scholarly journals Evaluation of Saline, Phosphate-Buffered Saline, and Minimum Essential Medium as Potential Alternatives to Viral Transport Media for SARS-CoV-2 Testing

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Kyle G. Rodino ◽  
Mark J. Espy ◽  
Seanne P. Buckwalter ◽  
Robert C. Walchak ◽  
Jeffery J. Germer ◽  
...  
Keyword(s):  
Minimum Essential Medium ◽  
Viral Transport ◽  
Phosphate Buffered Saline

scholarly journals Infectivity of Theileria parva sporozoites following cryopreservation in four suspension media and multiple refreezing : evaluation by in vitro titration

2006 ◽  
Vol 73 (3) ◽  
Author(s):  
V. Mbao ◽  
D. Berkvens ◽  
T. Dolan ◽  
N. Speybroeck ◽  
J. Brandt ◽  
...  
Keyword(s):  
East Coast ◽  
Calf Serum ◽  
Roswell Park Memorial Institute ◽  
Foetal Calf Serum ◽  
Theileria Parva ◽  
Minimum Essential Medium ◽  
Freezing Medium ◽  
In Vitro Technique ◽  
Phosphate Buffered Saline

Theileria parva sporozoite stabilates are used for immunizing cattle against East Coast fever and in in vitro sporozoite neutralization assays. In this study, we attempted to identify a cheaper freezing medium and quantified the infectivity loss of sporozoites due to refreezing of stabilates, using an in vitro technique. Pools of stabilates prepared using Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI 1640), foetal calf serum (FCS) and phosphate-buffered saline (PBS) were compared. All were supplemented with bovine serum albumin except the FCS. RPMI 1640 was as effective as MEM in maintaining sporozoite infectivity while the infectivity in PBS and FCS reached only 59 % and 67 %, respectively. In a second experiment, a stabilate based on MEM was subjected to several freeze-thaw cycles including various holding times on ice between thawing and refreezing. Refrozen stabilate gave an average sporozoite infectivity loss of 35 % per cycle. The results indicate that RPMI can be used as a cheaper freezing medium for T. parva stabilates and that refrozen stabilate doses need to be adjusted for the 35 % loss of infectivity.

scholarly journals Automated molecular testing of saliva for SARS-CoV-2 detection

2020 ◽  
Author(s):  
Nancy Matic ◽  
Tanya Lawson ◽  
Gordon Ritchie ◽  
Aleksandra Stefanovic ◽  
Victor Leung ◽  
...  
Keyword(s):  
High Throughput ◽  
Turnaround Time ◽  
Molecular Testing ◽  
Cross Contamination ◽  
Plasma Samples ◽  
Test Results ◽  
Clinical Laboratories ◽  
Viral Transport ◽  
Global Demand ◽  
Phosphate Buffered Saline

Introduction: With surging global demand for increased SARS-CoV-2 testing capacity, clinical laboratories seek automated, high-throughput molecular solutions, particularly for specimen types which do not rely upon supply of specialized collection devices or viral transport media (VTM). Saliva was evaluated as a diagnostic specimen for SARS-CoV-2 using the cobas SARS-CoV-2 Test on the cobas 6800 instrument. Methods: Saliva specimens submitted from various patient populations under investigation for COVID-19 from March-July 2020 were processed in the laboratory with sterile phosphate-buffered saline in a 1:2 dilution and vortexed with glass beads. The processed saliva samples were tested using a commercial assay for detection of the SARS-CoV-2 E gene (LightMix) in comparison to the cobas SARS-CoV-2 Test. Results: 22/64 (34.4%) of the saliva samples were positive for SARS-CoV-2. Positive and negative concordance between the LightMix and cobas assays were 100%. There was no cross-contamination of samples observed on the cobas 6800. The overall invalid rate for saliva on the cobas 6800 (1/128, 0.78%) was similar to the baseline invalid rate observed for nasopharyngeal swabs/VTM and plasma samples. Conclusions: Saliva is a feasible specimen type for SARS-CoV-2 testing on the cobas 6800, with potential to improve turnaround time and enhance testing capacity.

