Types, Techniques, Gram Stain and Hanging Drop Method
Introduction (E. Coli)
Commonly referred to as E. coli, Escherichia coli is a bacterium that is
typically found in a number of environments including various foods, soil and
animal intestines. E. coli is very diverse and belongs to the genus
Escherichia. While most of the strains are harmless (and also
important in the human intestinal tract), others are harmful and can cause very
serious health implications. A good example of this is E coli 0157:H7.
Apart from food poisoning that can cause
diarrhea and other problems in the digestive system, harmful strains of E coli
can cause the following health problems;
Infection of the urinary
In the intestinal tract, E coli plays an
important role in that it aids in digestion and supports the absorption of
vital vitamins from food in the body. In addition, it has been shown to be
beneficial in that it prevents the growth and proliferation of other harmful
species of bacteria that would otherwise cause health problems.
E. coli is described as a Gram-negative
bacterium. This is because they stain negative using the Gram
The Gram stain is a differential technique that
is commonly used for the purposes of classifying bacteria. The staining
technique distinguishes between two main types of bacteria (gram positive and
gram negative) by imparting color on the cells.
Being Gram-negative bacteria,
E. coli have an additional outer membrane that is composed of phospholipids and
lipopolysaccharides. The presence lipopolysaccharides on the outer membrane of
bacteria gives it an overall negative charge to the cell wall. Because of these
properties, E. coli does not retain crystal violet during the Gram staining
To determine whether a strain(s) is
present in a sample, it's necessary to stain the sample. Here, Gram stain is
used as it helps distinguish between the gram positive and gram
negative bacteria in a sample.
Being a differential stain,
Gram stain is more complex compared to more simple stains like methylene blue.
As such, more than one stain is used for the purposes of differentiating the its cellular components.
For this technique, 3 different stains are used.
Microscope glass slide
A collecting wire loop
A heater (Burner)
Sample (E. Coli Culture)
Gram stain (Crystal violet,
Iodine stain and Carbol Fuschin)
Before scooping the sample,
heat the wire loop and allow cooling (to make sterile).
Using the wire loop, scoop
and place the broth culture on to a clean, sterile slide.
Gently spread the inoculum
to about a centimeter in diameter (spreading the sample on the slide)
For plate cultures (of
bacteria) use a sterile wire loop to drop saline solution at the
middle/center of the slide
Using another sterile wire
loop (heat and allowed to cool) scoop a small amount of the sample and stir on
the drop of water to form an emulsion
Here, heat is used for fixing. Heat fixing procedure involves the following steps:
Air dry the slide for the smear to dry
Pass the slide through the flame a few times (2 or 3 times) smear side up
* During heat fixing, avoid overheating the slide.
Place the slide on a
staining rack and flood the smear with crystal violet for about one minute
Slightly tilt the slide and
rinse with water (distilled or tap water) gently - Avoid roughly washing the
Flood the sample with iodine
and allow the slide to stand for about 1 minute
Slightly tilt the slide
again and rinse using tap or
distilled water (gently).
Tilt the slide and decolorize using 95% ethyl
alcohol/acetone - decolorizing should be done by applying the alcohol in drops
for about 8 seconds
Rinse with water gently
Blot the slide dry
Place the slide on
microscope and view the sample
When viewed under the microscope, Gram-negative
E. Coli will appear pink in color. The absence of this (of purple color) is
indicative of Gram-positive bacteria and the absence of Gram-negative E. Coli.
The hanging drop method refers to a technique
that helps examine small, living and unstained organisms. It can be used for
such motile bacteria as Ps. fluorescens and E. coli.
Using the hanging drop
technique is also very important given that it helps distinguish between Brownian
movement (the movement of particles that are suspended in a fluid) and motility
Some of the requirements include:
Clean glass slides
A cover slip
Heater (Bunsen burner)
A young broth culture of
motile bacteria (Preferably within 24 hours)
Apply a paraffin ring on a clean
glass slide (an adhesive tape ring can be used for this)
Using a scraper/laboratory
scoop, dab Vaseline on the corners of a cover slip
Using a wire loop,
carefully place a loopful of the broth on to the center of the glass cover slip
Turn the glass slide over
the sample on the cover slip to have the sample lying between the slide and the
Place the slide on the
microscope for viewing (the cover slip should be on top)
Start with the lowest power
and view the specimen
To determine whether the bacteria is
really moving, it's important to ensure that it's moving in different
directions and changing position. This differentiates motility of the organism
from mere Brownian motion.
Dr Kaiser, G. E. (February 2009). Gram Stain of
Practical Microbiology for Secondary Schools, pp 22-23
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