1.

0 Title
Staining Techniques, Microbial Morphology
2.0 Objectives
1. To understand the chemical and theoretical basis for different staining procedure.
2. To perform bacteria staining techniques using basic dyes, acidic dyes, and Gram
staining.
3. To compare the morphological shapes and arrangement of bacterial cells under
microscope.
3.0 Introduction
3.1 Simple staining and their classifications
Staining is a technique apply to bacteria so that bacteria appeared as a contrast to surrounding.
This is because living bacteria are almost colourless. Objectives of staining was to provide
contrast between the microorganisms and background; help to differentiate organisms as some
microbes will retain the dyes of the bacteria; and to identify particular structure of microbes as
some dyes can be used to identify internal structures of the cell. Staining is classified according
to chemical nature and staining methods. There are several types of stains in different chemical
nature that were widely used for basic staining techniques, such as simple stains (basic dyes,
acidic dyes, and indifferent dyes), differential stains (Gram stain, acid-fast stain) and structural
stains (feulgen, endospore stain, cell-wall stain, capsule satin, and flagella satin).Besides, there
are several staining methods such as direct stain, indirect stain, differential stain and selective
stain (Supriya, 2019).
3.2 Bacterial shape and arrangement
There are three common shapes of bacteria: coccus, bacillus and spiral.
For coccus, bacteria usually appear spherical in 0.5 micrometer in diameter, some may appear
oval, elongated or flattened on one side. A diplococcus or streptococcus divide in one plane, a
tetrad divide in two planes, a sarcina divide in three planes and staphylococcus divide in
random planes.
For bacillus or rod appears as a hotdog-shaped bacterium in 0.5-1.0 μm wide and 1-4 μm long.
Bacillus is a simple hotdog-shaped bacterium, streptobacillus is bacilli in chains while
coccobacillus is oval shaped dividing bacillus that similar to coccus.
In addition, there is spiral shaped bacteria which appear in vibrio (comma shape), spirillum
(thick, rigid spiral), and spirochete (thin, flexible spiral). Spiral shaped bacteria oftern have a
width of 0.25-0.5 μm (Aryal, 2018).
Figure 1: Different bacteria shapes
3.3 Staining techniques
3.3.1 Direct staining with basic dyes
Dyes are salts in which one of the ions bringing colour and composed of positively charged
and negatively charged ions. Direct staining of basic dyes involved the coloured ions which is
positively charged combined with negatively charged of bacterium when the surrounding is
nearly neutral. Acidic stain involved the coloured ions which is negatively charged. For
example, methylene blue dye has positively charged methylene blue ions and negatively
charged chloride ions. From here, methylene blue ions is the ion that brought colour, hence
positively charged methylene blue ions will combine with negatively charged bacterial cell in
high affinity due to the different charges. Direct staining is the staining of the cells but nor
background (Supriya, 2019).
3.3.2 Negative or indirect staining
Acidic dyes with coloured compound in negatively charged ions normally will be used in this
method of staining. Since coloured dyes is in negatively charged ions, then it will not combined
with bacteria cell which is also negatively charged. Examples of negative staining are
Nigrosine and Eosin. Indirect staining is not staining the cells. Hence, a transparent cell with
coloured background will be observed which subsequently provide a clearer view of the cells
(Supriya, 2019).
3.3.3 Differential staining techniques- Gram stain
Gram stain divide the bacteria into two groups, which are Gram positive and Gram negative.
Gram stain requires four different solutions which are basic dyes, a mordant, a decolorizing
agent and a counterstain. Initial stain normally will be Crystal violet. A mordant is used to
increase the affinity of dyes and bacteria cells. Decolourizing agent was used to wash away the
stains dyes, alcohol is the decolorizing agent that was used. Counterstain aims to produce a
different colour from the initial dyes and Safranin red is commonly used as counterstain. Gram
positive bacteria will be stained purple while Gram negative cells will be stained red. This is
because Gram positive cells had thick peptidoglycan so it will retain the initial dyes while thin
peptidoglycan layer of Gram negative cells will be stained red because the crystal violet was
washed away by the alcohol (Supriya, 2019).
3.3.4 Endospore staining
Gram positive cells such as Bacillus spp. and Clostridium spp. will form endospores in
unfavourable environment such as nutrient depletion and dessication. Since endospore has a
thick mycolic acid, it cannot be simply stained by other dyes as the acid will exclude the dyes.
Hence, hot malachite green is used for initial staining as heat steam will melt the mycolic acid.
After that, safranin red is used as counterstain. The endospores will be stained green, while the
other vegetative cells will be stained red. There are three types of endospores formed, which
are central, terminal and lateral endospores (Acharya, 2015).

