Sterile Balanced Salt Solution Research Articles

Evaluation for endotoxin in intraocular materials used during phacoemulsification surgery using a recombinant factor C assay.

Cataract surgery remains the sole method to resolve blindness secondary to cataract formation. One complication includes fibrin web formation post-operatively. This study aimed to investigate the presence of endotoxin within materials used during cataract surgery as a possible cause of fibrin web phenomenon. Preservative-free epinephrine, heparin, viscoelastic devices, and intraocular lenses were collected for evaluation. Various manufacturers and manufacturing lot numbers were used when available. Viscosity of viscoelastics was reduced by incubating samples with human recombinant hyaluronidase. Intraocular product (IOL) packaging fluid was collected and stored for testing. The IOLs were then washed with a sterile balanced salt solution, incubated at 37°C for 48 h, and then fluid was collected for testing to mimic intraocular placement. Samples were tested using a commercially available rFC kit. Fluorescence was measured at time zero and after 1 h using a fluorescence microplate reader. The change in fluorescence was corrected for blank fluorescence and plotted to a standard curve. Endotoxin levels were below the limit of detection (0.05 EU/mL) in all samples. Incubation of IOLs at intraocular temperature did not increase extraction of endotoxin. Endotoxin was not identified in any tested sample, including those used in cases of fibrin web formation post-phacoemulsification. As fibrin webs are often observed episodically, it is possible that endotoxin levels may vary between batches, or that endotoxin is not related to fibrin formation.

Ultrasound Cyclo Plasty in Eyes with Glaucoma.

Glaucoma is a chronic disease caused by the progressive degeneration of the optical nerve fibers, resulting in decreased visual field that can lead to severe visual impairment, and eventually blindness. This manuscript describes a simple, surgeon-friendly, non-incisional technique, named Ultrasound Cyclo Plasty (UCP), for reducing intraocular pressure (IOP) in glaucoma patients. The technique determines a selective coagulation necrosis of the ciliary body; in addition, the stimulation of supra-choroidal and trans-scleral portions of the uveo-scleral outflow pathway has been recently proposed. UCP shows several technical improvements in ultrasound technology compared to previous techniques, providing more precise focusing on the target zone. The procedure is performed in the operating room under peribulbar anesthesia. Briefly, the coupling cone is put in contact with the eye and the ring probe, that contains six piezoelectric transducers which produce the ultrasound beams, is inserted inside it. Their proper centering over the ocular surface represents a crucial step for the correct targeting of the ciliary body. Sterile balanced salt solution is used to fill the empty spaces to ensure ultrasound acoustic propagation. Surgical treatment consists in the sequential automatic activation of each of the six transducers, for a total duration of less than 3 min. The patient leaves the hospital 1 h after the procedure with the treated eye patched. In the present study, 10 patients with open-angle glaucoma were followed-up during at least 12 months after the procedure. IOP was reduced at each interval compared to pre-operative, as well as the number of hypotensive medications. Twenty percent of patients did not respond to the treatment, and needed subsequent surgery to better control IOP. Treatment tolerability was good, with no cases of hypotony or phthisis. The UCP procedure is simpler, faster, safer, and less invasive than traditional cyclodestructive procedures with similar results in reducing IOP.

Evaluation of Microbial Flora in Eyes With a Boston Type 1 Keratoprosthesis

To evaluate the microbial flora of eyes with a Boston Keratoprosthesis (K-Pro). A prospective study was performed for 17 eyes of 15 patients who underwent a K-Pro implantation between September 2005 and June 2011. Preoperative diagnoses included failed corneal grafts, limbal stem cell deficiency, chemical burns, and Stevens-Johnson Syndrome. The patients used topical antibiotics after their surgery including a fluoroquinolone, polymyxin-trimethoprim, vancomycin, or a combination of the 3. The conjunctiva in the study eye was swabbed and cultured. A separate culture was taken of the contralateral eye as well. If available, the bandage contact lens was removed, and half of it was placed in thioglycolate broth, and half in 5 mL of a sterile balanced salt solution. The contact lens in the balanced salt solution was sonicated using a QSonica Q125 sonicator (Newtown, CT) for 1 minute, at an amplitude of 20%. Ten microliters of fluid was subsequently cultured. Of the patients who underwent the K-Pro surgery during that time period, 15 patients with 17 eyes were able to participate in the data collection. Nine of the 17 eyes implanted with the K-Pro (53%) had positive cultures. Two of the 13 (15%) of the control swabs exhibited bacterial growth. Eight percent (1/12) of the sonicated lenses were positive on culture, whereas 4/12 (33%) of the lenses placed in thioglycolate broth were positive for organisms. Despite being on antibiotics, eyes implanted with the K-Pro were more likely to have a positive conjunctival culture in our cohort as compared with that of fellow eyes.

