Annual Report July 2012 June 2013  Bangladesh Atomic Energy Commission

Radiation protection is required for the safety of the operator, worker, public and the environment. Before starting operation, necessary steps were taken to prevent the radiation hazards such as TLD badges and survey meters. The badges used by the workers were tested at regular intervals by the Health Physics Division to evaluate and to determine the exposed dose of the workers. According to the IAEA standard, the received dose by the worker should not exceed the minimum dose level of 2.5 µSv / hour. Emergency response plan were maintained to keep the worker free from any hazard and accident. Trained personnel were involved in operation services because they can take necessary steps in emergency situation and know how to tackle the situation and to control with the different authorities like Police, Fire brigade & Medical surveillance for emergency situation.

Gamma Source Division rendered sterilization services for Commercial products and earned

The division focuses in developing the methods of management of insect pests using nuclear and biotechnological approaches; developing quarantine treatment for fresh agricultural produces; and exploiting of beneficial insects.

Tephritidae is a large family that includes many fruit pests and these are usually adopted for housing large quantities of bacteria in their digestive tract. Explorations on different fruit fly’s associated bacterial community revealed that most of fly’s gut microbiota is dominated largely by free-living bacteria of the Enterobacteriaceae. These symbionts are known to play significant role in fly’s different fitness parameters. Therefore, efforts were made to isolate and to identify the mid-gut bacterial community of laboratory host reared pumpkin fly, Bactrocera tau (Walker) (Diptera:Tephritidae) using conventional biochemical techniques. Colony characterization of the isolated bacteria was conducted on Nutrient agar and MacConkey agar plates. Isolated gut-bacterial species viz., Proteus rettgeri and Klebsiella oxytoca were examined through incorporating with protein (casein:yeast extract:sugar, 1:1:2) and sugar diets to study the effect of bacteria supplemented diets on the ovariole number and fecundity of B. tau. A total of nine bacterial species were identified under the family Enterobacteriaceae. The bacterial species were Proteus rettgeri, Proteus vulgaris, Klebsiella oxytoca, Streptobacillus moniliformis, Alcaligenes faecalis, Haemophilus ducreyi, Erwinia sp., Chromobacterium lividum and Flavobacterium picketti. Mean ovariole number were 20.66 ±2.51, 20.56±3.53, and 22.41±3.75 for B. tau fed on P. rettgeri, K. oxytoca incorporated protein diets and only protein diet, respectively. Experimental result revealed no significant influence of gut bacteria added adult diets on the fecundity of B. tau fed on above mentioned diet treatments.

The pumpkin fruit fly, B. tau is one of the most destructive pests on cucurbit vegetables of Bangladesh. So far, very limited scientific data are available on the Sterile Insect Technique (SIT) approach for pumpkin fly, B. tau. The study aimed to determine the male sterility dose and to optimize the ratio of irradiated and unirradiated male pumpkin fly, Bactrocera tau (Walker) for field application of Sterile Insect Technique (SIT). The percent of sterility attained in F₁ generation was 33.84, 46.55, 64.21, 86.54, 90.63, 100 and 100 at 10, 20, 30, 40, 50, 60 and 70 Gy dose treatment of adult male respectively. Complete sterility of male pumpkin fly was observed at 60 Gy. Fixed numbers of virgin females were allowed to mate with unirradiated and irradiated (60 Gy) males at 1:1, 1:2, 1:5 and 1:9 ratios. Comparing with control group, a sharp decrease of pupal recovery was observed when females were allowed to mate with varying ratios of irradiated males. Minimum pupal recovery (42 fold less) was obtained at 1:9 ratio. Percent of viable adults and fliers were significantly decreased as the irradiated males were increased from 1:2 to 1:9 ratios mated with females. Significantly deformed adults were increased as the ratios of irradiated males were increased. All the above parameters suggest that 1:9 ratio of normal vs sterile males of pumpkin fly is apparently the effective ratio for releasing sterilized male melon fly in nature for possible field application of SIT.

