Newborn calf serum supplemented by tellurite as alternative transport medium for Corynebacterium diphtheriae

Indonesia is one of the five countries with highest diphtheria cases in the world. Laboratory confirmation by culture method as a gold standard requires bacterial survival. Indonesia’s geographical condition as an archipelagic country and difficulties in transporting clinical samples are often obstacles in maintaining bacterial survival. This study aims to evaluate the ability of several transport mediums to maintain the survival of Corynebacterium diphtheriae. A total of 90 isolates were divided into nine groups of transport mediums. Samples were divided into 2 treatment groups, namely room temperature and temperature 2-8 ºC. On day 2, 4, 8, 16, and 32, 1 isolate from each group with 2 different incubation temperatures was cultured on blood agar medium and incubated for 24 hours at 37 ºC. Bacterial survival was indicated by the growth of suspect colonies which were identified by microscopic and biochemical tests. Results show serum with tellurite can be used with viability lower than silica gel, but higher than other media. Meanwhile at a temperature of 2-8 ºC, there are 2 types of the best transport medium, namely serum with tellurite and open silica gel in aluminum foil. Newborn Calf Serum supplemented with Tellurite can be used as an alternative transport medium for Corynebacterium diphtheriae, both at room temperature and at 2-8 ºC.

Naringenin and ellagic acid reduce tetrazolium salt in the absence of cells

Background: Plant derived compounds, naringenin and ellagic acid might interfere in MTT (3-(4, 5- Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) assay in the form of reducing the tetrazolium salt in the absence of cells. Objective: Effect of naringenin and ellagic acid in MTT assay. Methods: MTT assay has been performed at different conditions like, varying the concentration of phytocompounds, incubation time, and changing the media. Results: Ellagic acid at the concentration of 80 μg/ml showed maximum absorbance of 0.579 in the presence of RPMI media (Roswell Park Memorial Institute Medium) that has 10 % newborn calf serum. Naringenin showed higher absorbance of 2.74 at 80 μg/ml in RPMI media that has 10 % newborn calf serum. Naringenin and Ellagic acid had better ability to reduce the MTT in the presence of RPMI media than anhydrous ethanol. Conclusion: Naringenin reduced the MTT better than ellagic acid in RPMI-1640 medium. These results show the need to be cautious of choosing MTT assay to measure the metabolic activity of cells treated with phytocompounds.

138 Effects of different treatments of donors on the efficiency of embryo production and conception in bovine ovum pickup-in vitro production

An in vitro-produced bovine embryo has a low conception rate compared with that of an in vivo embryo. The present study was conducted to examine the effects of different treatments delivered to donors before ovum pickup (OPU) sessions to improve the conception rate of in vitro-produced bovine embryos. In total, 351 OPU sessions were performed on 138 Holstein and 213 Japanese Black cows from January to December 2017. Donors were divided into 4 groups based on their pretreatment before OPU: (1) single injection of 2.5 AU of FSH 40h before OPU; (2) CIDR insertion on Day 0, injection of 2mg of oestradiol benzoate on Day 1, 4 injections of FSH (each 2.5 AU) every 12h beginning from Day 5 to 7, followed by removal of CIDR and OPU on Day 9; (3) injection of 50μg of gonadotropin-releasing hormone 72h before OPU; or (4) no pretreatment. The collected cumulus-oocyte complexes were matured for 22h in 25mM of HEPES buffered TCM-199 supplemented with 5% newborn calf serum and 0.02 AU mL−1 FSH. After 6h of gamete co-culture (5.0×106 sperm mL−1), the presumptive zygotes were washed and the remaining cumulus cells were denuded by pipetting. The presumptive zygotes were then cultured in KSOMaa supplemented with 5% newborn calf serum for 9 days in a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). Blastocyst formation rates were analysed 9 days after insemination, and the formed blastocysts were transferred to oestrous synchronized recipients on the seventh or eighth day after oestrus. The data were analysed by Chi-squared test with Yates correction. The average numbers of collected oocytes were 57.7±17.4 (n=136), 25.3±12.8 (n=20), 28.8±12.5 (n=18) and 24.3±12.9 (n=177) in groups 1 to 4, respectively. Groups 1 and 2 showed significantly (P<0.01) high percentages of Grade-1 oocytes (52.1 and 49.6%, respectively) compared with groups 3 and 4 (37.3 and 39.9%, respectively). The proportion of blastocysts in groups 1 (38.6%) was significantly different compared with that in groups 2 (32.1%) and 4 (35.3%), but the difference was insignificant in the case of group 3 (36.4%). The conception rates in groups 1 (43.5%, n=868) and 2 (59.1%, n=44) were significantly (P<0.05) higher than those in groups 3 (35.1%, n=57) and 4 (34.9%, n=768). These results suggest that although the efficiency of embryo production did not differ largely between donors pretreated with 4 FSH injections and those without any pretreatment, the conception rate in donors pretreated with 4 FSH injections was significantly higher than that in donors without pretreatment. Moreover, donors pretreated with a single injection of FSH showed significantly high efficiency of embryo production and conception rate than donors without pretreatment.

