細說ATCC細胞的孢子分離法

<p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">細胞是從事微生物學及生命科學研究的基本材料，在醫學領域中，診斷制品的制備，菌苗的生產、微生物致病性研究，藥物的抑菌試驗及藥品微生物檢驗等都有一套完整的菌種菌種分為母種</font><font face="Calibri">(</font><font face="宋體">一級種</font><font face="Calibri">)</font><font face="宋體">、原種</font><font face="Calibri">(</font><font face="宋體">二級種</font><font face="Calibri">)</font><font face="宋體">和栽培種</font><font face="Calibri">(</font><font face="宋體">三級種</font><font face="Calibri">)</font><font face="宋體">三級。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">工業發酵的有用菌種，其篩選步驟包括菌種分離、初篩和復篩，挑選具有某種能力的有用菌種。菌種的分離就是首先是從土壤或腐生植物中收集含菌樣品，用無菌水稀釋后，涂布于置有適宜細菌、放線菌或霉菌生長的瓊脂培養基平皿上，并將其倒置于恒溫箱中，培養一定時間，平皿上長出的許多單個菌落</font>(<font face="宋體">單一微生物的集落</font><font face="Calibri">)</font><font face="宋體">經分別分離后即為各種純種菌株，簡稱純種，移種至試管斜面培養基上，置</font><font face="Calibri">4</font><font face="宋體">℃冰箱備用。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">ATCC<font face="宋體">細胞的孢子分離法：</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">孢子分離法又可分為以下幾種方法</font>:</span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">( 1 ) </span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">褶上涂抹法</font><font face="Calibri">:</font><font face="宋體">選取新鮮、健壯、未開傘、未破裂的子實體，洗凈后用</font><font face="Calibri">0 . 1 %</font><font face="宋體">升汞或</font><font face="Calibri">75 %</font><font face="宋體">酒精表面滅菌</font><font face="Calibri">2 </font><font face="宋體">分鐘，然后連同培養基一起放入接種箱內，經福爾馬林熏蒸消毒</font><font face="Calibri">12 ~ 24 </font><font face="宋體">小時，</font><font face="Calibri">ATCC</font><font face="宋體">細胞再按無菌操作技術將接種環在子實體菌褶上涂抹，把涂抹后帶孢子的接種環在培養基上劃線接種，，</font><font face="Calibri">zui</font><font face="宋體">后置于</font><font face="Calibri">25 ~ 28 </font><font face="宋體">℃恒溫箱內培養，可得純菌種。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">( 2 ) </span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">孢子印采集法</font><font face="Calibri">:</font><font face="宋體">取滅過菌的載玻片、試紙或黑布，置于新鮮、未開傘或未開裂的子體菌褶的下方。經</font><font face="Calibri">24 </font><font face="宋體">小時后，便落下孢子，形成印痕</font><font face="Calibri">(</font><font face="宋體">即孢子印</font><font face="Calibri">)</font><font face="宋體">。然后，用接種環或玻璃棒蘸取孢子，接種在平面或斜面培養基上，進行恒溫培養，可得純菌種。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">( 3 )</span></strong><span style="font-family: 宋體; font-size: 9pt;"> </span></span><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">孢子收集器采集法</font><font face="Calibri">:</font><font face="宋體">孢子收集器由玻璃鐘罩、培養皿、三角架、搪瓷盤等組成。鐘罩下為一搪瓷盤，直徑</font><font face="Calibri">25 </font><font face="宋體">厘米左右，盤內先墊襯</font><font face="Calibri">4~6 </font><font face="宋體">層紗布，中央放</font><font face="Calibri">1 </font><font face="宋體">副</font><font face="Calibri">9 </font><font face="宋體">厘米直徑的培養皿，大蓋口朝下，上放小的，口向上。然后，在上面小的培養皿上放</font><font face="Calibri">1 </font><font face="宋體">只鉛絲繞成的三角架，再將帶孔的玻璃鐘罩蓋上，頂上罩孔用棉塞塞好，</font><font face="Calibri">zui</font><font face="宋體">后，用紗布包好整個孢子收集器，置于高壓滅菌鍋或蒸籠內滅菌</font><font face="Calibri">2</font><font face="宋體">小時。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">孢子收集器滅菌消毒后，放入無菌室或無菌箱內。然后，用小刀割取</font>1 <font face="宋體">塊</font><font face="Calibri">4~5 </font><font face="宋體">厘米大小的子實體，插在收集器內的三角架頂上</font><font face="Calibri">(</font><font face="宋體">若無孢子收集器，也可用培養皿代替</font><font face="Calibri">)</font><font face="宋體">。再置于溫度</font><font face="Calibri">24 ~26 </font><font face="宋體">℃下培養</font><font face="Calibri">24 </font><font face="宋體">小時，培養皿中便可見到白色粉狀物，此即為孢子。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">此時，可將孢子收集器再移至接種箱內，取出培養皿，蓋好，并在培養皿內加入</font>10 ~ 20 <font face="宋體">毫升的無菌水，使孢子散于本中，成為孢子懸浮液。然后，再用無菌打針筒或吸管吸取孢子懸浮液，接種于試管斜面培養基上，每管注</font><font face="Calibri">2 ~ 3 </font><font face="宋體">滴。置于</font><font face="Calibri">24 ~ 26 </font><font face="宋體">℃溫度下培養</font><font face="Calibri">5 ~ 7 </font><font face="宋體">天，培養基上便可長出白色菌落。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">待菌落直徑有</font>0 . 5 <font face="宋體">厘米時，選健壯的菌落，再移接于另一試管的斜面培養基上培養。</font><font face="Calibri">ATCC</font><font face="宋體">細胞當白色菌絲長滿試管時，便得純菌種。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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>