scholarly journals Evaluación de un medio de cultivo para el aislamiento selectivo de campylobacters termotolerantes

2018 ◽  
pp. 26-30
Keyword(s):  
Campylobacter Jejuni ◽  
Culture Medium ◽  
Culture Media ◽  
Campylobacter Coli ◽  
Selective Isolation ◽  
Basic Fuchsin ◽  
Average Growth ◽  
San Lorenzo ◽  
Significant Difference ◽  
Microbial Agents

Evaluación de un medio de cultivo para el aislamiento selectivo de campylobacters termotolerantes Evaluation of a culture medium for the selective isolation of thermotolerant campylobacter Álvaro Tresierra-Ayala, Juan Huanaquiri, Ramsés Perea, María Bendayán. Laboratorio de Microbiología. Centro de Investigación de Recursos Naturales, Universidad Nacional de la Amazonía Peruana. Psje. Los Paujiles S/N, San Lorenzo, distrito de San Juan Bautista, Iquitos-Perú. DOI: https://doi.org/10.33017/RevECIPeru2014.0004/ Resumen Se evaluó la capacidad de aislamiento selectivo de un medio de cultivo para campylobacters termotolerantes, el cual fue suplementado con sangre de porcino y agentes restrictivos (sales biliares, tioglicolato de sodio, fucsina básica), en lugar de la sangre de ovino o equino y diversos antibióticos que suelen ser componentes de los medios comúnmente empleados. Se logró aislar e identificar 20 cepas (13 de Campylobacter jejuni y 7 de Campylobacter coli) de un total de 70 muestras, determinándose que no había diferencia estadísticamente significativa respecto a la capacidad de aislamiento entre el medio de cultivo evaluado y el medio Skirrow modificado. Para el análisis comparativo de la cuantificación del crecimiento de las cepas en ambos medios, se empleó la técnica de Miles y Misra modificada [1] y se observó que las cepas no mostraban diferencia estadísticamente significativa en cuanto a su crecimiento en los medios ensayados, obteniéndose recuentos promedios de 23,4 x106 y 21,7x106 UFC/ml, para la especie de C. coli en el medio de cultivo evaluado y el medio Skirrow modificado y de 44,6 x106 y 40,1 x106 UFC/ml para la especie C. jejuni en ambos  medios de cultivo, respectivamente.  El medio de cultivo evaluado permite la misma capacidad de aislamiento selectivo para campylobacters termotolerantes que el medio Skirrow modificado; además, estos agentes microbianos crecen cuantitativamente de modo similar en ambos medios de cultivo. Descriptores: Campylobacter, medio de cultivo, sangre de porcino, sales y colorantes. Abstract The ability of selective isolation of a culture medium for thermotolerant campylobacters, supplemented with pig blood and restrictive agents (bile salts, sodium thioglycollate, basic fuchsin), instead of the blood of sheep or horses and various antibiotics which are often components of the means commonly employed was evaluated. Twenty strains (13 C. coli y 7 C. jejuni) were isolated and identified from a total of 70 samples There was not significant difference with respect to the isolation ability between the culture medium evaluated and the modified Skirrow medium. For the comparative analysis about quantification of the growth of strains in both media, Miles and Misra technique, modified [1], was employed. So, it was determined that the strains showed no significant difference with respect to their growth on both media studied. Average growth of 23,4 x 106 and 21,7 x 106 CFU/ml for C. coli in culture medium evaluated and the modified Skirrow medium, and 44,6 x 106 and 40,1 x 106 CFU/ml for  C. jejuni in both culture media, respectively, were detected. Culture medium evaluated allows the same ability selective isolation of thermotolerant campylobacter than modified Skirrow medium; moreover, these microbial agents grow similarly quantitatively in both culture media. Keywords: Campylobacter, culture medium, blood pig, salts and dyes.

