The Limited Utility of Routine Culture in Pediatric Pilonidal, Gluteal, and Perianal Abscesses.

BACKGROUND: Pilonidal, buttock, and perianal abscesses are common reasons for surgical consultation in the pediatric emergency department. Treatment typically includes a bedside incision and drainage, often followed by an abscess culture, and a course of oral antibiotics. We aimed to study the impact of culture data on changes in management and clinical outcomes. We hypothesized that management is unaffected by culture data, and therefore, fluid culture from pilonidal, buttock, and perianal abscesses in the pediatric population may represent an unnecessary laboratory test and cost. MATERIALS AND METHODS: A single institution's electronic medical record was searched between February 1, 2013 and August 1, 2017, identifying 249 pediatric patients meeting the inclusion criteria: age 0 to 18 y; diagnosis of pilonidal, buttock, or perianal abscess; bedside incision and drainage. Patients were divided into two different comparison groups for data analysis based on the presence or absence of culture and recurrence or no recurrence. RESULTS: Culture results directly altered management in only 5 patient encounters (2.7% of all cultured). When comparing groups by culture or no culture, no statistically significant difference in recurrence rate (P = 0.4) was noted. When comparing groups by recurrence versus no recurrence, we found no statistically significant difference between sex, resident type, vessel loop use, packing use, or antibiotic use (P > 0.05). CONCLUSIONS: We conclude that microbiological culture results are of limited utility in the management of pediatric pilonidal, buttock, and perianal abscesses as they do not appear to alter treatment, and omission of culture is not associated with failure of surgical management.

Next-Generation Sequencing vs Culture-Based Methods for Diagnosing Periprosthetic Joint Infection After Total Knee Arthroplasty: A Cost-Effectiveness Analysis.

BACKGROUND: Periprosthetic joint infection (PJI) after total knee arthroplasty is challenging to diagnose. Compared with culture-based techniques, next-generation sequencing (NGS) is more sensitive for identifying organisms but is also less specific and more expensive. To date, there has been no study comparing the cost-effectiveness of these two methods to diagnose PJI after total knee arthroplasty. METHODS: A Markov, state-transition model projecting lifetime costs and quality-adjusted life years (QALYs) was constructed to determine the cost-effectiveness from a societal perspective. The primary outcome was incremental cost-effectiveness ratio, with a willingness-to-pay threshold of $100,000/QALY. Sensitivity analyses were performed to evaluate parameter assumptions. RESULTS: At our base case values, culture was not determined to be cost-effective compared to NGS, with an incremental cost-effectiveness ratio of $422,784 per QALY. One-way sensitivity analyses found NGS to be the cost-effective choice above a pretest probability of 45.5% for PJI. In addition, NGS was cost-effective if its sensitivity was greater than 70.0% and its specificity greater than 94.1%. Two-way sensitivity analyses revealed that the pretest probability and test performance parameters (sensitivity and specificity) were the largest factors for identifying whether a particular strategy was cost-effective. CONCLUSION: The results of our model suggest that the cost-effectiveness of NGS to diagnose PJI depends primarily on the pretest probability of PJI and the performance characteristics of the NGS technology. Our results are consistent with the idea that NGS should be reserved for clinical contexts with a high pretest probability of PJI. Further study is required to determine the indications and subgroups for which NGS offers clinical benefit.

Implementation of a systematic culturing program to monitor the efficacy of endoscope reprocessing: outcomes and costs.

