A simple staining method for cryptosporidian oocysts and sporozoites

A useful staining method for detection of cryptosporidian oocysts and sporozoites was developed and described. The modified Kohn's one-step staining technique with an additional modification, i.e., longer staining time and higher staining temperature than those originally described, was tested on fecal smears from cats infected with Cryptosporidium sp. By this improved staining procedure, the oocyst appeared as a slightly oval body containing four internal sporozoites colored blue to blue-gray. The oocyst wall was stained dark green to black. The morphological feature of the oocyst was recognized much more clearly by this staining technique than by such currently used techniques as acid-fast and Giemsa staining. This staining procedure proved to be simple and less costly and to secure a good preservation.     

Simple and rapid staining for detection of Entamoeba cysts and other protozoans with fluorochromes

Three fluorochromes were applied to stain various parasitic protozoans. By double staining with 4',6-diamidino-2-phenylindole and propidium iodide, differentiation of the nuclei from the cytoplasm can easily be achieved within several seconds. The chromatoid bodies in Entamoeba cysts were stained bright red. Plasmodium yoelii at all stages except late trophozoites and young gametocytes was easily identified. In the oocysts of Cryptosporidium sp., the nuclei and cytoplasm of the sporozoites fluoresced bluish white and red, respectively, whereas the residual body appeared blue or green. The third fluorochrome, Calcofluor white M2R, was suitable for detecting the cysts of Entamoeba spp. and Chilomastix mesnili.     

Chemical and physical factors affecting the excystation of Cryptosporidium parvum oocysts.

Cryptosporidium parvum oocysts were examined to ascertain excystation requirements and the effects of gamma irradiation. Oocysts and excysted sporozoites were examined for dye permeability and infectivity. Maximum excystation occurred when oocysts were pretreated with acid and incubated with bile salts, and potassium or sodium bicarbonate. Pretreatment with Hanks' balanced salt solution or NaCl lowered excystation; however, this effect was overcome with acid. Sodium ions were replaceable with potassium ions, and sodium bicarbonate was replaceable with sodium phosphate. Oocysts that received 200 krad irradiation excysted at the same rates as nonirradiated oocysts (95%), the excystation rates were lowered (50%) by 2,000 krad, and no excystation was observed by 5,000 krad. No differences were observed between the propidium iodide (PI) permeability of untreated oocysts and oocysts treated with 200 krad, while 92% of oocysts were PI positive after 2,000 krad. Most of the sporozoites exposed to 2,000 krad were not viable as indicated by the dye permeability assay. The oocysts irradiated with 200 and 2,000 krad infected cells, but no replication was observed. The results suggest that gamma-irradiated oocysts may still be capable of excystation and apparent infection; however, because the sporozoites could not reproduce they must not have been viable.     

Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients

Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The oocyst recovery method utilized 2 sequential discontinuous sucrose gradients followed by 1 Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls.     

A comprehensive evaluation of imidazole‐zinc reverse stain for current proteomic researches

In this paper, we comprehensively evaluated the capability of imidazole‐zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2‐D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two‐third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.     

Utility of papanicolaou stain-induced fluorescence in the cytodiagnosis of extrapulmonary tuberculosis

OBJECTIVE: To study the utility of Papanicolaou stain-induced fluorescence (PIF) in the detection of tubercle bacilli and to compare its diagnostic efficacy with that of the conventional Ziehl-Neelsen (ZN) method. STUDY DESIGN: This prospective study was carried out at a tertiary health care center, over a period of 2 years between January 2001 and December 2002. A total of 500 cases offine needle aspiration cytology from lymph nodes and other extrapulmonary sites were studied. Only cases that were clinically and cytologically suggestive of tuberculosis were included in the study. The smears were stained with ZN and Papanicolaou stain and examined under light and fluorescence microscopes, respectively for detection of acid-fast bacilli (AFB). Mycobacterial culture was used as the gold standard to compare the results. RESULTS: Cytologic smears were categorized into 4 distinct cytomorphologic patterns: epithelioid granulomas without caseous necrosis (101 cases), epithelioid granulomas with caseous necrosis (268 cases), caseation or acute inflammatory exudate only (114 cases), and occasional epithelioid cells without necrosis or giant cells (17 cases). The overall AFB positivity was 30.8% with the ZN method, while it was 40.6% with PIF. Moreover, PIF was more effective in detecting bacilli in group I lesions (18.8% vs. 6.9% with ZN method), in which the bacillary load is very low. CONCLUSION: PIF is superior to the conventional ZN method in detecting tubercle bacilli, particularly when the bacillary load is low. It is a relatively inexpensive and fast technique.     

