Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

/pdf/using-flow-cytometry-for-counting-natural-planktonic-625gf2rrn7.pdf

Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

The only immunoglobulin heavy-chain classes known so far in teleosts have been mu and delta. We identify here a previously unknown class, immunoglobulin zeta, expressed in zebrafish and other teleosts. In the zebrafish heavy-chain locus, variable (V) gene segments lie upstream of two tandem diversity, joining and constant (DJC) clusters, resembling the mouse T cell receptor alpha (Tcra) and delta (Tcrd) locus. V genes rearrange to (DJC)(zeta) or to (DJC)(mu) without evidence of switch rearrangement. The zebrafish immunoglobulin zeta gene (ighz) and mouse Tcrd, which are proximal to the V gene array, are expressed earlier in development. In adults, ighz was expressed only in kidney and thymus, which are primary lymphoid organs in teleosts. This additional class adds complexity to the immunoglobulin repertoire and raises questions concerning the evolution of immunoglobulins and the regulation of the differential expression of ighz and ighm.

The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z

This review discusses photomodification of biological membranes and model membrane systems. Current concepts of membrane structure are first reviewed briefly. The role of preillumination association of sensitizer with membranes as it relates to photomodification rate is discussed, as well as the role of singlet oxygen in membrane photomodification. Finally the characteristics of singlet oxygen generation in membranes are considered. The evidence clearly indicates that membrane photomodification cannot be understood based only on the properties of sensitizers and singlet oxygen in aqueous solution. Rather the properties of sensitizers in association with membranes are the determinants of membrane photomodifcation. These properties differ significantly in aqueous solution and in membranes.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-1097.1987.tb04749.x

Photomodification of biological membranes with emphasis on singlet oxygen mechanisms

Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.

Applications of flow cytometry to clinical microbiology.

Critically ill post-surgical, post-trauma and/or septic patients are characterised by severe inflammation. This immune response consists of both a pro- and an anti-inflammatory component. The pro-inflammatory component contributes to (multiple) organ failure whereas occurrence of immune paralysis predisposes to infections. Strikingly, infectious complications arise in these patients despite the presence of a clear neutrophilia. We propose that dysfunction of neutrophils potentially increases the susceptibility to infections or can result in the inability to clear existing infections. Under homeostatic conditions these effector cells of the innate immune system circulate in a quiescent state and serve as the first line of defence against invading pathogens. In severe inflammation, however, neutrophils are rapidly activated, which affects their functional capacities, such as chemotaxis, phagocytosis, intra-cellular killing, NETosis, and their capacity to modulate adaptive immunity. This review provides an overview of the current understanding of neutrophil dysfunction in severe inflammation. We will discuss the possible mechanisms of downregulation of anti-microbial function, suppression of adaptive immunity by neutrophils and the contribution of neutrophil subsets to immune paralysis.

/pdf/the-role-of-neutrophils-in-immune-dysfunction-during-severe-1k9s6f1adv.pdf

The role of neutrophils in immune dysfunction during severe inflammation

A leukocyte cDNA library from Atlantic salmon, based on oligo-dT priming, was constructed in lambda-gt10. Immunoglobulin heavy chain (IgH) cDNA were isolated from the library using a specific probe generated by polymerase chain reaction (PCR) between two conserved areas within the variable region (second and fourth frame region). Two cDNA clones encoding the entire constant region of membrane-bound IgH, and one cDNA encoding the entire constant region of secretory IgH were sequenced, revealing messages from two isotypic IgM genes. Both genes were shown to be present in haploid embryos and have been isolated from a genomic library, the exons and deduced amino acid sequences of which are presented here (salmon CHA and CHB). The splicing of transcripts encoding the membrane-bound IgH excises the whole fourth exon as in other teleosts. The nucleotide and amino acid identity between salmon CHA and CHB are 98.2%, and 96.2%, respectively. Two subfractions of IgM from Atlantic salmon separated by ion chromatography can be explained by a net exchange of basic residues in salmon CHB compared to CHA. The finding of two closely related salmon CH genes is in accordance with the quasi-tetraploid state of the Atlantic salmon genome.

