.細(xì)胞計數(shù)方法-細(xì)胞計數(shù)板法實驗原理：當(dāng)待測細(xì)胞懸液中細(xì)胞均勻分布時，通過測定一定體積懸液中的細(xì)胞的數(shù)目，即可換算出每毫升細(xì)胞懸液中細(xì)胞的細(xì)胞數(shù)目。具體操作：1. 將計數(shù)板及蓋片擦拭干凈，并將蓋片蓋在計數(shù)板。2. 將細(xì)胞懸液吸出少許，滴加在蓋片邊緣，使懸液充滿蓋片和計數(shù)板之間，靜置3min，注意蓋片下不要有氣泡，也不能讓懸液流入旁邊槽中。3. 計算板四大格細(xì)胞總數(shù)，壓線細(xì)胞只計左側(cè)和上方的。然后按公式計算：細(xì)胞數(shù)/mL=四大格細(xì)胞總數(shù)/410個/ml(注：當(dāng)細(xì)胞很多時，可在四個格中選一定數(shù)目較平均的小格，由于每大格中有16個小格，然后計左側(cè)和上方的細(xì)胞數(shù)，求出每小格的細(xì)胞數(shù)，取平均值m，m16即每個格的平均值。所以，細(xì)胞密度=m1610個/ml)說明：公式中除以4，因為計數(shù)了4個大格的細(xì)胞數(shù)。公式中乘以10因為計數(shù)板中每一個大格的體積為：1.0mm（長）1.0mm（寬）0.1mm（高）=0.1mm 而 1ml=1000ul=1000mm（注意：鏡下偶見有兩個以上細(xì)胞組成的細(xì)胞團(tuán)，應(yīng)按單個細(xì)胞計算，若細(xì)胞團(tuán)10%以上，說明分散不好，需重新制備細(xì)胞懸液。）=細(xì)胞計數(shù)板的使用一、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(xì)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(xì)胞的數(shù)量。二、細(xì)胞計數(shù)板-使用方法1視待測菌懸液濃度，加無菌水適當(dāng)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(xì)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)沿蓋玻片的下邊緣摘入一小滴（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(xì)胞沉降到計數(shù)板上，不再隨液體漂移。將細(xì)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(xì)胞的折光率和水的折光率相近，觀察時應(yīng)減弱光照的強(qiáng)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(zhǔn)確性，避免重復(fù)計數(shù)和漏記，在計數(shù)時，對沉降在格線上的細(xì)胞的統(tǒng)計應(yīng)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(xì)胞計入本格，本格的下線和右線上的細(xì)胞按規(guī)定計入相應(yīng)的格中。見下圖：即本格中計數(shù)細(xì)胞為3個。6對于出芽的酵母菌，芽體達(dá)到母細(xì)胞大小一半時，即可作為兩個菌體計算。每個樣品重復(fù)計數(shù)2-3次（每次數(shù)值不應(yīng)相差過大，否則應(yīng)重新操作），按公式計算出每mL（g）菌懸液所含細(xì)胞數(shù)量。7測數(shù)完畢，取下蓋玻片，用水將細(xì)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(fēng)機(jī)吹干，放入盒內(nèi)保存。三、細(xì)胞計數(shù)板-計數(shù)公式1、16格25格的細(xì)胞計數(shù)板計算公式：細(xì)胞數(shù)/ml=100小格內(nèi)細(xì)胞個數(shù)/10040010000稀釋倍數(shù)1、25格16格的細(xì)胞計數(shù)板計算公式：細(xì)胞數(shù)/ml=80小格內(nèi)細(xì)胞個數(shù)/8040010000稀釋倍數(shù)四、血細(xì)胞計數(shù)板計數(shù)的誤差主要來自哪些方面?應(yīng)如何盡量減少誤差,力求準(zhǔn)確?血細(xì)胞計數(shù)的誤差分別來源于技術(shù)誤差和固有誤差。其中由于操作人員采血不順利，器材處理、使用不當(dāng)，稀釋不準(zhǔn)確，細(xì)胞識別錯誤等因素所造成的誤差屬技術(shù)誤差；而由于儀器（計數(shù)板、蓋片、吸管等）不夠準(zhǔn)確與精密帶來的誤差稱儀器誤差，由于細(xì)胞分布不均勻等因素帶來的細(xì)胞計數(shù)誤差屬于分布誤差或計數(shù)域誤差（filed error）。