The external colour, firmness and storage loss of cucumbers were determined at different quarantine treatment methods including the untreated control group. The effects of the treatment of 100 Gy gamma irradiation, 180 watt microwave energy (10 seconds) and both 100 Gy gamma irradiation and 180 watt microwave energy together were analysed against untreated fresh cucumbers. Treated and untreated fresh cucumbers were kept at 25°C and relative humidity was maintained between 80 and 90%. Up to 15 day storage period, highest damage (30%) was recorded from the controlled cucumbers (no treatment applied) while the lowest damage was recorded (13.3%) when cucumbers were subjected to both gamma irradiation and microwave energy together. Damage of 16.66% cucumbers was recorded when they were treated with gamma irradiation only. During the same period no significant difference of firmness were observed in both the treated and untreated cucumbers. The colour analysis showed no significant differences of L (darkness) and b (blue to yellow) values during the storage period. On the contrary, significant differences were observed on green to red values (a). Further works are on-going.

Female Aedes aegypti mosquitoes spread human pathogenic viruses that cause yellow fever, dengue fever, and Chikungunya. Much of the struggle against these diseases has relied on a combination of prophylactic measures such as vector control including insecticides and traps. At present, dengue transmission can be reduced or interrupted by controlling its mosquito vector, Aedes aegypti by Sterile Insect Technique. Sterile dose selection of A. aegypti mosquito was observed as a part of SIT. Radiation was applied to adult male which were mated with same number of females after emergence. Effect of different radiation dose (longevity, egg hatching rate, male and female ratio, pupation) determined up to F1 and F2 generation of Aedes aegypti. A. aegypti exhibited decreased fecundity and egg hatch success in relation to radiation dose up to F1 generation. Further works are on-going.

1. IAEA Cooridinated Research Project entitled Management of mosquitoes using sterile insect technique (CRP Contract No. 14686)

2. Use of Symbiotic Bacteria to Reduce Mass-rearing Costs and Improve Mating Success of Selected Fruit Pests in Support of SIT Application (CRP No. 17011/RO).

5. Microbiology and Industrial Irradiation DIVISION, AERE

5.1 Research and Development Work

5.1.1 Assessment of microbiological status of chanachur, a traditional snack and its quality improvement by gamma radiation

Chanachur is an Indian sub-continental traditional ready-to-eat spicy snack which is consumed mostly as one of the favourite street food. In this study microbiological status of chanachur was determined and its quality was improved by applying gamma radiation. To assess the microbial load in chanachur samples four types (non-branded coded as S1 and branded coded as S2, S3 and S4) were collected and Total Viable Bacterial Count (TVBC), Total Coliform Count (TCC) and Total Fungal Count (TFC) were determined by spread plate method using Nutrient Agar (NA), MacConkey Agar and Potato Dextrose Agar (PDA), respectively. TVBC, TCC and TFC values for four types of chanachur samples were ranged from 1.18×105 to 2.1×104 CFU/g, 1.2×104 to 3.0×103 CFU/g and 1.5×104 to 2.0×103 CFU/g respectively which were beyond the satisfactory or acceptable level. To improve the quality of chanachur samples, a series of doses of gamma radiation viz. 2.0, 5.0, 10.0 and 15.0 kGy were applied. Irradiation with 2.0, 5.0 and 10.0 kGy, microbial load was decreased by 1 to 3 log but not completely eliminated. After applying the radiation dose of 15.0 kGy, all kinds of viable microorganisms including spores were eliminated rendering the chanachur samples sterile and safe for consumption. Bacteria associated with the chanachur samples were also identified through cultural, morphological and different biochemical studies.

Potential acetic acid bacteria were investigated from different readily available sources. Seven different samples (sugarcane bagasse, sugarcane juice, sugarcane juice processing water, soil, rotten apples, rotten red grapes and rotten white grapes) were collected from local market. After processing and enrichment, samples were inoculated on Glucose Yeast Calcium carbonate (GYC) agar plates and incubated at 30C for four days. Nineteen different bacterial colonies were selected and isolated on the basis of clear zone formation on GYC medium. The bacterial isolates were identified on the basis of their morphological, biochemical and physiological characterization. Among nineteen isolates, one was identified as Acetobacter aceti, one as Acetobacter pasteurianus, one as Acetobacter orleansis, two were identified as Acetobacter cibinongensis, and the remaining fourteen isolates were identified as Gluconobacter spp. As potential acetic acid producers, only the Acetobacter isolates were further assessed for their acid production capability under different temperature and pH using ‘Potency Index’ as a potency determining parameter. Temperature 30C and pH 5.5 were found to be the optimum temperature and pH respectively for maximum acetic acid production by most of the species. Acetobacter pasteurianus with the highest P.I. value of 3.78 was the most potent acetic acid producer among these isolates.

