EMB Plate - This medium is selective as well as differential.
The medium favors the growth of gram negative organisms since
methylene blue and eosin in the medium inhibits gram positive organisms.
It also contains lactose which lets one distinguish a lactose
fermenter from a nonfermenter. The colonies of lactose
fermenting organisms can range in appearance from very pink in color to a
purple/blackish color. Sometimes the organism releases so much acid
during lactose fermentation that dyes in the medium begin to precipitate
over the colony surface to produce a green metallic sheen. This
is common with E.coli and can also be seen sometimes with
Citrobacter. Examples of lactose fermenters and nonfermenters on EMB
plates can be seen in the figures below.
Plate - The organism growing on this EMB plate does not ferment lactose
since the colonies are white. Sometimes colonies of a
nonfermenter will appear to have a little pink color due to the color of the
medium but do not confuse this with a much darker color due to fermentation.
Try to look at plates of a fermenter and nonfermenter side by
side to see the difference.
Right Plate - The organism growing on this EMB plate does ferment
lactose. The colonies have a purple/black color.
which is growing on the EMB plate below shows the green metallic sheen
that you sometimes see when cells release a lot of acid during lactose
Morphological and Physiological Characteristics of Enteric Microorganisms -
For this portion of lab you will work in pairs.
Each pair will be given one of seven different enteric organisms and will
use that organism to inoculate various media as described in your manual. Next week you will interpret the results of your inoculations and
as a class you will create a table of test results for all seven organisms. You will use this table as a reference when characterizing your enteric
"unknown" which you will be given next week in lab. You can make a
print-out of the following
template for recording class test results.
Your TA will describe how to perform the inoculations and review with you
what each test result will tell about the physiology of the organism. The
following is a list of the various media that you will be inoculating so
that you can review those specific sections of your manual.
Kligler's Iron Agar 2. Simmons
Citrate 3. Semisolid motility tube
Broths - 1. Glucose 2. Lactose 3. Mannitol
4. MR-VP 5. Sucrose 6. Nutrient 7. Tryptone 8.
Plates - 1. Nutrient 2. Eosin Methylene Blue (EMB)
You will also test for catalase and use the filter paper method to
test for oxidase.
Motlity (Exercise 18) -
you will test for motility using the
tube method described in the manual. If organisms are motile in
the semisolid tube they will move out from the stab line while the nonmotile organisms grow within the stab line. Sometimes it is
difficult to see movement clearly so 2,3,5 -Triphenyltetrazolium
chloride (TTC) was added to the semisolid medium. The TTC is used
by bacteria as an electron acceptor. In its oxidized form it is
colorless and will become red after it is reduced by the growing cells.
If you see a red stab line only that would mean cells are not moving
from the initial stab. If the tube has a diffuse red color
that would mean the cells are moving out from the stab line in all
directions. You can also check the surface of the agar and usually
there is bacterial growth all over the surface with motile cells whereas
the nonmotile cell will grow on the surface over the stab line only. See
the figure below which shows each type of organism.
It is important that your stab in the semisolid agar is in the
center of the tube and is as straight as possible. To do this you
might keep your elbows on the table to steady your hands as you stab the
agar. You also should make sure that your needle is straight.
Use the forceps in the drawer to straighten it if needed. Only
stab the medium once with the microorganism.
The organism in the tube on the left in the figure below is motile and
the organism in the tube on the right is nonmotile.