ISOLATION, GRAM STAINING AND
IDENTIFICATION OF BACTERIA
DR. TED PASS II
KENTUCKY MICROBIOLOGY LABORATORY
PROCEDURE FOLLOWED TO
OBTAIN A PURE CULTURE USING A
DIFFERENTIAL MEDIA SUCH AS
PREPARE A SLIDE USING GRAM STAIN
IDENTIFY BACTERILA ISOLATES USING
THE API OR ENTEROTUBE
MacConkey agar is probably the most popular solid
differential/selective medium in the world. It is
mainly used in isolation of lactose fermenting,
Gram-negative enteric pathogens and for inhibiting
growth of Gram-positive organisms. Bacterial
colonies that can ferment lactose turn the medium
red. This red color is due to the pH indicators
(Neutral Red) response to the acidic environment
created by the fermentation of lactose. MAC is
decolorized by NLF bacteria...as they utilize Amino
Acids alkaline metabolites are released and the
Neutral Red becomes colorless.
Lactose Fermentors vs Non Lactose
Examples of LF:
Examples of NLF:
*Gram Positive bacteria are inhibited by Crystal violet
and bile salts…i.e., Staphylococcus aureus
ISOLATION STREAK TECHNIQUE
ISOLATION OF BACTERIA ON MACCONKEY
FOR GRAM STAINING AND IDENTIFICATION
The reagents you will need to successfully
perform this technique are:
•Crystal Violet ( Primary Stain)
•Iodine Solution (Mordant)
•Decolorizer ( 95%Ethanol )
•Safranin ( Counterstain)
•Water (preferably in a squirt bottle)
STEP 1 STEP 2
STEP 3 STEP 4
STEP 1: Flood (cover completely) the
entire slide with Crystal Violet…CV
attaches to the cell wall of both G+ and G-
bacteria. Let the crystal violet stand for
about 60 seconds.
STEP 2: Now, flood your slide with
the iodine solution.
Rinse the slide with water after 60 sec. At
this point, the specimen should appear
brownish to blue-violet in color. Iodine
strengthens the bond between the CW
and the CV
STEP 3: Add the Decolorizer,
Ethanol for 15 sec.
To be safe, add the Ethanol dropwise until
the blue-violet color is no longer emitted
from your specimen…Removes 20% lipids,
i.e., LPS and CV-I complex (Decolorizing
the cell) Quickly, rinse the slide with the
water for 5 seconds.
STEP 4: The final step involves
applying the Counterstain,
Flood the slide with the dye as you did in
steps 1 and 2. Let this stand for about a
60 sec. Rinse with water for 5 seconds to
remove any excess dye. Safranin will
attach to G- Cell Wall
GRAM NEGATIVE AND GRAM
GRAM POSITIVE COCCI
GRAM NEGATIVE RODS
GRAM POSITIVE COCCI
The Gram stain differentiates between
bacteria that possess 2% and 20% Lipids in
their cell wall or "cell envelope“. The G- CW
has an outer membrane of
API IDENTIFICATION SYSTEM
This API-20E test strip (from
bioMerieux, Inc.) is used to identify the
enteric gram negative rods; 20 separate
test compartments are on the strip, all
dehydrated. A bacterial suspension is used
to rehydrate each of the wells. Some of
the wells will have color changes due to
pH differences: others produce end
products that have to be identified with
reagents. A profile number is determined
from the sequence of + and - test results,
then looked up in a codebook having a
correlation between numbers and bacterial
INOCULATION OF AN API STRIP
Prepare a suspension of the bacteria
in the saline tube
Inoculate a large colony (2-3mm
diameter)of the bacterium (pure culture)
into the 0.85% NaCl solution.
Use a McFarland barium sulfate standard
Holding the strip at a slight angle up from the table
top, you will now inoculate the bacterial suspension
into each well with the sterile pipette.
Touch the end of the pipette to the side of the cupule,
allowing capillary action to draw the fluid into the well
as you slowly squeeze the bulb. This should eliminate
any bubbles forming in the wells. Each well should be
filled up to the neck (see diagram).
CIT, VP, and GEL have boxes around their names.
These test wells will be filled all the way up to the top
of the well.
LDC, ODC, ADH, H2S, and URE are filled as described
in step B, but they will then be filled up to the top with
sterile mineral oil.
TILT AT ANGLE TO FILL CUPULES
Incubate the strip in its
The bottom of the incubation chamber has small
indented wells in the bottom: fill it with water
just enough to fill these indentations.
Place the strip into this bottom. There should
not be so much water that it spills onto the API
Place the top of the incubation chamber over the
bottom, and label it.
Place the strip at 35 to 37º C for 18-24 hours.
Add the proper reagents to the
1 drop of Kovac's to the IND (read within a
couple of minutes)
1 drop of Barritt's A and B to VP (a positive
reaction may take up to 10 minutes)
1 drop of FeCl3 to TDA
Record results on the diagram handed out to
you in lab (1, 2, or 4 points for + reaction, 0
points for - reaction). The oxidase test
reaction should be negative, and is added as
the last test result.
Three test reactions are added together at a
time to give a 7-digit number, which can then
be looked up in the codebook.
The Enterotube® II contains 12 different
agars enabling the performance of a total
of 15 biochemical tests as well as an
enclosed inoculating wire.
The Enterotube® II contains 12 different agars enabling the performance
of a total of 15 biochemical tests as well as an enclosed inoculating wire.
INOCULATION OF ENTEROTUBE
1. Remove both caps of the Enterotube® II and
with the straight end of the inoculating
wire, pick off the equivalent of a colony from
your unknown plate. A visible inoculum
should be seen on the tip and side of the
2. Inoculate the Enterotube® II by grasping
the bent-end of the inoculating wire,
twisting it, and withdrawing the wire through all
12 compartments using a turning motion.
3. Reinsert the wire into the tube (use a
turning motion) through all 12
compartments until the notch on the
wire is aligned with the opening of the
tube. (The tip of the wire should be seen
in the citrate compartment.) Break the
wire at the notch by bending. Do not
discard the wire yet.
4. Using the broken off part of the wire, punch
holes through the cellophane which covers
the air inlets located on the rounded side
of the last 8 compartments. Your instructor
will show you their correct location. Discard the
broken off wire in the SHARPS container.
5. Replace both caps and incubate the
Enterotube® II on its flat surface at 35-
37°C. for 18-24 hours.