Забыл одну вещь описать... странную.

[hamstermutant]

Мда... вчера был на кафедре Генетики... с Мутовиным всё как всегда, если он понял так, как понял, дальше бесполезно... я ему про практику, он мне про курсач и т.п.

В общем, Шеф озвучил гипотетическую тему курсовой на 5-м курсе -- "ПЦР-диагностика бактериальных инфекций, сопутствующих муковисцидозу"... ладно, здесь всё понятно. Документ для запроса с биохимии по мою душу он подпишет без проблем в любом случае, тьфу-тьфу.

Поговорили с завом генетического отделения. Далее начался эпик -- у меня поинтересовались как можно вытащить бактерии из мокроты, чтобы можно были делать ПЦР сразу по получению материала, без геморроя с посевами.......... причём ход и тон беседы к шуткам не располагал. И на мои мысли по поводу иммуносорбции была реакция человека по типу "слышу в первый раз"... в ахуе.
"Я врач, клиницист. Это по клинике я могу тебе рассказать много. А вся эта молекулярная технология -- это по твоей части"... МБФ МБФом, но пардон, мужику лет 40-50, пишет докторскую, до хрена опыта работы в клинике... перед ним сидит четверокурсник-биохимик, неуверенно выдаёт общие фразы и пытается объяснить, как можно опознать возбудитель(по типу "если мы будем знать маркерную последовательность ДНК возбудителя, то нам его достаточно выделить -- на ПЦР и дальше делать всё, что угодно" -- при этом я без понятия, известна ли эта последовательность(или придётся самим носом рыть), насколько доступны наборы моноклонАт на эти возбудители и т.п.)... при этом лицо у собеседника как у ребёнка, которому объясняют как играть с новой крутой машинкой... я в шоке, чтобы не сказать в ахуе...

В итоге, меня нагрузили "посмотри в интернете", нагрузят аспиранта, и вообще там, возможно, закрутится очень интересная вещь...

АПД: Таки погуглил(вернее, попабмедил)... если кому интересно, может заглянуть под кат.

выдернутые из статей методики\способы выделения и\или анализа синегнойной палочки из мокроты

In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections.
PMID- 18156255

AB- The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.

Bacterial isolates.
The P. aeruginosa strain collection examined is listed in Table 2. Clinical P. aeruginosa strains from sputum samples were isolated on Pseudomonas isolation agar (Difco) containing ampicillin (100 μg/ml). All P. aeruginosa isolates were genotyped by single-nucleotide polymorphism typing using AT biochips (Clondiag Chip Technologies, Germany) (22).

Growth media and measurements of growth rates in laboratory media.
Pure-culture growth studies of P. aeruginosa strains were performed under aerobic conditions at 37°C in Luria-Bertani (LB) medium; in pig mucus medium; or in ABT minimal medium (9) supplemented with either 0.2% glucose plus 1% Casamino Acids, 0.5% glucose, 2% Casamino Acids, or 10 mM sodium citrate. The mucus medium was prepared by rinsing pig lungs with 0.9% NaCl. This solution was diluted (1:1, vol/vol) in ABT minimal medium and used for growth studies. CF sputum medium was prepared by dissolving sputum samples from patient p16 in ABT (1:20, vol/vol) by vortexing for 2 min, kept at 4°C for 30 min, and sterilized by filtering through a 0.45-μm filter. All bacteria in the sputa were efficiently removed as evaluated by plating aliquots of the CF sputum medium. Anaerobic growth was performed in LB medium plus 1% nitrate at 30°C with a constant nitrogen flow. Growth rates were measured by monitoring the optical density at 600 nm during growth in 50 ml medium in 250-ml flasks with shaking at 150 rpm. Growth rates are expressed as generation times in minutes or as specific growth rates (ln2/hour [i.e., reciprocal hours]).

Isolation of bacterial cells from sputum samples.
Sputum samples were processed within an hour after expectoration or stored at 4°C for later analysis. The sputum samples could be stored for more than 5 days without effects on growth rate determinations. To determine in situ growth rates, P. aeruginosa cells from sputum samples were extracted by dissolving the sample in 0.9% NaCl. For very thick sputa, Sputaosol (Oxoid, Hampshire, United Kingdom) was used to help dissolve samples. The samples were centrifuged at 800 × g for 5 min at 4°C to remove mucus and epithelial cells. The supernatant which contained bacteria was removed and fixed for rRNA hybridizations as described below.

Oligonucleotide probe.
Probe PSEUDAER (5′-GGACGTTATCCCCCACTAT-3′, specific to P. aeruginosa 16S rRNA (21), was labeled with Cy3 (Molecular Probes, Eugene, OR). The probe has previously been reported to have a sensitivity and specificity of 1.000 (21). Potential cross-reactivity of the PSEUDAER probe is of minor concern, as the probe did not hybridize to Pseudomonas putida cells under the conditions used in this work (data not shown).

