KWAME NKRUMAH UNIVERSITY OF SCIENCE AND

TECHNOLOGY
DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY

NAME: OBENG KWADWO ABABIO

Preparation of slides for microscopic studies has long been a powerful tool in obtaining in-
depth insight into the structure of microbes. Fixation, labelling of slide, application of sample
by smearing, and staining are important in preparing a slide for observation (Cappuccino and
Welsh, 2017). Staining techniques form the cornerstone of microbiological studies, allowing
researchers to observe, identify, and differentiate microorganisms under a microscope.
Among these, the preparation and examination of stained smears are critical steps for
visualizing cellular structures, identifying morphological characteristics, and distinguishing
between different groups of microorganisms. By enhancing contrast through the application
of dyes, these methods reveal details that are otherwise invisible in unstained specimens. The
technique utilized specific dyes, such as crystal violet and safranin, to highlight variations in
cell wall structure. The successful preparation and staining of smears provide valuable
insights into bacterial classification and pave the way for further microbiological analyses.

MATERIALS AND METHODS

Materials and apparatus include: Grease-free slides dipped in alcohol, sterile/distilled water,
sterile loop, Bunsen burner, Gram stain (0.5% crystal violet, Iodine, absolute alcohol, Safranin),
forceps and filter paper, microscope.

Two plates, A and B with bacteria culture were provided. Two slides were removed from an
alcohol solution with forceps and wiped with a clean tissue paper. The slides were labelled A
and B and a very small amount of water was dropped on each slide. A little bacterial colony
from plate A was added to the drop of water on slide A with a sterile loop and mixed. The
mixture was spread across the slide in a circular form. Same procedure was applied to obtain
smear B. The two slides were allowed to air dry and were fixed by heating with Bunsen flame
2 to 3 times. Both smears were flooded with 0.5% crystal violet for 2minutes. After, the
smears were washed with water, drained and stained with dilute iodine for 2minutes.
Absolute alcohol was carefully dropped onto the smears (one slide at a time) and allowed to
run off until no primary stain dripped off (was done 3 times) and later washed off with water.
Both smears were counterstained with Safranin for about a minute, drained and washed off
with a gentle stream of water. Filter paper was used to blot off excess water around the slides
before they were viewed under the microscope. The slides were examined without a cover
slip under low power (x10) and high power (x40) objectives first and a drop of immersion oil
was directly placed onto the smears. The smears were examined using the oil immersion
(x100) lens. The results were recorded accordingly.

RESULTS

Table 1.0: Characteristics of Bacterial Colony as observed under the microscope

BACTERIUM GRAM REACTION SHAPE POSSIBLE NAME

Bacterium A Negative Bacillus Escherichia coli

Bacterium B Positive Coccus Staphylococcus

DISCUSSION

The preparation of bacterial smears and Gram staining are fundamental techniques in
microbiology for observing and identifying microorganisms. Smear preparation involves
transferring a small bacterial sample onto a clean slide, spreading it thinly, and heat-fixing it
to ensure the cells adhere and maintain structural integrity (Jorgensen and Pfaller, 2015).
Proper smear thickness is critical, as overly thick smears hinder observation as light from the
microscope is not able to penetrate the slide, while overly thin smears may lose significant
details. Gram staining differentiates bacteria into Gram-positive and Gram-negative groups
based on their cell wall structure (Moyes et al., 2009). The process involves four reagents:
crystal violet (primary stain), which penetrates the bacterial cell wall and cytoplasm, staining
all cells purple; iodine (mordant), iodine binds with the crystal violet to form a large crystal
violet-iodine complex that becomes trapped more easily within the thick peptidoglycan layer
of Gram-positive cells; alcohol (decolorizer), which dissolves the outer membrane of Gram-
negative cells and removes the crystal violet-iodine complex from their thin peptidoglycan
layer, leaving them colorless whiles in Gram-positive cells, it dehydrates the thick
peptidoglycan, tightening its structure and retaining the dye complex; and safranin
(counterstain), which counterstains the now colorless Gram-negative cells, giving them a
pink or red color, while Gram-positive cells remain purple as their thick peptidoglycan retains
the crystal violet stain. Gram-positive bacteria retain the violet-iodine complex due to their
thick peptidoglycan layer, while Gram-negative bacteria lose the complex and take up the
pink safranin stain (Bartholomew and Mittwer, 1952). Observations under a microscope
reveal not only Gram reactions but also bacterial morphology, aiding in species identification
and highlighting key differences vital to diagnostics and treatment strategies.

In bacteria A, the cells looked rod-like under the microscope while they also appeared pink,
under the influence of safranin. In bacteria B, the cells, appeared violet and clustered which
means it retained the crystal violet dye, hence gram positive. With the characteristics, they
are possibly the names assigned them (Cowan and Talaro, 2021)

CONCLUSION

Gram stain differentiates bacteria based on the differential staining properties of bacteria cell
walls. It also helps in diagnosing harmful bacteria for appropriate treatment. Gram positive
bacteria appear violet whiles Gram negative bacteria appear pink under the microscope based on
their retention of the stains (Clark, 1981)

REFERENCES

Bartholomew, J. W., & Mittwer, T. (1952). The Gram stain. Bacteriological Reviews, 16(1),
1–29.

Cappuccino, J. G., & Welsh, C. T. (2017). Microbiology: A laboratory manual (12th ed.).
Pearson.

Clark G. (1981). Staining Procedures, Fourth Edition. Williams And Wilkins, Baltimore, P.241.

Cowan, M. K., & Talaro, K. P. (2021). Microbiology: A systems approach (6th ed.). McGraw
Hill.

Moyes, R. B., Reynolds, J., & Breakwell, D. P. (2009). Differential staining of bacteria: gram
stain. Current Protocols in Microbiology, 15(1), A-3C

Jorgensen, J. H., & Pfaller, M. A. (2015). Manual of clinical microbiology (11th ed.). ASM
Press.