BACKGROUND AND AIMS: In 2015, the U.S. Food and Drug Administration and Centers for Disease Control and Prevention (CDC) issued guidance for duodenoscope culturing and reprocessing in response to outbreaks of carbapenem-resistant Enterobacteriaceae (CRE) duodenoscope-related infections. Based on this guidance, we implemented best practices for reprocessing and developed a systematic process for culturing endoscopes with elevator levers. The aim of this study is to report the outcomes and direct costs of this program. METHODS: First, clinical microbiology data from 2011 to 2014 were reviewed retrospectively to assess for possible elevator lever-equipped endoscope-related CRE infections. Second, a program to systematically culture elevator lever-equipped endoscopes was implemented. Each week, about 25% of the inventory of elevator lever-equipped endoscopes is cultured based on the CDC guidelines. If any cultures return bacterial growth, the endoscope is quarantined pending repeat culturing. The costs of the program, including staff time and supplies, have been calculated. RESULTS: From 2011 to 2014, none of 17 patients with documented CRE infection had undergone ERCP or endoscopic ultrasound in the previous 36 months. From June 2015 to September 2016, 285 cultures were performed. Three (1.1%) had bacterial growth, 2 with skin contaminants and 1 with an oral contaminant. The associated endoscopes were quarantined and reprocessed, and repeat cultures were negative. The total estimated cost of our program for an inventory of 20 elevator lever-equipped endoscopes was $30,429.60 per year ($1521.48 per endoscope). CONCLUSIONS: This 16-month evaluation of a systematic endoscope culturing program identified a low rate of positive cultures after elevator lever endoscope reprocessing. All positive cultures were with non-enteric microorganisms. The program was of modest cost and identified reprocessing procedures that may have led to a low rate of positive cultures.

Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood.

We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.

Cost analysis in laccase production.

In this paper the cost of producing the enzyme laccase by the white-rot fungus Trametes pubescens under both submerged (SmF) and solid-state fermentation (SSF) conditions was studied. The fungus was cultured using more than 45 culture medium compositions. The cost of production was estimated by analyzing the cost of the culture medium, the cost of equipment and the operating costs. The cost of the culture medium represented, in all cases, the highest contribution to the total cost, while, the cost of equipment was significantly low, representing less than 2% of the total costs. The cultivation under SSF conditions presented a final cost 50-fold lower than the one obtained when culturing under SmF conditions at flask scale. In addition, the laccase production under SSF conditions in tray bioreactors reduced the final cost 4-fold compared to the one obtained under SSF conditions at flask scale, obtaining a final price of 0.04 cent €/U.

Cost-effectiveness of rapid MRSA screening in surgical patients.

This study investigates the effectiveness of a same-day polymerase chain reaction (PCR) test for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) in a general screening of patients admitted to the trauma surgery and heart surgery department in a German university hospital. A total of 442 patients were screened over a 4-month period by using a PCR assay, compared to culture methods, for specimens from the nose and throat. The MRSA carriage rate on admission was 3.85% during the study period. The PCR results of 1,680 swabs showed a sensitivity of 85% and a specificity of 99.39% for swabs from the nares and for the throat 42.11% and 98.78%, respectively. A combination of specimens from the nose and throat from the same patient led to a sensitivity of 100% with a specificity of 98.29%. Cost calculation under the circumstances of a diagnosis-related groups (DRG) payment system found that the eight MRSA-positive patients created costs of 38,472 euros, i.e. 4,809 euros per patient, facing screening costs of 36.62 euros per sample. Screening patients by using the rapid PCR assay for a combination of specimens from the nose and throat would offer a safe and cost-effective way of MRSA screening on admission.

Determination of an economical medium for growth of Lactobacillus fermentum using response surface methodology.

AIM: Lactobacillus fermentum is a widely utilized probiotic compound fed as an alternative to antibiotics for growth promotion in a wide variety of livestock species. The objective of this research is to develop an economical and practical fermentation medium for the growth of Lact. fermentum using response surface methodology. METHODS AND RESULTS: A two-level Plackett-Burman design was used to determine which factors in the fermentation medium influence the growth of Lact. fermentum. Under our experimental conditions, peptone, urea and yeast extract were found to be major factors. Then, the steepest ascent method and the central composite design were applied to optimize the culture of Lact. fermentum. The following composition of the fermentation medium was estimated to be the most economical formula (per litre): 30 g corn syrup, 15 g glucose, 14.4 g peptone, 7 g (NH(4))(2)SO(4), 0.5 g urea, 3 g sodium acetate, 4 g sodium citrate, 0.1 g MnSO(4).4H(2)O, 0.5 g MgSO(4).7H(2)O, 7.3 g yeast extract, 0.5 g K(2)HPO(4). CONCLUSION: Based on 10 side-by-side comparisons, we found that the yield of Lact. fermentum using our fermentation medium was 64% greater than those using modified de Man, Rogosa and Sharp broth (MRS) medium (1.8 x 10(9) CFU ml(-1)vs 1.1 x 10(9) CFU ml(-1), respectively), while the cost was 89% lower than MRS. This research indicates that it is possible to increase bacterial yield by using inexpensive materials. SIGNIFICANCE AND IMPACT OF THE STUDY: It is more likely that the use of Lact. fermentum as a probiotic will increase. The low cost medium developed in this research can be used for large-scale, commercial application where economics are quite likely to be important.

