Tissue Culture Isolation Research Articles

Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method.

A multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine. The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media. Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result. Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation. The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively. In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively. Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination. In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks. Shedding of type 3 virus ceased in approximately 70% of vaccinees within a week after immunization. In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Prevalence and clinical aspects of congenital cytomegalovirus infection

OBJECTIVES: The objectives of the present study were to determine the prevalence of congenital CMV infection, as well as to evaluate the importance of this agent as cause of congenital disease, and to describe the clinical manifestations in children attended at a General Hospital in Ribeirão Preto, SP, Brazil. POPULATION AND METHODS: A first group including 189 newborns and their mothers was evaluated for the prevalence of the congenital CMV infection. A second group including 130 newborns and 74 infants who presented clinical manifestations of congenital disease were also investigated to evaluate the importance of the CMV as a cause of this disease and to describe the clinical findings. Diagnosis of congenital CMV infection was established by detecting the virus using viral isolation in tissue culture, polymerase chain reaction DNA amplification in urine samples and detection of specific anti-CMV IgM and IgG by immunofluorescence indirect test. RESULTS: The prevalence of congenital CMV infection was 2.6% and the prevalence of CMV antibodies in mothers was 95%. In the first group, none of the 5 congenitally infected presented clinical apparent disease at birth, although one of them had intracranial calcifications. In the second group, CMV was recognized as a causative of congenital disease in 12 children (5.9%). Of these, 10(83%) were identified after the neonatal period. The clinical findings included hepatosplenomegaly (75%), jaundice with direct hyperbilirubinemia (42%), neurologic disease consisting of microcephaly and intracranial calcifications in 42% of these children. CONCLUSIONS: The prevalence of congenital CMV infection was similar to that reported in other studies about highly immune populations. Infants with asymptomatic congenital CMV infection may have diseases of the central nervous system that are not clinically evident at birth, such as punctate calcifications. CMV infected patients who are symptomatic at birth have a multisystem disease, and the differential diagnosis of any newborn with clinical abnormalities including involvement of the hepatobiliary, hematopoietic and central nervous systems should include congenital CMV infection. CMV was an important agent of these abnormalities, and the majority of symptomatic patients were identified after the neonatal period, making the diagnosis more difficult.

Experimental infection of pigs with a thymidine kinase negative strain of pseudorabies virus

Sixteen 20 day old pigs, devoid of neutralizing antibody to pseudorabies virus (PRV), were divided into two groups of eight, and the animals of each group were housed in a separate unit. In each group 6 pigs were inoculated intranasally with the thymidine kinase (TK −) mutant (Group 1) or the field strain of PRV (Group 2), each pig receiving an inoculum of 4 ml. The remaining 2 pigs in each group served as uninoculated controls. The only clinical sign observed in the pigs of Group 1 was a transient febrile reaction, in the case of six pigs inoculated with the TK − mutant of PRV, whereas no signs of disease were seen in the uninoculated controls. The virus was isolated from the 6 infected pigs of the group only on post infection day (PID) 2, whereas it was never isolated from the controls. By contrast, the pigs of Group 2, had a severe clinical response and one, among those that were inoculated with the field strain of the PRV, died on PID 9. Virus was consistently isolated from all pigs of Group 2, inoculated and control. On PID 30 all pigs, i.e. the 8 of Group 1 and 7 of the Group 2 which survived to the infection, were subjected to dexamethasone (DMS) treatment. After DMS treatment virus was never isolated from the nasal swabbings obtained from the pigs of Group 1, whereas it was consistently isolated from pigs of Group 2. After 30 d from the start of DMS treatment the pigs were killed and several tissues were collected from each pig for virus detection, by isolation in tissue culture and by PCR analysis. At necropsy no lesions were found in pigs of Group 1, whereas acute pneumonia and gliosis in the trigeminal ganglia were observed in pigs of Group 2. Virus was never isolated from any of the tissues taken from pigs of both, Group 1 and Group 2, nevertheless sequences of PRV were detected by PCR analysis in the trigeminal ganglia of the pigs of both Groups.

