosynthesis) are used. HGPRT will recognize 8-AG as a substrate and convert it to the monophosphate nucleotide. The 8-AG-containing nucleotide is then processed further and incorporated into DNA and RNA, where it is toxic. Therefore, cells with a functional HGPRT enzyme grown in the presence of 8-AG will die. Cells that are deficient in HGPRTase cannot incorporate 8-AG in vivo and thus continue to grow. Cells that have been selected for resistance to 8-AG should be checked periodically to ensure that they maintain sensitivity to drugs that block the de novo synthesis of DNA. In addition, all myeloma cell lines should be checked periodically for reversion of their drug selection markers. Any line that is not killed completely by drug selection should either be reselected or replaced with a new line.

First Characterization and Description of Aspergillus Series Versicolores in French Bioaerosols

Air quality can be altered by fungal contaminants suspended in the air, forming bioaerosols. Aspergilli section Nidulantes series Versicolores are recurrent in bioaerosols and are mainly responsible for allergies and asthma aggravation. Phylogenetic studies recently identified 12 new species within this series. This study is the first to identify species of Aspergillus series Versicolores in French bioaerosols and to characterize them macroscopically, microscopically and molecularly. Bioaerosols were collected in a cancer treatment center, in contaminated homes and in agricultural environments. A total of 93 isolates were cultured on selective media, observed by optical microscopy and identified by benA amplification before sequencing. The field data (temperature and relative humidity) were statistically tested to explore the ecology of these species. Eight species were identified from bioaerosols: Aspergillus creber and A. jensenii, which represent more than 80% of the isolates, and A. protuberus, A. puulaauensis, A. sydowii, A. tabacinus, A. amoenus and A. fructus. Aspergilli series Versicolores are distributed differently depending on the sampling site and climatic determinants. Aspergillus protuberus was found in bioaerosols collected under significantly lower relative humidity (p = 3.899 × 10−4). Characterization and repartition of these isolates belonging to the Versicolores series constitute an important step to better assess exposure to fungal bioaerosols.