December, 1925
INDUSTRIAL AND ENGINEERING CHEMISTRY
1279
Laboratory Pure Culture Apparatus’ By F. M . Hildebrandt U. S. ISDUSTRIAL ALCOHOL Co., BALTIMORE, MD.
N LABORATORY work with microorganisms it is often desirable to have a relatively large quantity of active pure culture for experimental purposes. The usual method of inoculating tubes and flasks in order to obtain a culture is time-consuming and attended by risk of infection. The apparatus described here was accordingly designed to provide a means of keeping on hand a considerable volume of nutrient medium containing active organisms. The organisms may be withdrawn easily and supplied with fresh sterile nutrient when necessary. The apparatus may be kept in continuous operation as long as desired. Description
This apparatus consists of a culture tube connected with a Pasteur flask for holding sterile nutrient solution, both tube and flask being mounted on a wooden frame. The frame is made either to hang on the wall or stand on a table. The details are shown in the accompanying figure. A is the Pasteur flask. It is held in a ring, R1,clamped on a n iron rod, the latter being fastened to the wooden frame by metal straps as shown. A second ring, R, is clamped on the rod. B is the culture tube. This tube is drawn down to small diameter at the bottom, is provided with a side tube, T , and, a t the top, is furnished with a tube, T1,for a cotton plug, bent over to prevent the entrance of dust and spores. It is fastened to the wooden frame with metal straps. The side outlets of the flask and the culture tube are connected through a glass T-tube as shown. Short lengths of rubber tubing are slipped over the lower openings of the fermentation tube B and the T-tube, and are closed with glass rods P and PI. The T-tube is held in place by a spring clip, D, fastened to the wooden frame (a spring clothes-pin was used in the author’s apparatus). At C and CI glass beads, which take the place of stopcocks, are inserted in the rubber tubes. Organisms are kept out of the long tube of the Pasteur flask by a guard consisting of a short piece of test tube slipped orer a cotton plug, as shown in the drawing. Operation
To start the culture, the Pasteur flask is first filled about two-thirds full of the growing solution. The tube B and the flask are then connected through the T-tube with the glass beads at C and C1 and plugs P and PI in place. The upper outlet of the tube B is plugged with cotton and the guard tube is placed over the long tube of the Pasteur flask. The flask and tube are then removed from the frame and sterilized in the autoclave with all connections in place. They are then remounted on the frame, and test tubes containing a n antiseptic solution hung under the tube B and the T-tube. The test tubes are filled level full with the solution so that the plugs P and PI and the short rubber tubes are completely covered. To inoculate the tube B, the tube containing antiseptic is removed and the plug P is taken out and dropped in a beaker containing antiseptic solution. A suspension of the organisms is then blown into B by means of a sterile pipet slipped into the rubber tube on the lower end 1 Presented before the Division of Biological Chemistry a t the 69th Meeting of the American Chemical Society, Baltimore, Md., April 6 to 10, 1925.
of B. The rubber is pinched over the glass bead C to give a passage to the liquid from the pipet. The plug and the tube of antiseptic solution are then replaced. I n order to run nutrient solution from A into B, the former is put on the ring R and tipped so that the solution will run out of the short side tube. The rubber is pinched over the bead Cl and the desired amount of solution passed from A into B. The flask is then replaced in the ring R1until more nutrient is necessary for the culture. The culture is withdrawn by pulling the plug P and pinching the rubber over C, enough material being left in B to re-inoculate the fresh solution passed in from the flask A . When the solution i n A is exhausted the flask is refilled as follows: A second Pasteur flask containing the nutrient in use is sterilized. This flask is provided with a guard over the long tube, as shown in the drawing, and the short side tube of
the flask is plugged with cotton. After sterilization the flask containing fresh solution is brought to the apparatus, the guard removed, and the long side tube quickly connected to the T-tube a t its lower end after removing the plug PI. The T-tube is taken out of the clip and the flask containing the fresh solution is elevated and tipped so that the liquid runs out of the long tube into flask A . The plug PI is then replaced and the antiseptic put under the T-tube outlet. Whenever the plugs are removed they are dropped into an antiseptic solution. This apparatus has been used with success in growing yeast in pure culture. Since all the openings point down, there is little chance of infecting it if reasonable care is used in handling the plugs and if the tubes are kept filled with antiseptic. Five per cent formaldehyde solution has been used when yeast cultures were grown. Various adaptations of the apparatus suggest themselves. For instance, approximately anaerobic conditions could be secured by evacuating tube B through the upper opening, and tube B could be graduated if i t were desired to withdraw definite amounts of culture. The apparatus is also convenient for use in acclimatizing organisms to various ty)es of solutions. It can be made in any size. I n the author’s apparatus, flask A is of one liter capacity and tube B has a capacity of about 350 cc. The frame on which the parts are mounted is 46 cm. (18 inches) high, 38 em. (15 inches) wide, and with a bottom shelf 18 cm. (7 inches) wide.