Fundus Autofluorescence Findings in a Mouse Model of Retinal Detachment

Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD). RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4-5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis. Results. In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence. In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.

Toxic effect of using twenty percent alcohol on corneal epithelial tight junctions during LASEK

The aim of this study was to determine the effect of using 20% alcohol on corneal epithelial tight junctions during laser epithelial keratomileusis (LASEK). Sixty Sprague-Dawley rats were randomly divided into two equal groups. The central area of the rat corneas in one group were demarcated with a 3-mm trephine, treated with 20% alcohol for 45 sec and washed with sterile balanced salt solution. The epithelium was removed by an epithelial microhoe used in LASEK. In the other group, the rat corneal epithelium in the central area was mechanically scraped. The experimental animals were sacrificed at 24, 48, 72, 96 and 120 h after surgery. The levels of the tight junction proteins, claudin-1 and ZO-1, were determined by immunofluorescence and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses. We found that at approximately 48 h after surgery, the wounded corneas were replaced by corneals with regenerated epithelium. Immunofluorescence analysis demonstrated that the expressions of claudin-1 and ZO-1 in the corneal epithelium of the alcohol-treated group were weaker compared to the mechanical group at the 24 and 48 h time-points. Semi-quantitative RT-PCR analysis showed that the mRNA levels of claudin-1 and ZO-1 in the central cornea after alcohol treatment were lower compared to those in the mechanical group from 24 to 48 h, with no significant difference after 72 h. Thus, we conclude that the treatment with 20% alcohol during LASEK results in damage to the corneal epitheleal tight junctions and prolongs normal recovery time.

Profound postoperative hypotony with globe collapse after 25-gauge pars plana vitrectomy.

To report a series of cases of profound hypotony with globe collapse on postoperative day 1 after sutureless 25-gauge pars plana vitrectomy and to identify common risk factors for this complication. Retrospective chart review of three patients. Ocular history and surgeries, preoperative diagnosis, operative technique, postoperative and final visual acuities, reformation intervention, and need for additional surgery were reviewed. Three cases were reviewed. Patient ages were 22, 53, and 53 years. All patients had a history of a rhegmatogenous retinal detachment repaired by surgery and all had at least one vitrectomy. Two had a prior scleral buckle and two had pathologic myopia. All patients underwent sutureless 25-gauge pars plana vitrectomy. No patient had intraocular air, gas, or silicone oil tamponade. All patients presented on postoperative day 1 with globe collapse, hand motion vision, and scleral infolding. All patients underwent office-based injection of sterile balanced salt solution or air. One patient required an additional procedure-operative closure of sclerotomies and intraocular gas tamponade. All three patients attained a final Snellen visual acuity of 20/50 or 20/60. These cases represent a small fraction (0.12%) of patients who underwent 25-gauge pars plana vitrectomy by one surgeon. Patients had several risk factors, including severe eye disease, prior retinal detachment repair, prior vitrectomy, pathologic myopia, and lack of intraocular tamponade. In patients with several risk factors, consideration should be given for intraocular gas or air injection or sclerotomy closure.