儀器誤差和分布誤差統(tǒng)稱為固有誤差或系統(tǒng)誤差。技術(shù)誤差和儀器誤差可通過規(guī)范操作、提高熟練程度和校正儀器而避免或糾正，但細(xì)胞分布誤差卻難于徹底消除。因此，搞好紅細(xì)胞計數(shù)的質(zhì)量控制一般需采用以下措施。1避免技術(shù)誤差，糾正儀器誤差（1）所用器材均應(yīng)清潔干燥，計數(shù)板、血蓋片、微量吸管及刻度吸管的規(guī)格應(yīng)符合要求或經(jīng)過校正。計數(shù)板的鑒定：要求計數(shù)室的臺面光滑、透明，劃線清晰，計數(shù)室劃線面積準(zhǔn)確。必要時采用嚴(yán)格校正的目鏡測微計測量計數(shù)室的邊長與底面積，用微米千分尺測量計數(shù)室的深度。美國國家標(biāo)準(zhǔn)局（NBS）規(guī)定每個大方格邊長的誤差應(yīng)小于1%，即10.01mm，深度誤差應(yīng)小于2%，即0.10.002mm。若超過上述標(biāo)準(zhǔn)，應(yīng)棄之不用。血蓋片應(yīng)具有一定的重量，平整、光滑、無裂痕，厚薄均勻一致，可使用卡尺多點測量（至少在9個點），不均勻度在0.002mm之內(nèi)。必要時采用平面平行儀進(jìn)行測量與評價，要求呈現(xiàn)密集平行的直線干涉條紋。最簡單的評價方法是將潔凈的蓋片緊貼于干燥的平面玻璃上，若能吸附一定的時間不脫落，落下時呈弧線形旋轉(zhuǎn)，表示蓋片平整、厚薄均勻。同時，合格的蓋片放置在計數(shù)室表面后，與支持柱緊密接觸的部位可見到彩虹。精選出的蓋片與其他蓋片緊密重合后，在掠射光線下觀察，如見到完整平行的彩虹條紋表示另一枚蓋片質(zhì)量也符合要求。目前臨床實驗室多采用一次性微量采血管采集毛細(xì)血管血，除注意定點購買使用信譽(yù)較好廠家的產(chǎn)品外，還應(yīng)對每一批量的采血管進(jìn)行抽樣檢查，可通過水銀稱重法或有色溶液比色法進(jìn)行校正，誤差不應(yīng)超過1%。（2）紅細(xì)胞稀釋液應(yīng)等滲、新鮮、無雜質(zhì)微粒。（3）嚴(yán)格操作，從消毒、采血、稀釋、充池到計數(shù)都應(yīng)規(guī)范，尤其應(yīng)注意的是血樣稀釋及充池時既要作到充分混勻，又要防止劇烈震蕩為破壞紅細(xì)胞。必須一次性充滿計數(shù)室，防止產(chǎn)生氣泡，充入細(xì)胞懸液的量以不超過計數(shù)室臺面與血蓋片之間的矩形邊緣為宜。（4）報告法定計量單位。2縮小計數(shù)域誤差或分布誤差 由于血細(xì)胞在充入計數(shù)室后呈隨機(jī)分布或稱Poisson分布（ ），而我們所能計數(shù)的細(xì)胞分布范圍是有限的，由此造成的計數(shù)誤差稱為計數(shù)域誤差或分布誤差。縮小這種誤差的有效方法就是盡量擴(kuò)大細(xì)胞計數(shù)范圍和計數(shù)數(shù)目，一般先進(jìn)行誤差估計，然后決定所需計數(shù)的數(shù)目和計數(shù)范圍，只要能將誤差控制在允許范圍內(nèi)即可。Berkson指出，當(dāng)使用同一支吸管、同一面計數(shù)室，計數(shù)0.2mm2面積的細(xì)胞數(shù)，有望將 CV控制在可接受的7%以內(nèi)。對于紅細(xì)胞計數(shù)而言，由于紅細(xì)胞數(shù)量較多，在計數(shù)室中顯得比較“擁擠”，根據(jù)Poisson公式推斷， 。欲將誤差控制在變異百分?jǐn)?shù)5%以內(nèi)，至少需要在計數(shù)室中計數(shù)400個紅細(xì)胞，因此要求計數(shù)五個中方格的紅細(xì)胞。事實上Berkson還通過實驗證明，紅細(xì)胞的計數(shù)域誤差為s=0.92 ，較理論誤差（Poisson分布誤差）要小。 3排除異常標(biāo)本的干擾 白細(xì)胞數(shù)量在正常范圍時，相對于紅細(xì)胞數(shù)量來講，其影響可忽略，但如白細(xì)胞過高（100109/L），則應(yīng)對計數(shù)結(jié)果進(jìn)行校正。方法是：實際RBC=計得RBC-WBC。如當(dāng)紅細(xì)胞換算后為3.51012/L、白細(xì)胞換算后為100109/L時，病人實際紅細(xì)胞數(shù)應(yīng)為3.41012/L。在高倍鏡下計數(shù)時，避開有核細(xì)胞。有核細(xì)胞體積比正常紅細(xì)胞大，中央無凹陷，無草黃色折光，可隱約見到細(xì)胞核。此外，當(dāng)病人急性嚴(yán)重貧血時網(wǎng)織紅細(xì)胞可提前大量釋放，也給紅細(xì)胞計數(shù)帶來一定的干擾，而且影響網(wǎng)織紅細(xì)胞絕對值計算結(jié)果。其校正方法有待探討。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