細胞計數(shù)板的使用方法.doc

血球計數(shù)板-基本構(gòu)造血球計數(shù)板是一塊特制的厚型載玻片，載玻片上有四個槽構(gòu)成三個平臺。中間的平臺較寬，其中間又被一短橫槽分隔成兩半，每個半邊上面各刻有一小方格網(wǎng)，每個方格網(wǎng)共分九個大格，中央的一大格作為計數(shù)用，稱為計數(shù)區(qū)。計數(shù)區(qū)的刻度有兩種：一種是計數(shù)區(qū)分為16個大方格（大方格用三線隔開），而每個大方 格又分成25個小方格；另一種是一個計數(shù)區(qū)分成25個大方格（大方格之間用雙線分開），而每個大方格又分成16個小方格。但是不管計數(shù)區(qū)是哪一種構(gòu)造，它 們都有一個共同特點，即計數(shù)區(qū)都由400個小方格組成。計數(shù)區(qū)邊長為1mm，則計數(shù)區(qū)的面積為1mm2，每個小方格的面積為1/400mm2。蓋上蓋玻片后，計數(shù)區(qū)的高度為0.1mm，所以每個計數(shù)區(qū)的體積為0.1mm3，每個小方格的體積為1/4000mm3。使用細胞計數(shù)板計數(shù)時，先要測定每個小方格中微生物的數(shù)量，再換算成每毫升菌液（或每克樣品）中微生物細胞的數(shù)量。細胞計數(shù)板-使用方法1視待測菌懸液濃度，加無菌水適當稀釋（斜面一般稀釋100倍），以每小格的菌數(shù)可數(shù)為度。2取潔凈的細胞計數(shù)板一塊，在計數(shù)區(qū)上蓋上一塊蓋玻片。3將菌懸液搖勻，用滴管吸取少許，從計數(shù)板中間平臺兩側(cè)的溝槽內(nèi)沿蓋玻片的下邊緣滴入一小滴（不宜過多），讓菌懸液利用液體的表面張力充滿計數(shù) 區(qū)，勿使氣泡產(chǎn)生，并用吸水紙吸去溝槽中流出的多余菌懸液。也可以將菌懸液直接滴加在計數(shù)區(qū)上（不要使計數(shù)區(qū)兩邊平臺沾上菌懸液，以免加蓋蓋玻片后，造成 計數(shù)區(qū)深度的升高），然后加蓋蓋玻片（勿使產(chǎn)生氣泡）。4靜置片刻，使細胞沉降到計數(shù)板上，不再隨液體漂移。將細胞計數(shù)板放置于顯微鏡的載物臺上夾穩(wěn)，先在低倍鏡下找到計數(shù)區(qū)后，再轉(zhuǎn)換高倍鏡觀察并計數(shù)。由于生活細胞的折光率和水的折光率相近，觀察時應減弱光照的強度。5計數(shù)時若計數(shù)區(qū)是由16個大方格組成，按對角線方位，數(shù)左上、左下、右上、右下的4個大方格（即100小格）的菌數(shù)。如果是25個大方格組成的計數(shù)區(qū)，除數(shù)上述四個大方格外，還需數(shù)中央1個大方格的菌數(shù)（即80個小格）。為 了保證計數(shù)的準確性，避免重復計數(shù)和漏記，在計數(shù)時，對沉降在格線上的細胞的統(tǒng)計應有統(tǒng)一的規(guī)定。如菌體位于大方格的雙線上，計數(shù)時則數(shù)上線不數(shù)下線，數(shù) 左線不數(shù)右線，以減少誤差。即位于本格上線和左線上的細胞計入本格，本格的下線和右線上的細胞按規(guī)定計入相應的格中。見下圖：即本格中計數(shù)細胞為3個。（細胞壓線，僅計數(shù)相鄰的兩條線上的細胞）6對于出芽的酵母菌，芽體達到母細胞大小一半時，即可作為兩個菌體計算。每個樣品重復計數(shù)2-3次（每次數(shù)值不應相差過大，否則應重新操作），按公式計算出每mL（g）菌懸液所含細胞數(shù)量。7測數(shù)完畢，取下蓋玻片，用水將細胞計數(shù)板沖洗干凈，切勿用硬物洗刷或抹擦，以免損壞網(wǎng)格刻度。洗凈后自行晾干或用吹風機吹干，放入盒內(nèi)保存。細胞計數(shù)板-計數(shù)公式1、16格25格的細胞計數(shù)板計算公式：細胞數(shù)/ml=100小格內(nèi)細胞個數(shù)/10040010000稀釋倍數(shù)1、25格16格的細胞計數(shù)板計算公式：細胞數(shù)/ml=80小格內(nèi)細胞個數(shù)/8040010000稀釋倍數(shù)網(wǎng)摘：/blogger/post_read.asp?BlogID=1766764&PostID=17108229之前寫的細胞計數(shù)板使用，其中的圖片天涯都給刪了，我沒有備份。今天瀏覽網(wǎng)頁看到一個英文版的介紹，寫的很好。Using a Counting Chamber For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types. A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts. To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power. It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or an objective lens may be damaged if the user is not not careful. One entire grid on standard hemacytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly distributed. To perform the count, determine the magnification needed to recognize the desired cell type. Now systematically count the cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For large cells this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells you may wish to count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide on a specific counting patter to avoid bias. For cells that overlap a ruling, count a cell as in if it overlaps the top or right ruling, and out if it overlaps the bottom or left ruling.Here is a way to determine a particle count using a Neubauer hemocytometer. Suppose that you conduct a count as described above, and count 187 particles in the five small squares described. Each square has an area of 1/25 mm-squared (that is, 0.04 mm-squared) and depth of 0.1 mm. The total volume in each square is (0.04)x(0.1) = 0.004 mm-cubed. You have five squares with combined volume of 5x(0.004) = 0.02 mm-cubed. Thus you counted 187 particles in a volume of 0.02 mm-cubed, giving you 187/(0.02) = 9350 particles per mm-cubed. There are 1000 cubic millimeters in one cubic centimeter (same as a milliliter), so your particle count is 9,350,000 per ml. Cells are often large enough to require counting over a larger surface area. For example, you might count the total number of cells in the four large corner squares plus the middle combined. Each square has surface area of 1 mm-squared and a depth of 0.1 mm, giving it a volume of 0.1 mm-cubed. Suppose that you counted 125 cells (total) in the five squares. You then have 125 cells per 0.5 mm-cubed, which is 250 cells/mm-cubed. Again, multiply by 1000 to determine cell count per ml (250,000). Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In that case you will need to multiply your final count by the dilution factor. For e