160 COMPARATIVE STUDY OF IN VITRO CULTURE MEDIA AND ASSISTED HATCHING TECHNIQUES ON MOUSE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 188
Author(s):  
N. C. Negota ◽  
L. P. Nethenzheni ◽  
M. L. Mphaphathi ◽  
D. M. Barry ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Culture ◽  
Chorionic Gonadotropin ◽  
Culture Medium ◽  
Culture Media ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin ◽  
F1 Generation

The in vitro culture media and assisted hatching techniques remain challenging obstacles to be utilised widely. Mechanical, chemical, enzymatic thinning, and laser-assisted techniques have been used previously but information is still lacking on its application in livestock. The aim of this study was to compare the effect of 2 in vitro culture media (Hamster F10 and TMC-199) and 4 (mechanical, chemical, enzymatic, and laser) assisted hatching techniques on blastocyst formation and hatching rate using murine embryos as a model. The C57/b and Balb/c breeds were raised until they reached maturity and bred naturally to produce F1 generation. The light in the breeding house was controlled at 14 h light and 10 h dark. Feed and water were provided ad libitum for the mice. Superovulation of females were stimulated using equine chorionic gonadotropin and human chorionic gonadotropin. The F1 generation was used for the collection of the 400 blastocysts and randomly allocated into 4 assisted hatching techniques. Blastocysts were paired into a group of 10 and replicated 4 times for each assisted hatching technique. The general linear model of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyse the data. Assisted hatching techniques of laser, mechanical, enzymatic, and chemical yielded 46.9 ± 37.1, 51.1 ± 40.2, 39.1 ± 35.8, and 33.3 ± 4.5%, respectively, under in vitro culture of Hamster F10. The TCM-199, laser, mechanical, enzymatic, and chemical assisted hatching techniques yielded 56.3 ± 43.3, 52.6 ± 35.5, 49.2 ± 37.5, and 33.9 ± 35.5%, respectively, with a significant difference. There was no significant difference observed in assisted hatching techniques and Hamster F10 culture medium. However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM than cultured in Hamster F10. Hatching rate of blastocysts increased from chemical, enzymatic, mechanical, and laser with response to Hamster F10 and TCM; thus, laser is a suitable assisted hatching technique with TCM-199.

Development of a Novel Chromogenic Medium for Improved Campylobacter Detection from Poultry Samples

2015 ◽  
Vol 78 (9) ◽  
pp. 1750-1755 ◽  
Author(s):  
HAJIME TERAMURA ◽  
MIHOKO IWASAKI ◽  
HIROKAZU OGIHARA
Keyword(s):  
Escherichia Coli ◽  
Campylobacter Jejuni ◽  
Campylobacter Coli ◽  
Chromogenic Medium ◽  
Enrichment Broth ◽  
Selective Media ◽  
E Coli ◽  
Significant Difference

The presence of expanded-spectrum β-lactamase (ESBL)–producing Escherichia coli is a common problem in the isolation of Campylobacter from poultry samples using conventional cefoperazone-based selective media. A novel chromogenic medium (CM-HT), based on modified charcoal cefoperazone deoxycholate agar (mCCDA), has been developed as a solution for improved Campylobacter detection from poultry samples. Although the basic components of CM-HT are the same as mCCDA, CM-HT uses both granular charcoal and sodium cefoxitin to enhance viewability and inhibit ESBL-producing bacteria. All tested Campylobacter jejuni (n = 31) and Campylobacter coli (n = 6) strains grew and formed purple-colored colonies on CM-HT. In contrast, the growth of all other tested microorganisms, including ESBL-producing E. coli strains, was suppressed by this medium. Additionally, 84 poultry samples were examined for the presence of Campylobacter using the ISO 10272-1 method (enrichment with Bolton broth) and the NIHSJ-02 method (enrichment with Preston broth) with mCCDA and CM-HT media for the isolation. The numbers of samples from which Camplylobacter was detected on CM-HT using Preston and Bolton broth were 22 and 18, whereas the numbers on mCCDA were 22 and 13, respectively. Only Campylobacter was detected on CM-HT using both enrichment broths; however, there were 5 and 19 samples from which ESBL-producing E. coli was detected on mCCDA using Preston and Bolton broth, respectively. Thus, there was a significant difference between CM-HT and mCCDA in selectivity for ESBL-producing E. coli regardless of which enrichment broth was used. The results obtained demonstrated that CM-HT is a possible solution for the improved isolation of Campylobacter from poultry samples.

scholarly journals Prevalence of Campylobacter among goats and retail goat meat in Congo

2014 ◽  
Vol 8 (02) ◽  
pp. 168-175 ◽  
Author(s):  
Rosette Kabwang A Mpalang ◽  
Raphaël Boreux ◽  
Pierrette Melin ◽  
Khang'Mate Akir Ni Bitiang ◽  
Georges Daube ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Democratic Republic Of Congo ◽  
Campylobacter Coli ◽  
Sample Collection ◽  
Goat Meat ◽  
Faecal Samples ◽  
Significant Difference ◽  
Meat Goat ◽  
Open Air Markets ◽  
Campylobacter Spp