Improved detection of respiratory syncytial virus in nasal aspirates by seminested RT‐PCR

A seminested RT-PCR for amplification of Respiratory syncytial virus (RSV)-RNA in nasal aspirates has been developed and used to test nasopharyngeal aspirates (NPAs) from 132 infants hospitalized with acute respiratory tract infections during winter epidemics. The results were compared with those obtained by virus isolation in tissue culture and antigen detection with an enzyme-linked immunosorbent assay (Ag-ELISA). RSV-RNA was detected by seminested RT-PCR in 57 of the 59 samples that were positive by virus isolation and/or ELISA, as well as in 25 of 73 samples negative by virus isolation and ELISA. Eighteen of these 25 samples were obtained from children older than one year of age, 17 of whom were experiencing reinfection, as indicated by the presence of preexisting serum RSV-IgG antibodies. These results indicate that seminested RT-PCR is more sensitive than conventional methods for the detection of RSV in patients experiencing reinfections and suggest that this assay might also be useful for rapid diagnosis of RSV infections in older people. J. Med. Virol. 53:366–371, 1997. © 1997 Wiley-Liss, Inc.

Improved detection of respiratory syncytial virus in nasal aspirates by seminested RT-PCR

A seminested RT-PCR for amplification of Respiratory syncytial virus (RSV)-RNA in nasal aspirates has been developed and used to test nasopharyngeal aspirates (NPAs) from 132 infants hospitalized with acute respiratory tract infections during winter epidemics. The results were compared with those obtained by virus isolation in tissue culture and antigen detection with an enzyme-linked immunosorbent assay (Ag-ELISA). RSV-RNA was detected by seminested RT-PCR in 57 of the 59 samples that were positive by virus isolation and/or ELISA, as well as in 25 of 73 samples negative by virus isolation and ELISA. Eighteen of these 25 samples were obtained from children older than one year of age, 17 of whom were experiencing reinfection, as indicated by the presence of preexisting serum RSV-IgG antibodies. These results indicate that seminested RT-PCR is more sensitive than conventional methods for the detection of RSV in patients experiencing reinfections and suggest that this assay might also be useful for rapid diagnosis of RSV infections in older people.

Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction

Two polymerase chain reaction (PCR) assays are described for the detection of swine vesicular disease virus (SVDV) RNA, a reverse transcription PCR (RT-PCR) and a reverse transcription nested PCR (RT-nPCR). Both the RT-PCR and RT-nPCR were able to detect representative members of each of seven phylogenetically distinct groups of SVDV and gave negative results with a range of porcine enteroviruses and of viruses responsible for vesicular conditions in pigs. When combined with a commercial kit for rapid RNA extraction, the RT-PCR was useful for the detection of SVDV in samples of epithelium and faeces from animals with clinical SVD. The addition of a second amplification step to create a nested PCR (RT-nPCR) increased the sensitivity of the technique for the detection of viral RNA (vRNA) in SVDV infected tissue culture fluid by a factor of approximately 1000, from 100 TCID 50 for the RT-PCR to 0.1 TCID 50 for RT-nPCR. When combined with a more elaborate extraction procedure for RNA, the RT-nPCR was considerably more sensitive than virus isolation in tissue culture for detecting SVDV in nasal swabs, tissues, and faeces collected from pigs between 7 days and 176 days after infection with a recent European isolate of SVDV. However, stringent conditions are necessary for carrying out the RT-nPCR to minimise the possibility of contamination.

Serologic and molecular detection of granulocytic ehrlichiosis in Rhode Island.

A new indirect fluorescent-antibody (IFA) assay with antigen produced in vitro in the human promyelocytic leukemia cell line HL60 was used to identify the first recognized case of human granulocytic ehrlichiosis in Rhode Island. This IFA assay was used to detect granulocytic ehrlichiae in white-footed mice and in a dog inhabiting the area surrounding the patient's residence. Host-seeking Ixodes scapularis ticks found in the same habitat also were infected. I. scapularis ticks collected from other locations were fed on dogs and New Zealand White rabbits to assess the competency of these species as hosts of granulocytotropic Ehrlichia. Tick-induced infections of dogs were confirmed by serologic testing, tissue culture isolation, and PCR amplification, whereas several rabbits seroconverted but were PCR and culture negative. PCR amplification of the 16S rRNA gene and DNA sequencing of the PCR products or culture isolation was used to confirm granulocytic Ehrlichia infections in humans, dogs, white-footed mice, and ticks.

