Lab 3 Instructions
Chapter 11:
Streak for Isolation
Instead of doing everything from this chapter, we will only do a streak for isolation, as shown on
page 91.
Use the slant you inoculated last week and streak for isolation onto a nutrient agar plate. See
page 91 in your lab manual. Be sure to flame your loop after you inoculate the first quadrant,
then drag your loop ONCE across quadrant 1(through the middle) and steak it into quadrant 2.
Flame your loop and drag it ONCE across quadrant 2 and streak it into quadrant 3. Flame your
loop and repeat for quadrant 4. Turn your plate counter-clockwise after you perform each streak.
Then use the broth you inoculated last week and streak another nutrient agar plate in the same
way. Place both plates in the incubator and check it next time to see if you were able to
successfully isolate a single colony in the fourth quadrant of each plate.
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When a doctor takes a swab from an infected wound and sends it to the lab, he needs to know
what organisms are in the wound so he can select the proper antibiotic. There usually will be
more than one organism present, so the lab will need to take the mixture of bacteria and separate
them out into pure cultures before they can identify each organism. Here is an experiment that
illustrates this.
Add one pipette of each of the below three organisms to one sterile test tube. Now that we have a
mixture of 3 organisms, the challenge is to separate them out into three pure cultures again. Here
are the three organisms to use:
E. coli (forms white colonies)
Serratia marcescens (forms red colonies, but only at room temperature, 25°C)
Chromobacterium violaceum (forms purple-blue colonies, but only at 25°C).
All of these organisms are Gram negative, so if you only did a Gram stain on the tissue swab, it
would look like there is only one organism present. Therefore, you have to grow them on a plate
so you can examine the colony morphology (color and shape) of the colonies, and then in real
life, the lab would also run some tests on each pure culture to identify each organism. We
already know the organisms in the mixture, so we will just use this experiment as practice for
separating a mixed culture into pure cultures. You will need this skill when you get your
unknown organisms in the next lab unit.
Do a streak for isolation onto a nutrient agar plate. If you were successful, in the next lab period
you will see your red, white, and blue colonies as separate colonies in the last quadrant. It will be
easy to tell them apart because we chose organisms that form different color colonies. If you
were not successful, your red, white, and blue colonies will still be overlapped in the last
quadrant. What might have gone wrong? Perhaps you had too many organisms on the first
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quadrant, or maybe you dragged too long of a line from one quadrant to the next, or perhaps your
zig-zags were too narrow and did not cover enough surface area.
Remember to leave your plates upside down during the week, so the agar is on the top.
Why do we do this? It prevents condensation from developing on the inside lid, and then
dropping onto your agar, mixing the organisms. It also makes it easier to retrieve the plate from
the sleeve container. If you do not put the plate in upside down, and you try to lift it, you might
accidentally lift the lid off your dish, exposing the agar to the air and contaminating it. Keeping
the plates stored upside down also enables you to read the important writing on the bottom of the
plate. After you examine your plate next week, select the very best looking white colony which
is separated from the other colonies, touch it with a sterile needle, and zig-zag it across the
surface of a slant. Do the same for each of the other two color colonies, so you end up with three
slants, one of each organism. You will then let those grow until the next lab period. You will
know if your isolation technique was successful if there is only one color in each tube.
Remember that the organisms for this experiment need to stay at room temperature.
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Chapter 12: Special Media for Isolating Bacteria
Each group should take one of each of these Petri dishes:
Nutrient agar
Mannitol Salt Agar (light red plate)
Eosin- Methylene Blue (EMB) agar (dark red plate)
Use your wax pencil to mark 4 quadrants onto each of the three plates. Label each quadrant with
E, P, M, or S to correspond to the four organisms below. Each plate will have one organism per
quadrant.
We will work with 4 organisms for this experiment:
1) E. coli (Gram neg rod)
2) Pseudomonas aeruginosa (Gram neg rod)
3) Micrococcus luteus (Gram + cocci that makes yellow colonies)
4) Staphylococcus epidermidis. This is a Gram + that makes white colonies
The MSA plate contains a lot of salt. Only Gram positive organisms can tolerate this, so which
of your quadrants should have growth next time?
The Eosin and Methylene Blue plate contains those two substances. They are dyes that inhibit
Gram positive bacteria, but are attractive to Gram negative bacteria. Which of your quadrants
should have growth next time?
MSA and EMB agar are therefore SELECTIVE MEDIA because only certain types of bacteria
will grow on them. All of your organisms will grow on the Nutrient agar, so it is not selective.
MSA also contains a sugar called mannitol. Only certain organisms will use the sugar. If the
sugar is used, the waste product of its metabolism is an acid. The red color in the plate is from
methyl red, a pH indicator that will turn yellow if acids are present. If the media turns yellow
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next week, the organism fermented the mannitol sugar. You should have two organisms that
survive on this agar next week, and one is a mannitol fermenter (media will turn yellow in that
quadrant) and the other is not (media will stay orange-pink). This demonstrates that we can grow
more than one organism on that plate and see that one ferments mannitol and the other does not.
That means that this media is also DIFFERENTIAL media. Therefore, MSA is selective AND
differential media, while EMB is just a selective media. Salt makes MSA selective, and mannitol
makes it differential. When E. coli is grown on EMB agar, it will have a green metallic sheen as
it absorbs the dyes. Look for that next lab period.
Another type of media is ENRICHED media, such as blood agar. In this media, sheep’s blood is
added to nutrient agar. This plate would grow organisms that are fastidious (picky eaters), such
as pathogenic bacteria like Streptococcus pyogenes (causes strep throat). With this media we can
also see whether the organism demonstrates alpha hemolysis (blood turns green), beta hemolysis
(blood turns clear), or gamma hemolysis (no change in blood color).
SOIL PROJECT
Find the colonies that secrete antibiotics
1) Take another paper grid (supplied in lab), and draw it onto the bottom of a new
TSA plate and number the squares. This will be your Test Plate.
2) Spread E. coli (or a different ESKAPE organism) on that new plate
3) Transfer colony #1 from your Master Plate to grid square #1 of your Test Plate,
and repeat for all the colonies.
4) Incubate upside down at room temperature. The Tech will put them in the
refrigerator on Monday so they do not overgrow.
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