细说ATCC细胞的孢子分离法

<p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">ATCC<font face="宋体">细胞是从事微生物学及生命科学研究的基本材料，在医学领域中，诊断制品的制备，菌苗的生产、微生物致病性研究，药物的抑菌试验及药品微生物检验等都有一套完整的菌种菌种分为母种</font><font face="Calibri">(</font><font face="宋体">一级种</font><font face="Calibri">)</font><font face="宋体">、原种</font><font face="Calibri">(</font><font face="宋体">二级种</font><font face="Calibri">)</font><font face="宋体">和栽培种</font><font face="Calibri">(</font><font face="宋体">三级种</font><font face="Calibri">)</font><font face="宋体">三级。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">工业发酵的有用菌种，其筛选步骤包括菌种分离、初筛和复筛，挑选具有某种能力的有用菌种。菌种的分离就是首先是从土壤或腐生植物中收集含菌样品，用无菌水稀释后，涂布于置有适宜细菌、放线菌或霉菌生长的琼脂培养基平皿上，并将其倒置于恒温箱中，培养一定时间，平皿上长出的许多单个菌落</font>(<font face="宋体">单一微生物的集落</font><font face="Calibri">)</font><font face="宋体">经分别分离后即为各种纯种菌株，简称纯种，移种至试管斜面培养基上，置</font><font face="Calibri">4</font><font face="宋体">℃冰箱备用。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="background-color: rgb(255, 204, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">ATCC<font face="宋体">细胞的孢子分离法：</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">孢子分离法又可分为以下几种方法</font>:</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">( 1 ) </span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">褶上涂抹法</font><font face="Calibri">:</font><font face="宋体">选取新鲜、健壮、未开伞、未破裂的子实体，洗净后用</font><font face="Calibri">0 . 1 %</font><font face="宋体">升汞或</font><font face="Calibri">75 %</font><font face="宋体">酒精表面灭菌</font><font face="Calibri">2 </font><font face="宋体">分钟，然后连同培养基一起放入接种箱内，经福尔马林熏蒸消毒</font><font face="Calibri">12 ~ 24 </font><font face="宋体">小时，</font><font face="Calibri">ATCC</font><font face="宋体">细胞再按无菌操作技术将接种环在子实体菌褶上涂抹，把涂抹后带孢子的接种环在培养基上划线接种，，</font><font face="Calibri">zui</font><font face="宋体">后置于</font><font face="Calibri">25 ~ 28 </font><font face="宋体">℃恒温箱内培养，可得纯菌种。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">( 2 ) </span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">孢子印采集法</font><font face="Calibri">:</font><font face="宋体">取灭过菌的载玻片、试纸或黑布，置于新鲜、未开伞或未开裂的子体菌褶的下方。经</font><font face="Calibri">24 </font><font face="宋体">小时后，便落下孢子，形成印痕</font><font face="Calibri">(</font><font face="宋体">即孢子印</font><font face="Calibri">)</font><font face="宋体">。然后，用接种环或玻璃棒蘸取孢子，接种在平面或斜面培养基上，进行恒温培养，可得纯菌种。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">( 3 )</span></strong><span style="font-family: 宋体; font-size: 9pt;"> </span></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">孢子收集器采集法</font><font face="Calibri">:</font><font face="宋体">孢子收集器由玻璃钟罩、培养皿、三角架、搪瓷盘等组成。钟罩下为一搪瓷盘，直径</font><font face="Calibri">25 </font><font face="宋体">厘米左右，盘内先垫衬</font><font face="Calibri">4~6 </font><font face="宋体">层纱布，中央放</font><font face="Calibri">1 </font><font face="宋体">副</font><font face="Calibri">9 </font><font face="宋体">厘米直径的培养皿，大盖口朝下，上放小的，口向上。然后，在上面小的培养皿上放</font><font face="Calibri">1 </font><font face="宋体">只铅丝绕成的三角架，再将带孔的玻璃钟罩盖上，顶上罩孔用棉塞塞好，</font><font face="Calibri">zui</font><font face="宋体">后，用纱布包好整个孢子收集器，置于高压灭菌锅或蒸笼内灭菌</font><font face="Calibri">2</font><font face="宋体">小时。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">孢子收集器灭菌消毒后，放入无菌室或无菌箱内。然后，用小刀割取</font>1 <font face="宋体">块</font><font face="Calibri">4~5 </font><font face="宋体">厘米大小的子实体，插在收集器内的三角架顶上</font><font face="Calibri">(</font><font face="宋体">若无孢子收集器，也可用培养皿代替</font><font face="Calibri">)</font><font face="宋体">。再置于温度</font><font face="Calibri">24 ~26 </font><font face="宋体">℃下培养</font><font face="Calibri">24 </font><font face="宋体">小时，培养皿中便可见到白色粉状物，此即为孢子。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">此时，可将孢子收集器再移至接种箱内，取出培养皿，盖好，并在培养皿内加入</font>10 ~ 20 <font face="宋体">毫升的无菌水，使孢子散于本中，成为孢子悬浮液。然后，再用无菌打针筒或吸管吸取孢子悬浮液，接种于试管斜面培养基上，每管注</font><font face="Calibri">2 ~ 3 </font><font face="宋体">滴。置于</font><font face="Calibri">24 ~ 26 </font><font face="宋体">℃温度下培养</font><font face="Calibri">5 ~ 7 </font><font face="宋体">天，培养基上便可长出白色菌落。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">待菌落直径有</font>0 . 5 <font face="宋体">厘米时，选健壮的菌落，再移接于另一试管的斜面培养基上培养。</font><font face="Calibri">ATCC</font><font face="宋体">细胞当白色菌丝长满试管时，便得纯菌种。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>