DFA, EIA, PCR, LCR and Other Technologies: What Tests Should Be Used for Diagnosis of Chlamydia Infections?

For many years, isolation in tissue culture (TC) was considered the test of choice for diagnosis of Chlamydia trachomatis infection. Non-culture tests, such as direct fluorescent antibody (DFA) and enzyme immunoassay (EIA) which detected chlamydial antigens in clinical specimens, made chlamydia diagnostic tests more widely available. DFA and EIA were less sensitive than TC and had some false positive results which compromised our ability to use these tests in low prevalence settings.Direct nucleic acid probes are available, but do not appear to be more sensitive than EIA. It was only with the introduction of amplified DNA tests [polymerase chain reaction (PCR) and ligase chain reaction (LCR)] that non-culture tests became available that were actually more sensitive than TC. Unfortunately these tests are also more expensive than the antigen detection methods. Until there is a fairly sophisticated cost benefit analysis or a change in the pricing of these tests, it seems obvious that TC will remain the best choice where medical/legal implications are important, DFA will probably remain a widely used tests for laboratories that process relatively small numbers of specimens and EIAs will play a role where cost is major factor and large numbers of specimens require bulk processing. Where they are affordable, the amplified DNA tests are to be preferred as they are far more sensitive than these other non-culture tests.

Keeping up with new technology: new approaches to diagnosis of Chlamydia infection

Chlamydia trachomatis infections are among the most common sexually transmitted infections in the US today. One of the keys to the prevention of C. trachomatis infection rests on the ability to make this diagnosis on the basis of accurate laboratory testing. For many years the standard for diagnosis of C. trachomatis infections has been isolation in tissue culture. Numerous nonculture methods, including enzyme immunoassay, have been used as an alternative to cell culture. The performance characteristics of these tests have all been compared with a standard, cell culture, which at best will detect 90% of positive specimens. Nucleic acid amplification techniques, including PCR and ligase chain reaction, have been recently introduced. The advantage of these tests is their ability to detect 10-20% more positive specimens when compared with culture or confirmed nonculture methods performed with a single specimen. The sensitivity of amplified tests also allows us to test specimens from multiple sites (endocervix, urethra, urine), which expands our standard from an infected sample to detection of an infected patient. Tests based on amplified nucleic acid technology have greatly improved our ability to diagnose urogenital C. trachomatis infection. The use of an expanded standard will help us accurately define the true performance and clinical utility of nonculture Chlamydia diagnostic tests.

Prevalence of genital Chlamydia trachomatis infection in selected populations in China.

Chlamydia trachomatis is an important pathogen of the urogenital tract. Numerous epidemiologic studies have reported on the prevalence of chlamydia world wide. However, the prevalence of Chlamydia trachomatis in China has not been widely studied. To identify Chlamydia trachomatis in three distinct populations in China. Urogenital specimens were collected from men with no prior history of sexual intercourse (virgin), men seen at a sexually transmitted disease clinic, and female prostitutes. Two-hundred urethral swabs were collected from group one, and 100 urethral swabs from group two, and 109 cervical swabs from group three. All specimens were tested for chlamydia by tissue culture isolation using McCoy cells and an iodine stain. Chlamydia trachomatis was not isolated from the virgin males. The prevalence of chlamydia in men seen at a sexually transmitted disease clinic was 13% (13/100) and in female prostitutes, 37.6% (41/109). The prevalence of chlamydia seen in these three groups is similar to rates found in previous studies done in the United States and Europe in similar populations. Patients with multiple sex partners in China need to be screened for C. trachomatis.

Evaluation of antigen and antibody detection in urine specimens from children with congenital human cytomegalovirus infection.

