I.
Introduction
Bacteria in nature do not exist as pure cultures like the ones that we have used in lab. Instead, they are found in mixed populations with many other species. To accurately examine the characteristics of an individual species, the species must be isolated from the rest of the population. Isolation can be accomplished by streaking for isolation as described in Laboratory exercise #1. Also, certain bacteria can be isolated by streaking the population onto selective media. After isolation, bacteria can be identified using biochemical tests such as the ones as described in Laboratory exercises #5 and #6.
The nine bacterial species that we will be using in this laboratory exercise are
Staphylococcus aureus , Pseudomonas fluorescens , Bacillus polymyxa , Alcaligenes faecalis , Klebsiella pneumoniae , Staphylococcus epidermidis , Escherichia coli ,
Streptococcus fecalis , and Micrococcus luteus . You will be given a mixed culture containing two of the nine organisms listed above. Your first goal is to isolate the two organisms from each other and generate a pure culture of each. Your second goal is to identify each of the organisms in your pure cultures.
II.
Procedure
A.
Week #1
1.
Session 1 a) Obtain mixed culture from instructor. Record the code # of the culture. You MUST turn in this number with your lab write up. If you do not do so, you will not receive credit for identifying the organisms.
b) Streak the culture for isolated colonies to TSBA (tyrptic soy broth agar).
c) Incubate 24 hours at 37°C.
d) Do a gram stain of the mixed culture. Record results. This initial gram stain will tell you what to expect later and aid in eliminating any possible contaminants that are inadvertently selected for during your work.
e) Save the mixed culture by storing at 4°C.
2.
Session 2 a) Record observations of the colony morphologies that you see on the
TSBA plate.
b) You should have two colony morphologies present and isolated colonies of each. If you can only see one colony type, examine the primary phase; there are probably two species growing there.
(1) If you have two colony types of different morphologies that are isolated, proceed to c.
(2) If you have two colony types that are not isolated, make a new streak for isolation using the primary phase to inoculate the loop.
Incubate 24 hours at 37°C. Then proceed to c.
(3) If you can not tell that there are two colony types, see the instructor.
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c) continuing… …
(1) Pick one very isolated colony representative of morphology A and streak for isolation on TSBA.
(2) Pick one very isolated colony representative of morphology B and streak for isolation on TSBA.
(3) Incubate both plates 24 hours at 37°C.
3.
Session 3 a) Verify that all of the colonies on plate A have the same morphology and that their morphology is the same as the colony from which they were inoculated.
b) Verify that all of the colonies on plate B have the same morphology and that their morphology is the same as the colony from which they were inoculated.
c) Pick isolated colony from each plate and streak onto TSBA plate.
Incubate 24 hours at 37°C.
4.
Session 4
Store plates wrapped in parafilm in the crisper compartment of the refrigerator in
Dr. Villa's lab. This will be the master plate to start cultures from next week.
B.
Week 2
1.
The day before lab a) Inoculate 2 ml tryptone broth for tryptophanase test for each organism.
Incubate 37°C in shaker in Dr. Villa's lab approx. 24 hours.
b) Streak your two organisms to fresh TSBA plates. Incubate 37°C.
2.
The morning before lab
Inoculate 2 ml TSB with each organism. Incubate 37°C in shaker in Dr.
Villa's lab from 4-8 hours.
3.
Lab a) Do a gram stain on each culture using fresh TSB culture and record results.
b) You will be provided with the materials for one set of biochemical tests (Labs 5-6) per colony type. Set up the biochemical tests that you need in order to determine the identity of your organisms.
4.
Post lab a) Compare your results to the chart provided to identify your organisms b) If needed, request extra supplies to repeat a test. Do not take extras without consulting instructor.
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