A cytogenetical study of 7-day-old bovine embryos of poor morphological quality

Genome ◽  
1987 ◽  
Vol 29 (1) ◽  
pp. 160-164 ◽  
Author(s):  
W. A. King ◽  
P. Guay ◽  
L. Picard
Keyword(s):  
Follicle Stimulating Hormone ◽  
Type A ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Normal Type ◽  
Minimum Essential Medium ◽  
Cytogenetical Study ◽  
Serum Gonadotropin ◽  
Type C ◽  
Phosphate Buffered Saline

Seven-day-old embryos were collected from Canadian Holstein and Ayrshire heifers after superovulation with pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH). A total of 103 morphologically abnormal (type C) and 23 morphologically normal (type A) embryos were cytogenetically analyzed after 4, 20–24, or 44–48 h of culture in enriched phosphate-buffered saline or Eagles minimum essential medium. Twenty-one of 23 (91.3%) type A and 75 of 103 (72.8%) type C embryos had cells in metaphase. Among the 21 type C embryos produced by PMSG stimulation, 17 (80.9%) could be analyzed: 6 were mixoploid (two 2n/3n, three 2n/4n, one 2n/6n), 2 were aneuploid (61 XXY), and 9 were diploid. Among the 82 type C embryos produced by FSH stimulation, 58 (70.7%) could be analyzed: 6 were mixoploid (one n/2n, one 2n/3n, three 2n/4n, one 2n/4n/8n), 2 were polyploid (4n), and 50 were diploid. No abnormalities were observed in the type A embryos. Key words: bovine embryo, chromosome, mixoploid, aneuploid.

Ultrastructure of developing visual cortical synapses in the cat superior colliculus

1991 ◽  
Vol 49 ◽  
pp. 156-157
Author(s):  
Caroline A. Miller ◽  
Laura L. Bruce
Keyword(s):  
Superior Colliculus ◽  
Phosphate Buffer ◽  
Receptive Fields ◽  
Visual Cortical Area ◽  
Cortical Synapses ◽  
Visual Cortical ◽  
And Function ◽  
Phosphate Buffered Saline ◽  
The Brain ◽  
Cat Superior Colliculus

The first visual cortical axons arrive in the cat superior colliculus by the time of birth. Adultlike receptive fields develop slowly over several weeks following birth. The developing cortical axons go through a sequence of changes before acquiring their adultlike morphology and function. To determine how these axons interact with neurons in the colliculus, cortico-collicular axons were labeled with biocytin (an anterograde neuronal tracer) and studied with electron microscopy.Deeply anesthetized animals received 200-500 nl injections of biocytin (Sigma; 5% in phosphate buffer) in the lateral suprasylvian visual cortical area. After a 24 hr survival time, the animals were deeply anesthetized and perfused with 0.9% phosphate buffered saline followed by fixation with a solution of 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1M phosphate buffer. The brain was sectioned transversely on a vibratome at 50 μm. The tissue was processed immediately to visualize the biocytin.

Exogeneous factors are not necessary in attachment, spreading and polarization of hela cells

1978 ◽  
Vol 36 (2) ◽  
pp. 310-311
Author(s):  
W. Liebrich
Keyword(s):  
Hela Cells ◽  
Calf Serum ◽  
Glass Tube ◽  
Serum Free Medium ◽  
Nacl Solution ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Serum Free ◽  
All Solutions ◽  
Glass Support

HeLa cells were grown for 2-3 days in EAGLE'S minimum essential medium with 10% calf serum (S-MEM; Seromed, München) and then incubated for 24 hours in serum free medium (MEM). After detaching the cells with a solution of 0. 14 % EDTA and 0. 07 % trypsin (Difco, 1 : 250) they were suspended in various solutions (S-MEM = control, MEM, buffered salt solutions with or without Me++ions, 0. 9 % NaCl solution) and allowed to settle on glass tube slips (Leighton-tubes). After 5, 10, 15, 20, 25, 30, 1 45, 60 minutes 2, 3, 4, 5 hours cells were prepared for scanning electron microscopy as described by Paweletz and Schroeter. The preparations were examined in a Jeol SEM (JSM-U3) at 25 KV without tilting.The suspended spherical HeLa cells are able to adhere to the glass support in all solutions. The rate of attachment, however, is faster in solutions without serum than in the control. The latter is in agreement with the findings of other authors.

Artifact-free electron microscopic immunocytochemistry on rat brain tissue

1991 ◽  
Vol 49 ◽  
pp. 272-273
Author(s):  
Veronika Burmeister ◽  
N. Ludvig ◽  
P.C. Jobe
Keyword(s):  
Microscopic Examination ◽  
Free Electron ◽  
Electron Microscopic Examination ◽  
Ultrastructural Localization ◽  
Rabbit Serum ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Phosphate Buffered Saline ◽  
Rat Brain Tissue ◽  
Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

Modified gach technique vs critical point drying: Shrinkage analysis for SEM

1987 ◽  
Vol 45 ◽  
pp. 954-955
Author(s):  
B. Thompson ◽  
N. Sculov ◽  
R.E. Crang
Keyword(s):  
Critical Point ◽  
Calf Serum ◽  
Drying Shrinkage ◽  
Specimen Preparation ◽  
Cell Shrinkage ◽  
Whole Cells ◽  
Critical Point Drying ◽  
Prepared Material ◽  
Inverted Microscope ◽  
Phosphate Buffered Saline