Utility of the microcolony method for evaluation of multidrug-resistant tuberculosis patients in Karachi, Pakistan.

OBJECTIVE: To compare the microcolony method (MCM) with the reference culture method to evaluate culture conversion in multidrug-resistant tuberculosis (MDR-TB) patients.MATERIAL AND METHODS: Adult patients with Mycobacterium tuberculosis culture-positive MDR-TB undergoing second-line anti-tuberculosis treatment were recruited from two tertiary care chest clinics from January 2013 to October 2014. The MCM was performed in addition to MGIT™ and Löwenstein-Jensen medium (reference method) on sputum samples submitted on a monthly basis.RESULTS: Of 140 patients, culture conversion could be evaluated in 95 patients. The MCM showed 100% agreement with the reference M. tuberculosis culture in 83 of 95 patients who achieved culture conversion. In smear-positive and smear-negative cases, the mean time to positivity was 9.1 and 11.4 days for the MCM and 16.1 and 23.2 days for the reference M. tuberculosis culture respectively. The contamination rate for the MCM was 4.5% in comparison with 6.1% for the reference M. tuberculosis culture. The cost of MCM was estimated to be 30% that of the reference method.CONCLUSION: The MCM can be used in non-urban laboratories as a safe, rapid and cost-effective substitute for the reference M. tuberculosis culture to assess culture conversion in MDR-TB patients.Note: Abstract has been published in International Journal of Mycobacteriology 2015; 4: 159-160.

Comparative analysis of four commercial on-farm culture methods to identify bacteria associated with clinical mastitis in dairy cattle.

Several multiple-media culture systems have become commercially available for on-farm identification of mastitis-associated pathogens. However, the accuracy of these systems has not been thoroughly and independently validated against microbiological evaluations performed by referral laboratories. Therefore, the purpose of the present study was to evaluate the performance of commercially available culture plates (Accumast, Minnesota Easy System, SSGN and SSGNC Quad plates) to identify pathogens associated with clinical mastitis in dairy cows. Milk samples from the affected quarter with clinical mastitis were aerobically cultured with the on-farm culture systems and by two additional reference laboratories. Agreeing results from both standard laboratories were denoted as the reference standard (RS). Accuracy (Ac), sensitivity (Se), specificity (Sp), positive and negative predictive values (PPV and NPV, respectively) and Cohen's kappa coefficient (k) of on-farm plates were determined based on the RS culture of 211 milk samples. All four plate-systems correctly identified ≥ 84.9% of milk samples with no bacterial growth. Accumast had greater values for all overall predictive factors (Ac, Se, Sp, PPV and NPV) and a substantial agreement (k = 0.79) with RS. The inter-rater agreements of Minnesota, SSGN, and SSGNC with RS were moderate (0.45 ≤ k ≤ 0.55). The effectiveness to categorize bacterial colonies at the genus and species was numerically different amongst the commercial plates. Our findings suggest that Accumast was the most accurate on-farm culture system for identification of mastitis-associated pathogens of the four systems included in the analysis.

Development of an economical, autonomous pHstat system for culturing phytoplankton under steady state or dynamic conditions.