PHAs (polyhydroxyalkanoates) are a group of biodegradable plastics of biological origin. These are attractive substitutes for conventional petrochemical plastics due to similar material properties as thermoplastics e.g., polypropylene. Moreover, it is completely biodegradable upon disposal under specific environmental conditions. PHAs are accumulated as a carbon and/or energy storage material in various bacteria usually under the condition of excess carbon source and limiting nutritional elements such as N, P, S, O, or Mg. In this study, attempts have been made to isolate PHB (polyhydroxybutyrate) producing bacteria from rich natural microbial source such as compost. Six different types of compost samples were collected from Bhawal Mirzapur, Gazipur, Dhaka, Bangladesh. For isolation, 10 g of compost from several composting sites was mixed with 90 mL of distilled water. A dilution series up to 10^-9 was made using distilled water. Aliquots of 0.1 ml were plated onto nutrient agar and PHA agar plates and were incubated for 24–48 h at 37C. In this study, 48 isolates were initially isolated from six compost samples. Bioslurry showed the highest count 3.75×10⁹ cfu/g whereas Poultry slurry 2 did not support any bacterial growth. All the bacterial isolates were qualitatively tested for PHB production following the viable colony method of screening using nile red and Sudan Black B dye. After primary screening using Nile red, 16 isolates were found to give orange fluorescence under UV (360 nm). Then they were further grown on nutrient agar plate and tested with Sudan black. Based on the intensity of the fluorescence and Sudan black coloration, high PHB producers were selected. Out of 48 isolates, sixteen isolates were successfully screened as PHA accumulating bacteria. These bacteria can be a potential source for commercial production of biopolymer. Further studies are being continued to identify these strains.

Chitosan is typically obtained by partial deacetylation of chitin. The product is a copolymer of N-acetylglucosamine units and D-glucosamine units. Degree of deacetylation (DD) is one of the main parameters characterizing chitosan. The most precise measurements of DD require sophisticated equipment (NMR spectrometer), not available at many laboratories worldwide working on chitosan. Within the same method, there are usually many analytical procedures for performing measurements and calculations and many ways of interpretation of results. It is obvious that not every research laboratory has an NMR spectrometer. Moreover, this method, although probably the most precise, is not suitable for routine measurements of DD due to high costs (deuterated solvents, depreciation of equipment, specialized staff time) and time-consuming sample preparation procedure. There is a need for low-cost, simple, yet sturdy and reliable methods and procedures for DD determination. The aim of this work is to develop a new, simplified method and compare a few of the existing analytical techniques on the same set of chitosan samples. The method consists of titrimetric determination and online monitoring of chitosan samples of nimal DD in the range os ….%. Moreover, evaluation of the ease of performance and availability of reagents in the developed methods will also be performed.

Citric acid is a commercially valuable microbial metabolite which is produced mainly by submerged fermentation of starch- or sucrose-based media, using the filamentous fungus Aspergillus niger. Industrially, large-scale production of citric acid uses beet or cane molasses, sucrose or glucose syrup as carbon source. In recent years, the use of cheap substrates e.g., agro-industrial wastes or surplus materials has gained global attention due to its economical and environmental benefits. In this study, attempts have been made to produce citric acid using cheap carbon sources such as sugarcane juice under submerged condition. For this purpose, sugarcane juice was collected from different vendors and brought into laboratory. Sugar estimation was carried out immediately after collection by Anthrone ulphuric acid method. This process was repeated for several batches to get an average value on the sugar content of the substrate. It was found that the average sugar content of sugarcane juices were in the range of 7-10%. Experiments are ongoing to compare the citric acid production by Aspergillus niger using sucrose.

