PRACTICAL 3

TITLE: The Staining of Microorganisms - Simple Staining and Gram Staining.

OBJECTIVES
1. Explain the rationale and procedure for the Simple Staining and Gram Staining.
2. Differentiate between Simple Staining and Gram Staining.

INTRODUCTION
Microbiologists use a microscope to examine the features of microorganisms such as algae,
protozoa, bacteria, fungus, and viruses. While certain species, such as protozoa and yeast cells,
are simple to examine using a wet mount, bacterial cells are more difficult to detect. Staining is
required for improved visibility of bacterial cells and cellular features, scientists devised many
approaches such as Gram staining, acid fast staining, and fluorescence labelling.
It is feasible to discover structural traits that aid in the classification of bacteria using
such staining approaches. Bacterial organisms are so microscopic that most of them can only be
seen with a 1000X magnification microscope. However, just magnifying bacteria does not offer
enough clarity, thus bacteria must be stained before observation to achieve the clarity required
for viewing.
Differential staining is the process of staining bacteria to distinguish between various
species of bacteria. The Gram stain is one example of a differential stain that identifies bacteria
based on their cell wall composition. Bacterial cells react with a crystal violet stain to take on a
violet colour in this procedure. Some bacterial cells lose colour when a de- staining chemical is
added, whereas others do not. When safranin stain is added, the decolorized cells absorb the stain
and turn red, whilst the bacterial cells that did not lose colour stay violet. Bacterial cells that
absorb the red colour are known as Gram negative organisms, whereas those that do not absorb
the colour are known as Gram positive organisms. Gram staining is a quick method for
identifying bacteria that are involved in infections.
MATERIALS
Compound microscope
Student’s saliva
24-hours culture of E. coli
Glass slides
Cover slips
Inoculating loop
Crystal violet
Methylene Blue
Paper towel
Gram’s iodine
Safranin
Bunsen burner
Staining rack
Test tube
95% Ethanol

METHODOLOGY
A. Simple Staining Techniques
1. Microscope slides were cleaned and dried thoroughly
2. The slide’s surface in which the smear was to be spread was flamed.
3. The inoculating loop was flamed.
4. A loop full of tap water was transferred to the flamed slide surface.
5. The loop was reflamed to make sure the entire length of the wire that will enter
the tube has been heated to redness.
6. The tube cap was removed with the fingers of the hand holding the loop.
7. The tube mouth was flamed.
8. Make sure the inoculating loop was not so hot that it will distort the bacterial
cells, then a pinhead size sample of the bacterial growth was picked up.
9. The tube mouth reflamed, the cap was replaced,and the tube was put back in the
holder.
10. The bacteria on the loop was dispersed in the drop of water on the slide and the
drop was spread thinly and evenly over an area the size of a 10 cent coin.
11. The inoculating loop was reflamed to redness including the entire length that
entered the tube.
12. The smear was allowed to air dry thoroughly.
13. Heat-fix the smear cautiously by passing the underside of the slide through the
burner flame two or three times. Test the temperature of the slide after each pass
against the back of the hand. It has been heated sufficiently when it feels hot but
can still be held against the skin for several seconds. Overheating will distort the
cells.
14. The smear was stained by flooding it with one of the staining solutions and
allowing it to remain covered with the stain for the time designated below.
15. During the staining, the slide may be placed on the rack or held in the fingers.
16. At the end of the designated time the excess stain was rinsed off thoroughly with
running tap water gently.
17. The back of the slide was wiped and blotted the stained surface with a paper
towel.
18. The stained smear on the microscope stage was placed, smear sided up and focus
the smear using the low and high objectives lens.
19. An area of the smear in which the cells are well spreaded in a monolayer was
chosen. The coverslips were put at the centre area to be studied, oil was applied
and focused on the smear with the 100X objective.
20. All procedures was repeated with different type of bacteria and student saliva
(student have to spit their saliva inside a test tube first)
21. The observations were recorded.
B. Gram Staining Techniques
1. The smear was prepared. S. aureus culture broth tube was gently agitated to
disperse the bacteria.
2. The inoculating loop was sterilized and let it cooled for 20-30 seconds.
3. Loop was placed in the bacterial broth and the loopful of the broth was put onto
the glass slide. The drop rubbed into a 10 cent-sized smear.
4. The loop was sterilized again to kill any remaining bacteria and the smear was air
dried.
5. The slide was fixed by using a heat-fixing method.
6. The smear was stained. A slide rack was used to hold the slide. Cover the smear
with crystal violet and let sit for 1 minute. The slide was rinsed carefully with tap
water gently . The smear was covered with iodine and let it sit for 1 minute. The
slide was rinsed again with running tap water gently.
7. The slide was picked up off of the staining rack and was tilted downward over
the sink. Decolorizer (ethanol) was squirted onto the slide, directly above the
smear.
8. The decolorizing step was stopped when there was no longer any excess purple
running off. (5 – 10 seconds)
9. The smear immediately was rinsed with water and placed back onto a staining
rack to cover the smear with safranin and let sit for 30 seconds.
10. The slide was rinsed once last time with tap water and the excess water was
shaken off of the slide and carefully blotted between two layers of paper towel.
11. The stained slide was examined microscopically using the low, high and oil
immersion objectives to determine if the sample is Gram Positive or Gram
Negative.
DATA AND ANALYSIS
Results
A. Simple staining by using Methylene blue