Laboratory investigations of physiological processes in phytoplankton require precise control of experimental conditions. Chemostats customized to control and maintain stable pH levels (pHstats) are ideally suited for investigations of the effects of pH on phytoplankton physiology, for example in context of ocean acidification. Here we designed and constructed a simple, flexible pHstat system and demonstrated its operational capabilities under laboratory culture conditions. In particular, the system is useful for simulating natural cyclic pH variability within aquatic ecosystems, such as diel fluctuations that result from metabolic activity or tidal mixing in estuaries. The pHstat system operates in two modes: (1) static/set point pH, which maintains pH at a constant level, or (2) dynamic pH, which generates regular, sinusoidal pH fluctuations by systematically varying pH according to user-defined parameters. The pHstat is self-regulating through the use of interchangeable electronically controlled reagent or gas-mediated pH-modification manifolds, both of which feature flow regulation by solenoid valves. Although effective pH control was achieved using both liquid reagent additions and gas-mediated methods, the liquid manifold exhibited tighter control (±0.03pH units) of the desired pH than the gas manifold (±0.10pH units). The precise control provided by this pHstat system, as well as its operational flexibility will facilitate studies that examine responses by marine microbiota to fluctuations in pH in aquatic ecosystems.

Combination of commercially available molecular assays and culture based methods in diagnosis of tuberculosis and drug resistant tuberculosis.

Early diagnosis of tuberculosis is of major clinical importance. Among 4733 clinical specimens collected from 3363 patients and subjected to Ziehl-Neelsen microscopy, 4109 were inoculated onto Löwenstein-Jensen slants and 3139 in Bactec/9000MB. Polymerase Chain Reaction (PCR) was performed in 3139 specimens, whereas, a genotypic assay was directly applied in 93 Mycobacterium tuberculosis complex PCR-positive for isoniazid and rifampicin resistance detection specimens (GenoType MTBDRplus). Recovered M. tuberculosis isolates (64) as well as, 21 more sent from Regional Hospitals were tested for antimycobacterial resistance with a phenotypic (manual MGIT-SIRE) and a genotypic assay (GenoType MTBDRplus). PCR in the clinical specimens showed excellent specificity (97.4%) and accuracy (96.8%), good sensitivity (70.4%), but low positive predictive value (40.3%). MGIT-SIRE performed to M. tuberculosis did not confer a reliable result in 16 isolates. Of the remaining 69 isolates, 15 were resistant to streptomycin, seven to isoniazid, seven to ethambutol and five to rifampicin. GenoType MTBDRplus correctly detected isoniazid (seven) and rifampicin-resistant M. tuberculosis strains (five), showing an excellent performance overall (100%). Susceptibility results by the molecular assay applied directly to clinical specimens were identical to those obtained from recovered isolates of the corresponding patients. Combining molecular and conventional methods greatly contribute to early diagnosis and accurate susceptibility testing of tuberculosis.

Descriptive, Longitudinal Study Results Applied to Statistical Models to Assess the Impact of Early Microbiological Cultures on the Economic Burden of Treatment for Infected Diabetic Foot Ulcers at a Mexican Public Health Facility.

Infection plays a critical role in health care and impacts the cost of the treatment of diabetic foot ulcers (DFU). To examine the cost reduction associated with the multidisciplinary treatment of infected DFU (IDFU) by obtaining early (ie, within 48 hours of admission) microbiological culture results, a descriptive, longitudinal study was conducted. Data were collected prospectively from patient medical charts of a cohort of 67 patients (mean age, 56.14 ± 12.3 years; mean duration of diabetes, 14.95 ± 8 years) with IDFU treated at a Mexican public health facility from January 1 to April 30, 2010. Information included demographic data (age, gender, marital status, time elapsed since first diagnosis of diabetes mellitus type 2 [DM2]), and the following clinical records: Wagner classification, bacterium type, antimicrobial resistance, length of hospital stay, and the antibiotic schedule utilized, as well as number and type of laboratory tests, medications, intravenous therapy, surgical and supportive treatment, type and number of specialists, and clinical outcome. Microcosting was used to calculate the unit cost of each medical treatment element. Using the Monte Carlo and Markov predictive simulation economical models, cost reduction associated with early identification of the specific microorganism through bacterial culture in IDFU was estimated. Based on the statistical results, differences between real and estimated costs when including early microbiological culture were identified and the number and type of most common species of infectious bacteria were detected. The total cost observed in the patient cohort was $502 438.04 USD, mean cost per patient was $7177.69 ± $5043.51 USD, and 72.75% of the total cost was associated with the hospital stay length. The cost of the entire treatment including antibiotics was $359 196.16 USD; based on the simulation of early microbiological culture, the model results showed cost could be reduced by 10% to 25% (in this study, the cost could be as low as $304 624.63 USD). The use of early microbiological cultures on IDFU to determine the appropriate antibiotic can reduce treatment costs by >30% if hospital stay is part of the consideration.

