Streak Plates

For preparation of a streak plate, a single loopful of infectious material is streaked over the surface of the solid medium (agar) in a Petri dish. There are several ways to do this. The patterns are widely used and give good separation of colonies, which can then be used for subculturing.

Bacterial Plate Count

The accuracy of the bacterial plate count rests on the premise that when material containing bacteria are cultured, every bacterium present develops into a colony. This statement is not strictly true, since some bacteria may fail to multiply and two or more bacteria may cling together to form a single colony. Nevertheless, the method is of decided value in the examination of’ water and milk.

One method of doing a bacterial plate count using 4 tubes containing 9 ml. of sterile distilled water (dilution blanks) is as follows’: Use a sterile I ml. pipette to add 1 ml. of the test sample (water or milk. for example) to the first dilution tube and mix. Transfer I ml. from this tube (using another sterile pipette) to the second dilution tube and again mix. From the second tube, transfer (using another sterile pipette) I ml. to the third tube and again mix. From the third tube transfer (with another sterile pipette) 1 ml. to the fourth tube. Again mix. You now have in the four dilution tubes 1:10, 1:100, 1:1000, and 1:10,000 dilutions, respectively, of the original material. With a sterile pipette transfer exactly I ml. from each tube to• a Petri dish and add sufficient (about 20 ml) melted agar (cooled to 42° C.). Mix the contents of each dish by rotating and allow solidifying. After incubation for 24 to 48 hours, count the colonies that have grown. Let us say that the second plate shows 200 colonies. This means that at least 200 bacteria were present in the 1 ml. of material placed in the tube, and since this was a 1: 100 dilution, the original test material must have contained at least 20,000 bacteria per milliliter. More bacteria may- have been introduced into the dish, since some may have failed to grow and some may have clung together in groups of two or more that grew as single colonies. In other words, the number of bacteria indicated by the count is certainly present, and more may be also.

The bacterial plate count is an important part of the microbiologic evaluation of urine from a patient with possible urinary tract infection. A measured amount of urine is handled as indicated above. A colony count is made from the bacterial growth on solid media. Quantitation helps to indicate whether bacteria recovered from urine are disease producers or contaminants. Generally, with less than 1000 to 10,000 colonies per milliliter of specimen, the microorganisms isolated represent normal flora of the urethra or contaminants.

Culture of Anaerobic Bacteria

To grow some anaerobic bacteria in a routine laboratory, one may use a tube of suitable culture medium about 8 inches in length and about half full of medium, which tube is heated in a boiling water bath for several minutes to expel oxygen present in the medium. The tube is quickly cooled and inoculated; then enough sterile melted petroleum jelly to make a layer about 3l4 inch thick is poured onto the top of the medium; thus the culture is sealed off from the air. If agar is used, inoculation is made when the heated medium cools to a temperature of around 42° C.

Thioglycollate broth, a special medium containing thioglycollic acid, supports the growth of anaerobes within the depths of a partly filled tube without special seal. Methylene blue indicator colors the upper layers of the fluid medium as oxidation occurs.

Cultures may be made in the usual way and placed in a specially constructed anaerobic jar (for example, GasPak jar), a tightly sealed chamber from which the oxygen is either removed by some chemical reaction that is made to take place in the chamber or replaced by a gas, such as hydrogen, that does not affect the growth of the bacteria. Cultures may be placed in an anaerobic incubator. Better results are obtained with the more sophisticated methods indicated.

Slide Cultures

Slide cultures used to study fungi may be placed on the stage of the microscope and actual growth observed from time to time. The depression of a sterile culture slide is filled with suitable medium, inoculated, and covered with a sterile cover glass. Liquid cultures may be made by inoculation of a drop of medium on a sterile cover glass and proceeding as for a hanging-drop preparation.