Hi microgeek,
Determining unknowns was one of my favorite parts of medical
microbiology! It was so rewarding to solve the puzzle, using all the
resources available in the lab! I hope you find it educational and
interesting!
Usually, the lab assistant will pass out a broth of unknowns. Start by
having a selection of culture media available, at room temperature.
Never plate organisms on cold media. You will need non-selective media
such as sheep blood agar, and chocolate agar, and selective media such
as MacConkeys, Thayer-Martin, HE, PEA, and maybe some TSI slants.
Your broth itself can give you clues: Look for cloudiness, pellicles,
rings and sediment. Make notes if any of these are present.
http://www.austin.cc.tx.us/microbugz/04growthpatt.html
Using sterile technique, open the tube of unknowns in this manner:
Assuming you are right handed, hold the tube in your left hand, and
the sterile loop in your right hand. (You should have flamed the loop
in the Bunsen burner, or the electrical incinerator and allowed it to
cool a few seconds).
See a picture of loops and straight loops (needles)
http://www.austin.cc.tx.us/microbugz/02aseptictrans.html
Open the loosened cap of the broth tube with the pinky of your right
hand, keeping it curled in your pinky. Still using your right hand,
place the loop in the broth, so not too much of the needle in
submerged. Loosely replace the cap, still using your pinky.(I became
so accustomed to doing this, I opened catchup this way for years,
untill they invented the snap close bottles!) Place the drop on one
end of the slide, being careful not to let the liquid go to the edge.
Using the loop, *gently* smear the broth around, to the size of about
a dime. Let the smear air dry.
When the smear is completely dry, flame the slide, by quickly passing
it through the flame of the Bunsen burner, one or two times. Do this
rather quickly, as you don?t want to fry the organisms! This fixes the
organisms onto the slide, preventing them from washing off during the
staining process.
Once you lay the smear on the staining rack, it should be cool enough
to stain. I?m sure you will have directions for using Gram stain. I?m
sure your lab manual will have a Gram stain technique to follow. I
found remembering the mneumonic "VIAS" helped me remember the order of
the reagents, as your lab partners are sure to get all the bottles of
reagents out of order!
V == Violet
I == Iodine
A == Acetone
S ==Safranin
Use the tweezers, Gram stain reagents don?t wash off easily from
hands, and not at all from clothes!
Once your slide is stained and dried, looking at it under the ?scope
will give you a good clues as to how many different organisms you are
working with. Scan the slide on 40x (Hi dry) and then with oil, using
ONLY the oil immersion lens.
Note how many Gram positives and Gram negatives. Note how many rods
and cocci you have. (You probably won?t have other forms in a
beginning class)
If you see Gram negative rods, look in your text book for possible
gram negative species. Morphology: Notice if the rods are long, short,
fat, slender, if they are bean shaped, how they stick together and if
two seem to be stuck together (diplococci). For Gram positives, notice
if they are clumped together as in a grape cluster, or in chains. I
found this fascinating!
Once you determine the number of organisms in the slide, you can
select your media. Most organisms will grow on blood agar. Plating the
broth on HE agar, for example, will allow ONLY enteric organsisms to
grow. Using Thayer-Martin will allow only gram negative diplococci to
grow. (Some contaminants may grow, but this adds to the mystery!)
Here are photos of some different organisms after Gram staining.
Plate your broth on as many different kinds of media allowed. Streak
for isolation. This is important, so you don?t get an overgrowth. Your
lab manual will have an illustration. Flame your loop first, and allow
it too cool. In real life, we stab the blood agar with the hot loop to
cool it, but your instructor MAY take a dim view of this, so I
recommend not doing it in class.
Streak about ¼ of the plate, in a rapid back and forth manner. Then,
running the loop through that area one time, streak another ¼ of the
plate. Repeat the process on emore time. The final streak should make
a little tail-like line, never touching the previously streaked area
more than the first pass. You want that last streak to be a single
line, and very thin. This will give you some good colonies to use for
the next step.
After you are done with your broth, be sure to replace the cap
loosely. Organisms in broth can produce a lot of gas, and you would
hate for your tube to explode in the incubator!
Here is a similar procedure illustrated. (Do not use a toothpick!)
http://www.rhodes.edu/biology/biologyi/drop-o2.gif
Incubate per your lab. If you have access to a CO2 incubator and/or
an anerobic incubator, it will enhance growth of certain organisms.
Your lab assistant can help you with this. Before CO2 incubators, we
used to put the plates in a huge jar, add a candle, light it, and
tightly close the jar. The candle burned away enough O2 before it went
out, raising the CO2 to the optimum level, to make it very efficient!
Some organisms are aerobic, some are anerobic, and some are
microaerophilic, and the right atmosphere is imperative for growing in
the laboratory.
Allow your plates to incubate about 48 hours. Inspect each plate for
colonies. On the blood agar plate, you will see several types of
colonies. Note the color and morphology of each colony, and using a
sterile loop or straight loop, pick ONE colony of each type, one at a
time. In other words, if you see a white glistening colony, select an
isolated colony, where no other kinds of colonies are touching it.
With this colony, make a new smear. Stain and examine each slide. This
will help narrow down the organism species. Each organism grows in a
distinct colony?some white, some grey, some yellow, some irregularly
shaped, some round, etc. This gives us clues to the kind of bacteria
we are dealing with.
If your lab allows making a subculture, using a sterile loop, grab a
colony of each type, and streak for isolation on new media. Allow to
incubate 48 hours. This will give you a pure culture of each organism.
In order to run confirmatory tests, it is imperative to have a prue
culture, or your results will be erroneous.
Your lab will have follow up tests, according to their budget, to
distinguish like species from each other. These tests, along with the
smear, the colony morphology and your textbook will determine which
organisms you have.
I don?t recommend sticking your nose into the agar plate, but each
species has a distinct odor, that will become familiar to you as you
work with them. E.coli, for example, on MacConkey agar smells grapey,
while pseudomonas smells a bit like corn tortillas.
I don?t know what confirmatory testing your lab has available, but
your manual will tell you what your lab has available.
Here is a list of some confirmatory tests done in a micro lab at Towson
http://www.towson.edu/~cberkowe/medmicro/Lab%2013.doc
This page gives a less detailed description, but covers all the steps
I have written about.
http://webpages.chhs.niu.edu/williams/AHP318/microbiology.htm
This page describes one Gram stain method
http://www.bio.davidson.edu/people/dawessner/302/302Lab2.html
You can see some plated organisms here:
http://www.austin.cc.tx.us/microbugz/03morphology.html
I hope you enjoy your unknowns! Remember that a sterile, aseptic
technique is imperative to avoid contamination. One little stray
organism can ruin all your tests! Flame your loop often! Good luck!
If any part of my answer is unclear, please request an Answer
Clarification, before rating. This will enable me to assist you
further, if possible.
Regards,
crabcakes
Search Terms
Bacterial streaking
Agar microbiology
Personal knowledge and experience |
