Specimen storage in transport medium and detection of group B streptococci by culture

Recovery of group B streptococci (GBS) was assessed in 1,204 vaginorectal swabs stored in Amies transport medium at 4 or 21 degrees C for 1 to 4 days either by direct inoculation onto Granada agar (GA) or by culture in blood agar (BA) and GA after a selective broth enrichment (SBE) step. Following storage at 4 degrees C, GBS detection in GA was not affected after 72 h by either direct inoculation or SBE; however, GBS were not detected after SBE in the BA subculture in some samples after 48 h of storage and in GA after 96 h. After storage at 21 degrees C, loss of GBS-positive results was significant after 48 h by direct inoculation in GA and after 96 h by SBE and BA subculture; some GBS-positive samples were not detected after 24 h of storage followed by SBE and BA subculture or after 48 h of storage followed by SBE and GA subculture. Storage of swabs in transport medium, even at 4 degrees C, produced after 24 h an underestimation of the intensity of GBS colonization in most specimens. These data indicate that viability of GBS is not fully preserved by storage of vaginorectal swabs in Amies transport medium, mainly if they are not stored under refrigeration.     

Evaluation of transport and storage techniques for isolation of Campylobacter fetus subsp. jejuni from turkey cecal specimens

Immediate culturing of fecal specimens is not always possible, and appropriate methods for transport and storage of Campylobacter fetus subsp. jejuni specimens have not been fully evaluated. Using nine techniques, we studied the survival of C. fetus subsp. jejuni in cecal specimens from infected turkeys. The organisms survived in specimens held without transport medium for 3 to 15 days (median, 9 days) at 4 degrees C, and 2 to 9 days (median, 4 days) at 25 degrees C. Only 20% of specimens frozen for 24 h at either -20 or -70 degrees C yielded C. fetus subsp. jejuni. Specimens dried on filter paper strips were negative for C. fetus subsp. jejuni within 1.5 h. Cary-Blair medium with decreased agar was the best of the six transport media tested, it enabled recovery of the organism from 100% (3 days) and 71% (7 days) of cecal samples held at 4 degrees C and 94% (3 days) and 85% (7 days) of cecal specimens held at 25 degrees C. In contrast, more than half of all cecal specimens held at 4 or 25 degrees C in Culturettes or buffered glycerol saline were negative by 3 days, and all were negative at 7 days. Results with the other three media studied (Campy-thio, thioglycolate medium, and alkaline peptone water) were intermediate. Overnight incubation of specimens in alkaline peptone water at 37 or 42 degrees C did not enhance recovery of C. fetus subsp. jejuni. Therefore, refrigeration without a transport medium is satisfactory for up to 3 days for recovery of C. fetus subsp. jejuni from specimens, however, we recommend the use of Cary-Blair medium with decreased agar for specimens that must be transported or stored for longer than 3 days and for rectal swabs, to prevent drying.     

Evaluation of transport media for Campylobacter jejuni in human fecal specimens

It is not always possible to culture feces immediately, and appropriate methods for transport of human specimens, unlike those from animals, have not been fully evaluated. Therefore, we took serial subcultures in two phases from six transport media inoculated with human diarrheal stools known to be positive for Campylobacter jejuni. In phase 1, Cary-Blair medium and buffered glycerol saline did not preserve C. jejuni as well as did alkaline peptone-water (APW), modified Cary-Blair medium, thioglycolate broth (Thio), and Campy-Thio. The four best media (APW, Cary-Blair medium, Thio, and Campy-Thio) preserved 20 fecal samples with C. jejuni better at 4 degrees C (90% survival for 5 to 8 days) than at 25 degrees C (90% survival for 1.7 to 2 days). In phase 2, APW and Thio, along with four modifications of the best media in phase 1, were tested with 23 positive strains. The ranges of survival times with modified media at 25 degrees C were 1.3 to 2.2 days (90%) and 4.7 to 6.8 days (50%). APW with reducing agents preserved C. jejuni better than did APW alone, Thio plus ox bile, or Campy-Thio plus ox bile (P less than 0.05). Thio at pH 8.5 was better at preserving C. jejuni than was APW or Thio plus ox bile (P less than 0.05). If human fecal specimens cannot be refrigerated during transport or storage, we recommend the use of Thio at pH 8.5 or APW with reducing agents for preservation of C. jejuni at 25 degrees C.     