Fetal infection with human cytomegalovirus (HCMV) is the leading viral cause of brain damage among newborns at birth or later in life. Efforts to screen newborns routinely for shedding of the virus by immunoassay have been hampered by inhibitors in urine, reportedly the host protein beta2-microglobulin (beta 2m). An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HCMV antigen in which the reactivity was not affected by the presence of beta 2m, but nevertheless inhibition was observed when urine samples with high levels of virus were tested. The presence of antibodies to HCMV was demonstrated in these urine samples by antibody ELISA and immunoblot using the major antigenic protein of HCMV (pp150) expressed in Escherichia coli; this offers an alternative explanation for the inhibition in ELISA. The presence of HCMV antibodies correlated significantly with congenital HCMV infection (as detected by tissue culture isolation of virus from urine samples of newborns), especially with asymptomatic cases (sensitivity 70%; specificity 94%). The data indicate a local (renal) immune response to HCMV in congenitally infected children, which may have future diagnostic applications.

Efficacy of herpes vaccine and acyclovir (ACV) in a rabbit model following intraocular inoculation of herpes simplex virus.

It has been shown that injection of herpes simplex virus (HSV) type I into the vitreous body of the eye in 18-day-old albino rabbits consistently induced encephalitis. In the untreated group the lesions followed a defined anatomical pathway in the central nervous system and produced a chronic progressive disease with 95% survival. Detailed observations in the spread of HSV along the optic pathway determined the extent of damage at any given day. Some of the old rabbits developed typical herpetic lesions on nose and lips. HSV was demonstrated from these lesions by electron microscopy and also by tissue culture isolation. The combined efficacy of heat-killed herpes vaccine prepared from the same isolate and acyclovir (ACV) in this animal model was studied by starting treatment four days before or four days after the challenge. Ten animals immunised before the challenge were protected. However, immunisation after the challenge not only did not confer protection, but surprisingly, appeared to enhance the primary disease. All 10 rabbits immunised after the challenge developed weakness of the hind legs and progressed very rapidly to paralysis. ACV treatment alone did not completely abrogate the HSV infection, there appears to be reactivation of HSV which produced fresh small lesions. However, a combination of immunisation and treatment with ACV after the challenge of the 10 rabbits in the group prevented the development of weakness of the hind legs or paralysis. Detailed observations on the spread of HSV along the optic pathway revealed that pathological lesions and damage were limited in the ACV and combined treatment with ACV and vaccine group.

Application of ELISA for IgM, IgA and antigen detection for rapid diagnosis of respiratory syncytial virus infections: a comparative study

Background: Infection with Respiratory Syncytial Virus (RSV) can be rapidly diagnosed by detection of viral antigen in nasopharyngeal secretions (NPS) or serologically by detecting IgM and IgA antibodies. Objectives: To evaluate the above methods for reliability and rapidity, and compare them with the complement fixation (CF) test and virus isolation. Study design: Viral antigen was tested in 145 NPS samples by ELISA in parallel with tissue culture isolation. The geometric mean titer (GMT) of complement fixing antibodies in various age groups was determined from 92 individual serum samples by CF test. The diagnostic methods, CF test, ELISA-IgM and ELISA-IgA, were evaluated using 21 pairs of acute and convalescent sera. Appearance of IgM and IgA in serum samples with low or negative CF titers was studied in two age groups: 0–10 months ( n = 82), and 11 months–9 years ( n = 47). Results: From the 145 NPS samples, 20 samples were positive by both ELISA and virus isolation, 9 were positive only by ELISA and 5 were positive only by virus isolation. The GMT by age group for 92 sera evaluated by CF test were 40 (0–10 months), 195 (11–24 months), 269 (2–4 years), 173 (5–12 years) and 132 (adults). Among the 21 paired sera examined, CF test detected 13 RSV infections by antibody rise or seroconversion while the ELISA-IgM IgA tests identified all 21 infections, 7 of them in the first sample. The presence of IgM alone or IgM and IgA antibodies was demonstrated in both age groups examined; however, IgA alone was found only in the age group 11 months and older. Conclusions: ELISA for antigen detection is better than virus isolation because it is faster (6–20 h vs. 3–20 days, respectively) and more sensitive ( 29 34 vs. 25 34 positives, respectively). ELISA-IgM and ELISA-IgA was more sensitive and reliable than the CF test, particularly for the 0–10 month age group. Thus, when necessary, serological diagnosis of RSV infection can be based on the presence of IgM and/or IgA antibodies in serum samples obtained early after onset of symptoms.

Chlamydia trachomatis-specific antibodies in patients with pelvic inflammatory disease: comparison with isolation in tissue culture or detection with polymerase chain reaction.