Background: The prevalence of Campylobacter jejuni and Campylobacter coli was determined in goat and goat meat sold at retail outlets in Lubumbashi, Democratic Republic of Congo (DR Congo). Methodology: A total of 644 samples, including 177 goat meat, 86 goat stomachs, 139 ready to eat (RTE) goat skewers, and 242 goat faecal samples were examined for the presence of Campylobacter jejuni and Campylobacter coli using polymerase chain reaction. Results: Overall, Campylobacter spp. were found in 34.6% of the examined samples. C. jejuni was isolated in 10.1% and C. coli in 26.7% of samples. Only 2.2% of all samples were positive for both species. There was a significant association between the prevalence of C. coli and the type of sample (p < 0.05). The overall prevalence of Campylobacter in different sample groups was 41.2%, 37.2%, 23.7%, and 35.1% for goat meat, goat stomachs, RTE goat skewers, and goat faecal samples, respectively. There was no significant difference (p > 0.05) between the prevalence observed in the rainy season (16.7%) and the dry season (20.0%). Moreover, the overall prevalence of Campylobacter in slaughter sites, open-air markets, warehouses, and semi-open-air markets was 28.2%, 34.2%, 35.4%, and 42.9%, respectively. Statistically, there was no influence of the sample collection site on the frequency of isolation of Campylobacter (p > 0.05). Conclusion: This study shows that, considering the relatively high prevalence of this pathogen, live goat and goat meat are major sources of human and environmental contamination by Campylobacter spp. in Lubumbashi.

INFLUENCE OF CULTURE MEDIUM ON THE SPORULATION AND VIABILITY OF ASPERGILLUS SPP. AND TALAROMYCES SPP. ENTOMOPATHOGENIC FUNGI

2018 ◽  
Vol 18 (1) ◽  
pp. 12
Author(s):  
Yuyun Fitriana ◽  
Radix Suharjo ◽  
I Gede Swibawa ◽  
Purnomo . ◽  
Puji Lestari ◽  
...  
Keyword(s):  
Entomopathogenic Fungi ◽  
Culture Medium ◽  
Culture Media ◽  
Spore Production ◽  
Corn Meal ◽  
Spore Viability ◽  
Randomized Design ◽  
Honestly Significant Difference ◽  
Significant Difference ◽  
Completely Randomized Design

Influence of Culture Medium on the Sporulation and Viability of Aspergillus spp. and Talaromyces spp. Entomopathogenic Fungi. The purpose of this study was to determine the effect of three kinds of cultures media on the spore production and viability of Aspergillus spp. (AS1, 6, 7, 9) and Talaromyces spp. (AS2–5, 8, 10) entomopathogenic fungi. This study was arranged using Factorial-Completely Randomized Design (CRD) with 2 factors and 3 replications. The first factor was three kinds of cultures media (potato dextrose agar (PDA), corn meal agar (CMA), and sabouraud dextrose agar (SDA)) and the second one was isolates of Aspergillus spp. Or Talaromyces spp.. Data of spore production and spore viability were tested using ANOVA and if there was significantly difference, the data then further analyzed using Tukey‘s Honestly Significant Difference (HSD) test at 5% of significant level. The spore production of Aspergillus spp. were in the range of 0.58 - 14.27 x 108 spores mL-1 (PDA); 0.28 – 2.68 x 108 spores mL-1 (SDA) and 1.85 - 5.33 x 108 spores mL-1 (CMA). The highest spore production was achieved by AS1 isolate that was grown on PDA media. The spore produced by Talaromyces spp. were in the range of 2.15 – 28.62 x108 spores mL-1 (PDA); 0.28 – 29.43 x108 spores mL-1 (SDA); and 1.88 – 16.63 x108 spores mL-1 (CMA). The highest spore production was produced by AS8 isolate which were cultured on PDA. The spore viability among isolates of the two entomopathogenic fungi were not significantly different. The spore viability of Aspergillus spp. was in the range of 95.10 – 97.66% (PDA), 94.02 – 98.45% (SDA) and 92.86 – 98.20% (CMA). The spore viability of Talaromyces spp. was in the range of 95.83 – 100% (PDA), 85.83 – 100% (SDA), and 90.75 – 100% (CMA). Culture medium influenced spore production but not the spore viability. The best culture media used for spore production of both of the entomopathogenic fungi was PDA media.