Optimal survival of Helicobacter pylori under various transport conditions.

The ability of clinical isolates and type strains of Helicobacter pylori to survive in Stuart transport medium, isotonic saline solution, and urea-containing isotonic saline was evaluated. The influences of temperature (4, 10, 15, 20, and 30 degrees C) and holding time (6 to 48 h) and the effect of exposure to air on survival were also studied. The recovery rate of H. pylori was highest from Stuart transport medium in comparison with the recoveries from the other transport media tested. We found that at holding temperatures above 15 degrees C the organisms became noncultivable within 6 h or less, while they survived for 2 days or longer at 10 degrees C. The presence of urea at a concentration of 2% (wt/vol) in isotonic saline resulted in the loss of viability of the organisms tested.     

Suitability of new chlamydia transport medium for transport of herpes simplex virus.

A new chlamydia transport medium (ChlamydiaPort; Scott Laboratories, Inc., Fiskeville, R.I.) was evaluated for its suitability as a transport medium for herpes simplex virus (HSV). Two laboratory HSV strains (McIntyre and 333) and two clinical isolates (AO218 and AO301) were suspended in ChlamydiaPort, ViraPort (Scott Laboratories), and cell culture medium and maintained at 2 and 22 degrees C. Samples were tested at various time intervals to determine surviving virus. The range of half-lives of the HSV strains held at 2 degrees C in ChlamydiaPort medium was from 3.5 to 10 days, while virus stability was greater in ViraPort and less in cell culture medium. These HSV strains held at 22 degrees C in ChlamydiaPort had half-lives from 1.5 to 6 days, which were significantly greater than the half-lives of the viruses held in either tissue culture medium or ViraPort. Clinical specimens were tested for virus by using the Selecticult-HSV (Scott Laboratories) system to determine the performance of the transport medium under field conditions. Clinical specimens maintained up to 5 days at ambient temperatures in ChlamydiaPort medium appeared suitable for diagnostic testing without detectable loss of positive specimens. In addition, there was a significant decrease in the average time required for diagnosis when compared with a standard transport system, Virocult (Microdiagnostics, Cleveland, Ohio). These results show that HSV infections can be successfully diagnosed in distant virology laboratories by shipping specimens in ChlamydiaPort transport medium at ambient temperatures.     

Novel observation of hot-cold-hot hemolysis exhibited by group B streptococci

Six human isolates of group B streptococci (GBS) were cultured on blood agar anaerobically at 37 degrees C for 18 h and then at 4 degrees C for 6 h and reincubated anaerobically at 37 degrees C for 6 h. Three of the strains showed a marked enlargement of the hemolysis zone compared with that obtained after hot-only (37 degrees C for 18 h) or hot-cold (37 degrees C for 18 h and then 4 degrees C for 6 h) treatment. Subsequent broth culture experiments revealed that enhanced hemolytic activity due to hot-cold-hot treatment was observed in all 6 GBS strains when cultured in the presence of starch.     

