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Characteristics of penicillinase release by washed cells of Bacillus licheniformis.

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly pH-dependent, occurring at a faster rate at alkaline pH in anionic or cationic buffers than at neutral pH. A strongly pH-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the pH to 6.0. Within 30 min at pH 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at pH 9.0 for 90 min. Penicillinase released at pH 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the pH to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the pH-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10(-4)m. Deoxycholate and Triton X-100 were active at 10(-3)m, but tungstate, arsenate, and molybdate had small effects at 10(-1)m. The rate of exopenicillinase release at pH 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at pH 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.

Antimetabolites↗

Swab-wash method for quantitation of cutaneous microflora.

We describe a comparison of the scrub-wash method of Williamson and Kligman and a swab-wash method for the enumeration of cutaneous microflora. The swab-wash method provides a less traumatic alternative to the scrub-wash method and can be used to sample the cutaneous microflora of premature neonates.

Candida albicans↗

Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples.

There is a need to develop noninvasive methods for the diagnosis of Pneumocystis carinii pneumonia in patients unable to undergo bronchoscopy or induction sputum. Oral wash specimens are easily obtained, and P. carinii nucleic acid can be amplified and demonstrated by PCR. In routine clinical use, easy sample processing and single-round PCR are needed to ensure rapid analysis and to reduce the risk of contamination. We developed a single-round Touchdown PCR (TD-PCR) protocol with the ability to detect PCR inhibition in the specimen. The TD-PCR was evaluated in a routine diagnostic laboratory and was compared to a previously described PCR protocol (mitochondrial RNA) run in a research laboratory. Both PCR methods amplified a sequence of the mitochondrial rRNA gene of P. carinii. Paired bronchoalveolar lavage (BAL) and oral wash specimens from 76 consecutive human immunodeficiency virus type 1-infected persons undergoing a diagnostic bronchoscopy were included. The TD-PCR procedure was quicker than the mitochondrial PCR procedure (<24 versus 48 h) and, compared to microscopy, had sensitivity, specificity, and positive and negative predictive values of 89, 94, 93, and 91%, respectively, for oral wash specimens and 100, 91, 90, and 100%, respectively, for BAL specimens. Our results suggest that oral wash specimens are a potential noninvasive method to obtain a diagnostic specimen during P. carinii pneumonia infection and that it can be applied in a routine diagnostic laboratory.

AIDS-Related Opportunistic Infections↗

Flow cytometric immunophenotyping of serous effusions and peritoneal washings: comparison with immunocytochemistry and morphological findings.

AIM: To evaluate immunophenotyping by means of flow cytometry as a complementary method for the detection of malignant cells in serous effusions and peritoneal washings. MATERIAL AND METHODS: Frozen samples of 49 fresh serous effusions and peritoneal washings were analysed by flow cytometry, using monoclonal antibodies against CD45, Ber-EP4, and N-cadherin. Results were compared with smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks. RESULTS: Seventeen specimens were cytologically diagnosed as malignant, whereas 25 were interpreted as benign. The remaining seven specimens were diagnosed as indeterminate or suspicious for malignancy. Ber-EP4 positive cells were detected in 16 of the 17 cytologically malignant effusions, as well as in five of seven suspicious cases and five of 25 specimens with benign cytology. In the latter group, three specimens showed atypical or malignant cell groups that were missed in routine morphological evaluation. In two additional samples, obtained from patients with benign and borderline ovarian tumours, Ber-EP4 positive cells showed benign or mildly atypical features, and were interpreted as exfoliated benign or borderline malignant epithelial cells of tubal origin, or as endosalpingiosis. All five Ber-EP4 positive indeterminate specimens showed atypical or malignant cells on re-evaluation, and were Ber-EP4 positive in four of five cases using immunohistochemistry in cell block sections. Large numbers of CD45 positive and relatively few N-cadherin positive cells were detected in most specimens with the use of flow cytometry, when compared with morphological evaluation. CONCLUSIONS: Flow cytometry is a rapid and highly effective method for the evaluation of effusions and peritoneal washings. The detection of Ber-EP4 positive cells using flow cytometry is strongly indicative of the presence of carcinoma cells in effusions and peritoneal washings. Although false positives are relatively infrequent, all specimens should be carefully evaluated morphologically to prevent the diagnosis of benign epithelial clusters as malignant.