Enhanced IgG production in eRDF media with and without serum. A comparative study.

The performance of three basal media RPMI, DMEM/F12 (DF) and eRDF (enhanced RDF, RPMI:DMEM:F12 in 2:1:1) were evaluated in cultures with and without serum with respect to cell proliferation, metabolism and monoclonal antibody (Mab) productivity. Based on the ease of adaptation, growth rate, maximum cell density and Mab production, the media were ranked as follows: eRDF > DF > RPMI. This was true for serum-free (SF) and serum supplemented (SS) media in static and shaker cultures. Growth performances in static and shaker cultures were consistently 20-50% lower in all three SF media compared to the corresponding SS conditions. Antibody titres in DF/SF and RPMI/SF cultures, irrespective of the culture condition, were generally similar or slightly lower than their SS counterparts. However, eRDF/SF medium yielded a much higher Mab titre (193 mg l-1) compared to eRDF/SS medium (145 mg l-1). This was also six times higher than the lowest titre of 30 mg l-1 in RPMI/SF medium. Hybridomas in eRDF/SF were further adapted to media without bovine serum albumin (eRDF/SF-BSA). Maximum cell densities in these cultures improved with scale up, from 1.1 x 10(6) ml-1 in static, to 1.9 x 10(6) ml-1 in shaker flasks, to 2.5 x 10(6) ml-1 in bioreactors. However, Ig levels remained between 100-130 mg l-1 which were much lower than in eRDF/SF medium. Thus BSA appears to be necessary for Ig production. The manufacturing cost (excluding purification) of Ig using eRDF was calculated to be between 17-50% of the price of the other two media and therefore this is regarded as the best medium for Ig production.

A simple plant and tissue culture growth chamber for the regeneration of tobacco mesophyll protoplasts.

The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli.

Eukaryotic cells grown on microcarrier beads offer a cost-efficient way to propagate Chlamydia trachomatis.

The use of microcarrier cell culture as a method for the in vitro propagation of the obligate intracellular bacterial parasite, Chlamydia trachomatis, is described. The microcarrier beads proved to be a more cost-effective means to propagate C. trachomatis than traditional tissue culture flasks or roller bottles without sacrificing yields or infectivity. In addition, microcarrier cell culture was found to be a much simpler technique to study the intracellular development of these bacteria.

The importance of cell physiology to the performance of animal cell bioreactors.

This report has pointed out the following: (1) the new animal cell products that will be produced commercially will have large dollar markets and will, together with cost containment and competitive pressures, place a greater emphasis on the reduction of the cost of production through the selection of appropriate culture technology; (2) the benefits to be gained by working with basic biological processes in animal cell culture that will increase cell density, cell productivity, and product quality; (3) the need to work with reactor technologies that can affect the basic biology of the cell in these positive ways; (4) it appears worthwhile to explore immobilized, high cell density culture technologies as a possible means to achieve the objectives by affecting the basic regulation of the cell through fundamental cell/cell environment biological processes.

Safety and economic aspects of continuous mammalian cell culture.