Combination of Gomori and Ziehl-Neelsen Stains for Showing Acid Phosphatase Activity and Mycobacterium tuberculosis in Macrophages

Tubercle bacillus-infected macrophage monolayer cultures, after fixation in glutaraldehyde or without fixation, are stained by a Gomori method to show acid phosphatase activity. The method includes H,S to convert lead phosphate to sulphide. Only a minority of the mycobacteria is outlined by the black or brown stain; the similarity to the surrounding stained cytoplasmic particles makes identification difficult. The Gomori staining procedure is followed by a Ziehl-Neelsen method to stain acid-fast microorganisms; the temperature of the carbol fuchsin is just high enough to produce steaming, and the time of decolorisation is short. To avoid loss of the Gomori stain from the acid-fast procedure it is essential to repeat the exposure to H₂S between the decolorisation and the light counterstaining. This combined method preserves the Gomori stain, against which the red acid-fast bacilli stand out sharply, so that acid phosphatase activity and bacteria can be located easily in the cell.     

Influence of hydrogen peroxide on acid-fast staining of Cryptosporidium parvum oocysts

Infections by the protozoan parasite Cryptosporidium parvum are routinely diagnosed by modified Ziehl-Neelsen (acid-fast) staining of faecal preparations despite the counterstaining and ghost-like appearance of some oocysts. Quantitative studies demonstrated that only a small percentage of oocysts excreted by naturally infected newborn calves displayed acid-fast characteristics, but that percentage increased when the time between excretion and sample staining was increased. The treatment of faecal samples with hydrogen peroxide (10 min, 5 vol. final concentration) caused all oocysts to become acid-fast, with up to 40-fold increases in test sensitivity in samples treated and stained within 3 h of excretion. Flow-cytometry analysis of hydrogen peroxide-treated oocysts also demonstrated increased labelling of oocysts by a commercial monoclonal antibody preparation commonly used for diagnosis.     

Role of modified bleach method in staining of acid-fast bacilli in lymph node aspirates

OBJECTIVE: To correlate acid-fast bacilli (AFB) positivity with cytomorphologic patterns of tuberculous lymphadenitis and evaluate bleach concentration method in diagnosing lymph node tuberculosis compared to Ziehl-Neelsen (ZN) method. STUDY DESIGN: One hundred cases of tuberculous lymphadenitis diagnosed by fine needle aspiration cytology (FNAC) were analyzed and classified into 6 cytomorphologic patterns and correlated with bacillary load using routine and modified bleach methods of ZN staining. Smears were graded for AFB positivity. Sensitivity of routine ZN and modified bleach concentration was compared. RESULTS: The classic cytomorphologic pattern of tuberculosis showing epithelioid granulomas, Langerhans giant cells and caseous necrosis was seen in 23% of cases. Routine ZN staining detected AFB in 27% of cases and the modified bleach method in 72%. In 58 cases the modified bleach method had a higher grade of AFB positivity than the routine method. The modified bleach method did not miss any AFB positivity detected on routine ZN staining. CONCLUSION: The modified bleach method demonstrated AFB positivity in 72% of cases. AFB positivity grade was much higher than with routine ZN staining, making bacilli easily visible, with shorter screening time. The modified bleach method is inexpensive, easily performed and more sensitive and safe than routine ZN staining.     

Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry

A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%–30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.     

Recovery of Waterborne Cryptosporidium parvum Oocysts by Freshwater Benthic Clams (Corbicula fluminea)

Asian freshwater clams, Corbicula fluminea, exposed for 24 h to 38 liters of water contaminated with infectious Cryptosporidium parvum oocysts (1.00 × 10⁶ oocysts/liter; approximately 1.9 × 10⁵ oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used—acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne C. parvum oocysts. To optimize the detection of C. parvum oocysts in C. fluminea tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MERIFLUOR test kit).     

A supravital staining technique for honey bee spermatozoa

ABSTRACT A new supravital staining technique is described for honey bee, Apis mellifera L., spermatozoa using the fluorochromes, propidium iodide and Hoechst 33342 (H342), a bis-benzimidazole derivative. Propidium iodide binds to the DNA of sperm which lack membrane integrity and H342 binds to the DNA of all sperm. This assay is a simple and rapid method for determining the percentage nonviabiiity of a male honey bee's sperm. The recommended staining procedure is to incubate sperm in a solution of 5 μ.g/ml H342 and 10 μ.g/ml propidium iodide in modified Kiev solution for 15–20 min. After incubation, wet mounts of the sperm-stain suspension are examined using fluorescence microscopy. Percentage nonviabiiity is determined by the ratio of propidium iodide stained sperm to H342 stained sperm.     

Purification of Eimeria Sporozoites by DE-52 Anion Exchange Chromatography

An anion exchange column of DE-52 has been used to purify Eimeria sporozoites from a post-excystation mixture of oocysts, oocyst shells, sporocysts, sporocyst shells, and sporozoites. The mean recovery from several experiments was 94% and virtually all non-sporozoite material was removed. Infectivity studies in vitro with sporozoites showed that they were viable after purification and were at least as infectious as the unpurified sporozoites; furthermore, oocysts in the crude preparation could be recovered from the DE-52 cellulose by resuspending them in a 20% (w/v) sodium chloride solution.     

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.     

