314 LABORATORY WORK. 



always the case and therefore the culture must be purified. With the tip 

 of a platinum needle remove a minute quantity of the growth on the agar 

 and place it in a 9 c.c. water blank. Thoroughly shake and transfer two 

 platinum loopfuls of the water to a melted agar tube. Shake gently so 

 as to mix, but not to produce bubbles, and then transfer two loopfuls of 

 this agar to a second agar tube, mixing as before. Pour the contents of 

 each agar tube into a Petri dish, harden and incubate as usual. If the cul- 

 ture was pure the colonies should be all alike. Pick out one of them with 

 the platinum needle, inoculate it upon another agar slant and label it a 

 pure culture from milk or whatever may have been its source. In this 

 condition it may be set aside and preserved for a long time. As long as the 

 agar remains moist the bacteria will usually be alive. 



In the above manner isolate and purify a considerable number of cul- 

 tures of bacteria from the plates made in Nos. 46, and keep these in a cool, 

 dark place for use in various experiments given below. 



No. 8. Microscopic Study of Bacteria. Prepare one or both of the 

 following staining solutions: 



Methylene Blue. 



Saturated alcoholic solution of methylene blue, 1 5 cm. 

 Potassium hydrate (i: 10,000),* 50 c.c. 



Fuchsin Solution. 



Saturated alcoholic solution of fuchsin, 5 c.c. 



Five per cent, solution of carbolic acid, 45 c.c. 



In the middle of a clean microscopic slide place a drop of water (steril- 

 ized). With a platinum needle remove a very small quantity of the bac- 

 teria growth from the surface of one of the slant cultures prepared in No. 7 

 and place in the drop of water. Stir this drop with the needle, to distribute 

 the bacteria and then (a) spread it over the slide. Allow it to dry in the air, 

 and then pass the slide three times slowly through a gas flame. The pur- 

 pose of this is to (6) fix the bacteria firmly to the slide so that they will 

 not be washed away. It is necessary not to use too much heat. This may' 

 also be done by leaving the slide for a few minutes on a water-bath. After 

 fixing, cover the bacteria completely with several drops of one of the stain- 

 ing solutions and allow to (c) stain for several minutes. The length of time 

 necessary for this varies with conditions, one to five minutes being usually 

 sufficient. Wash off the stain in running water and then dry the slide by 

 gentle heat. Place a drop of immersion oil upon the stained bacteria and 

 place the slide under the microscope. Use a i /i2 inch immersion, lowering 

 the objective into the immersion oil and focusing very carefully. If the 

 microscope has an Abbe condenser or a diaphragm it is best to have this 

 widely open. The bacteria are so minute that it is hardly possible to study 

 them with a lower magnifying power than a 1/12, although they can be 



* To make this solution add i c.c. of a 10 per cent. KOH solution to 99 c.c. of water, and then 

 add 5 c.c. of this to 45 c.c. of water. 