Adult↗

Clinical features and management of recurrent balanitis; association with atopy and genital washing.

OBJECTIVE: To evaluate clinical features and diagnostic investigations in patients with recurrent or unresponsive balanitis in order to institute rational management. DESIGN: Forty-three patients presenting to a genitourinary medicine clinic with recurrent or persistent balanitis were studied. All patients were asked whether they had a history of atopic illness and about their practice of genital washing. All patients were investigated by taking a swab specimen from the preputial area for bacterial and viral culture and 30 underwent biopsy of the affected skin. Follow-up was between three and six months. SETTING: Outpatient genitourinary medicine clinic, St Mary's Hospital, London, UK. RESULTS: In 31 (72%) of the patients a diagnosis of irritant dermatitis was made. In comparison with the remaining patients, they had a greater lifetime incidence of atopic illness and more frequent daily genital washing with soap. For 28 (90%) of these patients, use of emollient creams and restriction of soap washing alone controlled symptoms satisfactorily. For the remaining 12 patients, a variety of diagnoses were made. Biopsy proved a well tolerated and diagnostic investigation, but the isolation of microbial pathogens from preputial swabs was irrelevant to management. CONCLUSION: A history of atopic illness and of the practice of penile washing are important aspects in the evaluation of patients with recurrent balanitis. Biopsy is an important investigation in the condition when it does not seem to be caused by irritant dermatitis.

Adult↗

Semen characteristics in HIV-1 positive men and the effect of semen washing.

We have undertaken an analysis of semen from HIV infected men with regard to sperm counts and motility, non-spermatozoal cells, and viral nucleic acid. Regression analysis showed that sperm concentration and motility were positively associated with blood CD4 cell count. By contrast, non-spermatozoal cell concentration (round cells) was inversely related to CD4 count. Extracellular HIV RNA was detected in the majority of semen samples and proviral DNA in a minority. Percoll gradient washing of 12 semen samples yielded six samples containing adequate sperm concentration for analysis. This washing procedure reduced prewash extracellular RNA to below detectable limits in all cases; proviral DNA present in two of the six prewash samples was also reduced to below detectable limits after washing. We conclude that semen washing before artificial insemination may reduce the risk of HIV transmission from an infected man to an uninfected woman. However, further evidence from prospective analyses of such an approach is required.

CD4 Lymphocyte Count↗

Effect of pre-slaughter washing of lambs on the microbiological and visible contamination of the carcases.

Studies in four slaughterhouses with contrasting dressing systems showed that microbiological contamination with total aerobic bacteria and Escherichia coli was greater on carcases which had been washed before slaughter, irrespective of wool length, and was generally higher on carcases derived from woolly lambs than on those derived from shorn lambs. The effects of pre-slaughter washing on the forequarters compared with the hindquarters were not consistent, and this was attributed to differences between the dressing systems applied in the slaughterhouses. Over all trials the mean total aerobic count ranged from 3.45 to 5.36 log10/cm2, and the mean counts of E coli ranged from 0.39 to 2.11 log10/cm2. There was less visible contamination on the carcases of washed lambs than on those of unwashed lambs. The differences in contamination with wool were largely dependent on the level of contamination of individual carcases, whereas the differences in contamination with faecal material and dirt or sand were largely dependent on the prevalence of affected carcases. The use of pre-slaughter washing to compensate in visual terms for poor pre-slaughter presentation results in a detrimental effect on microbiological loads on ovine carcases.