Spectroscopic study of explanted opacified hydrophilic acrylic intraocular lenses

To report a detailed spectroscopic analysis of explanted hydrophilic acrylic intraocular lenses (IOLs) that were removed because of postoperative opacification of the lens optic. Thirteen Hydroview H60M (Bausch & Lomb Surgical) IOLs were explanted from 13 different patients on average 56 months after phacoemulsification and IOL implantation. All patients had decreased visual acuity because of a fine granularity of the optical surface of the IOLs. The surface was investigated by gross, microscopic, histochemical and scanning electron microscopic analysis, and the elemental composition of the opacified IOLs was determined by X-ray fluorescence spectroscopy (XRF). The spectrograms were compared to three different originally packed and never-implanted hydrophilic acrylic IOLs. Light and scanning electron microscopy of the optical surface of explanted IOLs revealed multiple fine granular deposits varying in size and shape that were positive for alizarin red. XRF confirmed that the explanted IOLs contained not only the previously reported calcium and phosphorous (calcium apatite), chlorine, silicone, sodium, aluminum and magnesium but also iron, sulfur, potassium as well as lesser amounts of iodine, zinc, strontium and yttrium. This is the first spectroscopic analysis determining the content of more than 10 elements of explanted and originally packed never-implanted hydrophilic acrylic IOLs. The possible origin of the different elements obtained from the spectrograms and their implications are discussed.

Implementation of Combined Storage of the Hematopoietic Cord Blood Cells and Fetal Mesenchymal Stromal Cells (umbilical cord stromal cells, UCSC) from the Same Donors in Warsaw Cord Blood Bank

Project design and rationale: Expanding clinical evidence suggests, that combined transplantation of allogeneic hematopoietic stem cells (HSC) and mesenchymal stromal cells (MSC) improves the results when compared to the transplantation of HSC only by the enhancement of engraftment or reduction of the frequency and gravity of graft versus host (GVH) disease. We have modified our method [1] of the processing and liquid nitrogen–storage of umbilical cord stromal cells (UCSC, the analogue of adult bone marrow MSC) for the combined collection and banking of cord blood hematopoietic stem cells (CB HSC) and UCSC obtained from the same donor for the hematopoietic transplantation purposes. Practical outcome of the project is the availability of cryopreserved combinations of cord blood samples plus the same donor-derived fetal MSC-type cells for clinical applications.Methods: Cord blood (CB) samples were collected and processed according to the routine protocol of Warsaw cord blood bank. Cord blood samples (vol.≥90 ml) were collected before or after placenta delivery, and viability and sterility-tested suspensions of mononuclear cells were volume-reduced and frozen in 10% DMSO/5% albumin using controlled-rate freezer, and stored in liquid nitrogen storage tanks. Umbilical cords, collected from the same deliveries as CB, were transported for processing in sterile balanced salt solution (PBS) supplemented with Penicillin/Streptomycin (22–25°C, time until processing ≤24h). Following mounting on the dissecting table, they were cut into 7 cm pieces, and surgically dissected in sterile conditions. After removal of cord amnion and blood vessels, Wharton jelly was cut into strips of thickness < 2 mm, sealed in 2 freezing bags in 10% DMSO/5%albumin and frozen in control-rate freezer. For experimental purposes, another strip of Wharton jelly was frozen in third bag, stored for 14 days, thawed, and the UCSC were in vitro expanded throughout consecutive 5 passages, phenotype-analysed, and tested for their ability for adipo-chondro-, and osteogenesis. Remaining fragments of umbilical cord (blood vessels, Wharton jelly pieces) were sterility-tested, and the contaminated frozen samples were eliminated at the quarantine stage. Maternal blood examination, HLA-typing, family history analysis etc. obligatory tests for CB banking were done only once, and the results were valid both for CB HSC and UCSC, reducing the labor time and costs of tests.Results: Both in cord blood samples, and umbilical cords, the frequency of bacterial/fungal infections was <1.5%, and viability of cells was>99%. The frequency of successful expansion of frozen/thawed UCSC was >90%, and average UCSC number after expansion was 2.5× 1011cells. All UCSC were plastic adherent, expressed CD34−, CD45−, Lin1−, CXCR4−, CD29+, CD73+, CD90+, CD105+ surface markers, and were able to differentiate into adipo-, chondro-, and osteogenic lineages, thus confirming their MSC characteristics. Before the full validation of the procedures, CB HSC and UCSC obtained from the same donor are stored in Warsaw cord blood bank in separate storage tanks in −170°C in liquid nitrogen vapors. If the transplantation of cord blood cells plus umbilical cord MSC-like cells will be accepted as the standard clinical protocol, the double cassette for combined CB HSC/UCSC storage will be implemented.Conclusion: The practical result of the presented study is the implementation of the new protocol allowing for availability of cord blood cells and mesenchymal stromal cells from the same donor for clinical transplantation purposes.