Cloning and sequence analysis of two isotypic IgM heavy chain genes from Atlantic salmon, Salmo salar L

Human polymorphonuclear neutrophilic leukocytes (PMNLs) phagocytosed fluorescein-isothiocyanate (FITC)-labelled Staphylococcus aureus. Free bacteria, phagocytes, and nonphagocytes were discriminated and quantified by flow cytometry (FCM). The relative fluorescence of phagocyte-associated and free bacteria (Nf:N) was calculated by dividing the mean phagocyte fluorescence by that of the free bacteria and the number of phagocytosed bacteria. Bactericidal capacity and chemiluminescence were measured by standard methods. The red-to-green fluorescence ratio of acridine orange-stained PMNLs (R/G) was measured by FCM. Degradation of bacteria was monitored by the reduction in FITC and ethidiumbromide fluorescence of bacteria liberated from the phagocytes. Bacterial FITC fluorescence was pH dependent. Nf:N was 0.5 to 0.7. Using a standard curve for the interrelationship between bacterial fluorescence and pH, phagosomal pH was 5.0-5.5. Phagocytes, kept at 4 degrees C for 24 h had Nf:N approximately 1, did not degrade bacteria, but killed them and emitted chemiluminescence. NH4Cl increased phagocyte fluorescence by 27% and decreased R/G by 50%. Cyanide and azide did not affect Nf:N. Nf:N of phagocytes from a patient with chronic granulomatous disease was 32% below, and R/G was 32% higher than the controls. Acidification of the phagosomes seems to be related to discharge of PMNL granule contents and independent of the respiratory burst.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.990040311

Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: role of polymorphonuclear neutrophilic leukocyte granules in phagosome acidification.

Factors important for the measurement of chemiluminescence production by polymorphonuclear leukocytes.

Human neutrophils were irradiated with light at 340-380 nm in the presence of low concentrations of protoporphyrin or uroporphyrin. At increasing light doses or increasing concentrations of protoporphyrin, the neutrophils rapidly lost the ability of locomotion. Also, neutrophil chemiluminescence and hexose-monophosphate shunt activity rapidly declined. An early event was leakage of endogenous K(+) followed by lactate dehydrogenase and at a later stage leakage of particle-bound acid phosphatase. A number of cellular enzymes were inactivated, the susceptibility to inactivation decreased in the order: succinate dehydrogenase greater than lactate dehydrogenase greater than glutamate dehydrogenase greater than acid phosphatase. Uroporphyrin had no effect on neutrophil functions, leakage of K(+), or cellular enzymes. The results suggest that photodamage to the plasma membrane and the mitochondria are earlier events than photodamage to lysosomes.

Porphyrin‐induced photodamage to isolated human neutrophils

Chemiluminescence (CL) production by polymorphonuclear leukocytes (PMNLs) was examined in 63 patients with bacterial infections and 63 healthy controls. The production was significantly higher in the patients (mean +/- standard error = 134.5 +/- 5.0 X 10(3) cpm) than in the controls (118.9 +/- 2.5 X 10(3) cpm; p less than 0.05). In 38 patients CL values were within the normal range and in 19 patients above. CL production below that of any control occurred in 6 patients: 3 (of 4) with staphylococcal endocarditis, 2 (of 4) with pneumococcal meningitis and 1 with salmonella septicaemia and osteomyelitis. PMNL hexose monophosphate shunt activity as measured by glucose metabolism correlated with CL production. Patients with low CL production more often had large numbers of juvenile and immature myeloid cells in the peripheral blood than patients with normal or high CL values. 3/6 patients with low CL values died, 2/38 with normal and 0/19 with high values. Directed and spontaneous PMNL migration was examined in 39 of the 63 patients with bacterial infections. 13 patients had PMNLs with higher directed and 16 with higher spontaneous migration capacity than their corresponding controls. The remaining patients had PMNLs with lower migration capacity. 2 of the 39 patients died. Each had PMNLs with low migration capacity. CL production by PMNLs was examined in 16 patients with viral infections and 16 healthy controls. The production was significantly lower in the patients (mean +/- standard error = 105.5 +/- 6.6 X 10(3) cpm) than in the controls (129.1 +/- 5.3 X 10(3) cpm; p less than 0.01). 15 patients had lower values than their corresponding controls. The PMNL migration capacity was also lower in the patients. These findings indicate that the majority of patients with bacterial infections have PMNLs with normal or increased function. However, some patients have reduced PMNL function and this reduction may contribute to a fatal outcome of the disease. Patients with viral infections usually have reduced PMNL function.

Polymorphonuclear leukocyte function in bacterial and viral infections.

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