Figure 2: Location of different endospores formed

4.0 Materials
Micrococcus luteus, Bacillus subtilis 168 (overnight culture), Agrobacterieum tumefaciens,
Bacillus subtilis 168 (5 days incubated in 30 ᴼϹ), Methylene blue dye, Nigrosine dye, Crystal
violet iodine, Safranin red, Malachite green dye, 85 % NaCl solution
5.0 Methods
5.1 Part A: Methylene blue staining
First, less amount of viable Micrococcus luteus was transfered onto a glass slide. One drop of
85% NaCl was added subsequently. The cells were evenly smeared by using a loop. Bacteria
smear was heat fixed by Bunsen burner. After that, Methylene blue was added to flood the
bacteria smear. Cover slide was put onto the bacteria smear. Lastly, the bacteria smear was
observed under microscope.
5.2 Part B: Negative staining by using Nigrosine
First, two loopfuls of overnight culture Bacillus subtilis 168 was transfereed onto a glass slide.
Next, one drop of Nigrosine dye was added to the bacteria smear. A cover slide was used to
spread the nigrosine dye in thin layer. The bacteria smear was let dry under the room
temperature. Finally, the glass slide was viewed under microscope.
5.3 Part C: Differential stain- Gram stain
First, less amount but viable Gram positive Agrobacterium tumefaciens was transferred from
glass slide onto glass slide by using needle loop. Heat fixed the thin smear by Bunsen burner.
The bacteria smear was flooded by crystal violet about 30 seconds. After that, iodine was added
for 30 seconds. 95% ethanol was added subsequently for 10 seconds. Finally, the bacteria smear
was counterstain by using safranin for 30 seconds. The smear was rinsed by water and tissue
was used to blot dry the bacteria smear. Finally, the glass slide was observed under microscope.
The whole procedure was repeated by using the Gram negative Escherichia coli.
5.4 Part D: Structural stain- Endospore stain Malachite green
First, Bacillus subtilis 168 that was incubated 5 days at 30 ᴼϹ was transferred onto the glass
slide by using needle loop. The glass slide was placed on boiling water for one minute. After
that, Malachite green was used to flood the bacteria smear for 10 minutes which was used to
satin the endospores. A filter paper was used to cover the bacteria smear when it was flooded
with malachite green to prevent it from drying out. Malachite green was washed away
subsequently. Safranin red was added as counterstain to stain the vegetative cells.
6.0 Results
Part A: Methylene blue staining

Rod shaped
Micrococcus
luteus

Figure 3: Micrococcus luteus under Methylene stain

Magnification: 10 x 40 x

Part B: Negative staining by using Nigrosine

 Bacillus
subtilis 168

Figure 4: Bacillus subtilis 168 under Nigrosine stain

Magnification: 10 x 40 x
Part C: Differential stain- Gram stain

Rod-shaped
Bacillus subtilis
168

Figure 5: Bacillus subtilis 168 (overnight culture) under Gram staining

Magnification: 10 x 100 x
 Agrobacterium
tumefaciens

Figure 6: Agrobacterieum tumefaciens under Gram staining

Magnification: 10 x 100 x

Part D: Structural stain- Endospore stain Malachite green

Vegetative cells of
Bacillus subtilis 168

Endospores

Figure 7: Bacillus subtilis 168 (5 days incubated at 30 ᴼϹ)

Magnification: 10 x 40 x

7.0 Discussion
There were some precautionary steps that was practiced in this experiment. For the Gram
staining, young culture was used because old cultures of crystal violet may lose their ability
to retain the crystal violet dyes because old culture of thick peptidoglycan of Gram positive
cells had already shrink. The amount of alcohol for the decolourization of Gram staining
should be added in appropriate amount and proper duration to avoid excessive decolorizing
that may affect the normal results.
8.0 Conclusion
Methylene blue staining is a direct staining that stain the specific cells blue. Nigrosine dye is
an indirect staining as it cannot stain the cells and the specific cells appeared transparent. For
the Gram staining, Gram positive bacteria will be stained purple while Gram negative cells
will be stained red. Malachite is used to stain endospores, so it may stain the endospores
green and stain the other vegetative cells red.
9.0 Reference
Acharya, T., 2015, Endospore Staining: Principle, Procedure and Results - Microbeonline.
[Online] Microbeonline. Available at: https://microbeonline.com/endospore-staining-
principle-procedure-results/ [Accessed 13 Jul. 2019].
Aryal, S., 2018, Different Size, Shape and Arrangement of Bacterial Cells [Online].
Microbiology Info.com. Available at: https://microbiologyinfo.com/different-size-shape-and-
arrangement-of-bacterial-cells/ [Accessed 13 Jul. 2019].
Supriya, N., 2019, What is Staining? Definition, Objectives and Types - Biology Reader
[Online]. Biology Reader. Available at: https://biologyreader.com/staining.html [Accessed 13
Jul. 2019].