Methotrexate Reduces the Complications of Endophthalmitis Resulting from Intravitreal Injection Compared with Dexamethasone in a Rabbit Model

The incidence of infectious endophthalmitis associated with intravitreal injection (IVI) of steroid has been reported to be as high as 0.87%. This study was designed to investigate whether the antimicrobial activity of intravitreal methotrexate (MTX) alters the incidence or course of bacterial endophthalmitis associated with IVI in a rabbit model. A rabbit model of endophthalmitis induced by Staphylococcus epidermidis (S) was established. Six groups of rabbits had IVI of sterile balanced salt solution (BSS), MTX (400 microg), dexamethasone (Dex, 200 microg), S, S and MTX (S-MTX), or S and dexamethasone (S-Dex). On days 0, 1, 3, 6, 10, and 14, total intraocular inflammation was measured in each animal. Vitritis was graded by the degree of vitreal haze. An intravitreal tap was performed on two animals from groups S, S-Dex, and S-MTX. A histopathologic study was performed on day 14. No endophthalmitis was observed in the control groups BSS, MTX, and Dex. Group S-Dex had the highest and group S-MTX had the least total ocular inflammation and vitritis scores from days 3 to 14. The difference in total inflammation and vitritis among groups S, S-Dex, and S-MTX is significant (P = 0.046 and P = 0.001, respectively). Live bacteria were isolated only from groups S and S-Dex. Pathology revealed severe ocular destruction in groups S and S-Dex and intact structures in group S-MTX. MTX appears to reduce the risk of development of bacterial endophthalmitis and ocular destruction associated with IVI compared with Dex. IVI of MTX may be a safer alternative than steroid injection in treating noninfectious uveitis.

Fluoroquinolone therapy in Mycobacterium chelonae keratitis after lamellar keratectomy

To characterize a rabbit model of Mycobacterium chelonae keratitis after lamellar keratectomy and assess the effectiveness of fluoroquinolone therapy. University Laboratory, University of California, Irvine, California, USA. Twenty-eight New Zealand white rabbits had unilateral lamellar keratectomy with placement of 2.5 x 10(5) colony-forming units of log-phase M chelonae under each flap. Eyes (7 per group) were randomized and treated with sterile balanced salt solution, gatifloxacin 0.3%, ciprofloxacin 0.3%, or levofloxacin 0.5% 4 times daily. Two masked observers examined all eyes on days 2, 5, and 7 and weekly for 4 weeks. Severity of disease and bacterial culture results were the main outcomes measured. The means and standard deviations were calculated, and differences between the groups were statistically analyzed. All eyes developed clinical disease. At the time the rabbits were killed, eyes treated with balanced salt solution, ciprofloxacin, levofloxacin, and gatifloxacin were culture positive in 6 (85.7%), 7 (100%), 6 (85.7%), and 3 (42.9%) of 7 eyes per group, respectively. Frequency of positive culture and the severity of clinical disease in gatifloxacin-treated eyes were significantly less (P < .05) than in the other groups combined. The rabbit model of M chelonae keratitis was successfully developed in our study. A fourth-generation quinolone (gatifloxacin) showed the best performance among the fluoroquinolones tested in our experimental approach. The fourth-generation fluoroquinolone, gatifloxacin, could be effectively used for the treatment of mycobacterial keratitis.