Routine services for microbiological analysis of food, food supplement, water and medical products were rendered for different pharmaceuticals and food industries. The division earned

To obtain fixed mutant(s) with photoperiod-insensitive, short day-length and shorter plant height with higher yield from local T.aman Ashfal. Heights were measured randomly from ten M₁ plants of each irradiated dose at 3 weeks after transplanting. M₂ seeds were harvested individually from three fertile M₁ plants at 5 months after transplanting. The M₂ seeds were spread on a seed bed on July 16, 2009. Survived seedlings were transplanted on August 17, 2009 and they were grown during Aman season. Number of days to heading was recorded as the number of days required from sowing time when 50% of plants of each line headed. Days of maturity was recorded as the number of days required from sowing time when 90% of plants of each line appeared with yellowish grains. These two data were recorded through visual observation by visiting the plots every alternate day. Plant heights, number of effective tillers per plant and panicle length were measured at the time of maturity with randomly selected 5 competitive plants. M₃ seeds were harvested separately from each M₂ plant. M₃ plants were grown as plant progeny during next Boro (long day) season (December 2009 to May 2010). Data were recorded as the same as described in M_2. Grain characteristics such as length (L) and width (W) of unhusked and husked grain were recorded in October 2012. Survival rate and plant height was determined to examine the effect of carbon ion irradiation over the plant growth cycle. Survival rate of the irradiated seeds were significantly decreased at 60 Gy and remains gradually at higher doses. Seedling height also gradually decreased with the application of increased rate of irradiation dose. The height of the seedlings were reduced almost half compared to unirradiated seeds and it was started at 80 Gy and gradually goes to at higher doses. Since the cultivar Ashfal is a highly photoperiod sensitive in which, none of the control plants headed during the Boro season. However, we have found nine M₁ plants headed under the same growing conditions. It was confirmed that photoperiod sensitivity was genetically altered in the fertile M₁ plants. It was also observed that in M₂ generation, the plant height of mutant lines were markedly shorten and yield also increased.

To develop an efficient and reproducible in vitro propagation protocol for M. paniculata L. through shoot tip and nodal explant culture. Shoot tips and nodal segments of garden grown plants were used as explants and cultured on MS supplemented with different concentrations and combinations of BAP, Kin and NAA for shoot multiplication. Maximum shoot regeneration was found in MS supplemented with 2.5 mgl^-1 BAP. With repeated subculture and addition of 100 mgl^-1casein hydrolysate to the medium enhanced the number of shoots per culture and incorporation of 100 mgl^-1 urea to the medium increased the length of shoots. Individual elongated shoots were rooted well on half-strength MS supplemented with 1.0 mgl^-1each of IBA and IAA within four weeks of culture. Well-rooted plantlets were transferred to pots containing soil and compost (2:1) for hardening. During hardening 70% plantlets survived, which were subsequently transferred to the experimental field.

The R & D activities of Radiation Entomology and Acarology Division is primarily focused on development of environmentally safe integrated pest management techniques against insect and mite pests of economic importance by using gamma radiation, biodegradable botanicals, hormones, pheromones, bio-control agents and chemical pesticides.

In the present study growth and developmental parameters of blowfly reared on natural (Tilapia fish) and artificial diets (composed of wheat bran, whole milk powder, chicken egg, agar and water) were recorded and compared. The larval and pupal periods lasted almost the same time with the both diets tested. The larval and pupal periods on artificial diets were slightly longer than those on natural diet but the differences were not statistically significant ( p>0.05). Egg to pupa transformation rate (63-68%) and adult emergence rate (97-98%) were found almost similar when larvae reared on natural diet and those reared on artificial diets. In the present study, the mean generation time, the time from the oviposition until female offspring were mature enough to oviposite their own eggs, was 14.70 days for artificial diet and that was 13.72 days for natural diet. Although the development of L. cuprina on artificial diet was a bit slower than on natural diet but the differences between natural and artificial diets were not statistically significant (p>0.05). The larval and pupal mortality and developmental durations were not significantly different when larvae reared on natural and on artificial diet. These results suggest that the nutritional quality of artificial diets is comparable with those of natural diet.