The detection of acute phase antibodies against C trachomatis and its comparison with tissue culture or polymerase chain reaction (PCR) on samples of cervix and urethra obtained from patients with pelvic inflammatory disease (PID). In the academic hospital Dijkzigt, Rotterdam, The Netherlands, prospective investigations were performed on 49 consecutive patients who were admitted with the diagnosis of PID. Infections with C trachomatis were traced using tissue culture, PCR or by determining acute phase IgG and IgM antibodies. Differences between the sensitivities of serology and tissue culture or PCR were calculated using the Fisher exact test. C trachomatis infection was detected more often in PID patients using serology in comparison with PCR (p < 0.05) or tissue culture (p < 0.05). All patients who were positive in tissue culture also had acute phase antibodies. The establishing of acute C trachomatis infections in PID patients on the basis of serology was superior to either tissue culture or PCR on samples obtained from cervix and urethra.

Comparison of in vivo plasma and peripheral blood mononuclear cell HIV-1 quasispecies to short-term tissue culture isolates

To determine whether the HIV-1 genomes that grow out in vitro from peripheral blood mononuclear cells (PBMC) better represent the in vivo quasi-species present in plasma or PBMC. For one patient (9606), PBMC culture represented more accurately the plasma rather than the in vivo PBMC quasi-species distribution, because a large number of tat-defective proviruses present in PBMC in vivo were not detected in plasma nor in the PBMC cultures. For a second patient (9605), PBMC culture was representative of both in vivo PBMC and plasma tat sequences, but selection of C2-V3 env sequences was observed in PBMC cultures compared with sequences present in both plasma and PBMC in vivo. This selection consisted of the absence in vitro of genomes with certain amino-acid substitutions at or near conserved glycosylation sites of the C2 region at positions 276 and 289. Site 276 has been reported to be important for viral infectivity, and these substitutions may therefore have affected infectivity. In the third patient (10095), selection of both tat and C2-V3 sequences was observed in culture as compared to plasma and PBMC in vivo. In contrast to the first two patients, this third patient contained V3 sequences in vivo that were predicted to impart syncytium induction and enhanced replication capacity. It was these sequences that grew out preferentially in vitro. This study suggests that short-term PBMC culture is representative of HIV-1 genomes present in PBMC and plasma in vivo to the degree that they are infectious.

Quantification of cytomegalovirus DNA in blood specimens from bone marrow transplant recipients by the polymerase chain reaction

A nested PCR system for cytomegalovirus (CMV) DNA in blood specimens from bone marrow transplant recipients is described, in which the biotinylated tritium-labelled product from the second round of PCR is quantified using streptavidin-coated fluorometric Scintillation Proximity Assay (SPA) beads (Amersham, UK). This assay has been compared with a PCR procedure based on limiting-dilution, in which the end-point is determined visually following electrophoresis in agarose gel. The two systems were shown to be equivalent in sensitivity and specificity on testing stored serial blood samples from six CMV antibody-positive allogeneic bone marrow transplant patients who developed viraemia as detected by conventional methods of virus isolation in tissue culture.

Detection of Chlamydia trachomatis in first catch urine samples from symptomatic and asymptomatic males.

Non-invasive tests are needed to detect Chlamydia trachomatis in the genital tract. For men, urine appears to be a useful specimen for chlamydial antigen or nucleic acid detection. To evaluate enzyme immunoassays (EIA) for chlamydial antigens in first catch urine (FCU) from symptomatic and asymptomatic men. We conducted five different studies; FCUs and urethral swabs were collected from 1,341 symptomatic and 816 asymptomatic males. Four EIAs (SureCell, Chlamydiazyme, MicroTrak and IDEIA) were tested on the FCU sediments. Prevalence of chlamydia by tissue culture isolation was 6% for asymptomatic and 14% for symptomatic men. With symptomatic males, the EIA sensitivities and specificities were: SureCell 85% and 97%, IDEIA 82% and 98%, MicroTrak 86% and 99%, and Chlamydiazyme 91% and 95%. For asymptomatic men, Chlamydiazyme sensitivity was 35% with frozen urine vs 77% with fresh urine. Overall, tissue culture sensitivity was about 90%. EIA results on FCU sediments are comparable to that of tissue culture isolation on urethral swabs. In many settings, FCU may be the specimen of choice for diagnosing chlamydial infections in men.

