實驗1 Streaking isolation of bacteria 細菌劃線分離 第20章， 129頁

目的 原理 材料與方法

目的 Use streaking technique to obtain pure bacterial isolates from mixed culture. 使用重複劃線的技巧由混合菌液中得到單一細胞形成的單一菌落。

原理 Bacteria exist as mixed cultures in natural environment and food samples. However, it is necessary to obtain pure culture for identification of bacterial species as well as other microbiological tests. In other word, obtaining pure culture is the first step for most microbiological studies. A basic microbiological technique, streaking, is the mostly used method to obtain pure well-isolated colonies, which are generated from a single bacterial cell.

Thus, a well-isolated colony is composed of one bacterial species and can be used for further identification. During streaking, bacterial population is diluted by serial streaking on a plate. At the final streaking, bacteria population should be reduced to a level which bacterial cells were singled on streaking and well-isolated colonies can be generated from those singled cells. 稀釋細菌 濃度至單一細胞，而獲得由單一細胞形成的菌落。

The colony which is visible contain millions cells. Colony forming unit (CFU) Not every bacterial cell can form a colony, only biological active cell can multiply to form a colony. A well-isolated colony theoretically is formed by from a single cell.

材料 大豆胰蛋白酶洋菜培養基(trypic soy agar, TSA) 大豆胰蛋白酶培養液(trypic soy broth, TSB) 蒸餾水（或去離子水） Bacterial species: Escherichia coli (E. coli) Staphylococcus aureus (S. aureus) Both were incubated in TSB at 37℃ for 18-24 hours. These two species are mixed into a tube. Each table should have one tube.

Inoculation loop (接種環), alcohol lamp (酒精燈), laminar hood (無菌操作台), incubator (培養箱).

Procedures: 去除手上(含手腕)所有物品，洗手。 清潔操作面(噴酒精、擦拭)。 點燃酒精燈，將接種環燒至成紅色，環後端也需以酒精燈過火消毒(不需燒至紅色)。 將燒紅的接種環置入TSA培養基中(盡量靠在邊緣)以冷卻接種環。

打開裝有菌液的試管，試管口需以酒精燈火焰滅菌。 將接種環放至於菌液中，略為晃動後取出。 將有菌液的接種環放置于TSA上開始劃線。 重複劃四遍，如下圖。第一及第二遍的範圍較小而第四遍(最後一遍)的範圍需最大。 待菌液乾後，將劃過的TSA培養皿倒置放入37℃培養箱培養。 培養18-24 後觀察，E. coli 應為白色半透明菌落，S. aureus應為黃色。

1st streaking 2nd streaking

3rd streaking 4th streaking

Obtain an isolate coming from a single cell. Thus, biochemical and genetic characteristics should be the same. Isolation and characteristics observation

special characteristics Observation of special characteristics β-hemolysis