193-nm Excimer Laser–Induced Fluorescence Detection of Fluoroquinolonesin Rabbit Corneas

To measure the 193-nm excimer laser-induced fluorescence of fluoroquinolone-treated cadaver rabbit corneas. Prior to ablation with a commercially available ophthalmic excimer laser (Nidek EC-5000; Nidek Technologies, Pasadena, Calif), 35 cadaver rabbit corneas were treated with topical sterile balanced salt solution, 0.3% tobramycin sulfate, or the fluoroquinolones-0.3% ofloxacin, 0.5% levofloxacin, 0.3% ciprofloxacin hydrochloride, or 0.3% gatifloxacin. The fluorescence generated from each ablated corneal layer was measured and used to identify the presence of antibiotic. This was achieved by training a partial least-squares model to discriminate between the fluorescence spectra of antibiotic-treated and antibiotic-free (healthy) cornea. Antibiotic concentrations down to 0.06 microg/mL were detected with high accuracy. Assuming a constant ablation rate of 0.3 microm per laser pulse, the number of corneal layers ablated to reach antibiotic-free cornea is used to calculate the penetration depth of the antibiotic. The mean +/- SD penetration to a detectable depth was as follows: 0.3% ofloxacin, 7.1 +/- 3.0 microm; 0.5% levofloxacin, 6.7 +/- 1.4 microm; 0.3% ciprofloxacin, 1.2 +/- 0.6 microm; and 0.3% gatifloxacin, 7.0 +/- 1.9 microm. The penetration depth of 0.3% tobramycin could not be determined because its fluorescence spectrum overlapped with that of the native cornea. Topical administration of fluoroquinolone-containing solutions results in measurable differences in laser-induced corneal fluorescence. Under these experimental conditions, 0.3% ofloxacin, 0.5% levofloxacin, and 0.3% gatifloxacin all appear to penetrate the epithelium significantly more than 0.3% ciprofloxacin (P<.02). Clinical Relevance Monitoring of laser-induced fluorescence may be helpful in determining the penetration depths and concentrations of topically applied fluoroquinolones within the cornea.

Fluoroquinolone therapy in multiple-drug resistant staphylococcal keratitis after lamellar keratectomy in a rabbit model

PurposeTo assess the effectiveness of a fourth-generation fluoroquinolone for prophylaxis against multiple drug-resistant staphylococcal keratitis after lamellar keratectomy in a rabbit model. DesignExperimental study. MethodsTwenty-eight New Zealand white rabbits underwent unilateral lamellar keratectomy using a manual microkeratome followed by the placement of 1,000 colony-forming units (CFUs) of log-phase Staphylococcus aureus bacteria under each flap. Eyes (seven in each group) were randomized and treated with one of the following agents: sterile balanced salt solution, gatifloxacin (0.3%), ciprofloxacin (0.3%) or levofloxacin (0.5%) immediately and 6, 12, and 18 hours after surgery. Inflammation was graded by two masked observers at 24 and 48 hours, and the presence or absence of infectious infiltrates was determined. The means and standard deviations were calculated, and differences among the groups were statistically analyzed. ResultsThere were no flap complications encountered during surgery. Eyes treated with ciprofloxacin, levofloxacin, and balanced salt solution developed infectious infiltrates in five of seven eyes per group. Gatifloxacin-treated eyes did not develop clinical infection and exhibited lower mean inflammation scores (P < .01 compared with the other groups). ConclusionsThe fourth-generation fluoroquinolone, gatifloxacin, is an effective prophylaxis against the development of keratitis after lamellar keratectomy in rabbits with an organism resistant to methicillin, levofloxacin, and ciprofloxacin.

The effectiveness of a topical antibiotic irrigating solution in a model of staphylococcal keratitis after lamellar keratectomy