Rapid detection of vesicular stomatitis virus New Jersey serotype in clinical samples by using polymerase chain reaction

Vesicular stomatitis virus of the New Jersey serotype (VSV-NJ) causes vesicular disease in cattle, pigs, and horses throughout the Americas. Vesicular disease is clinically indistinguishable from foot-and-mouth disease (FMD). Therefore, outbreaks of vesicular disease in FMD-free areas must be rapidly diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm the absence of FMD. Diagnosis is currently performed in high-containment (biosafety level 3) laboratories by using complement fixation and virus isolation in tissue culture. We describe here an alternative method for the detection of VSV-NJ RNA in clinical samples. This method includes a rapid acid guanidine-phenol RNA extraction procedure coupled with a one-tube polymerase chain reaction (PCR) using reverse transcriptase. By using this test, we were able to detect the largest number of positive samples (53 of 58), followed by complement (48 of 58) and isolation in tissue culture (43 of 58). The primers chosen for this assay amplify a 642-nucleotide region of the phosphoprotein gene of VSV-NJ but not of VSV-IN. Sequencing of the PCR product enables genetic typing of virus isolates and epidemiological studies. Since no infectious materials are necessary to perform this test and any infectious virus in clinical samples is destroyed by acid guanidine-phenol treatment, diagnosis can be safely performed in regular diagnostic laboratories.

Immunocytologic Findings in a Case of Vogt-Koyanagi-Harada Syndrome

Vogt-Koyanagi-Harada (VKH) syndrome is a form of chronic panuveitis that often has a poor long-term visual outcome. To the authors' knowledge, there have been only two previous reports describing immunocytologic findings in the eyes of patients with VKH syndrome, all studied in relatively late stages of the disease. The authors performed immunocytologic studies on the enucleated eyes of a patient with active VKH syndrome, using monoclonal antibodies to mononuclear subsets, major histocompatibility antigens, and viral and treponemal agents, to better understand the pathophysiologic mechanisms underlying the disease. Emulsified chorioretinal tissue also was processed for viral isolation in tissue culture and for Treponema pallidum by rabbit inoculation studies. The dense uveal infiltrates in active VKH syndrome are composed predominantly of T lymphocytes and HLA-DR+ macrophages. Scattered T cells and plasma cells were present in the retina. In addition, nondendritic-appearing CD1 (Leu-6) positive cells were localized in the choroid, in close proximity to choroidal melanocytes. No microbial agents were identified. Whereas the etiology of the VKH syndrome remains an enigma, the authors' immunocytologic findings are consistent with a T-cell-mediated disorder. In demonstrating CD1 (Leu-6) positive cells in the infiltrates, the authors propose a potential role for these cells in the etiology of VKH syndrome.

TISSUE-CULTURE AND PROTOPLAST ISOLATION IN ASPARAGUS DENSIFLORUS L.

We have developed tissue culture and protoplasts isolation protocols for Asparagus densiflorus in order to use this genetic material in the breeding of Asparagus officinalis. For tissue-culture of A. densiflorus, the conditions which optimize the induction and the production of callus are a full MS medium with 1 mg/L of both pCPA and BAP and 0.5 mg/L of thiamine. HCL in the dark. on this medium, we obtained a friable white callus. Indirect organogenesis was obtained if pCPA was omitted from the medium. Replacement of the plant growth regulators by 2,4-D and Kinetin produced a hard and compact callus which did not differentiate. Protoplast have been isolated from 10 days old friable callus. cell wall was digested with 0.3% macerase, 1% cellulase and 0.8% rhozyme for a period of 16h at a temperature of 27°C in a CPW medium. Protoplast yield was 2 ×106 protoplasts/g callus. osmolarity of the digestion solution was 0.8 M provided with a mixture of glucose (0.6 M) and mannitol (0.2 M). cells were then plated at a density of 1 × 105 cells per ml. Microcolonies formed on a 1/2 MS medium with 0,5 mg/L NAA and ZEA and 1 g/L glutamine in the dark.