Metabolic and ionic requirements for the axoplasmic transport of dopamine beta-hydroxylase

1. Metabolic and ionic requirements for the intra-axonal transport of dopamine beta-hydroxylase (DBH) were investigated in the cat hypogastric nerve-inferior mesenteric ganglion preparation in vitro by monitoring the enzyme accumulation above a crush, 2-2.5 cm distal to the ganglion.2. DBH accumulation in the proximal segment immediately above the crush increased linearly up to 6 h, during incubation in normal Krebs solution at 37 degrees C. The rate of transport of the enzyme was about 4 mm/h.3. Removal of the ganglion, electrical stimulation or reserpine pretreatment (1-6 days before the experiment) did not modify the rate of DBH accumulation.4. Anoxia and glucose deprivation, singly, did not affect accumulation of DBH; however, the combined treatment of anoxia plus glucose deprivation, or dinitrophenol plus glucose deprivation, very markedly interfered with accumulation.5. Removal of sodium or potassium from Krebs solution markedly inhibited the transport of DBH. Preincubation of the nerve in a high-calcium Krebs solution at 4 degrees C, and then reincubation at 37 degrees C, prevented the enzyme accumulation.6. N-ethylmaleimide, ouabain and oligomycin markedly inhibited the transport of DBH.7. The results suggest that transport of DBH, and therefore of noradrenaline storage vesicles, within the hypogastic nerve is dependent on metabolic energy derived from either glycolysis or oxidative phosphorylation. It is also suggested that the sodium-potassium-activated ATPase may play an important role in the intra-axonal transport of storage vesicles.     

Evaluation of a medium (STGG) for transport and optimal recovery of Streptococcus pneumoniae from nasopharyngeal secretions collected during field studies

Field studies of Streptococcus pneumoniae (pneumococci) nasopharyngeal (NP) colonization are hampered by the need to directly plate specimens in order to ensure isolate viability. A medium containing skim milk, tryptone, glucose, and glycerin (STGG) has been used to transport and store NP material, but its ability to preserve pneumococci has not been evaluated. Our objective was to qualitatively and semiquantitatively evaluate the ability of STGG to preserve pneumococci in NP secretions. Entwined duplicate calcium alginate NP swab samples were obtained from children. One swab was plated directly onto a gentamicin blood agar plate; the other was placed in STGG. Growth from the directly plated specimen was compared with growth from an STGG aliquot immediately cultured or stored at -70 degrees C for 9 weeks, -20 degrees C for 9 weeks, or 4 degrees C for 5 days. Of 186 specimens, 96 (52%) were positive for pneumococci from the direct plating; 94 (98%) of these were positive from the fresh STGG specimen. Pneumococci were recovered from all 38 positive specimens frozen at -70 degrees C, all 18 positive specimens frozen at -20 degrees C, and 18 of 20 positive specimens stored at 4 degrees C. Recovery of pneumococci after storage of NP material in STGG medium at -70 degrees C is at least as good as that from direct plating. Storage at -20 degrees C is also acceptable. Storage at 4 degrees C for 5 days is not ideal.     

Evaluation of optimal storage temperature, time, and transport medium for detection of group B Streptococcus in StrepB carrot broth

The performances of the ESwab and Amies transport media were evaluated for optimal survival of group B streptococcus (GBS) in StrepB carrot broth. ESwab was superior to Amies at all temperatures evaluated but was optimal at 21°C and 24°C, whereas recovery in Amies was significantly decreased at these temperatures.     

In vitro evaluation of the performance of Granada selective enrichment broth for the detection of group B streptococcal colonization

A broth for the screening of group B streptococcal (GBS) carriage during pregnancy is about to be introduced. Simulating conditions in everyday practice, we have compared the sensitivity of this Granada tube broth (GT) with that of classical Amies transport medium (AT) in vitro. A total of 1,485 GT and 1,485 AT were tested with 33 well-characterized GBS strains in three different concentrations, five different incubation times, and three different temperatures. After initial incubation at room temperature (RT) or 4°C, GT were placed at 37°C. GT were scored for the presence of orange pigment. GT and AT were subcultured on blood agar (BA). Pigment was observed in 98% of GT incubated at 37°C. GBS could be cultured in 91%, 73%, and 55% of GT incubated at 37°C, RT, or 4°C, respectively. For AT, these percentages were only 20% at 37°C, 52% at RT, and 59% at 4°C. When GT initially incubated at RT or 4°C were subsequently incubated at 37°C, the sensitivity improved significantly. We conclude that GT is a more sensitive GBS transport and culture medium than the conventional method, especially for low inocula and prolonged transport/incubation times. GT does not exclude the presence of GBS, and should always be incubated at 37°C and subcultured on solid agar for optimal sensitivity.     