Abattoirs↗

Development of a cell wash buffer that minimizes nucleic acid loss from Clostridium perfringens 10543 A.

Autolytic activity and nucleic loss from Clostridium perfringens 10543 A was demonstrated during successive cell washes in hypotonic TES buffer. Autolysis increased nearly sixfold and nucleic acid loss nearly twofold when 10 mM EDTA was added to 0.3 M Tris-sucrose buffer. Attempts to minimize both autolysis and nucleic acid loss from C. perfringens during routine washing steps were unsuccessful when the effects of sucrose concentration, pH, CaCl2 addition, or wash temperature were examined independently. However, autolytic activity was eliminated and nucleic acid loss reduced to less than 5% when C. perfringens cells were washed at 4 or 25 degrees C in 1.0 M sucrose, 50 mM Tris--HCl, and 25 mM CaCl2 at pH 5.7.

Bacteriological Techniques↗

Bladder transitional cell carcinomas: a comparative study of washing and tumor bioptic samples by DNA flow cytometry and FISH analyses.

OBJECTIVE AND METHODS: We compared information obtained from samples of tumor biopsy and bladder washing to evaluate the representatives of the latter type of sampling. Both types of samples from 44 cases of superficial bladder TCCs and one papilloma were analyzed by FCM, to evaluate cellular DNA content, and FISH, to detect numerical aberration of chromosome 9. RESULTS: The use of both tumor and washing samples by FCM and FISH analyses evidenced alterations in 95% of cases. FCM evidenced higher aneuploid frequency in bladder washing than in bioptic specimens. In bladder washing, FISH analysis showed higher frequency of monosomy and lower frequency of trisomy than in biopsy. No correlation was found between histological grade and centromeric chromosome 9 loss while correlation was evidenced with centromeric 9 gain. CONCLUSION: Irrigation specimens, analyzed by FCM and FISH, can complement information obtained by biopsy in cytodiagnosis and follow-up of patients with bladder TCC.

Biopsy↗

Effects of washing and gel filtration on the ultrastructure of human platelets.

The ultrastructure of washed and gel-filtered platelets has been examined by scanning- and transmission-electron microscopy. Three different washing procedures previously established for functional studies have been employed. Each washing method resulted in typical morphologic alterations of the platelets. Gel filtration of platelet-rich plasma appeared less harmful than washing. Specimens fixed immediately after elution contained numerous platelets with typical shape changes which were restored almost completely after incubation at 37 degrees C. Our study emphasizes the need for cautious interpretation of results obtained with separated platelets and mandates morphologic controls for such experiments.

Blood Platelets↗

Influence of the freezing-thawing-washing procedure on the cytomegalovirus DNA content of cellular blood products.

There is a proven relation between cytomegalovirus (CMV) infection in seronegative immunocompromised patients and transfusion of random CMV unscreened cellular blood products (BP), where the leukocytes are thought to be the vector of transmission. We examined whether freezing, thawing, and washing, in attempt to reduce the quantity of leukocytes, also reduces the CMV-DNA-carrying cells of BP of CMV-seropositive donors and their infectivity for fibroblasts, using the polymerase chain reaction (PCR). The leukocyte contamination of the thawed-washed erythrocyte concentrates was < 1 x 10(8)/l and of the thawed-washed platelet concentrates, approximately 2.5 x 10(8)/l. All plasma samples and most samples of BPs of the CMV-seronegative controls were PCR-CMV-DNA-negative. The differences of the PCR results with the samples of CMV-seropositive donors before and after freezing were not significant. Thus we concluded that freezing-thawing-washing of cellular BP could not eliminate all CMV-DNA-bearing leukocytes. Plasma carries low risk for CMV infection, if at all.

Blood Component Removal↗

Paranuclear blue inclusions of small cell carcinoma of the stomach: report of a case with cytologic presentation in peritoneal washings.

