فصلنامه زیست فناوری میکروبی
پیاپی 9 (تابستان 1390)

The in vitro techniques of cell culture are important in biological sciences, medicine, and biotechnology. The presence of many biological substances in cell culture is necessary. Fetal bovine serum (FBS) is the most popular cell culture additive for the stimulation of cellular proliferation and biological production. Since there is a possibility of contamination of animal sera with viruses, bacteria, fungi and immunoglobulins, the application of methods for the inactivation or removal of these pathogens is extremely important in plasma production. The gamma irradiation has been used in sterilization of FBS and other biological materials. In the current study, the effect of variable doses of gamma irradiation on the sterilization of FBS was investigated and compared to filtration method. Raw fetal bovine serum was prepared from Academic Center for Education, Culture, and Research (ACECR). FBS samples were divided into eight groups including Raw sera, filtrated sera, and 5, 10, 15, 20, 25 and 35 KGray gamma receivers (in 5.6 Gray/s dose rate). Sampling was repeated three times. The specimens were assessed for the sterility. The results showed that there were no bacterial, mycological, mycoplasmal or viral microorganisms in 10 KGray and higher doses of gamma receivers. However, the filtration method failed to complete elimination of the virus. According to the results, it can be concluded that gamma irradiation is a confident and useful method in FBS sterilization, and the applicable dose varies based on the product preparation terms and application.

Synthetic polymers obtained from petrol causes soil pollution, for this reason, PHB has gained importance since it can easily be dismantled in nature. The microbial poly hydroxyalkanoate family of polyesters is synthesized by a wide variety of bacteria as intracellular carbon and energy storage materials. PHB has been identified in more than 20 bacterial genera, including Azotobacter, Bacillus, Alcaligenes, Pseudomonas and Rhodospirillum. In this study, 11strains of the genus Mesophile Bacillus and 7 strains of the genus Thermophile Bacillus isolated from Golestan forest soil. The samples were heated at 75̊c for 20min in water bath, than were diluted in sterile water and were placed on nutrient agar media. Plates were incubated at 30̊c and 50̊c for 24-48h. PHB production by these strains was determined by the spectrophotometric method and confirmed by GC-Mass and FT-IR. PHB production was found in Mesophile strains ranged from 6/72-48/70 % (W/V) and in Thermophile strains 3/84-39/21%. PHB was found in all strain, among them B11 and B4 were capable to produce maximum biopolymer 48/70% and 39/21%. The best shaker rotation and temperature for B11 are 200rpm and 32°c and for B4 are 200rpm and 45°c. B11 and B4 strains are capable to produce biopolymer and suggested that in the future these strains can be used as one of the producer of biopolymer. Using of isolated Bacillus sp in biosynthesing PHB which can make them suitable for applications in the packaging industry and as substitute for hydrocarbon-based plastics.

Mycotoxins are poisons which produce by soil mould funguses and are abundant in the environment and food stuff. The most important group of these toxins is Aflatoxins which are produced by some mold species from the Aspergillus genus. The most dangerous and strongest is B1 Aflatoxin with different toxic impressions including human being liver cancer. Researchers have revealed that a group of none pathogenic bacteria named as Lactic Acid Bacteria are able to connection the B1 Aflatoxin and other Aflatoxins and Mycotoxins discharge them from environment and food stuff. The purpose of this research to obtain Lactic Acid Bacteria that their ability to help in connection and isolation B1 Aflatoxin is used them as food supplements or probiotic products for prevent of absorption B1 Aflatoxin in human and animal body. In the present research the bacteria are isolated from five groups of different sources. For survey their bacteria ability for isolation of B1 Aflatoxin used ELISA method and used for identification the result strains 16S rRNA sequencing method by apply universal primers. Among the strains isolated two strain Lactobacillus pentosus and Lactobacillus berevis ability of B1 Aflatoxin absorption and isolation that respectively are abile to absorb and discharge 17.4 and 34.7 percent of B1 Aflatoxin from the experiment solution. Isolated Lactobacillus pentosus from human feces and Lactobacillus berevis from local milk sample. For survey significant of the result Used SPSS 16 software.

Production of pigments by microorganisms has significant importance than other sources; because microorganisms are grow quickly, easier extraction and higher yields. The purpose of this study was isolation and identification of pigment-producing microorganisms from soils in regions of West Mazandaran province in the second half of 1389 and toxicity effect pigments. 70 soil samples from the Mountain West Mazandaran province (Chalus, Tonekabon and Ramsar) were collected and after of prepared serial dilution, each of samples was cultured on Nutrient agar and Sabouraud Dextros Agar. After, colonies were selected with the ability to produce pigment. Pigment extraction was done by Methanol extraction on Sediment microorganisms. Different color compounds were identified by TLC. Molecular identification of microorganisms was performed by rDNA PCR Sequencing. Also, oral acute toxicity of pigments were evaluated on the albino mice. Seven microorganism were identified include Aspergillus sp, viridans Aerococcus, Methylobacterium sp, Bacillus sp, Eurotium rubrum, Microbacterium testaceum and Acinetobacter johnsonii. Oral acute toxicity study of the Pigments on rats was no mortality in the maximum dose. However, studies of liver enzymes and liver histopathology were showed the pigments reversible toxic effect on the liver tissue in maximum dose. As regards the natural pigments in the proper doses are healthier than their chemical, their application is useful in different industries. Also to determine the structure of this pigment and to removal of toxic groups of pigments can be used in various industries.