Fixation of bacterial cells.
Sputum samples, isolated cells from sputum samples, and cells from laboratory cultures were fixed in 4% paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS) (pH 7.4). Fixed cells were stored at −20°C in storage buffer (50% ethanol, 10 mM Tris [pH 7.5], 0.1% Nonidet P-40) until use.

Whole-cell hybridization.
Fixed cells were applied homogenously on poly-l-lysine (Sigma Chemical, St. Louis, MO)-coated slides (36) and air dried. Hybridizations were carried out on the slides using 30 μl of solution I (30% formamide, 100 mM Tris [pH 7.5], 0.1% sodium dodecyl sulfate, 0.9 M NaCl) and 80 ng of the PSEUDAER-Cy3 probe. For FISH carried out directly on fresh sputum, hybridization solution was added directly to the sample. The slides were incubated in the dark in a humidified chamber for 3 h at 37°C, followed by washing of the slides with 45 μl of prewarmed (37°C) solution I for 30 min at 37°C and subsequently with 45 μl of prewarmed (37°C) solution II (100 mM Tris [pH 7.5], 0.9 M NaCl) for 40 min at 37°C. The slides were then quickly rinsed in distilled water and air dried. For sputum samples, 50 μl Calcofluor white (CFW) (fluorescent brightener 28; Sigma-Aldrich) was added for staining of alginate and incubated for 3 h in a humidified chamber before washing with distilled water. For FISH counterstained with the fluorescent nucleic acid dye DAPI (4′,6′-diamidino-2-phenylindole), the slides were then incubated in the dark with 14 mM DAPI in PBS at room temperature for 5 min and washed with PBS for 5 min. The slides were then rinsed and air dried.

Microscopy and image analysis.
Microscopic observations of cells within sputum samples were completed using a Zeiss LSM510 scanning confocal laser microscope (Carl Zeiss, Jena, Germany) equipped with an NeHe laser as well as an UV lamp and detectors and filter sets for simultaneous monitoring of red fluorescence emitted from the Cy3 probe (excitation, 543 nm; emission filter, 565 to 615 nm) and CVW fluorescence from fluorescent brightener 28 (UV lamp excitation; emission filter, 395 to 465 nm). Images were obtained using a 40×/1.3 Plan-Neofluar oil objective. Images were processed using the IMARIS software package (Bitplane AG, Zürich, Switzerland).
Visualization of hybridized cells isolated from sputum samples or from laboratory cultures was done using an Axioplan epifluorescence microscope (Carl Zeiss) equipped with a 100-W mercury lamp and a Cy3 filter. A 63×/1.25 Plan-Neofluar oil objective (Carl Zeiss) was used for inspection and image acquisition. Image analysis was done in 12 bits with PMIS-S200 software version 4.1.4 and the Unix-based CELLSTAT image analysis program (38). Hybridized cells were automatically identified by use of the CELLSTAT program, providing cell volume, fraction of dividing cells, and mean fluorescence intensity. In some cases, unfocused cells or images and extremely high-intensity signals from crystals were deleted manually before further analysis.


Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis
PMID- 17504792
Sputum processing
An aliquot of raw sputum free of salivary contamination was weighed and an equivalent volume to weight of dithiothreitol (10%) was added. The sample was then gently vortexed and placed in a water bath at 38°C for 30 min. At 10-min intervals the sample was removed and gently vortexed once again. If the sample appeared particularly tenacious, further mixing was undertaken with gentle pipetting to ensure homogenisation. The sample was diluted a further five times with PBS, ensuring a constant final dilution of 10 times, and then centrifuged (350xg for 15 min). Following centrifugation, the cell-free supernatant was decanted and stored at -80°C in 1 mL aliquots for later analysis, and the cell pellet re-suspended in PBS for the TCC.
Routine bacterial culture
At the time of sputum processing, an aliquot of raw sputum was sent for routine bacterial culture by the Hospital Pathology laboratory (Rayal Hobart Hospital, Hobart, Australia). P. aeruginosa colonies were identified as being oxidase positive and with typical morphology, i.e. mucoid or nonmucoid, following culture on blood agar aerobically.
Sputum analysis
Sputum TCC were performed on 20 µL of re-suspended cell pellet using a Neubauer haemocytometer (Neubauer, Wertheim, Germany). In addition to inflammatory cells, red blood cells (RBC) and buccal squamous cells were quantified to examine the potential contribution of occult haemorrhage to iron content and the degree of salivary contamination. Sputum samples with any evidence of blood contamination were excluded from further analysis.
Bacterial isolation and quantitative bacterial load determination
The total and P. aeruginosa bacterial loads were determined using an adaptation of the method described by Wong et al. 11. Briefly, CF sputa homogenates were serially diluted in PBS-gelatin (0.1%) and spread onto a variety of culture media, including Pseudomonas selective agar (PSA), an enteric selective agar (MacConkey or Cysteine lactose electrolyte deficient medium) and two horse blood agar (HBA) plates (one used for aerobic and the other for anaerobic incubation). PSA facilitates the isolation and quantification of P. aeruginosa, and characteristic colonies are identified according to their appearance (blue-green or brown-pigmented), having been incubated under aerobic conditions at 37°C. Anaerobic incubation of HBA plates allowed the isolation and quantitation of most other relevant pathogens, such as Haemophilus influenzae, Staphylococcus aureus and Streptococcus pneumoniae (P. aeruginosa does not grow on HBA under anaerobic conditions without nitrogen supplementation). The selection of sputum plugs free of saliva ensured that contamination with bacteria from the oropharynx was minimised. Colony-forming units (CFU) were counted on all plates after incubation for 48 h. Total anaerobic bacterial and P. aeruginosa loads were expressed as CFU•mL–1 of sputum.
Метод изолирования бактерий:
Wong K, Roberts MC, Owens L, Fife M, Smith AL. Selective media for the quantitation of bacteria in cystic fibrosis sputum. J Med Microbiol 1984;17:113–119.[CrossRef][ISI][Medline] [Order article via Infotrieve]