Optimization of Culture Media and ITS Identification of Tricholoma matsutake

2013 ◽  
Vol 634-638 ◽  
pp. 1574-1580
Author(s):  
Ji Zhong Wang ◽  
Zhong Hai Jiang ◽  
Shang Yong Zheng
Keyword(s):  
Growth Rate ◽  
Culture Medium ◽  
Culture Media ◽  
Fruit Body ◽  
Tricholoma Matsutake ◽  
Its Sequence ◽  
Average Growth Rate ◽  
Average Growth ◽  
Rdna Its ◽  
Rdna Its Sequences

Aims: To optimize the culture media of 0288 Tricholoma matsutake and to identify it according to its rDNA ITS sequence. Methods: Fruit body, collected from Heilongjiang province, is used as the material, and the mycelium is isolated and radiated under ultraviolet. Then the radiated mycelium is cultured in five different media and its morphology of fungus colony and growth rate are investigated. After that, the rDNA ITS sequences of 0288 and the natural fruit body of T. matsutake are analyzed and identified by PCR and sequencing. Results: The radiated mycelium grew faster in formula A culture medium than others and its average growth rate is up to 0.575mm/d. Results also showed that the homology of the rDNA ITS sequence is almost up to 99% between 0288 and the natural fruit body. Through this experiment, the best growth condition of 0288 is obtained and its fruit body is induced as pure cultural product. It would lay strong foundation for the artificial domestication of the wild T. matsutake.

Prevalence and Diversity of Campylobacter jejuni in Pig Herds on Farms with and without Cattle or Poultry

2005 ◽  
Vol 68 (4) ◽  
pp. 722-727 ◽  
Author(s):  
J. BOES ◽  
L. NERSTING ◽  
E. M. NIELSEN ◽  
S. KRANKER ◽  
C. ENØE ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Mixed Infection ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Campylobacter Coli ◽  
Poultry Production ◽  
Pulsed Field ◽  
Follow Up Study ◽  
Significant Difference

The prevalence and diversity of Campylobacter jejuni was investigated in pig herds on farms with and without cattle or poultry production. A bacteriological screening of pig cecal samples from 247 finisher herds was carried out at the slaughterhouse. Subsequently, a follow-up study was conducted in 24 herds (either with or without prior C. jejuni isolation from pigs) in which fecal samples were collected from pigs and, if present, cattle and poultry. Samples were analyzed for presence of Campylobacter, and subsequent analysis included species identification, serotyping, and, for selected strains, pulsed-field gel electrophoresis typing. In the slaughterhouse screening, C. jejuni was isolated from pigs in 21 (8.5%) herds, but no significant difference in prevalence was found between herd types (pigs, pigs and cattle, pigs and poultry). At the slaughterhouse, C. jejuni and Campylobacter coli prevalence in pigs was 2.3 and 90.1%, respectively. In the follow-up study, herd prevalence of C. jejuni was 8.3%, whereas C. jejuni and C. coli were isolated from 0.8 and 92.0% of pigs, respectively. In mixed production herds, C. jejuni predominated in cattle (42.7%) and poultry (31.6%), whereas C. jejuni was only isolated from 1.3 to 2.5% of pigs in these herds. There were no significant differences in C. jejuni or C. coli prevalence in pigs, cattle, and poultry between herds with and without prior C. jejuni isolation at the slaughterhouse. Pulsed-field gel electrophoresis typing did not yield evidence of C. jejuni transmission between cattle or poultry and pigs in mixed production herds. In contrast, pulsed-field gel electrophoresis analysis showed indistinguishable serotypes of C. coli in pigs and cattle in two herds. Verification of C. jejuni–positive pig samples showed that individual pigs can excrete high levels of C. jejuni and that mixed infection with C. jejuni and C. coli was common in C. jejuni–positive pigs. The results of our study suggest that transmission of C. jejuni between pigs and cattle or poultry in mixed production herds occurs infrequently. Detection of indistinguishable C. coli isolates in two herds, however, might indicate the existence of low-level transmission between pigs and cattle in herds of mixed production.