BACKGROUND: Primary gastric small cell carcinoma is a rare but important entity. We describe a case that we diagnosed by peritoneal washing cytology. CASE: A 70-year-old male presented with upper abdominal discomfort and underwent endoscopic evaluation. Gastric endoscopy revealed a diffuse, infiltrating tumor from the body to the antrum. Total gastrectomy with lymph node dissection and intraoperative peritoneal washing cytology were carried out. Peritoneal washing cytology showed the presence of many undifferentiated malignant small cells with a necrotic background. The tumor cells were small and round, with naked, hyperchromatic nuclei and finely granular chromatin. Some tumor cells contained paranuclear blue inclusions (PBls) in the cytoplasm. The tumor cells were positive for neuron-specific enolase and synaptophysin on immunocyto-chemistry. Carcinoembryonic antigen, alpha-fetoprotein (AFP) and leukocyte common antigen were negative. Pathologic diagnosis after the operation was moderately to poorly differentiated adenocarcinoma and small cell carcinoma containing AFP-positive cells. CONCLUSION: The prognosis of primary gastric small cell carcinoma is usually poor. Our patient died of multiple liver metastases and peritonitis carcinomatosa 69 days after surgery. When a gastric small cell carcinoma is suspected in peritoneal washings, immunocytochemical demonstration of neuroendocrine differentiation is required to arrive at the final diagnosis.

Aged↗

Value of bladder wash cytology in the follow-up of patients with bladder carcinoma receiving intravesical chemoprophylaxis with mitomycin C.

511 bladder wash cytologic findings in 121 patients receiving intravesical chemoprophylaxis with mitomycin C after transurethral resection of stage pTa to stage pT2 bladder carcinoma were retrospectively analyzed. In 42 recurrent tumors, sensitivity of detection by cytology amounted to 67% in GIII tumors, 56% in GII tumors, and 20% in GI tumors. 16% of G0 tumors were cytologically positive. 33% of a total of 30 endoscopically detected recurrent GI-GIII tumors yielded negative results at bladder wash cytology, the findings were suspicious in 27%, while only 40% of all recurrences had a positive bladder wash cytology. Positive cytologic findings combined with normal endoscopic results were obtained in 24 patients. 15 of these (62%) developed recurrences after a mean interval of 15 months (3-60 months) during or after metaphylaxis. When suspicious findings were included, 20 of 30 carcinomas were detected, while 7 of 12 G0 tumors were overgraded. Bladder wash cytology has its merits for early detection of recurrences, since almost two thirds of the patients with positive cytologic findings and negative endoscopy developed a recurrent tumor at a later date. Whether or not positive cytology combined with negative endoscopic results should lead to therapeutic consequences remains to be discussed.

Administration, Intravesical↗

Presence of a bradykinin-like substance in pulmonary washings.

Pulmonary washings were studied for the presence of bradykinin-like substances and the capacity for their formation and degradation. Bronchopulmonary lavage with saline was performed in anesthetized, exsanguinated Syrian golden hamsters with a cell yield of 99 per cent alveolar macrophages. In the presence of inhibitors of both kinin formation (hexadimethrine) and destruction (1,10-phenanthroline), immunoreactive kinin was found in bronchopulmonary lavage fluid (12.5 ng per 5 x 10(6) cells lavaged), washings of pooled macrophages (5.62 ng per 5 x 10(6) cells washed), and lysates of alveolar macrophages (3.97 ng per 5 x 10(6) cells lysed). Higher kinin concentrations were obtained by selective inhibition of kinin-destroying enzymes with phenanthroline. Inhibition of kinin formation with hexadimethrine alone rendered nearly all concentrations below the limit of detectability. These findings demonstrated that the capacity for kinin formation and degradation exists in pulmonary washings. The precise origin of this kinin and its forming and destroying enzymes (whether from the pulmonary alveolar macrophage, other sites in the bronchopulmonary tree, or plasma) is not yet known.

Animals↗

Hand washing. Simple, cost effective, evidence based ... lip service!