Brucellosis is a zoonotic disease which has an extremely economic importance. Prevention and diagnosis of human and cattle infected with the organism both are challenging researches. Immunologic evaluation of diverse antigens of Brucella cells has a key role in development of prevention and diagnosis programs. Here, we report the production and immunologic evaluation of recombinant 18kDa outer membrane lipoprotein of Brucella abortus (Omp19). Brucella omp19 gene was amplified with PrimSTAR® HS DNA polymerase, cloned in pJET1.2 and sequenced. The omp19 gene was cut by BamHI and HindIII from confirmed vectors and subcloned in pET28a(+). pET28a(+) vectors cloned with omp19 were transformed into E. coli BL21(DE3) as expression host. Expression of recombinant protein was induced by IPTG and verified by western-blotting. Recombinant protein was purified using Ni-NTA resin and was applied to detect antibodies in human and animal brucellosis cases serum samples via western-blotting. Recognition of antibody to the recombinant Omp19 (rOmp19) in serum samples of infected cases suggests the stimulation of immune response to this protein during brucellosis. The study indicates rOmp19 of Brucella abortus is an appropriate antigen for further investigations in vaccines and serologic diagnosis programs.

Indiscriminate use of antibiotics has resulted in resistant microorganisms. In order to find new antibiotics to defense pathogenic microorganisms resistant to common antibiotics, An antibiotic-producing bacterium isolated from skin of the frog Rana ridibunda. In sterile conditions samples from skin of frog were transferred to broth medium. Antimicrobial agent-producing bacteria were isolated using serial dilution method on nutrient agar. One of them was selected for more investigation. Isolated bacterium identified based on 16SrDNA and biochemical and morphological characteristic. Antimicrobial activity of culture supernatant was examined against laboratorial standard Bactria by disc diffusion and MIC methods. In order to characterization of produced antimicrobial compound, Culture supernatant of bacterium was washed by chloroform and then antimicrobial substance was extracted by methanol and chloroform and detected by bioautography on silica gel plates.The dialysis tube was used to find the molecular weight of this substance. Isolated bacterium identified as strain of Bacillus atrophaeus. The anti-microbial substance exhibited heat stability between 25 and 100°C, and activity under both acidic and basic conditions from pH 2 to 11. Bioauotography assay showed that methanol is optimum solvent for the extraction. The dialysis tube indicated that the antimicrobial substance weight is less than 1 kDa and Ammonium sulfate was not capable to precipitate the compound. This study showed that antimicrobial substance differs from peptide antibiotics produced by bacteria of the genus Bacillus such as bacitracin, in some properties that increases the likelihood of being novel.

Staphylococcus aureus is one of the most important community-acquired infections which caused significant infections such as bacteremia, endocarditis, osteomyelitis, toxic shock syndrome and dermal infections. This survey was conducted with the aim of determining resistance pattern of Staphylococcus aureus strains; because of there are different reports about susceptibility of these bacteria against different. In this descriptive study, which was done during the first six months of 2011, 100 strains of Staphylococcus aureus were isolated from clinical samples of Imam Reza and Shohada hospitals in-patients, in Tabriz and susceptibility test was done according to Kerby-Bauer method by using Ceftazidime, Clindamycin, Ciprofloxacin, Tetracycline, Cefazolin, Gentamicin, Cotrimoxazole, Cephalexin, Carbenicillin, Methicillin and Oxacillin disks. The Staphylococcus aureus ATCC25923 was applied as quality control in this test. In this study, susceptibility against antibiotics were used such as Cotrimoxazole, Carbenicillin, Gentamicin, Ciprofloxacin, Clindamycin, Cephalexin, Cefazolin, Tetracycline, Methicillin and Oxacillin, were 74%, 73%, 69%,67%, 63%, 61%, 59%,42%, 4% and 0%, respectively. The most resistance strains were resulted against Oxacillin 100%, Methicillin 91%, Tetracycline 44%, Cephalexin 30%, Cefazolin 29%, Gentamicin and Carbenicillin 20%, Cotrimoxazole 16%, Clindamycin 15%, and Ciprofloxacin 12%. By noting the increasing rate of antibiotic resistance, it seems that rapid and timely diagnosis of resistance in strains is crucial in order to select proper therapeutic choices and prevent development of resistance.

Considering the high environmental stability of lead and its easy transmission through food chains as well as its well-documented destructive impacts on living cells, the present study was aimed at investigating the applicability of effective bacteria for elimination or reduction of lead through the development of microbial flocculation. In order to isolation of lead-resistant bacteria, the sample of activated sludge was cultivated on BHI Agar medium with 1000ppm lead standard solution, incubated at 30°C for 24 hours. Resistance and reduction of lead was investigated by Atomic Absorption analysis. MIC and MBC of the samples were determined and the most lead-resistant bacterial strain was evaluated. The ability of the selected strain in development of bio-floc and lead reduction by fish food (fish color) as the bacterial nutrition source and bed was studied. 16S rRNA sequencing was used to identify and confirm of selected strain. from 25 isolated strains, the most lead-resistant bacterium was demonstrated the MIC and MBC of 8280 and 12420ppm, respectively. Atomic Absorption analysis showed this strain was reduced 98% of lead. The ability of bacterium to produce bio-floc and lead reduction was evaluated 120g/L and 84%, respectively. Biochemical and molecular analysis of the selected strain was confirmed 99% as Bacillus subtilis. Bacillus subtilis isolated in this research can be a good alternative to production of biological sludge for treatment of wastewater and ponds of fish and shrimp farming pools contaminated with this metal.