Oxygen levels affect the sensitivity of insects to radiation. The increased radiation damage in a high-oxygen environment is a general phenomenon in radiobiology. Damage induced by radiation is typically lower in an oxygen-reduced environment (hypoxia) than in air, so usually higher doses are needed to produce comparable reproductive sterility. However, we have no data about what happened if the blowfly pupa is irradiated in absence of oxygen. The present study was thus undertaken to determine the effect of radiation on some biological aspects (percent adult emergence, longevity) of blowfly pupa in presence of oxygen (aerobic condition) and absence of oxygen (anaerobic condition). The percent adult emergence under anaerobic and aerobic condition for 25Gy, 35Gy, 45Gy and 55Gy was 96, 97, 97.5, 95 % and 97, 97.5, 94.5, 97.5 % respectively. Longevity under anaerobic and aerobic condition for 25Gy, 35Gy, 45Gy and 55Gy was 27.83, 28.84, 24.37, 24.41 day and 27.82, 27.85, 22.96, 24.33 day respectively. The above data revealed that irradiated blowfly pupa either in presence or absence of oxygen did not affect significantly on percent adult emergence and longevity.

Blow fly, Lucilia cuprina (Diptera: Calliphoridae) is a major pest of sun-drying fish industry in the costal area of Bangladesh. Bangladesh Atomic Energy Commission has taken R & D programme to implement Sterile Insect Technique (SIT) in the off shore Islands for suppressing blow fly population. The major component of SIT is the production of high quality blow fly in laboratory mass rearing. The quality as expressed by larval and puapl weights as well as F1 adult longevity and fecundity were found significantly different in natural and artificial diets. Larval and pupal weights were relatively greater (Larva 36.57±3.75, Pupa 29.11±8.07 mg) in those resulted from artificial diet than those of natural diet (Larva 34.58±1.80 mg and Pupa26.28±3.29 mg ). Adults emerged from heavy pupa reared on artificial diet lived longer life as well as laid significantly more number of eggs per female than those from natural diet. In conclusion, both the diets tested, the production quality of L. cuprina showed significantly higher with artificial diet than natural diet. Thus artificial diet can be used for mass rearing of blowfly larvae for several basic and applied studies.

In the present study we have collected a good number of moths by using a light trap in the AERE campus and succeeded to identify them up to species level. Among the collected 1489 moths 683 belong to the family Pyralidae, followed by Noctuidae (470), Arctiidae (207), Geometridae (41), Lymantridae (33), Hypsidae (18), Lymacodidae (17), Ctenuchidae (8), Drepanidae (7), Sphingidae (5), Notodontidae (4) Psychidae (1), Nolidae (1) and Lasiocampidae (1). The highest 58 species of moth was recorded from the family of Noctuidae followed by 44 species of Pyralidae, 16 species of Geometridae, 13 species of Arctiidae, 6 species of Lymantridae, 4 species of Sphingidae, 3 species of Ctenuchidae, 2 species of Lymacodidae, 2 species of Hypsidae and only 1 species was recorded in each family of Drepanidae, Psychidae, Nolidae, Notodontidae and Lasicampidae. The above mentioned results have shown that the largest and the 2nd largest numbers of moths were recorded from the family of Pyralidae (683) and Noctuidae (470) in contrast the largest and the 2nd largest numbers of species belongs to the family Noctuidae (58) and Pyralidae (44). Considering these results the family of Noctuidae showed highest species diversity among the recorded families in the study area.

The effect of three photoperiod regimes on the reproductive and developmental parameters and adult survival rate in Dermestes maculates (Coleoptera: Dermestidae) were investigated under laboratory conditions (30±2^oC temperature and 65±5% relative humidity) on dried Katchki fish. The photoperiods tested were: 12 hours light and 12 hours dark period (LD 12:12), 24 hours light and 24 hours dark period (LD 24:24). The mean incubation period was significantly longest in the light regime. The fecundity and fertility was significantly higher in the LD 12:12 regime and lower in 24 hours light period. Pupal weight was significantly higher in the dark period. The pre-oviposition period was significantly longer in the 24 light regimes and lowest in the LD 12:12. Oviposition period was insignificantly high in LD 12:12, low in 24 hours light period and intermediate in 24 dark condition. These results suggest that continuous photo phase is not suitable for the development and reproduction of Dermestes maculatus.

