FACULTY OF APPLIED SCIENCES

KUALA LUMPUR CAMPUS

Semester:​ [ / ] May [ ] September [ ] March (​please “​✓​”)
Academic Year:​ 2019/2020
Course Code & Title: ​AACB 3233 MICROBIOLOGY
Programme: ​Diploma in Science (Chemistry and Biology )
Student’s Name (Registration Number): 1. Jerome Liew (18WLD03129)
2. Chee Yong Chun (18WLD01499)
3. Wong Pui Mun (18WLD03931)
4. Chin Jia Yue (18WLD03979)

Submission Date:​ ​ ​1 August 2019

Declaration
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We declare that this assignment is free from all forms of plagiarism and for all intents
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Signature(s):
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Name(s): Jerome Liew, Chee Yong Chun, Wong Pui Mun, Chin Jia Yue
Date: ___________________
Experiment 4 Techniques of Preparing Smear and Methods of Staining
Introduction:
Basic stains, such as methylene blue, Gram safranin, or Gram crystal violet are useful for
staining most bacteria. These stains will readily give up a hydroxide ion or accept a
hydrogen ion, which leaves the stain positively charged. Since the surface of most bacterial
cells is negatively charged, these positively charged stains adhere readily to the cell surface.
Gram staining is a common technique used to differentiate two large groups of bacteria
based on their different cell wall constituents. The Gram stain procedure distinguishes
between Gram positive and Gram negative groups by coloring these cells red or violet.
Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in
their cell walls, which retains the crystal violet these cells are stained with. Alternatively,
Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which
does not retain the crystal violet during the decoloring process.
Endospores staining is used for the purposes of differentiating and classifying bacteria. On
the other hand, it is also very important in medicine and the food industry.
Because they are tough and hard to destroy it is very important to determine whether they
are present in canned food and thus avoid food poisoning to protect consumers.
Capsule stain is a type of differential stain which uses acidic and basic dyes to stain
background & bacterial cells respectively so that presence of capsule is easily visualized.
Capsule is synthesized in the cytoplasm and secreted to the outside of the cell where it
surrounds the bacterium. Most of the capsulated bacteria have a capsule made up of
polysaccharide layer but some bacteria have capsule made up of polypeptide, or
glycoprotein.
The main purpose of Negative staining is to study the morphological shape, size and
arrangement of the bacteria cells that is difficult to stain. eg: Spirilla. It can also be used to
stain cells that are too delicate to be heat-fixed.
It is also used to prepare biological samples for electron microscopy. It is used to view
viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein
aggregates, which all have a low electron-scattering power. It is also used for the study and
identification of aqueous lipid aggregates like lamellar liposomes (le), inverted spherical
micelles (M) and inverted hexagonal HII cylindrical (H) phases by Negative staining
transmission electron microscopy.
In this experiment we prepared multiple smears to examine, we performed simple stains,
gram staining, negative staining, endospores staining and capsule staining​.

Objectives:
1. To prepare bacterial smear from broth and solid cultures.
2. To perform gram staining on ​Escherichia coli​ and ​Bacillus sp.
3. To perform negative staining on ​Bacillus subtilis​.
4. To perform special staining, endospores staining and capsule staining for ​Bacillus
sp.​ and ​Klebsiella pneumoniae​.
Materials:
Stock cultures (broth and agar):
● Staphylococcus aureus
● Escherichia coli (E. coli)
● Bacillus subtilis
● Klebsiella pneumoniae
Transfer loops
Slides
Distilled water
Methylene blue (simple staining)
20% copper sulfate solution (capsule stain)
Nigrosin stain
Gram staining reagents:
● Crystal violet
● Iodine
● Decolorizer- 95% ethyl alcohol
● Safranin
Schaeffer-Fulton endospores staining reagents:
● Malachite green, 5% aqueous (aq.)
● Safranin, 5% aq.

