Calibrated Information and Courses from MediaLab, Inc.
These are the MediaLab courses that cover Calibrated and links to relevant pages within the course.
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| Diluted Specimens Calibrated automatic pipets should be used on specimens that require a dilution. Mouth pipetting must of course always be avoided.
Automated cell counters may not be the best choice for cell counts on CSF because of the variation in background counts.
A high background count could cause a false increase in normal or slightly elevated count. The dilution required is based on the appearance of the sample.
AppearanceDilution RatioVolume of SampleVolume of Diluentslightly hazy1:1030 ul270 ulhazy1:2030 ul570 ulslightly cloudy1:10030 ul2970 ulslightly bloody1:10030 ul2970 ulcloudy1:20030 ul5970 ulbloody1:20030 ul5970 ulturbid1:100000.1 ml of a 1:100 dilution9.9 ml | View Page |
| Examining a Diluted Specimen Examining a diluted CSF specimen involves the following steps:
Mix the CSF sample manually 6 - 10 times or place it on a mechanical mixer for 5 - 10 minutes.Use a calibrated automatic pipet and place the appropriate volume of sample and diluent in a tube. Mix the diluted sample well.Use a Pasteur pipet and fill both sides of the hemacytometer. Allow the cells to settle for 5 minutes in a moist environment.Count cells in the four corner squares and the center square on both sides of the chamber. The number of cells counted times the dilution factor is then equal to the number of cells per microliter.
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| Which of the following is the safest and most accurate tool for diluting spinal fluids? | View Page |
| What are Standards and Calibrators? Before controls and patient samples can be run, testing instruments must first be calibrated. This requires standards. Standards are materials which contain accurately determined concentrations of an analyte that are used to either confirm a testing method's validity, or to make sure an instrument reads correctly. Calibrating an instrument allows every unknown patient sample or control to be analyzed from a measured starting point. Standards should not be used in place of daily controls because they do not measure or control other variables in the testing process such as operator technique or sample appropriateness.
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| Possible Corrective Action (4) Review calibration of the test instrument.What was the date of the last calibration? Test instruments need to be calibrated according to the manufacturer’s instructions or more frequently if necessary. Federal requirements call for analytic tests to be recalibrated at least every six months to verify the accuracy of the testing procedure. | View Page |
| Materials Needed The following is a list of materials needed for semen analysis. Laboratories will differ slightly in the equipment used. Use of this equipment will be described further in the later pages of this course.
Materials needed include:graduated test tube or serological pipets with safety bulb to measure volumepH paper in neutral to basic range (e.g. 7.2-8.8)counting chamber and/or automated counting machineglass slides and coverslips for wet mount if motility and sperm count are to be assessed separatelyhand counterif dilution is donediluting fluid calibrated automatic pipetspositive pressure pipets and glass boreslight microscope with phase contrast objectives for sperm count and bright field objectives for morphology assessmentglass slides and fixative for morphology slidesset-up for performing Papanicolaou or other morphology stainingEvery laboratory should also have a copy of the "WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction", published on behalf of the WHO by Cambridge University Press. The fourth edition was published in 1999. | View Page |
| Diluting a specimen for counting on a hemacytometer Following liquefaction (20-30 minutes), mix the sample manually by swirling the container several times. Thorough mixing is essential for accurate counting. Calibrated automatic pipettes are used to prepare a dilution. Because of the viscosity of semen, the semen should be added to the diluent using a positive pressure pipettor.
The dilution often used for routine sperm counts is 1:20 but the actual dilution factor will vary depending on the total sperm count. For high concentration specimens a greater dilution will be necessary. For low concentrations an undiluted or minimally diluted specimen may be required. The appropriate dilution is determined by estimating the concentration needed to do a count of at least 100 cells per side of the loaded hemacytometer.
The diluent that may be used for sperm counts on a hemacytometer can be as follows: 5 gm of sodium bicarbonate in 100 ml of distilled water, plus 1ml of formalin (neutral). | View Page |