Introduction stain, which stains Gram-positive bacteria blue-purple and Gram-negative


The identification of bacteria and other microbes is a core aspect of
microbiology, and allows us to research, diagnose, and treat a variety of
diseases. A key general method for bacterial and microbe identification is
staining. Staining methods consist of two broad types of stains: simple and
differential. Both simple stains and differential stains are essential to
identifying bacteria, whether by adding contrast to better visualize the
bacteria to identify them based on morphology, or by differential methods to identify
bacteria similar in morphology from each other based on other essential
characteristics such as cell wall structure. Simple staining uses a single dye,
with examples of common dyes including crystal violet, methylene blue,
malachite green, and safranin (Anderson,
Salm, Allen, & Nester, 2016, p. 54). In the case of a simple stain, the dye provides
contrast to cells that would otherwise appear opaque, and thus gives more
detailed definition to their morphology.

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Differential stains are multi-step processes, and can be used to
identify different types of bacteria based on unique traits not visible by
standard light microscopy. One of the most common and medically important
methods of differential staining is the Gram stain, which stains Gram-positive
bacteria blue-purple and Gram-negative bacteria pink-red. The Gram stain
separates the two types of bacteria using differences in cell wall structure:
the thick peptidoglycan of the Gram-positive cell wall dehydrates during an
alcohol rinse applied after a crystal violet stain and prior to a safranin
stain, and the crystal violet remains locked inside these Gram-positive cells. As
the difference in cell wall structure of Gram-positive and Gram-negative
bacteria is important in the selection of antibiotics, the Gram stain is often
used to inform the choice of treatment for bacterial infections.


Simple stains and differential stains are used for a wide variety of
unique purposes, and new applications of stains are frequently discovered to
aid in the process of identification of microbes. In some cases, researchers
are exploring simpler staining methods that may achieve faster or
higher-quality results than standard methods. In a paper on a modified version
of Field’s staining for identification of Trichomonas vaginalis, researchers
discovered that a simple rapid stain using this method provided a preferable
alternative to the two standard methods of Giemsa staining and Gram staining (Afzan, Sivanandam, & Kumar, 2010). Staining
methods aid in rapid identification of microbes as compared to other
identification methods, and are often accessible in settings where complex
testing is unavailable, for instance in rural settings and developing countries
(Moemen, Bedir, Awad, & Ellayeh, 2015; Wang
& Murdoch, 2004). The efficiency of identification via staining
methods is also essential when a suspected infection requires rapid treatment
in order to achieve a favorable outcome for the patient (Moemen et al., 2015).


Materials and Methods 

The goal of this lab was for students to conduct gram stains on three
different species of bacteria: Escheria coli, Staphylococcus saprophyticus, and
Bacillus subtilis. A previously scheduled lab relevant to this report on
simple staining procedure was not conducted. Initially, six slides were
wet-mounted (two of each sample), allowed to air-dry until no liquid was
visible, and then heat fixed. Aseptic technique was used to transfer a single
loop of each species to the slides. After heat fixing, a gram stain of the E.

coli sample was attempted, using the following standard process provided:

Crystal violet applied to flat slide
(60 seconds)

Water rinse drop-wise (10 seconds)

Iodine applied to flat slide (60 seconds)

Gentle water rinse drop-wise (10

Decolorizing alcohol rinse drop-wise
(10 seconds)

Gentle water rinse (10 seconds)

Safranin applied to flat slide (60

Gentle water rinse (10 seconds)

Upon inspection of the initial E. coli slide, no bacteria could
be visualized. Multiple people in the lab inspected the slide via microscope,
and none could locate bacteria. This process was then repeated twice with the B.

subtilis species, and no bacteria could be visualized. Upon conferring
between partners and instructors in the lab, it was decided that an inadequate
amount of substance might have been added to the slides. During the first three
gram stain attempts, it seemed that the visible sample was fully washed away
from the slides during the alcohol rinse. Thus, two changes were made to
attempt a successful stain: the use of additional culture on the smear, and a
reduction in time and volume of the alcohol rinse. Two additional slides, one
of S. saprophyticus and one of E. coli, were wet-mounted with three
loops of sample and heat fixed. Following heat fixing, the revised
method of staining was used on each slide.



Of the five total gram stains attempted,
bacteria were visualized only for a single slide of S. saprophyticus. This
was the fourth slide prepared, and the first prepared using the modified
technique. These bacteria stained purple in color and were spherical in shape.

The bacteria clustered together, primarily in chains but occasionally in larger
clusters, as indicated in Photograph 1, below.

The final slide was also prepared using the modified technique, but
yielded no visible bacteria. Table 1, below, summarizes the results of the five
attempted stains. The deviations described were the only changes made between
the first three and the last two samples.Conclusion  This lab highlighted that differential staining requires a high degree
of precision in the multi-step process. While we found a solution to our
initial problem, we were unable to replicate the solution a second time. There
are numerous variables that could explain this. As we obtained a successful
stain of one species but not the others, some of the samples may have been
sparse in terms of bacterial growth. However, due to the visibility of the
smear prior to the alcohol rinse and the full disappearance of the smear
following the alcohol rinse in each unsuccessful stain, this is not the most
likely explanation. Human error due to a learning curve with the Gram stain
procedure is a more likely explanation, as is the possibility of an overly
concentrated or otherwise incorrect alcohol solution.  

The results we did obtain were as expected. Staphylococcus
saproyphyticus is a Gram-positive coccus, and thus we would expect it to
stain blue-purple. While we were unable to successfully stain the other
bacterial samples, we would have expected Escheria coli, a Gram-negative
bacterium, to be rod-shaped and stain pink, and the Bacillus subtilis, a
Gram-negative bacterium, to be rod-shaped and stain purple. The most
meaningful takeaway from this lab is that differential staining procedures may
take practice to execute. We also learned the importance of familiarity with
and understanding of equipment and materials, as we left the lab still unsure
of which type of alcohol we had used during the alcohol rinse step where our challenges