Effects of transport temperature and medium on recovery of Bordetella pertussis from nasopharyngeal swabs.

We compared relative recoveries of Bordetella pertussis from simulated nasopharyngeal (NP) specimens incubated in three separate transport media at different temperatures. Transport media included one-half-strength Regan-Lowe (RL.5), Regan-Lowe with one-half-strength agar (RL.5A), and buffered charcoal-yeast extract agar supplemented with alpha-ketoglutarate, lincomycin, and anisomycin (BCYE alpha LA). For each transport medium, recovery of B. pertussis was least efficient after storage at 25 degrees C. The highest recovery of B. pertussis from a mixed culture was achieved with RL.5 at 4 degrees C. Overall, RL.5 and RL.5A were comparable as transport media whether held at 4 or 25 degrees C, but fewer organisms were recovered from BCYE alpha LA. In addition, Regan-Lowe (RL), Bordet-Gengou, and cyclodextrin media were compared as primary isolation media for recovering B. pertussis from simulated NP swabs held at 4 and 35 degrees C in RL.5 medium. The highest recovery of B. pertussis was obtained on RL primary isolation medium. Bordet-Gengou medium recovered only 80% and cyclodextrin medium recovered less than 60% of the numbers recovered on RL medium. Based on these results, refrigeration (4 degrees C) of NP swabs shipped in RL.5 transport medium and using RL as the primary isolation medium are recommended for recovering B. pertussis from swab specimens.     

Evaluation of swabs, transport media, and specimen transport conditions for optimal detection of viruses by PCR

Depletion of swabs and viral transport medium during epidemics may prompt the use of unvalidated alternatives. Swabs collected and transported dry or in saline were compared to commercially available swab/medium combinations for PCR detection of influenza, enterovirus, herpes simplex virus, and adenovirus. Each was detected at an ambient temperature (22°C) and 4°C for 7 days. Detection of influenza on dry or saline swabs is important because of its capacity to cause outbreaks involving large numbers of cases.     

Mailed, home-obtained urine specimens: a reliable screening approach for detecting asymptomatic Chlamydia trachomatis infections

The use of mailed, home-obtained urine specimens could facilitate screening programs for the detection of asymptomatic Chlamydia trachomatis infections. Since transport time could have an adverse effect on the sensitivity of C. trachomatis detection by PCR, the influence of DNA degradation on amplification was monitored over the course of 1 week. Therefore, urine specimens were aliquoted on the day of collection or arrival. Two groups of urine specimens were investigated. Group I contains first-void C. trachomatis-positive and -negative urine samples. DNA degradation was monitored in group I samples for 7 days at room temperature (RT) and at 4 degrees C by amplifying different lengths of the human beta-globin gene and the C. trachomatis plasmid target. DNA degradation was observed only for the larger human beta-globin fragments at days 5 to 7 at RT. In contrast, at 4 degrees C all targets could be amplified. Urine specimens were also frozen and thawed before aliquoting to mimic freezing during transport. This resulted in a lower sensitivity for the detection of C. trachomatis after thawing and 3 to 4 days at RT. In addition, mailed, home-obtained C. trachomatis-positive urine specimens (group II) were analyzed for 7 days after arrival by two commercially available C. trachomatis detection systems (PCR and ligase chain reaction [LCR]). The C. trachomatis plasmid target in mailed, home-obtained urine specimens could be amplified by both PCR and LCR after 1 week of storage and/or transport at RT. In conclusion, our findings indicate that mailed, home-obtained urine specimens are suitable for the sensitive detection of asymptomatic C. trachomatis infections by amplification methods, even if the transport time is up to 1 week at RT. These findings support the feasibility and validity of screening programs based on mailed, home-obtained urine specimens. Larger studies should be initiated to confirm our results.     