Effect of iron concentration on toxin production in Campylobacter jejuni and Campylobacter coli

1986 ◽  
Vol 32 (5) ◽  
pp. 395-401 ◽  
Author(s):  
Barbara A. McCardell ◽  
Joseph M. Madden ◽  
John T. Stanfield
Keyword(s):  
Campylobacter Jejuni ◽  
Ferrous Sulfate ◽  
Culture Media ◽  
Iron Concentration ◽  
Toxin Production ◽  
Enzyme Linked Immunosorbent Assay ◽  
Campylobacter Coli ◽  
Casamino Acids ◽  
Culture Supernatants ◽  
Campylobacter Spp

The effect of iron concentrations in culture media on supernatant yields of Campylobacter cytotonic toxin (CCT) was studied. Of the 118 Campylobacter spp. strains surveyed, 78.8% produced toxin in brucella broth or in casamino acids – yeast extract (CYE) broth. When the iron concentration of CYE was increased from 0.44 μg/mL (7.9 μM) to 0.65 μg/mL (11.6 μM) by the addition of ferric chloride, 94.9% of the strains were positive for toxin in a ganglioside GM1 based, enzyme-linked immunosorbent assay, using antibody to affinity-purified CCT. The addition of iron as ferrous sulfate was less effective. When four toxin-positive strains were grown in a deferrated medium of conalbumin-treated CYE with 0.04–0.08 μg iron/mL (0.72–1.43 μM), two of the culture supernatants became negative (absorbance at 410 nm, < 0.1 and < 10 ng CCT/mL), and two produced about 90% less CCT but were still classified as positive (absorbance, ≥ 0.1 and ≥ 10 ng CCT/mL). It was therefore concluded that the production of CCT by Campylobacter spp. is influenced by iron concentration.

scholarly journals Aquaculture biological waste as culture medium to cultivation of Ankistrodesmus gracilis (Reinsch) Korshikov

2017 ◽  
Vol 78 (3) ◽  
pp. 579-587 ◽  
Author(s):  
L. H. Sipaúba-Tavares ◽  
T. Florêncio ◽  
B. Scardoeli-Truzzi
Keyword(s):  
Chlorophyll A ◽  
Cell Density ◽  
Total Phosphorus ◽  
Biochemical Composition ◽  
Eichhornia Crassipes ◽  
Culture Medium ◽  
Culture Media ◽  
Conventional Culture ◽  
Significant Difference ◽  
Biological Wastes

Abstract Current study investigated the effectiveness of different macrophytes as culture media for Ankistrodesmus gracilis in laboratory conditions. Significant difference (p < 0.05) was reported in cell density with regard to conventional culture medium (CHU12) and macrophytes culture media. Mean cell density in NPK, Eichhornia crassipes and E. azurea media was higher (p < 0.05) than in conventional culture medium. Chlorophyll-a was higher than 1 g.L-1, except in CHU12 (0.7 ± 0.4 g.L-1) and T. domingensis (0.8 ± 0.3 g.L-1) media. Nitrate decreased sharply as from the 7th-day of the experiment. Ammonium and total phosphorus were highest in culture media and ranged between 0.4 g.L-1 (P. cordata medium) and 1.7 g.L-1 (CHU12 medium) for ammonium, and between 0.8 g.L-1 (CHU12 medium) and 1.9 g.L-1 (T. domingensis medium) for total phosphorus. Results revealed inorganic fertilizer and macrophytes combined with vitamins may be effective as culture media and strongly supports the growth of Ankistrodesmus gracilis, since cell density and biochemical composition are similar to or higher than conventional culture medium (CHU12). Macrophyte is a tool for aquaculture since biological wastes may be used with nutrients to improve the cultivation of microalgae.

scholarly journals Evaluation of Culture Media for Selective Enrichment and Isolation of Salmonella in Seafood

2010 ◽  
Vol 93 (5) ◽  
pp. 1468-1471 ◽  
Author(s):  
Rakesh Kumar ◽  
Poothuvallil K Surendran ◽  
Nirmala Thampuran
Keyword(s):  
Brilliant Green ◽  
Culture Media ◽  
Performance Difference ◽  
Selective Isolation ◽  
Salmonella Spp ◽  
Selective Enrichment ◽  
Significant Difference ◽  
Agar Media ◽  
Isolation Media