Transdifferentiation Potency of Fibroblast Cell to Neuron Cell in Vitro

The aim of this study was to examine the potency of fibroblast cells transdifferentiated to neuron cells in vitro. Newborn rat neuron conditioned medium (NBRN CM) was collected from neuron cells cultured with mDMEM without serum for 48 hours. Fibroblast cells werecollected from fetal rat muscle treated with trypsin. Fibroblast cells were culture with 3 kind of culture medium: mDMEM + 0.01 mM β-mercaptoethanol; mDMEM + 50% NBRN-CM and mDMEM + 0.01 mM β-mercaptoethanol + 50% NBRN CM . As control, cells was culturedwith mDMEM +10% newborn calf serum (NBCS). The addition of NBRN CM into culture medium resulted in 12.97% newborn cells in fibroblastculture medium passage I. Newborn rat neuron conditioned medium in fibroblast culture medium resulted 12.97% neuron cells at passage 1. Thepercentage was increased (14.60%) when β- mercaptoethanol added into medium. The same result was found at passage 3 (12.67%; 13.17%). Itshowed that fibroblast cells has potency to transdifferentiated into neuron cells when cultured with NBRN CM. Further research is needed toknow the fibroblast transdifferentiation potency.Key words: fibroblast, transdifferentiation, conditioned medium, neuron cells, in vitro

Optimization of Conditions for In Vitro Culture of the Microphallid DigeneanGynaecotyla adunca

In vitro cultivation of digeneans would aid the development of effective treatments and studies of the biology of the parasites. The goal of this study was to optimize culture conditions for the trematode,Gynaecotyla adunca. Metacercariae of the parasite from fiddler crabs,Uca pugnax, excysted in trypsin, were incubated overnight to permit fertilization, and were cultured in different conditions to find those that resulted in maximum worm longevity and egg production. When cultured in media lacking serum, worms lived longer in Hanks balanced salt solution and Dulbecco’s Modified Eagle medium/F-12 (DME/F-12) than in RPMI-1640 but produced the most eggs in DME/F-12. Worm longevity and egg production increased when worms were grown in DME/F-12 supplemented with 20% chicken, horse, or newborn calf serum but the greatest number of eggs was deposited in cultures containing horse or chicken serum. Horse serum was chosen over chicken serum due to the formation of a precipitate in chicken serum. The optimal concentration of horse serum with respect to egg production ranged from 5 to 20%. Infectivity of eggs deposited by worms in culture was tested by feeding eggs to mud snails,Ilyanassa obsoleta. None of these snails producedG. aduncacercariae.

PROLIFERASI DAN DIFERENSIASI SEL TULANG TIKUS DALAM MEDIUM KULTUR IN VITRO YANG MENGANDUNG EKSTRAK BATANG Cissus quadrangula Salisb. (SIPATAH-PATAH)

Penelitian mengenai pengaruh ekstrak batang Cissus quadrangula Salisb. (sipatah-patah) terhadap tingkat proliferasi dan diferensiasi sel-sel tulang tikus (Sprague Dawley) prepuber umur empat minggu menggunakan sistem kultur in vitro. Sel-sel tulang dikultur dalam medium dulbecco’s modified eagle’s medium (DMEM) yang diberi tambahan newborn calf serum (NBCS) 10%, non essential amino acid (NEAA) 10%, NaHCO3, ITS 1 µl/ml (mengandung insulin 5 μg/ml, transferin 10 μg/ml, selenium 5 μg/ml; Sigma St Louis USA), dan 50 µg/ml gentamisin (mDMEM), dengan dan tanpa ekstrak Cissus quadrangula (CQ). Penelitian terdiri atas lima perlakuan yakni kontrol positif (mDMEM + deksametason 10-8 M), kontrol negatif (mDMEM), dan tiga konsentrasi CQ: mDMEM + CQ 0,3 mg/ml; mDMEM + CQ 0,6 mg/ml; dan mDMEM + CQ 1,2 mg/ml. Kultur dilakukan dalam inkubator CO2 5%, pada suhu 37° C selama tujuh hari. Parameter yang diamati adalah konsentrasi sel, komposisi, diameter osteoblas, dan diameter osteosit. Konsentrasi sel dihitung menggunakan hemositometer Newbauer. Komposisi osteoblas dan osteosit ditentukan berdasarkan pengamatan morfologi di bawah mikroskop cahaya. Diameter sel diukur menggunakan eyepiece micrometer. Data dianalisis menggunakan analisis varians dan uji Duncan. Hasil penelitian menunjukkan bahwa pemberian ekstrak Cissus quadrangula Salisb. pada konsentrasi 0,3 mg/ml; 0,6 mg/ml; dan 1,2 mg/ml secara signifikan dapat meningkatkan proliferasi sel tulang; dan pada konsentrasi 0,6 mg/ml mampu menginduksi diferensiasi osteoblas menjadi osteosit (P<0,05). Disimpulkan bahwa pemberian ekstrak Cissus quadrangula pada konsentrasi 0,6 mg/ml ke dalam medium kultur dapat meningkatkan proliferasi dan diferensiasi osteoblas.