Printer friendly version [26 pages]Microbiology laboratories commonly receive cerebrospinal fluid (CSF) or blood specimens from patients with meningitis, pneumonia, or unexplained febrile illness.
The test is thus as an optimal basic test for culture confirmation the foundation for cost-effective MRSA diagnostics. Rapid result: Performing the test from the overnight culture provides you with a result in only 4 hours and thus guarantees rapid diagnosis. Laboratories may also receive joint fluid, pleural fluid, or other sterile site specimens from these patients.
Differentiation of the clinically most significant pathogens causing staphyloccocal infections is thus assured.
In contrast to nosocomial MRSA, CA-MRSA have a higher pathogenicity, but do not demonstrate any multiresistance to antibiotics. Distinguishing CA-MRSA from nosocomial MRSA therefore makes sense from a therapeutic and epidemiological viewpoint. RCF is dependent on the length of the radius of the rotor, thus the same RPM may not generate the same g force in another centrifuge. Typically, centrifugation at 1000 x g for 10-15 minutes is sufficient to sediment bacteria.After the specimen has been centrifuged, the supernatant should be drawn off with a Pasteur pipette and reserved if antigen detection by latex agglutination is planned. Once it has been well-mixed, one or two drops of sediment should be used to prepare the Gram stain and one drop should be used to streak the primary culture media.Cytological examination of the CSFLaboratory examination of the CSF is usually the first step to confirm the presence of bacterial meningitis. Positive results for any of these tests can rapidly provide evidence of infection, even if cultures fail to grow.Performing a Gram stainThe Gram stain is an empirical method for differentiating bacterial species into two large groups based on the chemical and physical properties of their cell walls.


Gram-positive bacteria retain the primary stain while gram-negative bacteria take the color of the counterstain. Proper smear preparation using the CSF sediment should produce a monolayer of organisms sufficiently dense for easy visualization but thin enough to reveal morphological characteristics.
Positive and negative quality control (QC) strains should be tested along with the unknown specimens.
Use one section for the unknown CSF and the other section for a known organism for QC.Prepare a smear by placing 1-2 drops of the well-mixed CSF sediment on the slide, allowing the drop(s) to form one large slightly turbid, uniform suspension.
To prepare a smear using an isolate, add a small drop of sterile water or physiological saline to the slide and create a slightly turbid, uniform suspension of cells from an overnight culture.Let the suspension air dry.
The suspension MUST be completely dry before proceeding.Fix the smear by the flooding the slide with 95% methanol for a minimum of 2 minutes (3).
If methanol is not available, heat-fix the smears by quickly passing the slide through a flame three times.
Shake off excess water.Decolorize with 95% ethanol until no more stain washes off (5-10 seconds may be enough). Negative and positive latex agglutination reactionsRapid diagnostic tests (RDTs) RDT for meningococcal meningitisRDTs have been developed for direct testing of CSF specimens without prior heat or centrifugation (2). The test is based on the principle of vertical flow immunochromatography in which gold particles and nitrocellulose membranes are coated with monoclonal antibodies to capture soluble serogroup-specific polysaccharide antigens in the CSF.
The test consists of 2 duplex paper sticks (also called dipsticks), which together enable identification of four serogroups of N.


Initial evaluation using RDTs on stored CSF from patients in Niger showed correct identification of the meningococcal serogroup 97% of the time (4). The position of that red line indicates the specific serogroup based on the RDT that was tested.
See Figure 5 for result combinations for each serogroup.A negative result consists of a single upper pink control line only.
If the T-I medium cannot be transported to a microbiology laboratory the same day of inoculation, insert a venting needle (sterile cotton-plugged hypodermic needle) through the rubber stopper of the T-I bottle, which will encourage growth and survival of the bacteria. Upon arrival, wipe the rubber stopper with 70% alcohol, insert a venting needle into the T-I bottle, incubate at 35-37°C with ~5% CO2 (or in a candle-jar), and observe daily for turbidity in the liquid phase for up to 7 days. Any turbidity or lysis of erythrocytes may be indicative of growth, and subcultures onto primary culture media should be made immediately. MacConkey agar is designed to grow gram-negative bacteria and stain them for lactose fermentation, in order to distinguish those that can ferment the sugar lactose (Lac+) from those that cannot (Lac-). Encapsulated strains appear more mucoidal than non-encapsulated strains, which appear as smaller compact grey colonies. Older cultures (> 24 hours) become more opaquely grey and sometimes cause the underlying agar to turn dark.



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