Radiation sterilized tissue allografts are frequently used in the field of orthopaedic surgery, oral & maxillofacial surgery, opthalmology and burn & cosmetic reconstruction. Tissue Banking and Biomaterial Research Unit (TBBRU) of Bangladesh Atomic Energy Commission is regularly providing high quality radiation sterilized amnion membrane allografts and bone allografts to different hospitals and clinics throughout the country to treat different sorts of health problems, such as burns, acid violence, leprotic ulcer, bedsore, traumatic open wound, diabetic wound, opthalmological defects, degenerative bone diseases, congenital deformities, bone fractures, gap non-union from traumatic accidents, oral and maxillofacial defects etc.. At present, 130 hospitals/clinics and more than 350 surgeons & physicians are involved with these activities through procurement of tissues and utilization of radiation sterilized tissue allografts. Moreover, this tissue bank arranges public and professional awareness programmes to increase tissue donation and applications. Above all, researchers of this unit are working on the development of new biomaterial and allograft substitutes to mitigate the huge demand of graft material.

Amniotic sacs were collected aseptically from labor room after normal vaginal deliveries in a sterile plastic container containing sterile physiological saline (0.9%) and were preserved temporarily in freezer (-20ºC). At the time of collection, each container was labeled with donor information, hospital registration number and date of collection. The sacs were collected only from the healthy and diseases free donors. The containers were placed in a cool box and transported to the tissue-banking laboratory as early as possible. After transportation to the tissue banking laboratory, amniotic membranes were separated aseptically from sacs using sterilized surgical instruments. The membranes were then placed in a sterile conical flask and shaken with sterile physiological saline using a mechanical platform orbital shaker for several times (5/6 times). Then the membranes were spread on sterile surgical gauge and placed on the plastic frame racks under sterile condition. The membranes were then oven dried at the temperature 40±1ºC for over night (14-16 hours). The dried membranes were then cut into different standard sizes e.g., (20x10 cm, 10x10cm) and special size for eye (5x5 cm), triple packed in polythene envelopes, vacuum-sealed and labeled properly under laminar flow cabinet. The membranes were then sterilized by gamma irradiation at the dose of 25 kGy. The radiation sterilized grafts were supplied to different hospitals after sterility test in accordance with the requirements. During the reporting period, 957 pieces of amniotic sacs were collected and 3936 pieces of amniotic membrane allografts were prepared for clinical use in rehabilitative surgery.

Human femoral heads were collected in sterile poly pack from clinically acceptable live donors undergoing surgical treatment, such as excised femoral head in fracture neck of femur (FNF), in replacement hemiarthoplasty or in total hip replacement (THR). After procurement, bone tissues were properly labeled and stored temporarily at -40ºC. Bones were then transported to the tissue banking laboratory (TBL) in cool box. In TBL, bones were stored in a freezer (-40 or -80ºC) temporarily until processing. During processing, bones were removed from freezer for thawing and were kept in a glass beaker containing sterile distilled water and were placed in a temperature controlled water bath for three hours at 60ºC for pasteurization. After pasteurization, the bones were preserved at-40ºC deep freezer for at least overnight. All soft tissues were removed from bones and then cut into pieces of different shapes and sizes using electric bandsaw and were placed in a sterile conical flask. All these works were done aseptically. The bone chips/segments from individual donors were washed separately with sterile distilled water several times at room temperature (±25ºC) using an electric shaker to remove blood. The bone pieces were then washed with warm (50ºC) sterile distilled water to remove fatty material using water bath shaker at 50ºC. Finally, the bone pieces were rinsed with cold sterile distilled water for 2-3 times. After washing, the bone pieces were transferred to freeze drying rack and kept in a freezer (-40ºC) till freeze drying. The bone pieces were freeze dried at -50⁰C. Freeze-dried bone pieces were triple packed in polyethylene envelope, properly labeled under laminar flow cabinet and sterilized by gamma irradiation at the dose of 25 kGy. After sterility test, the bone grafts were supplied to the hospitals as per requirements for use in reconstructive surgery. During the period, 620 raw bone tissues were collected from different hospitals and 9776 cc of freeze dried radiation sterilized bone chips, blocks, dowels etc. were prepared for clinical use in orthopaedic reconstruction.