PURPOSE: To create a model of Staphylococcus aureus keratitis after lamellar keratectomy; to assess the toxicity of an antibiotic irrigating solution on the corneal stromal bed; and to test the chemotherapeutic effectiveness of a topical antibiotic, both alone and with an antibiotic-containing irrigating solution in preventing S. aureus keratitis after lamellar keratectomy. METHODS: The right eye of each of 38 rabbits were used in this study. In 18 eyes, a lamellar flap was created with a microkeratome, and an inoculum of S. aureus (either 1,000, 5,000, or 50,000 CFUs) was instilled under each flap; the eyes were examined for signs of infection and inflammation at 24 and 48 hours. In another five eyes, a lamellar flap was created in the same manner and the stromal bed was irrigated with 0.3% ofloxacin; the eyes were assessed for ocular inflammatory changes and evidence of crystalline deposits. Finally, in each of 15 additional eyes, 1,000 CFUs of S. aureus were instilled under a lamellar flap to create experimental infectious keratitis. The keratitis was treated according to three regimens: irrigation of the stromal bed with sterile balanced salt solution; irrigation of the stromal bed with 0.3% ofloxacin, followed by application of topical ofloxacin four times a day; application of topical ofloxacin only, four times a day. Eyes were examined for infection and ocular inflammatory changes at 24 and 48 hours. RESULTS: Staphylococcus aureus keratitis can consistently be produced under the stromal flap by inoculation of relatively few organisms. Irrigation of the stromal bed with commercial-strength topical ofloxacin does not appear to be toxic to the stromal bed, with no evidence of crystalline precipitates of the antibiotic. In our model of infectious keratitis after lamellar keratectomy, both topical ofloxacin alone and the combination of topical ofloxacin and irrigation of the stromal bed with 0.3% ofloxacin were effective at preventing S. aureus keratitis. However, the combined treatment of antibiotic irrigation plus topical antibiotic was more effective at preventing inflammation than topical ofloxacin alone. CONCLUSIONS: In this model of S. aureus keratitis after lamellar keratectomy, irrigation of the stromal bed with antibiotic plus topical antibiotic appears to be both safe and effective for preventing infection.

INTRAVITREAL INJECTION OF HYALURONIDASE CANNOT INDUCE POSTERIOR VITREOUS DETACHMENT IN THE RABBIT

To investigate whether intravitreal injection of hyaluronidase can induce posterior vitreous detachment (PVD) in the rabbit. One eye each of 12 New Zealand white rabbits received intravitreal injection via the pars plana of 20 IU of hyaluronidase (0.1 mL reconstituted in sterile balanced salt solution [BSS]) into the midvitreous cavity. The fellow eye of each rabbit received a vitreous injection of 0.1 mL of BSS. At 3 and 6 months after intravitreal injection, four and eight rabbits were killed, respectively, and the eyes were enucleated. After fixation, scanning electron microscopy was performed to study the vitreoretinal interface. At 3 and 6 months after injection, scanning electron microscopy showed that the retinal surfaces in eyes that received either hyaluronidase or BSS were covered with vitreous collagen fibers. No eyes, even those that received hyaluronidase over a period of 6 months, had the smooth retinal surface consistent with a bare internal limiting lamina that suggests the development of PVD. Hyaluronidase cannot induce PVD in the rabbit over a 6-month period after vitreous injection.

Comparative Intravitreal Antibiotic Therapy for Experimental Enterococcus faecalis Endophthalmitis

To evaluate the most effective intravitreal antibiotic treatment of vancomycin-sensitive and -resistant Enterococcus faecalis endophthalmitis. Animal experiment. Seventy-eight New Zealand white rabbits received an intravitreal injection of 10(5) vancomycin-sensitive or -resistant E faecalis organisms in one eye. Infections were allowed to proceed 3 hours before dividing animals randomly into the following treatment groups (n = 6, each): the vancomycin-sensitive E faecalis model--(1) vancomycin (1 mg/0.1 mL), (2) combined vancomycin (1 mg/0.1 mL) and amikacin (0.4 mg/0.1 mL), (3) combined vancomycin (1 mg/0.1 mL) and gentamicin (0.1 mg/0.1 mL), (4) combined vancomycin (1 mg/0.1 mL) and ceftazidime (2 mg/0.1 mL), (5) combined ampicillin (5 mg/0.1 mL) and gentamicin (0.1 mg/0.1 mL), and (6) pristinamycin (1 mg/0.1 mL); and the vancomycin-resistant E faecalis model--(1)same as above, excluding group 4. Control groups received sterile balanced salt solution. Twenty-four hours following intravitreal treatment, vitreous humor was collected for quantitative bacteriological studies. Intravitreal therapy with combined vancomycin and amikacin provided the most effective reduction of vancomycin-sensitive E faecalis organisms compared with combined vancomycin and gentamicin therapy (P =.10, Wilcoxon's rank sum test) or any other treatment group (P < .01, Wilcoxon's rank sum test). For vancomycin-resistant E faecalis endophthalmitis model, the combined ampicillin and gentamicin therapy was the most effective, followed by the combined vancomycin and amikacin therapy (P < .01, Wilcoxon's rank sum test). Treatment with intravitreal vancomycin plus amikacin and with intravitreal ampicillin plus gentamicin provide an effective bactericidal therapy for severe experimental vancomycin-sensitive and -resistant E faecalis endophthalmitis, respectively.