Evaluation of bacteriological swabs and transport media in the recovery of group B streptococci on laboratory media.

The survival of group B streptococci on a variety of swabs, held either in their containers or in transport media for periods of up to 48 hours, at room temperature and at 4 degrees C, has been assessed. Results indicated that holding swabs in transport media did not favour prolonged survival of the streptococci and that the yield of organisms was much greater from swabs held in their ordinary plastic tubes. A holding temperature of 4 degrees C rather than room temperature is recommended if any delay in plating out swabs is anticipated.     

Use of swabs without transport media for the Gen-Probe Group A Strep Direct Test

For several years we used rayon or Dacron swabs with liquid transport media for collection and transport of throat swab specimens for testing with the Gen-Probe Group A Strep Direct Test (GASDT). A report of favorable results with a Dacron swab without any transport media for GASDT by another laboratory prompted us to compare detection of group A streptococci (GAS) with and without transport media (referred to as "wet" and "dry" swabs, respectively). Phase one of this study used swabs seeded with GAS. Initially, six recent clinical isolates of GAS were inoculated onto wet and dry swabs and stored at room temperature (RT). After 1, 2, and 3 days of storage, colony counting and GASDT were performed with the swabs. The results, expressed as the mean percentage of the results at zero time, were as follows: for GASDT with wet swabs at 1, 2, and 3 days, 62, 51, and 56%, respectively; for GASDT with dry swabs at 1, 2, and 3 days, 105, 80, and 85%, respectively; for colony counts with wet swabs at 1, 2, and 3 days, 52, 26, and 13%, respectively; for colony counts with dry swabs at 1, 2, and 3 days, 10, 0, and 0%, respectively. An additional six strains of GAS were tested in a similar manner, except that extracts of pharyngeal flora (PF) were added to the inocula. The results obtained with extracts of PF were comparable to those obtained with GAS alone. We also compared the performance of GASDT with wet and dry swabs stored at RT and 4 degrees C. Ten strains of GAS were inoculated onto wet and dry swabs, and GASDT was performed each day for 9 days. The GASDT results for swabs on day 9, expressed as the mean percentage of the results obtained at zero time, were as follows: dry swab and 4 degrees C, 59%; wet swab and 4 degrees C, 31%; dry swab and RT, 33%; and wet swab and RT, 19%. In phase two of this study we conducted a clinical evaluation to determine whether the differences observed with seeded specimens would also be evident with patient specimens. We used a single dry Dacron swab paired with a single rayon Bacti-Swab with liquid Stuart transport medium for the clinical evaluation. Specimens were collected from 1,005 outpatients, plated onto a Strep Selective Agar plate, and then tested within 30 min by GASDT. If culture of GAS from the same swab is used to define a true-positive test result, the sensitivities and specificities were as follows: GASDT with wet swabs, 86.2 and 98.5%, respectively; GASDT with dry swabs, 90.7 and 98.1%, respectively. However, the use of culture as the "gold standard" may understate the actual performance characteristics of GASDT, particularly for the dry swabs. In conclusion, for GASDT the use of swabs without transport media may be preferable to the use of swabs with transport media.     

Preservation of tissue specimens during transport to mycobacteriology laboratories.

Chloramine-T and sodium borate solutions were evaluated for their effectiveness in preserving Mycobacterium bovis and controlling the growth of non-mycobacterial contaminants on tissue specimens during transport to laboratories. The number of culturable M. bovis cells in suspension was reduced by 5.1 log10 upon exposure to chloramine-T solution and by less than 1 log10 upon exposure to sodium borate solution for 7 days. Reinoculation of laboratory media (because of overgrowth by non-mycobacterial contaminants) was required for 52.6% of 190 routine bovine tissue specimens shipped refrigerated in chloramine-T solution and for 6.1% of 520 specimens shipped unrefrigerated in sodium borate solution. M. bovis was isolated from bovine tissue stored in sodium borate solution at 23 degrees C for 17 weeks and at 4 degrees C for 25 weeks. Unrefrigerated sodium borate solution has been used successfully to ship tissue specimens to our laboratory for the past 11 years.     