Long bone, such as femur, tibia, fibula etc. were collected from seronegative live donor after total knee replacement (TKR) operation, corrective osteotomy and primary traumatic limb amputation surgery. After procurement, the bones were temporarily preserved at –40ºC in the hospitals/clinics and were then transported to the tissue banking laboratory in insulated cool box and preserved under frozen condition (–80ºC) until the processing began. For processing, the frozen long (massive) bones were first thawed to room temperature and then the bone marrow as well as the remnants of muscles attached to the bones were removed using sterile surgical instruments under aseptic condition. Then the bones were treated with providone iodine solution for 30 minutes and washed several times with plenty of sterile distilled water to remove blood. After proper washing, the bones were first packaged and vacuum sealed in polyethylene package and wrapped with fabric and again vacuum sealed in another outer layer of polyethylene package and labeled with graft identification number, dose and date of gamma irradiation, preservation conditions, expiry date etc. Finally, the bones were packaged and vacuum-sealed in a third layer of polyethylene. The bones were then placed in an insulated cool box and the cool box was kept in deep freezer (–80ºC) for at least overnight. For sterilization at the dose of 25 kGy, the cool box with the frozen bones was transferred to the gamma irradiator. After irradiation, the bones were preserved at –80ºC and supplied to hospitals/clinics after sterility testing. During the period, 10 massive bones were processed for clinical use in limb salvage surgery.

To provide high quality allografts, tissue procurement, processing, abelling, quality control practices were documented and were performed in accordance with approved standard operating procedure and instructions. After sterilization of tissue allografts, gamma irradiated tissues from every donor were checked for the presence of viable microorganisms. For this purpose, small bone pieces from each donor were gamma sterilized under similar conditions and was inoculated in test tubes containing sterile media viz., nutrient broth, brain-heart infusion broth, thioglycollate broth, and Sabouraud Dextrose broth. The tubes were observed up to 14 days for any type of microbial growth. After comparing with the control tubes, if no contamination found, then the particular batch was released for the dispatch to the hospitals. During the period, 735 amnion samples from 52 batches and 512 bone samples from 27 batches were tested. All processed samples were found microbiologically safe.

Cultured keratinocytes grafting has become an alternative therapeutic approach for the treatment of acute cutaneous wounds that are unresponsive to traditional treatments. Keratinocytes were cultured from human skin by traditional feeder-dependent method and explants outgrowth technique using serum containing media. A comparative study was performed between these two methods and effect of different parameters (donor age, sample storage time, attachment factor, and serum concentration) on keratinocyte culture was also examined. In feeder-dependent method, keratinocytes were isolated by cold trypsinization technique and were cultured on mitotically inactivated mouse embryonic fibroblasts. Highest Keratinocytes count (1.6×10⁶ cells/ml) and percentage of cell viability (93%) was obtained when cells were isolated after 24 hours of skin collection from youngest (3 years old) donor. In explants culture, cell proliferation rate was higher in 20% serum containing medium in comparison with 10% serum. During this period, keratinocyte cells were cultured experimentally from thigh skin of two donors (age: 3 and 10), two foreskin samples (age: 5 and 7 years) and abdomen skin of one 29 years old donor.

In recent years, the demand of bone allografts has increased drastically. But the supply of allografts is very limited. In this context, TBBRU is trying to introduce the use of xenograft in reconstructive surgery. Therefore, a research work has been initiated to determine the microbiological quality, biochemical composition, bone mineral density, and mechanical competence of calf and human bone samples. During the period, research work on microbiological aspects and bone mineral contents of the bone tissues were performed. Initial bioburden level of calf bones was 3.10×10³ to 2.95×10⁵ cfu/g and it was 3.10×10² to 3.20×10⁵ cfu/g in human bone samples. D₁₀ values of the isolates ranged from 0.32 to 2.72 kGy and 0.70 to 1.49 kGy in calf and human bones respectively. 8 kGy gamma radiation was found effective to eliminate all microorganisms of human and calf origin. The concentrations of Zinc (Zn) and Sodium (Na) were slightly higher in human bone in comparison with calf bone. Other bone minerals determination is in progress.