Anterior chamber fluid contamination after uncomplicated phacoemulsification

This study determined the rate of anterior chamber aerobic bacterial contamination in 103 eyes that had uncomplicated posterior chamber phacoemulsification. Anterior chamber fluid was aspirated on completion of surgery. Cultures of sterile balanced salt solution served as a control. Of 103 anterior chamber cultures, 4 (3.9%) showed contamination; however, 3 of 103 control cultures (2.9%) also grew micro-organisms. These findings suggest a very low rate of aerobic bacterial contamination after uncomplicated phacoemulsification and underscore the importance of control cultures when determining contamination rates.

Prevention of Keratocyte Loss After Corneal Deepithelialization in Rabbits

To determine if keratocyte loss, after removal of the corneal epithelium, can be prevented by a collagen shield used alone or in combination with a topically applied corneal preservation medium. Twenty-four New Zealand white rabbits were divided into six groups of four rabbits each. The central 6 mm of corneal epithelium was removed from one eye of each animal by means of a blunt spatula. Postoperatively, these eyes were treated every 4 hours for 24 hours with the following: group 1, balanced salt solution (BSS) drops (Akorn Inc, Metairie, La); group 2, Optisol drops (Chiron IntraOptics, Irvine, Calif); group 3, a collagen shield soaked in sterile BSS plus BSS drops; group 4, the same collagen shield soaked in Optisol plus Optisol drops; group 5, a different collagen shield soaked in sterile BSS plus BSS drops; and group 6, the same collagen shield soaked in Optisol plus Optisol drops. The animals were killed at 24 hours after surgery, the corneas were fixed, and keratocytes within the anterior and posterior cornea, beneath the epithelial defect, were quantitated. Four untreated rabbits served as controls. Optisol drops alone, applied every 4 hours after deepithelialization, did not prevent keratocyte loss to a greater extent than did BSS drops alone (P = .96). Both collagen shields soaked in sterile BSS plus BSS drops every 4 hours were associated with less keratocyte loss than were BSS or Optisol drops alone. Both collagen shields soaked in Optisol plus Optisol drops every 4 hours were most successful at minimizing keratocyte loss (P = .0002 and P = .001). After corneal deepithelialization, use of a collagen shield in combination with topical application of a corneal storage medium may minimize keratocyte loss and may thus be beneficial after refractive surgery.

Separation of Gametocytes of Leucocytozoon smithi from Whole Peripheral Turkey Blood

A technique that uses a Ficoll density-gradient system was devised for isolating gametocytes of the parasitic blood protozoan Leucocytozoon smithi from whole turkey blood. Turkey blood containing gametocytes was layered onto a 35% solution of Ficoll and centrifuged. A white band, containing gametocytes and leukocytes, was observed just below the Ficoll-plasma interphase, clearly separated from the underlying packed erythrocytes. The gametocyte-leukocyte suspension was then subjected to a second centrifugation on a continuous gradient of Ficoll ranging from 12% at the top of the tube to 35% at the bottom. The second centrifugation separated the white cells containing the gametocytes into 5 distinct layers: 1) thrombocytes; 2) gametocytes of L. smithi; 3) lymphocytes; 4) a band of monocytes; and 5) granulocytes. Recovered gametocytes, stored at 4 C in a sterile balanced salt solution, retained their morphological integrity for a minimum of 4 weeks.