Evaluation of the virocult transport tube for isolation of herpes simplex virus from clinical specimens.

Herpes simplex virus survived in Virocult transport tubes and had a half-life of 3.5 days at 2 degrees C and 2.75 days at 22 degrees C. Of 2,000 consecutive clinical specimens transported on Virocult tubes and cultured for herpes simplex virus, 448 (22.4%) were positive. Comparison of the holding times between positive and negative cultures, up to 12 days, revealed no significant loss of positive cultures with time.     

Maintenance of viability and comparison of identification methods for influenza and other respiratory viruses of humans.

A comparison of Hanks balanced salt solution, veal infusion broth (VIB), and charcoal viral transport medium for maintaining viability of type A influenza virus indicated approximately equal survival of virus on all three media at -70 and 4 degrees C, whereas at 25 degrees C virus survived best in VIB. VIB supplemented with bovine serum albumin was used as transport medium in a community-wide surveillance of febrile respiratory disease for influenza viruses. Unfrozen throat swab specimens were placed in VIB and stored at 4 degrees C for up to 5 days without effect on isolation frequencies of either type A or type B influenza virus or type 1 or type 3 parainfluenza virus. Comparison of indirect immunofluorescence with hemadsorption for detection of type A influenza virus in rhesus monkey kidney cultures revealed a requirement for at least five fluorescing cells to eliminate false positive indirect immunofluorescence tests and at least 3 days of incubation to eliminate false negative tests when compared with hemadsorption at later times. Detection frequencies for the two methods after 2 and 3 days of incubation were not significantly different.     

Sorting and transepithelial transport of adsorbed protein tracers: effects of temperature

The epithelium of the guinea pig yolk sac is involved in the selective transport of macromolecules to the fetus. We studied the compartments involved in sorting and transepithelial transport of protein tracers and the effect of lowered temperature (18 degrees C) on these events. Explants of yolk sac were incubated with a mixture of cationized ferritin (CF) and horseradish peroxidase (HRP, Sigma type VI). At 4 degrees C, both tracers were bound to the cell surface and binding of an HRP-gold complex was shown to be inhibited by mannan. At 37 degrees C and 18 degrees C, both tracers were taken up into tubules and vesicles in the apical cytoplasm. Usually the tubules contained a mixture of tracers, but they often showed a polarized distribution with CF and HRP at opposite ends. The vesicles also contained mixtures of the tracers, but some contained only one. In addition, there were some irregularly shaped vacuoles composed of saccules that contained either a mixture, HRP alone, or CF alone. These results suggest that these adsorbed ligands are binding to unique microdomains of the endocytic complex. After 20 min at 37 degrees C coated vesicles 100 nm in diameter were located in the apical cytoplasm and coated vesicles of the same size were located at the lateral cell membrane. Usually they contained only HRP or CF, although occasional mixtures were seen. At 18 degrees C, HRP was transported across the cells in 100 nm vesicles. However, transport of CF was completely inhibited at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transport and culture conditions for optimum transformation responses of wildfowl lymphocytes to mycobacterial antigens

A method has been developed for increasing the survival of wildfowl lymphocytes during transport over considerable distances. Blood in an equal volume of heparinised RPMI was maintained at close to avian body temperature, i.e., approximately 40 degrees C. Using this system lymphocyte transformation in the presence of antigen (mycobacterial) has been successfully performed with wildfowl mononuclear cells for the first time. Duck cells were cultured in 10% autologous sera with 8 x 10(5) cells/well for 4 days. Cells from Hawaiian geese (Branta sandvicensis) required similar culture conditions but were incubated for 3 days.