Researchers of TBBRU are working on the development of new biomaterial and allograft substitutes to mitigate the huge demand of graft materials. To develop biocompatible scaffold for bone tissue engineering aiming to use as an alternative to bone allografts in the treatment of skeletal defects, systematic studies on this research programme was undertaken including: (i) extraction of hydroxyapatite from available biological waste materials (e.g. bovine cortical bone, egg shells etc.), (ii) extraction of chitosan from shrimp shell, (iii) isolation and purification of collagen from natural sources (e.g. bovine tendon), (iv) fabrication of biocompatible composite scaffold using gamma radiation, (v) determinnation of the effects of the scaffold on osteogenic cell attachment and proliferation, ( vi) evaluation of the potency of the new biomaterial as an alternative to bone allografts alone or in combination with bone morphogenic protein (BMP). During the reporting period, Hydroxyapatite was extracted from bovine cortical bone by both low and high temperature sintering and also synthesized from eggshell by hydrothermal process and biomimetric synthesis. For characterization of the as-prepared hydroxyapatite, quantitative elemental analysis, phase identification, particle size determination and microstructural analysis are in progress.

Tissue transplant surgeries improve life quality, even save life of thousands of people. But these miraculous surgeries are possible only because of the generosity of donor families and the commitment of people who make the decision to donate. In our country, the majority people are familiar with the concept of blood transfusion and organ transplantation but the concept of tissue transplantation is relatively new to them. To make the concept familiar and to inspire tissue donation, seminars were arranged at different hospitals. Beside these, discussion meeting with physicians were arranged regularly to increase professional awareness.

During this period, TBBR Unit has organized 05 seminars in Chittagong Medical College Hospital, Comilla Medical College Hospital, Mymensingh Medical College Hospital, Sylhet Medical College Hospital and Bangladesh Atomic Energy Commission, Agargaon, Dhaka for the promotion of public & professional awareness to increase tissue procurement and clinical use of radiation sterilized tissue allograft in reconstructive surgery.

9. Veterinary Drug Residue Analysis DIVISION, AERE

9.1 Research and Development Work

Veterinary Drug Residue Analysis Laboratory (VDRAL) is a newly established division of IFRB. VDRAL deals with Development and validation of screening/analytical methods for detecting veterinary drug residues in foods of animal origin. The specific aims of this division is to monitor residues of veterinary drugs in foods of animal origin to protect public health and enhance international trade through utilization of nuclear and allied analytical methods.

The aim of the present study was to compare the efficiency of several SPE cartridges for tetracycline determination from animal tissue by ELISA and to develop optimal experimental conditions that can later be applied for screening and the quantification of the veterinary drug residue analysis. Solid Phase Extraction is an important step of sample preparation for the screening method of tetracyclines in animal tissues by ELISA. Efficiency of four different cartridges were compared. The SPE cartridges tested were r-biopharm No. R2002 (RB), Chromabond C18ec (3ml/200mg) (CB), Biotage Isolute C18(ec) (6ml/1g) (IS) and Mega Bond ELUTE C18 (6ml/1g) (MB). In the search of the best recovery and clean-up, extraction conditions and solvents were same for all four (SPE) cartridges tested. There were no significant differences in the performance (evaluated as % binding of SPE cartridges for determination of tetracyclines by ELISA.

An appropriate confirmatory (HPLC) method for the determination of tetracyclines analytes (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) using HPLC (Agilent 1260 infinity series) were developed and the following parameters were measured.

(i) Standard solutions of the analytes (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) were prepared. A standard curve was prepared using the concentration of mixed standard solution of the analytes for the determination of concentration of those analytes present in an unknown sample.

(ii) Optimized chromatography (HPLC) and Diode Array Detector (flow of mobile phases, gradient, injection volume, column temperature, range of UV spectra).

(iii) Reproducibility of retention times and peak areas were checked.

10. NUCLEAR Medicine

10.1 NATIONAL INSTITUTE OF NUCLEAR MEDICINE & ALLIED SCIENCES

NINMAS (former INMU) is the apex Nuclear Medicine facility in Bangladesh and it is located in Bangabandhu Sheikh Mujib Medical University (BSMMU) premise. The Institute provides medical imaging using nuclear techniques and therapeutic treatment services.