Sequential genotyping of Pseudomonas aeruginosa from upper and lower airways of cystic fibrosis patients
A. Jung1, I. Kleinau1, G. Schönian2, A. Bauernfeind3, C. Chen1, M. Griese4, G. Döring5, U. Göbel2, U. Wahn1 and K. Paul1
PMID- 12503704
The bacterial cultures were diluted on selective media and P. aeruginosa was identified with the API 20 NE system (Bio Mérieux, Marcy-Etoile, France). A positive culture was defined as growth of the organism at any density. Single colonies of any phenotypically different isolates, if obtained from the same culture, were chosen for further investigations. Antimicrobial susceptibility testing was performed by micro-broth dilution. The isolates were frozen at –80°C, recultured on McConkey agar overnight at 37°C and characterised by genotyping techniques.
DNA macrorestriction and PFGE of P. aeruginosa isolates were carried out as previously described 14, with a few modifications. Bacterial isolates were grown overnight at 37°C in tryptone soya broth agar to an optical density of 0.7 ( =590 nm). The suspensions were incubated with 0.5 M ethylenediamine tetraacetic acid (EDTA) and centrifuged for 3 min at 10,000 rpm (Eppendorf Centrifuge 5415D; Eppendorf, Hamburg, Germany). After washing and resuspending the pellets in 100 µL of a sodium chloride/EDTA (SE) buffer (75 mM NaCl, 0.5 M EDTA, pH 7.4), the suspensions were heated to 55°C, and 100 µL of a 2% low melting agarose (Bio-Rad Laboratories, Hercules, CA, USA) was added. The suspensions were pipetted into a plug mould and hardened at 4°C. Plugs were incubated in 1 mL of sodium chloride/N-lauryl-sarcosine (SLS) buffer (0.5 M EDTA, 1% SLS, pH 9.5) and proteinase K was added to a final concentration of 0.5 mg•mL–1. The mixtures were incubated overnight at 55°C with gentle shaking. After washing with Tris/EDTA (TE) buffer (5 times over a period of 10 h), 2x5 mm pieces were cut from the plugs and incubated overnight at 37°C with 15 U of the restriction enzyme Spe I (Boehringer, Mannheim, Germany). The reaction was stopped by adding TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Plugs were loaded onto a 1% agarose gel (Seakem Gold Agarose for PFGE; Bio-Products, Rockland, ME, USA). A clinical P. aeruginosa reference strain, obtained from the authors' laboratory, had been added to each gel for internal control. A molecular size standard (DNA size ladder; Boehringer, Mannheim, Germany) was also included. The slots were filled up with 2% low-melting agarose, and the electrophoresis (Chef-DRII System; Bio-Rad Laboratories, Richmond, CA, USA) was run in 0.5xTris/borate/EDTA (TBE) buffer using the following ramp conditions: 1–25 s for 24 h at 12°C, with amperage=0.1 ampere (6 volt (V)/cm), voltage=200 V, included angle 120°C. Thereafter, the gels were stained with ethidium bromide, photographed with a Polaroid camera and compared by eye.
All samples were typed in duplicate by reculturing isolates from the frozen stocks. The relationship between typing patterns and interpretation of the results was determined using criteria suggested by Tenover et al. 17. Thus, isolates were considered to be closely related if their PFGE patterns differed in not more than three restriction bands. Isolates that showed fragment differences from four to six bands were judged to be possibly related, whereas strains with more than six different restriction fragments were considered to be genetically unrelated.
Data from both investigations (t1 and t2) were pooled for statistical analysis and sensitivity, specificity and predictive values of respiratory specimens were calculated.