Mammalian cell cultures are the most appropriate host cells for recombinant DNA derived products if complex protein structures have to be synthesized in their native form. Due to their physiological behaviour they grow either adherent or in suspension. For the attachment of adherent cells, microcarriers or wire springs can be applied to increase the internal surface of the bioreactor. Both systems provide a simplified media exchange but, however, show some limitations in scale up. In contrast, suspension culture systems as homogeneous systems independent of any carrier have not shown any limitation in scale up. Because most cell lines which are of commercial interest grow in suspension, this technology is best advanced and used in batch and continuous mode. Although mammalian cell cultures are sensitive to hydrodynamic shear forces, technologies for deep tank production are developed which allow stirrer tip speed of up to 1.5 m s-1 sufficient for oxygen uptake, suspension of cells and homogeneous supply with nutrients. For long term bioprocesses without selection pressure it has to be considered that transformed cell lines might show genetic instability due to their variations of chromosomes. In addition, sterile technology becomes an important factor in long term bioprocesses. The decision as to which cell culture system should be chosen, whether batch or continuous processes should be applied essentially is based on the capital investment, the amount of material to be produced, genetic stability of the production cell line, reliability of sterile technology and the flexibility required in the production plant. Under the assumption that 20 kg of a protein have to be produced per year and the same product concentrations in the harvest fluid are reached in the batch process and for instance in the chemostat, it can be considered that the capital investment for one 10,000 l batch process and a 2 x 2,000 l continuous process, necessary to produce the amount of material, is comparable. Risk of microbial contamination or technical failure can be considered to be fairly low in the batch process. The economic risk for long term bioprocess in the chemostat can be expected to be medium and high in the perfusion system which is in the large scale not technically fully satisfactory. In addition, due to the longer down time period after contaminations and the start up of the continuous process, the annual yield of the batch process can be considered to be higher.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of cultured epidermal autografts in the treatment of large burns.

Mortality in patients with large areas of full skin thickness burns is, in part, due to complications developing during the period of prolonged delay required to obtain enough wound healing to permit skin grafting from limited donor sites. Cultured epithelial autograft (CEA) has become available as an alternative measure to the use of expanded skin autografts and regrafting. Small biopsies are taken and transported to the laboratories of BioSurface Technology where keratinocytes are grown to cover large areas during a 3-week period. The cultured keratinocytes are then available on petroleum jelly gauze which is applied to the patient. The gauze is used as a temporary dressing. To date, 37 patients have been biopsied. Grafts have been applied in 15. Graft 'take' averaged 71.5 per cent at our institution. Two of the patients grafted with CEA died of sepsis. One patient had a 100 per cent loss of the CEA grafts. In 12 patients, the use of CEA probably contributed significantly to wound coverage and survival. Such grafts are more susceptible to mechanical loss than routine autograft, although long-term coverage after several years is considered to be satisfactory. The cost of the process is high.

Plant regeneration of indica rice (Oryza sativa) cultivars from mature embryo-derived calli.

Plant regeneration from seven-week-old callus cultures derived from mature embryos of several indica rice cultivars was achieved with frequencies of morphogenic calli from 10 to 47%. Three media were tested both for callogenesis and plant regeneration. For 3 of the 7 genotypes examined, the best combination of media for plant regeneration was Murashige & Skoog basal medium: MSC (callogenesis) and MSR (regeneration). The rates of callogenesis were not related to the capacity for plant regeneration. Two genotypes CR-1113 and CR-5272 produced the highest number of regenerated green plants. The results of this study suggest that genetic differences could be directly linked to the ability to regenerate in these plant cultivars.

In vitro propagation of cauliflower using curd microexplants.

Cauliflower (Brassica oleracea var. botrytis) with its distinctive pre-inflorescence or curd is a remarkable member of the Brassica cabbage group. During curd development, intense and repetitive branching leads to a spectacular increase in size and the accumulation of millions of meristems at its surface. Although destined to produce flowers, most of these meristems are capable of regenerating vegetative shoots in vitro, making curd fragments an excellent material for the micropropagation of cauliflower. Most reported methods using these tissues were devised for the production of small clones of vitroplants as the true potential of curd fragments remained highly underestimated. We describe a technique exploiting fully this abundance of meristems and optimized for the large-scale in vitro propagation of cauliflower. The curd surface is first mechanically disrupted to break up the meristem clusters and generate microexplants carrying 1-3 meristems. These microexplants are then cultured at high density 1:100 (v:v) (microexplants:medium) in liquid medium containing Kinetin and indole-3-butyric acid (IBA) and produce thousands of microshoots in 12 days. After selecting the best quality microshoots on a sucrose pad, they are transferred en masse to a rooting medium supplemented with IBA. Four weeks later, rooted microshoots are carefully acclimatized before transfer to the field. This semi-automated protocol is rapid, cost effective, and well adapted for the production of clones of several thousands of plants by a single worker in a short space of time.