Abstract Seafood, including fish, shrimp, clam, crab, mussel, oyster, lobster, squid, octopus, and cuttlefish samples, was used to compare the recovery of Salmonella serovars by different selective enrichment and isolation media. The samples were selectively enriched in Rappaport-Vassiliadis (RV) broth and tetrathionate broth (TT), followed by selective isolation on Hektoen enteric (HE) agar, xylose lysine desoxycholate (XLD) agar, bismuth sulfite (BS) agar, and Brilliant Green (BG) agar media. Of 443 seafood samples analyzed, 108 were found to be contaminated with Salmonella. The role of selective enrichment in Salmonella spp. recovery with RV medium was distinctly high (70%) compared to TT broth (30%). The selective enrichment in RV broth followed by selective isolation on XLD, HE, BS, and BG agar recovered Salmonella at levels of 56, 41, 28, and 16%, respectively. Similarly, after enrichment in TT broth, XLD and HE agars recovered 27 and 23 respectively. The recovery of Salmonella with enrichment in TT followed by isolation on BS and BG was abysmally low at 4.6 and 5, respectively. There was no significant difference (P &gt; 0.05) in the recovery of Salmonella using the combinations of XLD and HE media with selective enrichment in RV broth. However, performance difference (P &lt;0.05) was observed in the recovery when BS and BG with RV, and XLD, HE, BS, and BG agars with TT broth were used. The present study showed that the combination of RV with XLD was the most efficient media for isolation of Salmonella from seafood when compared to other isolation media combinations.

P–115 Supplementation of healthy Sertoli cells into culture media containing follicle stimulating hormone (FSH)/testosterone (T) has no advantage in germ cell maturation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Aydos ◽  
Y Yukselten ◽  
T Ozkan ◽  
S Ozkavukcu ◽  
M Erdogan ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Annexin V ◽  
Hormonal Control ◽  
Obstructive Azoospermia ◽  
Inadequate Response ◽  
Tissue Samples ◽  
Significant Difference ◽  
Protamine 2

Abstract Study question In nonobstructive azoospermia (NOA) cases, whether supplementation of healthy Sertoli cells (SCs) has an effect on spermatogenic differentiation in culture medium containing FSH/T. Summary answer Expression of Crem and Acrosin increased significantly in both medium with FSH/T and medium with additional healthy SCs but there was no difference between them What is known already In NOA the induction of spermatogonial stem cells (SSCs) proliferation and differentiation has been demonstrated using different culture systems. SCs have vital roles in the regulation of spermatogenesis. Hormonal control of spermatogenesis is through FSH and T activity on SCs. Growth factors secreted by SCs via FSH, stimulate proliferation and colonization of SSCs. Although germ cells do not express androgen receptors, FSH receptors are localized on spermatogonia. It is not clear whether native SCs are sufficient for FSH/T added to the culture medium to be effective in induction of spermatogenesis, and whether supplementation of healthy SCs will increase this activity. Study design, size, duration 34 NOA and 12 obstructive azoospermia (OA) cases were included. Testicular tissue samples were taken with testicular sperm extraction (TESE) in the study and control groups. In a group of fertile cases, healthy Sertoli cells were identified and purified and then cryopreserved. Tissue samples of each case prepared in standard DMEM/F12 medium were processed in 2 separate environments containing FSH/T and FSH/T plus thawed healthy SCs for 7 days. Participants/materials, setting, methods The characterization of healthy SCs isolated from fertile cases was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. FC was used to measure the 7th day levels of specific markers expressed in spermatogonia (Vasa), meiotic cells (Crem) and post-meiotic cells (Protamine–2 and Acrosin). Main results and the role of chance In Annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT exhibited that cryopreservation didn’t inhibite the SCs proliferation compared to the pre-freezing state. Vasa and Acrosin basal levels were found to be lower in infertile patients compared to the control group (8.2% vs. 30.6% and 12.8% vs. 30.5%, p &lt; 0.05). Compared to day 0 measurements, on the 7th day in FSH/T environment, Crem level increased by 58.8% and Acrosin level increased by 195.5% (p &lt; 0.05). Similarly, in medium supplemented with healthy SCs, by day 7, the Crem and Acrosin levels were increased to 92.2% and 204.8%, respectively (p &lt; 0.05). Although Vasa and Protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the 7th day increase rates of Crem, Vasa, Acrosin and Protamine–2 in either FSH/T-containing medium or in medium additionally supplemented with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8% and 232.3% vs. 198.4%, respectively, p &gt; 0.05). Our results suggest that freezing-thawing process would not impair the viability and proliferation of SCs, and adding healthy SCs to the culture medium to correct impaired gene expression does not have an advantage over FSH/T. Limitations, reasons for caution The 7-day culture period we determined might be not sufficient for spermatogenic differentiation completion. This period could be extended in order to see further morphological differentiation may need. Wider implications of the findings: The failure of the culture media containing FSH/T to show the expected effectiveness could be thought to be due to the SCs’ inadequate response to these hormones. Therefore, healthy SCs supplementation would be needed, but this could pose ethical issues. Our findings show that it is not necessary. Trial registration number 214S532

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