Neutrophil myeioperoxidase standard 3,3'-Diaminobenzidine Tetrahydrochioride (DAB) procedure:

When staining neutrophils, a cell suspension would be preferred, but a finely minced buffy coat can be used, if you thick section and locate the stained cells before thin sectioning. Staining should be carried out as soon as possible after fixation, The myeioperoxidase (MPO) activity falls off such that we process samples within 2-3 weeks of fixation.

Note that fixation is for 1 hour. Enzyme activity may be destroyed if tixed for longer periods.

Uranium compounds, especially uranyl acetate, have been widely and routinely used as transmission electron microscopy centrist stains for biological materials since 1958. Those of us who do TEM of biologicals use small quantities of uranyl acetate, nitrate, formate, sulfate and perhaps other uranium compounds almost daily and therefore keep inventories of these salts and their solutions.

In the 1980's growing concerns about medical and research wastes entering regional dump sites prompted state radiation officials in Oregon to begin tightening the regulations for monitoring and controlling all radioactive substances including the uranium compounds commonly used in processing biological specimens for TEM.

The use of fluorescence microscopy to investigate fungal growth in wood often causes interference due to the strong autofluorescence of wood lignin, unless fluorescent probes which specifically react to fungal hyphae, are used. Techniques to enable differentiation of hyphae from wood have been recently reported (Singh et al., 1997; Xiao et al., 1997). The authors demonstrated that while glutaraldehyde can be used to detect fungal native proteins, wheat germ agglutinin (WGA) which reacts with cell wall chitin of hyphae, considerably improved detection of fungi growing in wood.

When CD-ROM was introduced in 1983, it started a revolution in data storage that is still going on. But it was not until 1992, when the first desktop CD Recorders were shipped, that the real power of compact disc reached consumers. The ability to make on ones own desktop as few as one disc with up to 650 Megabyte of data in a formal that was readable en any computer with a CD-ROM drive, made massive data storage and file interchange a reality. That revolution is continuing with the advent of DVD-Recordable in 1998, allowing up to 3.9 Gigabytes on a single disc. In the next year or so, storing the full 4.7 Gigabytes specified in the DVD standards will be possible.

Small particle contamination analysis requires consideration of secondary contamination of our sampie. Protection of such samples is very important. The procedure for sampling with freshly pulled lengths of clear 3M Magic™ tape is a favorite procedure among forensic microscopists. The tape must be immediately pressed onto a clean surface; usually, a clean microscope slide. There it is protected from further contamination.

Microscopical study and identification is usually possible utilizing the tape backing as a coverslip. Its thickness Is about 56 μm, one-third of the recommended 170 μm, but quite adequate for objectives 4-20X, If you feel persnickety, you can use two thicknesses of the tape, a second one over the sampling tape, This will give you optimum images for 4-40X objectives. Three thicknesses of tape, at 168 μm, is very close to the recommended thickness of 170 μm for any or all objectives.

Let's face it. Looking through a microscope is not what our bodies were built for, and looking through a microscope for an extended period of time requires an unnatural rigidity of the body. You're not moving around, even a bit, as you normally would. The result can be cramped muscles, especially in the neck and shoulders, and a whole body fatigue Hi at can make 10:00 AM feel like 5:03 PM. The solution is correct posture and a proper arrangement of microscope, chair, and body.

The use of antigen retrieval techniques has been the foundation of the development of immunocytochemistry (ICC). After the pioneering work done on cryostat sections, the use of enzyme digestion made formalin-fixed archival and current material available for study by ICC. However, the relationship between the duration of fixation and the digestion time necessary for optimum results is a major drawback associated with enzyme digestion. The longer the tissue has been in fixative, the longer the required digestion time will be. Even after a standardised fixation time, some antigens require greatly extended digestion times. Whereas 10-15 minutes in trypsin may be a standard protocol after overnight fixation, immunoglobulin deposits in renal glomeruli may require 60-90 minutes.

Paraffin wax is a mixture of (virtually) straight chain hydrocarbons. Note the word “mixture”. Unless one goes to the enormous lengths of purifying or searching for a fine chemical supplier, the wax will always be a mixture. I used a “pure” wax once as a standard for gas chromatographic studies on paraffin waxes. There is a relationship between hydrocarbon chain length and melting point, but as the waxes are always mixtures, melting points are never exact, either in the compounding or the measuring.

Creutzfeldt-Jakob disease (CJD) resembles two other diseases, kuru (found in New Guinea tribesmen) and scrapie (found in sheep). CJD is a slow progressive dementia of the central nervous system. The transmissible agent of CJD is unusual for several reasons: it has a long incubation period, up to eight years in some cases; it does not provoke an inflammatory reaction within infected tissues; it has never been isolated; it is resistant to routine paraffin processing. Histotechnologists need to be informed of the potential hazards and of the correct handling of tissues infected with this unconventional agent, even though it has low transmissibility. Treatment of CJD tissues with long sterilization at high temperatures or prolonged exposure to bleach has proven effective in deactivating the agent. This article outlines the precautionary procedures used in our laboratory when processing CJD tissues. (J Histolechnol 12:214, 1989).

Project MICRO (Microscopy in Curriculum - Research Outreach) is the Microscopy Society of America's educational outreach program for middle schools. The idea began in 1993, but has taken a lot of time and effort to implement. MSA's early decision to collaborate with experienced science educators at the Lawrence Hall of Science (LHS) of the University of California at Berkeley was a wise one as their educational materials have a well-earned national reputation for excellence. The first phase of MICRO has just been completed, with the publication of a LHS teacher's manual that will support the MSA/MICRO effort to get microscopist-volunteers into classrooms nationwide.

We have successfully sectioned decalcified paraffin embedded “inner ears” from mice, rats and humans. We have also prepared guinea pig cochlea for SEM, So, briefly:

Paraffin:

1. 1) Immediately after dissection, place the cochlea in 10% neutral buffered formalin (NSF) and let fix with gentle agitation 24 hours per 2 mm thickness at room temperature.

2. 2) Decalcify by immersing in 5% formic acid at least 4X specimen volume.

3. 3) Gently agitate at room temperature and change at least once per 24 hours. Check for completeness of decalcification by decanting 4 mLs of “spent” formic acid into a clean, clear test tube, Add 1 mL of 5% ammonium oxaiate in distilled water. Let stand undisturbed for 15 to 20 minutes, White precipitate of calcium oxaiate indicates that the sample is not completely decalcified, Change formic acid at least once per 24 hours until the spent formic tests clear.