MKBN316/317 Date: 11 March 2025

Report – Physical Practical 3

Bacterial cell staining techniques –
Gram staining, Acid fast staining and Endospore staining

Name, Surname: SIDNEY PHIRI

Student #: 42912318

TOTAL: 65
Mark: _____

1
1.0 Abstract [10
marks]
In microbiology, differential staining techniques are more commonly employed than basic
stains to gain a deeper understanding of bacteria. The reason differential staining techniques
—which usually entail several steps and multiple stain—are called such is because they
enable the differentiation of various cell types or cell structures. Examples of these
techniques include endospore staining, acid-fast staining, and the most crucial, gram staining.
The primary goal of this experiment was to prepare a bacterial smear—also referred to as
Gram stain—for examination under a microscope. Learning how to prepare acid-fast and
endospore staining for a microscope examination and being proficient in distinguishing
between acid-fast, endospore, and gram staining were the other two goals. Gram-staining
was the sole differential staining technique employed in the lab.The method consisted of four
steps: first, cells were stained with crystal violet (primary stain); next, iodine (mordant) was
added to the stained cells; third, 95% ethyl alcohol was added as a decolorizing agent; and
finally, safranin (counterstain) was applied. Note that the two supplied bacterial cultures in
agars were spread onto cleaned and dried microscope glass slides using a sterile inoculating
loop prior to the gram-staining procedures.Gram-staining was done twice.
Following the gram-staining procedures, the outcomes were examined under a light
microscope. Gram-positive bacteria are indicated if they appear purple in the visual
representation. The presence of pink bacterial cells indicates that the bacteria are gram-
negative. Since the findings were observed, the experiment's objectives were met. The
experiment worked out well.
effectively, indicating that the procedures were carried out exactly and accurately.

2.0 Introduction or Background

Since many microbes cannot absorb, reflect, refract, or diffract light, light
microscopes find it difficult to examine them. One technique for improving the
appearance of cells and cell components under a microscope is cell staining (Campbell,
2018). Using different stains makes the cell wall or nucleus visible. Only a few types of
stains can be used on living cells, but the majority of stains can be used on non-living
cells. Differential staining is a technique that uses multiple dye applications to help
distinguish between distinct cell types and their architecture, according to Mienie and
Bezuidenhout (2022).Hans Christian Gram created the basic differential staining
method known as the Gram staining procedure, which is extensively used to divide
bacteria into two main categories: Gram-positive and Gram-negative. The unique
structural features of bacterial cell walls serve as the foundation for this differentiation.
According to Gram, Gram-positive bacteria have a thick covering of peptidoglycan that
makes them appear purple under a microscope and maintains the crystal violet stain
employed in the process. Gram-negative bacteria, on the other hand, have a thinner
outer membrane and peptidoglycan layer, which causes them to absorb the
counterstain, safranin, and look red or pink instead of violet (Smith and Johnson, 2019).

Beyond simple classification, Gram staining is significant. In clinical contexts, it is a

rapid and efficient diagnostic technique. For example, as Gram-positive and Gram-
negative bacteria frequently exhibit distinct antibiotic sensitivity, knowing the kind of
bacteria causing an infection can help choose the best antibiotic therapies (Brown et al.,
2021). Additionally, the method offers insights into the metabolic and structural
characteristics of bacteria, enabling more focused studies in microbial disease and

2
microbiology (Kim and Lee, 2020).Other staining methods, such acid-fast staining and
endospore staining, provide other means of differentiating bacterial species according to
distinct structural elements in addition to Gram staining. Because mycobacteria have
waxy cell walls from a high lipid content, which makes them resistant to conventional
staining methods, acid-fast staining is very helpful for identifying them (Jones and
White, 2020). Staining with endospores
[10 marks]

3
3.0 Results [25
marks]
3.1. Fill in the differential table below: 8 marks

E. coli Staphylococcus aureus

Drawing of the
representative field

Cell morphology shape

1. Arrangement Straight,rod -shaped cells.Cells Spherical-shaped,cells arranged
in pairs and some in single in clusters
2. Cell colour pink Purple(crystal violet)
3. Gram reaction Gram negetive Gram positive

3.2. What is the the advantage of differential staining procedure over simple staining

technique (1

helps to differentiate between different species of bacteria whereas simple staining can only

tell about the cell morphology

3.3. Why is it essential that the primary stain and the counterstain be of contrasting colors?

(1)

- So that cell types or their cell structures can be distinguished from each other on the basis of

the stain tha is retained

3.4. Which is the most crucial step in the performance of the Gram staining procedure and

why? (2)

Decolourization is the most important step in gram staining because if the decolourizing

agent is left on the cells for too long, both gram-positive and gram-negative cells will lose

their crystal violet stain.