Figure 1: Image of E.coli and saliva under the microscope

Figure 2: Image of s. Aureus under the microscope

DISCUSSION
E.coli from 24 – hours cultures are observed under microscope by using a simple staining. The
bacteria E.coli is significant and has a significant impact on human health. It is frequently found
in warm-blooded animals' and humans' guts. E. coli strains in general are not harmful. However,
some strains, such Shiga toxin-producing E. coli (STEC), can result in serious foodborne illness.
It is mainly spread to people by eating contaminated foods like raw or undercooked ground meat,
unpasteurized milk, contaminated raw vegetables, and sprouts. In figure 1, the rod shaped of E.
coli can be seen under 4X magnification (from group 3). However, samples of E. coli from our
group cannot be seen under a microscope. This is because students tend to overheat the bacteria
that cause cell distortion. Image of saliva in figure 1 can be seen under 10X magnification. In
order to get a clear result from the experiment, it is consequently important to avoid making
personal mistakes in the lab.
For gram staining by using crystal violet from figure 2, the cocci shapes of s.aureus can
be seen under 40X magnification and 100X magnification (from group 3). By gram staining
using crystal violet, we can observe the s. Aureus is a gram positive bacteria which has a thick
layer of peptidoglycan layer. However, the picture used for the result had to be replaced with the
s.aureus for gram staining taken from another group which was from group 3. This is due to a
personal error during the experiment. While attempting to complete the gram staining procedure,
the e coli sample accidentally fell into the sink. This mistake resulted in no sign of e coli when
seen under the microscope. Hence, why the results had to be replaced with another group’s
results. Therefore, when carrying out the experiment, it's necessary to adhere strictly to the
instructions in the lab manual to avoid mistakes like overheating bacteria, which could result in
cell distortion and personal errors.

CONCLUSION
The objective of this experiment is to differentiate between simple staining and gram staining.
During the experiment, we can see that simple staining requires a few different chemicals to
perform it and the slides were placed on the rack before it was observed while gram staining was
performed to determine whether the sample was Gram positive or Gram negative. It can be
concluded that the objective of this experiment is obtained as the result shown.

QUESTION

1. Are there any bacteria in your saliva sample?

2. Are there any differences between the cell wall of Gram positive and Gram negative
bacteria, which might explain differences in the rate of decoloration?
Gram-positive bacteria keep their crystal violet colour and stain purple, while
gram-negative bacteria lose their crystal violet colour and stain red. Gram-positive
bacteria have a cell wall that is composed of thick layers of peptidoglycan. On the other
hand, gram-negative bacteria have thin layers of peptidoglycan.
 3. Based on your opinion, do you think gram staining is a simple procedure?
Based on my personal opinion, gram staining is not a simple procedure. Although it is a
most widely used staining technique in bacteriology, the staining technique has a
complex and differential staining procedure.

4. Can you add iodine before crystal violet when preparing a Gram stain?
No. According to the staining technique, iodine must be applied after the staining by
crystal violet. This is because crystal violet stains can only bind to peptidoglycan in cell
walls with the help of iodine.

REFERENCE

1. Aryal, S. (2015, April 7). Gram staining: Principle, procedure, interpretation, examples
and animation. Microbiology Info.com.
https://microbiologyinfo.com/gram-staining-principle-procedure-interpretation-examples
-and-animation/
2. Major difference between Gram-positive and Gram-negative bacteria. (2022, September
22). BYJUS.
https://byjus.com/biology/difference-between-gram-positive-and-gram-negative-bacteria/
#
3. Staphylococcus aureus - StatPearls - NCBI bookshelf. (2022, July 18). National Center
for Biotechnology Information.
https://www.ncbi.nlm.nih.gov/books/NBK441868/