Materials

pancreas slides, H&E stain
immersion oil
lens tissue and cleaner
eyepiece reticules
stage micrometers
dissecting stereo-zoom microscope

Examine a histological specimen to practice proper focusing of the condenser and setting of the field stop and condenser diaphragms. A 1 m thick section of pancreas or other tissue stained with hematoxylin and eosin is ideal. A typical histological specimen is a section of a tissue or organ that has been chemically fixed, embedded in epoxy resin or paraffin, sectioned, and stained with dyes specific for nucleic acids, proteins, carbohydrates, and so forth. In hematoxylin and eosin (H&E) staining, hematoxylin stains the nucleus and cell RNA a dark blue or purple color, while eosin stains proteins (and the pancreatic secretory granules) a bright orange-pink. When the specimen is illuminated with monochromatic light, the contrast perceived by the eye is largely due to these stains. For this reason, a stained histological specimen is called an amplitude specimen and is suitable for examination under the dissecting-zoom microscope using bright field optics. A suitable magnification is 10–40.

Equipment and Procedure –for upload.

Three items are required: a focusable eyepiece, an eyepiece reticule, and a stage micrometer. The eyepiece reticule is a round glass disk usually containing a 10 mm scale divided into 0.1 mm (100 _m) units. The reticule is mounted in an eyepiece and is then calibrated using a precision stage micrometer to obtain a conversion factor (m/reticule unit), which is used to determine the magnification obtained for each objective lens. The reason for using this calibration procedure is that the nominal magnification of an objective lens (found engraved on the lens barrel) is only correct to within 5%. If precision is not of great concern, an approximate magnification can be obtained using the eyepiece reticule alone. In this case, simply measure the number of micrometers from the eyepiece reticule and divide by the nominal magnification of the objective. For a specimen covering 2 reticule units (200 m), for example: 200 _m/10__20 _m.

The full procedure, using the stage micrometer, is performed as follows:

To mount the eyepiece reticule, unscrew the lower barrel of the focusing eyepiece and place the reticule on the stop ring with the scale facing upward. The stop ring marks the position of the real intermediate image plane. Make sure the reticule size matches the internal diameter of the eyepiece and rests on the field stop. Carefully reassemble the eyepiece and return it to the binocular head. Next focus the reticule scale using the focus dial on the eyepiece and then focus on a specimen with the dissecting zoom microscope focus dial. The images of the specimen and reticule are conjugate and should be simultaneously in sharp focus.

All materials and media used for culturing microorganisms (especially bacteria) must be sterilized before using. This is most efficiently done in a device called an autoclave that heats the material to high temperature (250 F) under pressure (~15 PSI) for a time long enough (15-20 min) to kill all microorganisms that may be present. Autoclaves are also used to sterilize old, contaminated materials prior to safe disposal via normal waste streams.
If you are not paying attention carefully, it is easy to cross-contaminate cultures by forgetting to flame a tool or changing swabs. Never use the same tool in two different cultures without first flaming it to sterilize - when in doubt, flame it. With disposable swabs or loops, when in doubt, use a new one.

• Examine the stage micrometer slide, rotating the eyepiece so that the micrometer and reticule scales are lined up and partly overlapping. The stage micrometer consists of a 1 or 2 mm scale divided into 10 _m units, giving 100 units/mm. The micrometer slide is usually marked 1/100 mm. The conversion factor we need to determine is simply the number of _m/reticule unit. This conversion factor can be calculated more accurately by counting the number of micrometers contained in several reticule units in the eyepiece. The procedure must be repeated for each objective lens, but only needs to be performed one time for each lens.• Returning to the specimen slide, the number of eyepiece reticule units spanning the diameter of a structure is determined and multiplied by the conversion factor to obtain the distance in micrometers.

Exercise

1. Calibrate the magnification of the objective lens/eyepiece system using the stage micrometer and an eyepiece reticule. First determine how many micrometers are in each reticule unit.

2. Determine the mean diameter and standard deviation of a typical cell, a nucleus, and a cell organelle (secretory granule), where the sample size, n, is 10. Examination of cell organelles requires a magnification of 40–100X.

3. Why is it wrong to adjust the brightness of the image using either of the two diaphragms? How else (in fact, how should you) adjust the light intensity and produce an image of suitable brightness for viewing or photography?