Hand washing is so simple that we teach our toddlers to do it. It's so important in our every day lives that facilities for hand washing can be found in every conceivable environment. Scientists, literature and the media give evidence on the benefits and in the art of good techniques. So why, in an environment where the management of infection is a matter of health and safety for all, is hand washing so low on our ever-growing list of priorities? This article examines the legal and moral issues of hand washing in relation to health and safety, concentrating on possible reasons for non-compliance and how these can be addressed.

Cost-Benefit Analysis↗

Collection of semen from marmoset monkeys (Callithrix jacchus) for experimental use by vaginal washing.

Assisted reproductive techniques make an important contribution to the conservation of endangered primate species. In our laboratories marmoset monkeys (Callithrix jacchus) are used as a model species for developing assisted reproductive technologies for New World primates. The studies require a reliable method for collecting functional sperm from these small animals. For this purpose a minimally invasive procedure, vaginal washing after natural mating, was evaluated for its suitability as a routine method of obtaining ejaculated sperm of high quality. The objective of the first series of tests was to identify a behavioural pattern which was easily discernible by the observer and provided a reliable indication that ejaculation had occurred. In a second series of tests the influence of length of separation of the male prior to copulation on the quality of the ejaculate was evaluated. Six adult ovariectomized and five adult intact females were used for vaginal washing, while their mates served as donors of ejaculates. Matings at specific times were achieved by separating the males from their females for a certain time period and subsequently introducing either their own mates or unfamiliar males to the females. After each observed mating vaginal washing was performed on the unsedated females. The seminal samples obtained were analysed for sperm concentration, the proportion of motile sperm and the proportion of live sperm. The results have shown (a) that ejaculation was indicated with a reliability of 86.1% by the behaviour of the female, who terminates a copulation by moving away from her partner; (b) that separation of the male for up to six days had no negative effect on the sperm quality; (c) that the sperm samples were of high quality. Vaginal washing after natural matings appears to be a practical, reliable and gentle method for routinely collecting ejaculates from marmoset monkeys and possibly other New World primates.

Animals↗

Removal of cat major allergen (Fel d I) from futon (Japanese bedding) with a home washing machine.

We evaluated the removal of a cat major allergen (Fel d I) from futons (Japanese bedding) with the use of a large-sized home washing machine. Before and after washing a futon that had been used in a home with a cat, a small amount of cotton was collected from the futon and Fel d I was extracted from the cotton. The levels of Fel d I were assayed by a sandwich enzyme-linked immunosorbent assay (ELISA). We found that washing reduced the Fel d I level in futons by more than 95%. In conclusion, washing of futons is an effective method for elimination of their cat allergens.

Allergens↗

Evaluation of hypochlorous acid washes in the treatment of chronic venous leg ulcers.

OBJECTIVE: Hypochlorous acid (HOCl) is a highly microbiocidal agent active against bacteria, viruses and fungi. Using quantitative microbiology, preliminary studies showed it achieved an appreciable reduction in the bacterial burden in chronic venous leg ulcers. The study aimed to determine whether it has a role as an additional treatment for chronic venous ulcers that have not healed with conventional treatment. METHOD: On the basis of previous reports we designed a study in which patients acted as their own controls, in that only patients who failed to achieve a 44% reduction in wound size with standard treatment (compression bandaging) received HOCl washes. RESULTS: Of 30 patients admitted to the study, 10 achieved a 44% ulcer reduction after three weeks of standard treatment. In addition to the standard compression treatment, the remaining 20 patients were given HOCl washes over 12 weeks. Of the 20 ulcers, nine (45%) healed and five (25%) reduced in size by over 60%. All patients became free of pain. CONCLUSION: These findings confirm the clinical efficacy of treating venous leg ulcers with hypochlorous washes. Use of HOCl washes as an adjunctive therapy for recalcitrant venous leg ulcers appreciably increases healing and rapidly relieves pain.

Aged↗