2.1 Objectives
(1) Learn to prepare blood smear slide and observe blood cells under optical microscope.
(2) Learn to prepare human oral epithelial cell slide and observe epithelial cells under optical microscope.
(3) Learn to measure the cell size by the use of graticules under optical mieroscope.
2.2 Introductions
The technique of preparing blood cell smear slide is generally used for morphologic observation, cellular classification and cell number counting in clinic diagnosis of diseases and physical examination.
2.3 Principles
Different cells have their special morphology structures and functions. In animal blood, there are erythrocytes, also called red blood cells, and leukocytes, also called white blood cells. Mature human red blood cells are oval biconcave disks without cell nuclei and most organelles, but amphibian's red blood cells still keep their nuclei. Human white blood cell sinclude monocytes, neutrophils, eosinophils, basophils and lymphocytes. Monocytes have abundant cytoplasm and kidney shaped nuclei. which are typically granulated. Once they move out the bloodstream, they become tissue macrophages. Neutrophils constitute 60%-70% of the circulating white blood cell with a distinctive feature of multi-lobed nuclei, which consists of three to five lobes connected by slender strands. Eosinophils compose about 2%-4% of the circulating white blood cell with bi-lobed nuclei. Basophils are less than 0. 5% of the circulating white blood cell with bi- or tri- lobed nuclei. They are characterized by several coarse, dark violet granules. Lymphocytes have a relatively small amount of cytoplasm and deeply staining nuclei. Hemocytometer is used to count cell numbers in a specific volume of fluid under microscope. lt consists of a thick glass microscope slide with a rectangular indentation that creates a chamber with a laser-etched grid of perpendicular lines,
In addition, muscle cells, also known as muscle fibers or myocytes, are long, tubular cells to form muscles, including cardiac, skeletal, and smooth muscle cells. Nerve cells, also known as neurons, are electrically excitable. Each neuron is divided into three parts: soma or cell body, dendrite, and axon.
Cell structures are of importance for disease diagnosis and treatment since many diseases are attributed to abnormalities of the cellular structures and functions. For example, abnormality response to insulin by the receptors on the liver or muscle cells leads to type Ⅱ diabetes.
Since general animal, plant and bacterial cells are too tiny to be observed by naked eye, a slide preparation before microscopic observation is necessary, which includes fixing the cells to be observed and staining them by dyes in some cases. Then the prepared slide is put in a holder, a thin piece of glass, to be observed under optical microscope.
General instant slide preparation can be operated by students themselves. It contains four main types such as dry mount slide preparation, wet mount slide preparation, smear slide preparation and squash slide preparation. To prepare a dry mount slide, a small specimen is thinly sliced and placed on the slide, covered with a slide cover and observed under a microscope directly. Wet mount slide preparation method is commonly used for observation of aquatic samples and living organisms. To prepare a wet mount slide, the specimens are suspended in fluids such as water, brine, glycerin or immersion oil. Smear slide preparation is performed by smearing the liquid specimen, such as blood sample using two glass slides. The angle between the slides determines the length of the smear. Squash lens tissue slide is used for examining soft substances by preparing a wet mount and placing over the cover glass. Pressure is gently applied so as not to destroy the specimen or break the cover glass. Excess water is removed.
In most of the cases, the prepared slides are often necessary to be stained so as to enhance contrast and make them more visible under the microscope. A variety of methods are used for staining microscope slides, including non-vial or in vitro stains of non-living cells and vital or in vivo stains of living tissue. Staining can make the detail structures visible compared to unstaining slide preparation techniques.
Microscope graticule, also known as reticle, is a standard tool for microscopic length measurement consisting of a net of fine lines or fibers in the eyepiece and stage. It can be divided into two types of eyepiece graticule and stage graticule. Eyepiece gratieule is a device that is used with microscope to help in determining the size of fibrous dust. Eyepiece graticule is circular piece of glass slide, wherein the central engraved with accurate calibration, usually 5 mm is divided into 50 lattices, the actual per lattice is equal to100 pm. Stage graticule is a special piece of slide, wherein the central has a small circle with a micro scale. The circle is inscribed with the scale, in which the linear length of 1 mm is divided into 100 units and each unit is equal to 10 μm.
2.4 Apparatuses and materials
Apparatuses: optical microscope, eyepiece micrometer, stage micrometer, microscopic slides, cover slip, dropper, dish, toothpick, scissors, forceps and tweezers.
Materials: toad, frog or human blood, 1% toluidine blue, methylene blue, 1% of Ringer’s solution.
2.6 Protocols
Procedure 1:
1. Collect drops of blood from a frog or toad's leg.
2. Prepare two blood smear slides: place the blood on one end of a slide, and bring the edge of a cover slip in contact with the drop to bank evenly behind the spreader. The angle between the slides has to be 30。-40。. The cells on the slides should be spaced far enough apart to be counted and differentiated.
3. Leave the slides to air-dry.
4. Take one smear slide to observe under microscope directly.
5、Fixing: immerse the other smear slide into jar containing 70% ethanol for 5-10 min, then take out the smear slide and exposure it to air until drying. .
6. Staining: put a drop of staining solution on the slide and fully cover the smear slide. Keep staining for 15-20 min. Then rinse the slide with distilled water. Drain off water and leave the slide to air-dry.
7、Observing and recording: observe the cells of smear slide under the compound microscope and record the results correctly.
Procedure 2:
1、Prepare human oral epithelial cell slide: use a toothpick to scrape the oral epithelial cells, and smear evenly onto a clean slide.
2. Stain the cells with a drop of toluidine blue over the cell smear for 5 min.
3. Find out the oral epithelial cells with a flat oval shape and blue nucleus under an optical microscope.
4. Measure the cell length by microscope graticule: remove the eyepiece lens, unscrew the oculars, and install the eyepiece graticule into oculars. Then fix the eyepiece and put it back to microscope. Place stage graticule on the microscope stage. Make sure the scaled side is up and the circle part is placed in the stage center.
5. Focus first with low magnification lens to identify the scale of stage graticule. Secondly, rotate the eyepiece graticule until eyepiece graticule and objective graticule are parallel. Thirdly, adjust the first lines of each graticule in order to coincide with each other, and then find the line of two graticules where they overlapped to the right.
6. Finally count and record the unit numbers of eyepiece graticule and stage graticule separately between the two coincidence lines and calculate one unit of eyepiece graticule representing the length of stage graticule.
7. Measure the oral epithelial cells size: take the stage graticule away, put the prepared oral epithelial cell slide on the stage, and then measure both the cell and nucleus sizes.
8. Calculate volumes of cell and nuclear and the ratio of cell nucleus to cytoplasm based on the following formula:
For sphere formula: V= 4/3Πr3 (r is the radius)
For oval formula: V=4/3Πab2 (a, b are long and short radius)
For nucleus to cytoplasm (N/C) ratio:
N/C=Vn/ (Vc-Vn) (Vn is the nuclear volume, and Vc is the volume of the cytoplasm)