Influence of pH, Serum, and Cell Cultures on Development of Ascaris suum to Fourth Stage In vitro

Ascaris suum larvae hatched from decoated eggs developed to parasitic stages in media KW-1, KW-1A, KW-1S, and KW-1SA, when each was used alone or as the culture fluid with swine kidney cell cultures. Media KW-1 and KW-1S were formulated (Douvres et al., 1966) with 50% bovine or swine serum, respectively, and medium NCTC 109, yeast extract, peptone, and dextrose; media KW-1A and KW-1SA are variations that were made by adjusting the pH to 6.9. Based on a culture period of 30 days, larvae developed to early or late phases of third stage in all 8 combinations, but to fourth stage only in systems with medium KW-1A. Despite yields of up to 90% late thirdstage larvae, only a few fourth stage were produced. The most rapid development, highest rate of survival, and largest yields of advanced stages were obtained in cultures of medium KW-1A with cells. The primary factors influencing development in the 8 culture combinations were pH, serum source, and the presence of cultivated cells. Previous attempts to cultivate Ascaris suum in complex cell-free media have had limited success (Pitts and Ball, 1953, 1955; Pitts, 1960, 1962, 1963; Cleeland and Laurence, 1962; Cleeland, 1963; Levine and Silverman, 1969). Since ascaris larvae traverse host tissues, it occurred to us that the cell culture system developed for in vitro cultivation of Stephanurus dentatus (Douvres et al., 1966) might provide a more favorable environment. Morphological criteria used to identify larval stages grown in vitro were developed chiefly from observations made during the present study and, to a lesser extent, on reevaluations of previous in vivo work (Roberts, 1934; Nichols, 1956). These criteria were subsequently used as the basis for a study of morphogenesis in vivo. Since the detailed descriptions arising from this work (Douvres et al., 1969) pertain equally to in vitro-grown larvae, we have limited descriptions in this report to certain features (stunting, delayed organogenesis, abnormalities) which are peculiar to larvae grown in vitro. MATERIALS AND METHODS Host tissues and sera used in the present study were obtained from animals raised and maintained as previously described (Douvres et al., 1966). Decoated embryonated ascaris eggs were from Stock I, prepared as previously described (Douvres et al., 1969). Media and cell cultures Eight combinations of media and cell cultures were used (Table I). Media KW-1 and KW-1S Received for publication 18 July 1969. were prepared as previously described (Douvres et al., 1966). After preliminary work with these media indicated that a lower initial pH was more favorable for ascaris, media KW-1A and KW-1SA were made by adjusting the pH from 7.9 to 6.9 with HC1. All pH measurements of stock media and of transferred and terminated cultures were made with a glass electrode in an open vessel. Primary cell cultures in 4-oz prescription bottles and in milk dilution bottles were prepared essentially as described by Douvres et al. (1966). However, they were grown and maintained in medium 199 (Morgan et al., 1950) supplemented with 20% calf serum. A cell line, subcultured from primary cells, was also grown and maintained in this medium in 32-oz prescription bottles. All vessels were provided with teflon-lined screw caps. Cell cultures were used when they showed a confluent sheet of cells, usually after 4 to 6 days. Adherence of the cells to the vessel was the criterion of viability. Hatching and preparation of the larval inoculum These procedures, with some exceptions, were essentially identical to those described by Cleeland and Laurence (1962). Eggs stored in 2% formalin at 5 C for 5 to 8 months were transferred to centrifuge tubes and placed in a water bath at 37 to 39 C for 2 hr. After 6 to 8 washes in sterile balanced salt solution (Fenwick, 1939), they were washed twice with medium NCTC 109 containing 1,000 units penicillin G potassium, 1 mg dihydrostreptomycin sulfate, and 10 jug Amphotericin B per ml. This preparation was also used as the hatching fluid. About 2 to 4 million eggs were transferred to 125-ml screw-capped flasks containing a spinner bar and the volume brought to 20 ml. The flasks were then held in a water bath (37 to 39 C) over a magnetic stirrer. After about 3 hr, 40 to 50% of the eggs had hatched. The contents of the flasks were transferred to tubes, centrifuged at 700 g for 5 min, and the supernatant fluid dis-