Methods:
Procedure 1: Preparing the Smears
1. Preparation of the slide- The slide was cleaned and dried to remove oils. Aseptic
technique must be practiced every time before the instruments were used.
2. Preparation of the smear- For the broth culture, one or two drops of specimen were
taken and it was spread in the center of the slide. For solid media cultures, a very
small drop of distilled water was put on the center of the slide. A loop of bacteria
from the surface of solid media was then mixed and spread in the water drop.
3. Fixation- the attachment of the organisms to the slide can be done by air-drying or
heat-drying. Slides must be completely dried and fixed before staining.

Procedure 2: Simple Staining

1. The smear was prepared using ​E. coli​ based on the steps in Procedure 1.
2. The slide was placed on the stain rack over the sink.
3. The slide was covered with methylene blue. The stain was allowed to stay on the
slide for 1 minute.
 4. After 1 minute, the slide was rinsed with distilled water until all excess stain was
removed. Rinsing should not be done too long as it may result in the removal of the
smear.
5. The excess water was shaken off from the slide. ( Water is a decolorizer and it
should not be allowed to sit on the slide for very long. )
6. The slide was blotted carefully and was allowed to be dried.
7. The stained smear was examined under the microscope on every power and the
observation was recorded.

Procedure 3: Gram Staining

1. The smear of ​Staphylococcus aureus was prepared. The smear was placed on the
staining rack over the sink.
2. The smear area was covered with the crystal violet stain and it was left for 15
seconds. Then, the slide was rinsed with distilled water.
3. Gram’s iodine was applied on the smear and rinsed after 15 seconds.
4. Gram’s decolorizer was applied on the smear area until no more colour on the area.
The smear was rinsed with distilled water quickly to stop the decolorizing process.
5. Safranin was applied to the smear and rinsed with distilled water after 15 seconds.
6. The smear was placed on the drying and fixation tray to be dried.
7. The smear was observed under microscope on oil immersion (100 × ) and the
observation was recorded.
8. The steps were repeated with the smear of ​E. coli.​

Procedure 4: Endospore Staining of ​Bacillus sp.

1. A smear was prepared using ​Bacillus subtilis​.
2. The smear was allowed to air dry and heat-fix.
3. Malachite green was applied to the slide. The slide was heated over a beaker with
heating water for 5 minutes.
4. The slide was cooled briefly and rinsed with distilled water until no green colour
left.
5. Safranin was applied on the smear and rinsed with distilled water after 1 minute.
6. The slide was blotted and observed under microscope on oil immersion.
7. The observation was recorded.

Procedure 5: Capsule Stain

1. The smear of ​Klebsiella pneumoniae was prepared and allowed to air dry. ( Heat-fix
was not suggested since it would distort the capsules.)
2. The slide was flooded with crystal violet for 4-7 minutes.
3. The slide was rinsed with 20% copper sulfate solution.
4. The slide was blotted and examined under microscope on oil immersion.
5. The observation was recorded.
Procedure 6: Negative Staining with Nigrosin
1. A small drop of nigrosin was placed at the end of a clean slide.
2. A drop of ​Bacillus subtilis​ was mixed into the drop of nigrosin.
3. The drop was spread out along the slide by using another slide so that a gradient of
dye formed.
4. The dye smear was allowed to air dry.
5. The smear was observed under the microscope and the observation was recorded.

Result:
Table 1: Characteristics of the bacteria observed

Specimen Gram reaction Cell shape Cell Colony

Arrangement Morphology

Staphylococcus Gram-positive Cocci Clusters Round, regular,

aureus smooth and
slightly convex

Escherichia Gram-negative Bacilli Clusters Smooth and

coli circular

Bacillus subtilis Gram-negative Bacilli Singles Round and

entire.

