Science Buddies: "Ask an Expert"

"Stir a pound of freshly ground hamburger into a liter of distilled water and put it in the icebox (at about 40 degrees F.) for 10 hours. Then skim off the fat which rises to the top and filter the remaining liquor through a single thickness of clean muslin. Add distilled water to bring the liquor back to a full liter, then add five grams of peptone and five grams of ordinary table salt and stir until the salt is dissolved. Pour 50 milliliters into a second flask and set it aside [this is what I would be using] . Then add 15 grams of agar to the 950-milliliter portion.

"Bacteria, like other organisms, are sensitive to the acid-base balance of the medium in which they grow. Those grown in this experiment prefer a neutral medium. The two solutions just prepared will be slightly acid; they must accordingly be adjusted to neutrality (pH 7) by adding precisely enough sodium hydroxide to counteract the acid. Mix 10 grams of sodium hydroxide in a liter of distilled water. Test the beef broth with a piece of blue litmus paper. An acid broth will turn the blue paper red. The sodium-hydroxide solution will turn red litmus blue. Add a drop or two of sodium hydroxide to the liquor, stir, and with the glass rod put a drop of the solution on a piece of blue litmus. The paper in contact with the drop will probably turn pink. Add more sodium hydroxide to the liquor and test again. Continue this until the test drop causes no change in the color of either red or blue litmus.

"Each container of liquor is then heated almost to 212 degrees F. for half an hour. This will precipitate the proteins in the liquor. The proteins are removed by passing the hot liquor through coarse filter paper. Each filtrate is again brought up to volume by adding distilled water

"One hundred milliliters of the hot agar medium are poured into each of six Erlenmeyer flasks, which are then stoppered with wads of absorbent cotton. Five milliliters of the liquor containing no agar are poured into each of 10 test tubes, which are similarly stoppered.

"The media are now sterilized. The containers may be placed in boiling water for half an hour on each of three successive days. They may alternatively be sterilized in a pressure cooker. Put the containers inside the cooker, add two inches of water and pressure-cook for 20 minutes. Be sure to cool the cooker slowly. Rushing the job by quenching the cooker with cold water will cause the internal pressure to drop suddenly and the vessels of hot medium to boil over. Stoppered tubes of tap water and other solutions may be sterilized by either of these methods.

Purchased cultures can be perpetuated indefinitely by keeping them in beef broth at room temperature and inoculating a fresh tube of medium (by putting a drop of the old culture into it) every other day. If the culture can be stored at 40 degrees F., the growth of the bacteria is slowed and new media need be inoculated only once every six days.