The use of co-polymerized glutaraldehyde-carbohydrazide (GACH) was proposed for specimen preparation in scanning electron microscopy (SEM) as a means of avoiding dehydration in organic solvents, and to provide dimensionally stable biological specimens through a process of air-drying. It has been assumed that shrinkage of specimens prepared by the GACH technique should be less than that of conventionally-prepared material by critical point drying (CPD). In a previous study, Bell has reported significant shrinkage of whole cells for SEM. This report compares cell shrinkage in GACH and CPD preparations.Fibroblasts from newborn rats were grown on collagen-coated glass cover-slips (with alpha numeric grids etched onto the surface of the coverslips) in Eagle's minimum essential medium + 10% fetal calf serum for 7 d. (3). Using an inverted microscope with phase-contrast optics, micrographs were taken of the cultures in their live state and 1 h. after fixation with 2.5% glutaraldehyde in Dulbecco's phosphate buffered saline (Figs. 1 and 3).

Cell surface changes during aging of human bladder epithelium examined by lectin probes and SEM

1990 ◽  
Vol 48 (3) ◽  
pp. 966-967
Author(s):  
J. Jacob ◽  
M.F.M. Ismail
Keyword(s):  
The Elderly ◽  
Ultrastructural Changes ◽  
Pokeweed Mitogen ◽  
Asymmetric Structure ◽  
Luminal Surface ◽  
Bladder Epithelium ◽  
Aldehyde Groups ◽  
Cell Surface Changes ◽  
Phosphate Buffered Saline ◽  
Surface Changes

Ultrastructural changes have been shown to occur in the urinary bladder epithelium (urothelium) during the life span of humans. With increasing age, the luminal surface becomes more flexible and develops simple microvilli-like processes. Furthermore, the specialised asymmetric structure of the luminal plasma membrane is relatively more prominent in the young than in the elderly. The nature of the changes at the luminal surface is now explored by lectin-mediated adsorption visualised by scanning electron microscopy (SEM).Samples of young adult (21-31 y old) and elderly (58-82 y old) urothelia were fixed in buffered 2% glutaraldehyde for 10 m and washed with phosphate buffered saline (PBS) containing Ca++ and Mg++ at room temperature. They were incubated overnight at 4°C in 0.1 M ammonium chloride in PBS to block any remaining aldehyde groups. The samples were then allowed to stand in PBS at 37°C for 2 h before incubation at 37°C for 30 m with lectins. The lectins used were concanavalin A (Con A), wheat germ agglutinin (WGA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) at a concentration of 500 mg/ml in PBS at pH 7.A.

Application of EM to differentiate herpes virus from pox virus in genital specimens

1994 ◽  
Vol 52 ◽  
pp. 262-263
Author(s):  
K. Rekrut ◽  
K. Schleuter
Keyword(s):  
Tissue Culture ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fresh Tissue ◽  
Culture Cells ◽  
Viral Transport ◽  
Carbon Coated ◽  
Immuno Assay ◽  
Simplex Virus ◽  
Enzyme Immuno Assay

Confirmation of herpes simplex virus (HSV) from genital lesions of obstetrical (OB) patients may affect both the management of the delivery and of the neonate.(l,2) During 1992 and 1993, 4,450 genital specimens from OB patients were submitted in viral transport media for herpes culture. The specimens were inoculated into MRC-5, Vero, and A-549 tissue culture tubes, incubated, and examined daily for 7 days for cytopathic effect (CPE). The original specimens were frozen at −70° C until final reports were issued. Culture tubes with CPE were tested by the Dupont Herpchek enzyme immuno assay (EIA) to confirm the presence of herpes simplex virus (HSV). (3,4) 170 OB patient specimens were positive by culture and confirmed by EIA.There were also 63 cultures exhibiting CPE ressembling HSV which were negative by EIA testing, which failed to pass in fresh tissue culture cells or progress to more enhanced CPE in culture. These original specimens were screened by electron microscopy after direct ultracentrifugation employing the Beckman airfuge with the EM 90 rotor on to formvar carbon-coated 300 mesh copper grids and negatively stained with 2% PTA.

Immunocytochemical localization of p65 with one-nanometer gold particles following silver development

1989 ◽  
Vol 47 ◽  
pp. 1042-1043
Author(s):  
Richard W. Burry ◽  
Diane M. Hayes
Keyword(s):  
Plasma Membrane ◽  
Bovine Serum Albumin ◽  
Calf Serum ◽  
Specific Protein ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Pass Through ◽  
Label Distribution ◽  
Phosphate Buffered Saline

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.

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