Klebsiella Gram-negative Bacilli Singles Circular,

pneumoniae convex and
smooth
 Figure 1: simple staining of E.coli
Magnification: 1000x

Figure 2.1: Gram staining of e coli. Figure 2.2: Gram staining of Staphylococcus
Magnification: 1000x. Magnification: 1000x

​ Figure 3: Endospores staining of. Figure 4 : Capsule staining of

Bacillus.sp. Klebsiella pneumoniae
Magnification: 1000x. Magnification: 1000x
 Figure 5: Negative staining with nigrosin of Bacillus.sp
Magnification: 1000x

Discussion:
Importance of staining
Staining is a method of enhancing the contrast in microscopic images of microorganisms.
Coloured chemicals, such as stains and dyes are used to highlight our interested species and
biological tissues. Stain is a chemical that adheres to the cell or biological tissue, and thus
enhancing the contrast of the bacteria and the background. The significant contrast allows
observer to clearly discern the interested species and/or biological tissues under the
microscope. In general, staining is used to increase our visibility on the specimen, to
accentuate the specimen’s accentuate morphological features, and to preserve the specimen.

Consequences of overheating bacterial smear

Heating the bacterial smear is a technique known as heat fixing. The technique serves to kill
the bacteria and to firmly adheres the stain onto the slide so that it will not be washed off
when it is being rinsed by any other agents. Overheating the smear could possibly distort
the cell wall of the bacteria and thus altering cell morphological features.

Alternative methods for each of the staining methods

A. Gram staining of ​E. coli​ and ​Staphylococcus sp.
Gram staining is a staining method to differentiate gram negative and gram positive
bacteria. Nonetheless, the drawbacks of the technique are that over decolorization of
gram positive bacteria and poor decolorization of gram negative bacteria caused by
chemical factors may defect classification (Dash C, Payyappilli RJ, 2016).
 KOH string test can be an alternative to gram staining identification. A loopful of
bacteria are to be emulsified over glass slide over a 3% KOH suspension. Then stir
the suspension thoroughly and continuously for one minute and pull the loop out of
it gently. The test is considered positive if string is seen within the first 30 seconds
after mixing in KOH solution (Dash C, Payyappilli RJ, 2016).

B. Endospore staining of ​Bacillus sp.

Instead of staining ​Bacillus ​endospore with malachite green via heat, staining them
fluorescent with thioflavin T (ThT) is feasible. The specimen can be observed under
Bright field or epifluorescence microscopies to observe the specimen as the
endospore glows (Xia B, et. al., 2011). Staining endospore was always challenging
as it has an almost impenetrable cell wall.

C. Capsule stain of ​Klebsiella pneumoniae

The technique of performing capsule stain in the experiment was Anthony’s Capsule
Stain that uses crystal violet, and an alternative to that is Maneval’s Capsule
Staining, using Ruthenium Red (or congo red). Replacing crystal violet with congo
red gives pink to red capsule of bacteria under the microscope (Springer EL, Roth
IL, 1973).

​ ith nigrosin
D. Negative staining of ​Bacillus subtilis w
Uranyl acetate and uranyl formate stains are stains that provide superior image
resolution than any other stains used in negative staining. They are smaller particles
with fine grain size (4 to 5 Å) and are used when the specimens are viewed under
high precision and accuracy microscopes (Scarff CA and et. al., 2018).

Conclusion:
Bacillus ​and ​E. coli a​ re gram-negative bacteria while ​Staphylococcus aureus i​ s a
gram-positive bacterium. Most of the bacteria in the experiment have capsules but ​Bacillus
sp. f​ orm endospores. All the features of these microbes can be observed using different
staining methods.

Reference(s):
1. Dash C, Payyappilli RJ, 2016, ​KOH string and Vancomycin susceptibility test as an
alternative method to Gram staining​, Journal of International Medicine and
Dentistry.
2. Xia B, Upadhyayula S, Nuñez V, Landsman P, Lam S, Malik H, Gupta S, Sarshar
M, Hu J, Anvari B, Jones G, Vullev VI., 2011, ​Amyloid Histology Stain for Rapid
Bacterial Endospore maging.​ J Clin Microbiol.
3. Springer EL, Roth IL, 1973, ​The Ultrastructure of the Capsules of D@lococcus
pneumoniae and Klebsiella pneumoniae Stained with Ruthenium Red,​ Journal of
General Microbiology.
4. Scarff CA, Fuller MJG, Thompson RF, Iadaza MG, 2018, ​Variations on Negative
Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems​,
Journal of Visualized Experiment.
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Education Limited, China.
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