Eimeria tenella: effects of immunity on sporozoites within the lumen of the small intestine

The effect of immunity on the numbers of sporozoites of Eimeria tenella recoverable from the lumen of the small intestine 1 hr after an oral challenge inoculum of oocysts was examined. The experiments were carried out in chickens which had been given an immunizing inoculum of oocysts 9 or 18 days earlier, and the results were compared with those obtained in a control, unprimed, but similarly challenged, group. Similar numbers of "challenge" sporozoites were found in the intestinal washes of control and 18 day primed chickens but there were fewer in the 9 day primed groups. The titers of antisporozoite IgA antibodies (measured by indirect fluorescence) were higher in the gall bladder bile of the 9 day primed groups but resistance to reinfection (measured by the output of oocysts in the feces after challenge with oocysts orally or with sporozoites intracecally) was greater in the 18 day primed group. Although fewer in number, the challenge sporozoites recovered from the intestinal washes of 9 day primed chickens appeared to be morphologically normal when examined by light microscopy. Also, they were as infective as sporozoites recovered from unprimed control, or 18 day primed, groups when injected intracecally into naive chickens. The findings indicate that, whereas reduction of the number of sporozoites of E. tenella in the lumen of the small intestine (presumably caused by the action of secreted antibodies) can be a means of reducing the effective challenge inoculum, this mechanism does not play a major role in the expression of immunity.     

A simple and reliable method of producing in vitro infections of cryptosporidium parvum (apicomplexa)

Abstract A variety of techniques have been used to infect cell monolayers in culture with the protozoan, Cryptosporidium parvum . However, most of these methods rely on the use of trypsin and/or bile salts to excyst sporozoites in vitro, followed by washing sporozoites free of excystation solution prior to their addition to subconfluent monolayers. This method not only increases the amount of time required to establish infections in vitro, but also results in prolonged exposure of free sporozoites to environmental conditions. Here we report a simple, fast, and efficient method of obtaining consistent infections of C. parvum in cell monolayers. This technique relies on the ability of the parasite to excyst at 37°C but not at room temperature following pretreatment with sodium hypochlorite. By adding surface-sterilized oocysts directly to monolayers, sporozoites have access to host cells immediately upon excystation.     

Observations on Eimeria tenella in Feces of Inoculated Chickens

SYNOPSIS. In studies on coccidial excystation, Eimeria tenella sporulated oocysts were fed to 5 individually caged chickens, and during the next 4.5 hours, all droppings were collected immediately after excretion, mixed with 0.85% NaCl solution, then promptly examined for the parasites. Some samples were placed in cold storage at 4 C and examined at intervals thereafter. Three of the birds were necropsied after 5 hours and the intestinal contents examined. Apparently unbroken oocysts containing active sporozoites were in chicken droppings beginning 1–2.25 hours postinoculation; some activated forms were inside and others outside the sporocysts. Free sporozoites, sometimes numerous, first appeared in the feces 1.1–2 hours postinoculation. At necropsy, the state and condition of the parasites in the lumen of bird intestines were similar to those in its last fecal dropping. After being in cold storage for 48 hours, free sporozoites inside and outside the oocysts became active and moved about when placed on the warming stage of the microscope.     

Species-specific immunocytochemical localization of Mycobacterium tuberculosis complex in fine needle aspirates of tuberculous lymphadenitis using antibody to 38 kDa immunodominant protein antigen

OBJECTIVE: To examine immunocytochemical localization of Mycobacterium tuberculosis (MTB) complex antigen in fine needle aspiration (FNA) smears of tuberculous lymphadenitis (TBLN) using species-specific monoclonal antibody MTSS to 38-kDa immnunodominant protein antigen as a diagnostic adjunct to conventional cytomorphology and its advantage over Ziehl-Neelsen (ZN) microscopy. Study Design FNA smears from 340 cases-174 TBLN; 34 negative controls from nontuberculous, positive controls of 13 known acid-fast bacilli (AFB)-positive sputum smears; 50 blind controls; and 69 other controls (smears from stock cultures of bacterial, atypical mycobacteria and fungal species) were subjected to ZN and immunocytochemical staining using MTSS by the streptavidin-biotin method. RESULTS: Immunocytochemical staining was positive in 59 of 61 (96.7%) archival and 110 of 113 (97.3%) fresh FNA smears; ZN positivity for AFB was observed in 27 of 61 (44.2%) archival and 48 of 113 (42.4%) fresh FNA smears of TBLN. CONCLUSION: The immunostaining using MTSS showed a definite advantage over conventional ZN staining for detection and specific diagnosis of TBLN in FNA smears with 0% false positive results. Immunostaining of cytosmears with species specific antibody to MTB would prove to be a good diagnostic adjunct to morphologic diagnosis.     

Annexin V staining during reperfusion detects cardiomyocytes with unique properties

With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8-10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+ (111 +/- 14 nM) was elevated in reperfused annexin V-negative cells (214 +/- 22 nM), and further elevated in annexin V-positive myocytes (382 +/- 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in approximately 3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.     

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.