3.5. Why do we have to heat the slide when performing endospore staining (1)

- to fix the bacterial smear.

3.6. What is the purpose of heating the acid-fast bacterial smear slide over a steaming water

4
bath.? (2)

The heat melts the waxy cell wall and permits the absorption of the dye by the cells.

3.7. What is the need for filter paper which is used to stain the sample with Malachite Green?

(2)

To take on extra stain and keep the stain from drying out.

3.8 Mention two different reagents used in endospore staining protocol as well as their

function. (4)

Malachite green- used to entice the endospores to take up the primary stain. Counter stain

with safranin- Used to stain those cells that have been previously decolorized.

3.9 Mention any two different reagents used in gram staining protocol as well as their

function. (4)

Primary stain(crystal violet)- used to stain all cells purple. Decolorizing agent( alcohol) –used as a

protein- dehydrating agent and in gram- negative it increases the porosity of thne cell by disolving

the lipids in the outer layer.

5
4.0 Discussion [5 marks]
Different techniques can be used to stain different species of bacteria. In the lab, only the
Gram staining method was applied. The four reagents needed for gram staining and a
bacterial culture were quickly provided by the lab assistants. A hot inoculation loop from the
culture was used to disseminate the bacterial isolates from the culture onto the slide, from
which the sample was taken. Alcohol application is exempt from the rinsing time
requirement because water is used to rinse it fewer than ten seconds after
application.Reagents were applied to the slide in order, with a 60-second pause between each
application. pointing out that beneath a microscope, gram-positive (Crystal Violet) or gram-
negative (Pink) cells will probably exhibit the anticipated outcomes. The results were
grouped, spherical, and crystal violet in color when viewed under a microscope. This
suggests that the germs utilized were Staphylococcus aureus. The use of 95% ethyl alcohol, a
decolorizing agent, is a crucial step in the Gram staining process (Carson & Hladik, 2009).
The counterstain cannot be absorbed if the initial stain remains after decolorization.(Kiernan,
2008), and the primary stain's color will be retained by the cell or any of its constituent
elements. The decolorization cellular components will accept and acquire the opposite color
of the counterstain if the original stain is eliminated. The stain that is preserved allows for
the differentiation of various cell types or their structures.

5.0 Conclusion [ 5 marks]

In summary, the Gram staining technique has been effectively applied in experiments to
distinguish between different species of bacteria based on the characteristics of their cell
walls.
Because it can distinguish between Gram-positive and Gram-negative bacteria, this
method is still a fundamental tool in microbiology and provides crucial details
regarding the structure and function of bacteria (Smith and Jones, 2019). Gram
staining, which includes iodine application, alcohol decolorization, crystal violet
staining, and safranin counterstaining, was successfully carried out, demonstrating its
usefulness in bacterial analysis (Wang et al., 2021).Although the laboratory only used
Gram staining, the fundamental ideas of differential staining are applicable to other
important techniques like acid-fast and endospore staining, which improve bacterial

6
identification in a variety of scientific and medical settings (Taylor and Brown, 2020).
The practical experience obtained while being ready

7.

Understanding bacterial shape and physiology through the interpretation of Gram

stains is crucial for directing future clinical diagnostics and microbiological research
(Johnson and Lee, 2023). Thus, our hypothesis was confirmed.

6.0 References [10

marks]
Campbell, J. (2018). Microscopic Techniques: An Overview. 3rd edn. New York: Academic
Press.
Johnson, L. and Lee, H. (2023). 'Advancements in Gram Staining Techniques for
Clinical
Applications', Journal of Microbiological Methods, 12(3), pp. 245-258. doi:
10.1016/j.jmbm.2023.02.005.
Mienie, A. and Bezuidenhout, D. (2022). Differential Staining in Modern Microbiology. 2nd
edn. London: Springer.
Smith, R. and Jones, T. (2019). 'Applications of Differential Staining in Microbial
Classification', Microbiology Today, 18(2), pp. 134-146.
Taylor, G. and Brown, S. (2020). 'Comparison of Staining Methods: Gram, Acid-Fast,
and
Endospore Techniques', International Journal of Microbial Research, 5(4), pp. 300-315.
Wang, Y., Chen, X. and Liu, Z. (2021). 'Refining the Gram Staining Method:
Innovations
and Clinical Implications', Clinical Microbiology Reviews, 34(5), pp. 789-805. doi:
10.1128/CMR.00521-20.

7
Mini guide

Abstract is a summary of the whole report.Introduction is the background and

significance of the methods. Discuss and interpret your findings in your discussion
(Remember, we only performed gram staining in class).Conclusion highlights results
obtained and if the aim or hypothesis was achieved.

All the best!