細胞計數(shù)方法------細胞計數(shù)板法實驗原理：當待測細胞懸液中細胞均勻分布時，通過測定一定體積懸液中的細胞的數(shù)目，即可換算出每毫升細胞懸液中細胞的細胞數(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.計算板四大格細胞總數(shù)，壓線細胞只計左側(cè)和上方的。然后按公式計算：細胞數(shù)/mL=四大格細胞總數(shù)/4×10個/ml(注：當細胞很多時，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)和上方的細胞數(shù)，求出每小格的細胞數(shù)，取平均值m，m×16即每個格的平均值。所以，細胞密度=m×16×10個/ml)說明：公式中除以4，因為計數(shù)了4個大格的細胞數(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胞組成的細胞團，應(yīng)按單個細胞計算，若細胞團10%以上，說明分散不好，需重新制備細胞懸液。）================================================細胞計數(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。二、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(yīng)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）。方法有待探討。UsingaCountingChamberFormicrobiology,cellculture,andmanyapplicationsthatrequireuseofsuspensionsofcellsitisnecessarytodeterminecellconcentration.Onecanoftendeterminecelldensityofasuspensionspectrophotometrically,howeverthatformofdeterminationdoesnotallowanassessmentofcellviability,norcanonedistinguishcelltypes.Adeviceusedfordeterminingthenumberofcellsperunitvolumeofasuspensioniscalledacountingchamber.Themostwidelyusedtypeofchamberiscalledahemocytometer,sinceitwasoriginallydesignedforperformingbloodcellcounts.

Topreparethecountingchamberthemirror-likepolishedsurfaceiscarefullycleanedwithlenspaper.Thecoverslipisalsocleaned.Coverslipsforcountingchambersarespeciallymadeandarethickerthanthoseforconventionalmicroscopy,sincetheymustbeheavyenoughtoovercomethesurfacetensionofadropofliquid.Thecoverslipisplacedoverthecountingsurfacepriortoputtingonthecellsuspension.ThesuspensionisintroducedintooneoftheV-shapedwellswithapasteurorothertypeofpipet.Theareaunderthecoverslipfillsbycapillaryaction.Enoughliquidshouldbeintroducedsothatthemirroredsurfaceisjustcovered.Thechargedcountingchamberisthenplacedonthemicroscopestageandthecountinggridisbroughtintofocusatlowpower.Itisessentialtobeextremelycarefulwithhigherpowerobjectives,sincethecountingchamberismuchthickerthanaconventionalslide.Thechamberoranobjectivelensmaybedamagediftheuserisnotnotcareful.OneentiregridonstandardhemacytometerswithNeubauerrulingscanbeseenat40x(4xobjective).Themaindivisionsseparatethegridinto9largesquares(likeatic-tac-toegrid).Eachsquarehasasurfaceareaofonesquaremm,andthedepthofthechamberis0.1mm.Thustheentirecountinggridliesunderavolumeof0.9mm-cubedSuspensionsshouldbediluteenoughsothatthecellsorotherparticlesdonotoverlapeachotheronthegrid,andshouldbeuniformlydistributed.Toperformthecount,determinethemagnificationneededtorecognizethedesiredcelltype.Nowsystematicallycountthecellsinselectedsquaressothatthetotalcountis100cellsorso(numberofcellsneededforastatisticallysignificantcount).Forlargecellsthismaymeancountingthefourlargecornersquaresandthemiddleone.Foradensesuspensionofsmallcellsyoumaywishtocountthecellsinthefour1/25sq.mmcornersplusthemiddlesquareinthecentralsquare.Alwaysdecideonaspecificcountingpattertoavoidbias.Forcellsthatoverlaparuling,countacellas"in"ifitoverlapsthetoporrightruling,and"out"ifitoverlapsthebottomorleftruling.

HereisawaytodetermineaparticlecountusingaNeubauerhemocytometer.Supposethatyouconductacountasdescribedabove,andcount187particlesinthefivesmallsquaresdescribed.Eachsquarehasanareaof1/25mm-squared(thatis,0.04mm-squared)anddepthof0.1mm.Thetotalvolumeineachsquareis(0.04)x(0.1)=0.004mm-cubed.Youhavefivesquareswithcombinedvolumeof5x(0.004)=0.02mm-cubed.Thusyoucounted187particlesinavolumeof0.02mm-cubed,givingyou187/(0.02)=9350particlespermm-cubed.Thereare1000cubicmillimetersinonecubiccentimeter(sameasamilliliter),soyourparticlecountis9,350,000perml.Cellsareoftenlargeenoughtorequirecountingoveralargersurfacearea.Forexample,youmightcountthetotalnumberofcellsinthefourlargecornersquaresplusthemiddlecombined.Eachsquarehassurfaceareaof1mm-squaredandadepthof0.1mm,givingitavolumeof0.1mm-cubed.Supposethatyoucounted125cells(total)inthefivesquares.Youthenhave125cellsper0.5mm-cubed,whichis250cells/mm-cubed.Again,multiplyby1000todeterminecellcountperml(250,000).Sometimesyouwillneedtodiluteacellsuspensiontogetthecelldensitylowenoughforcounting.Inthatcaseyouwillneedtomultiplyyourfinalcountbyth