Throughout Labs 8-11, a number of biochemical tests have been introduced that can be helpful in the identification of an unknown microorganism. In this experiment, each student will be assigned two unknown bacterial isolates. One of the unknown isolates will be a Gram-positive organism and the other will be a Gram- negative organism. The first step in identifying these unknowns will be the Gram stain. In the next lab period, working and stock cultures will be prepared. Morphological and physiological criteria will be used to identify the genus and species of each isolate. During the next few lab periods, various tests will be performed on each unknown. After sifting through all the results of each test, it will be possible to deduce the true identity of each unknown organism.
Bacterial Unknowns Procedure:
Procedure: (each student) Collect 1 TSA plate and 1 MacConkey plate from the side
bench.
1. Collect an A & B unknown culture pair from the TA or instructor. Be certain that both the A & B unknowns have the same number! Tell the TA the number of the unknown so that he or she will have an accurate record.
Unknown "A" is presumed to be Gram-positive; Unknown "B" is presumed to be Gram-negative. It is very important to keep careful and detailed records. Record all observations, test results, etc. in the table following Lab 20. Be careful not to reverse the A and B unknowns!
2. Mix the A and B cultures by flicking. Be certain that both cultures are visibly turbid indicating that they have grown and are viable. If either one of your unknowns did not grow, inform the TA or instructor and obtain a replacement culture.
3. Prepare two microscope slides by labeling each slide with the unknown number and either A or B. Prepare a smear of each unknown on the appropriately labeled slide. Following are some tips for obtaining a high quality smear preparation. (Slide Prep Procedure)
a. Clean the slide thoroughly before beginning the smear preparation.
b. Do not dilute the culture. Simply use one to two loops of the concentrated broth
culture.
c. Patiently let the smear air dry and then be certain to heat fix well!
4. Perform a Gram stain, click to review Gram Stain procedure, to verify the Gram reaction of each unknown and to determine the morphology and arrangement. A focus line drawn on the top of the slide will assist in locating the smear. Save these slides and record all observations in the Lab 18 Unknown Charts.
5. Label both the TSA and MacConkey plates each with your initials and lab section number. Label the TSA plate with your unknown number A and the MacConkey plate with your unknown number B.
6. Using these labeled plates, streak both unknowns for isolation using the T-streak method, click to review T-streak procedure. Invert the plates and place them in the tray on the side bench to be incubated at 37ºC.
7. Return the original unknown cultures to their rack on the side lab bench.