STAINING TECHNIQUES

Prado, R., Tarun, N.A.

Department of Biology
College of Science
University of the Philippines Baguio

ABSTRACT
Bacterial cells are minute and colorless. To observe forms and structures of bacteria better,
different staining methods are can be done. However, smear preparation must be done first
before the staining techniques. Bacterial cells must be killed or immobilized in order to examine
the structure of the cells better. There are two main types of stain, the simple stain that uses only
one stain or dye and the differential stains that includes the gram staining, endospore staining,
and negative staining for capsule. Gram staining is to distinguish if the bacterial cell is gram-
negative or gram-positive while the endospore staining is to distinguish if endospore is present
and to locate where it is. On the other hand, negative staining for capsule is used to identify if the
culture is encapsulated. In order to proceed with the staining techniques, smear preparation is
accomplished. First, primary stains are applied for each corresponding staining techniques before
proceeding to the counterstains. Then, the stained smears are examined under the preferred
objective. Results show that the isolated culture A and B are both cocci and using gram staining,
it is found out that the isolated culture A is gram-positive while the isolated culture B is gram-
negative. Moreover, both isolated cultures do not comprise of endospore and are not
encapsulated. Thus, with the use of staining techniques bacterial cells are examined well.

INTRODUCTION

Bacteria are microscopic organisms. They are colorless for the most part and hard to view

with the naked eye or even under the bright field microscopy because the cells are motile and

live microorganisms (Weistreich, 1997). In order to visualize them to study their structure, shape,

and other structural characteristics, it becomes necessary to make them more easily visible thus

the structures have to be contrasted from their environment so that they can be seen easily and

the bacterial cells must be immobilize through some pre-treatments (Kumar, 2015). One of these

pre-treatments is bacterial smear in which microorganisms from a loopful of medium are spread

onto the surface of a glass slide that can be used to view killed organisms. Bacterial smear must
be prepared carefully and properly in order to sufficiently separate bacterial cells from one

another, will not disrupt cell arrangements, will adhere to the slides and the bacterial cells are

more readily accept stains (Birge, 1992).

Staining techniques make the individual cells stand out against their backgrounds for

structural and chemical differences in cellular structures examination and to look for the parts of

the cell (Sandle, 2016). With a stain or dye, cells were bind to a cellular structure and were given

colors. There are two kinds of dyes, the catinonic or basic dyes, which are attracted to any

negatively charged cell components and samples of these dyes are methylene blue, crystal violet,

safranin, and malachite green, and the other type of dyes is the anionic or acidic dyes such as

eosin and picric acid, that are attracted to any positively charged call materials (Ahern, 2016).

The usage of a single dye to contrast the cell of the bacteria against its background is

called as simple staining. It reveals desired basic cell shapes, sizes and cell arrangements when

employed. On the other hand, differential stain makes use of two or more dyes and distinguishes

between two kinds of organisms or between two different parts of an organism. The most

frequently used differential stains are Gram stain. Gram staining helps to identify bacterial

pathogens in specimens and cultures by their Gram reaction if it is Gram positive and Gram

negative and morphology (Acharya, 2013).

A few types of bacteria produce resistant cells referred to as endospores. Endospores are

dormant forms of living bacteria and should not be confused with reproductive spores produced

by fungi. These structures are produced by a few genera of Gram-positive bacteria in response to

adverse environmental conditions such as heat and chemicals that permits survival of the

bacterial species for very long period of time. Endospore walls are very resistant to penetration
of ordinary stains thus a special method called endospore staining was developed to make them

easier to see with a bright field microscope. When a simple stain is used, the spores will be seen

as clear, glassy, and easily recognizable areas within the bacterial cell. Thus, this method uses

heat or long exposure time to entice the endospores to take up the primary stain, usually

malachite green (Bauman, 2004).

A layer of polysaccharide material that surrounds many bacterial cells and can act as a

barrier to host defense mechanisms pertains to capsule. It also repels stains. By contrast, acidic

dyes are repulsed by the negative charges on the surface of cells and therefore do not stain them.

This stain is referred to as negative stains because they stain the background and leave cells

colorless. Negative stains are used primarily to reveal the presence of negatively charged

bacterial capsules. Encapsulated cells appear to have a halo surrounding them (Black, 2013).

Certainly, bacteria have different characteristics and structures differentiating species

from another species and some characteristics and structures such as Gram-negative and Gram-

positive, and if it possesses endospore and capsule are distinguished and carried out. Similarly,

this experiment also inseminates among students the proper procedure for preparing a bacterial

smear.

MATERIALS AND METHODS

A 24-hour old of the pure culture of bacterial isolate is emulsified until it is thin enough

to a sterile slide with a loopful of sterile water. After air drying the smear, it was fix through

passing the slide smear side up over flame making sure that the slide does not become too hot

that can distort cells. Then, the prepared smear is flooded with methylene blue for one minute
and was rinse with tap water. It was then examine under the microscope after draining off excess

water.

For Gram staining, smear of the bacterial pure culture is prepared. First, smear is flooded

with crystal violet for one minute then it was rinse with tap water. Next, it was stain with iodine

solution for another one minute and is rinsed with tap water and drained. Third, using the 95%

ethanol the smear was decolorize for eight seconds until the crystal violet was wash off, it was

rinse and drain. The usage of the 95% ethanol is strictly for just a short period of time because if

it lasts long, the bacterial cells will be affected or worst will be killed. Lastly, it is counterstained

with safranin for a minute and was rinse. The reaction was then determine under oil immersion

objective and was distinguish if it is gram (-) or gram (+).

In endospore staining, another smear of atleast 48-hour old bacterial culture is prepared.

The smear is flooded with malachite green. While the smear is flooded with malachite green, it

was pass gently over the flame for five minutes without allowing it to boil and it was rinse with

tap water and was drain. Then, with the use of safranin, it is counterstained for a minute. It was

rinse and drain then. The location of the endospore is located when it is examined under the oil

immersion objective.

To determine if the culture is encapsulated, a drop of nigrosin was place near one edge of

a clean and strile slide and a loopful of bacterial culture was mix with the nigrosin ink. Using

another sterile slide, the mixture was run across the slide 1-3 times and the newly formed smear

is air dried. For 30 seconds to one minute, the smear is covered with safranin and was rinse

gently with running water. Heat is not used because the nigrosin is an example of anionic stain
which is used to stain the background not the vegetative cell thus the dye should not penetrate

the bacterial cell. It was then examine under oil immersion objective.

RESULTS AND DISCUSSION

Cell staining in microbiology is an important and useful technique in visualizing cell

morphology and structure under a microscope (Hu et. al, 2015). Staining involves coloring the

microorganism with a dye to increase the degree of contrast between the organisms to be

observed and the background, resulting to more accurate observation of microbes’ shape and

cellular arrangement (Mehrotra &Sumbali, 2009).

Simple and differential staining are two main types of staining techniques employed in

the laboratory, where simple staining involves the use of only a single dye, while differential

staining utilizes two or more dyes to differentiate the morphology of microbial species. Simple

stain, moreover, is usually an aqueous or alcohol solution of a single basic dye in low

concentration applied in the fixed smear to highlight the shape, size, and arrangement of cells

(Mehrotra &Sumbali, 2009).

In the laboratory experiment, simple staining was done using methylene blue dye applied

to previously fixed bacterial smear. Results show after observation in the microscope that the

cells from the bacterial isolate are stained blue, circular in shape, and exist as single cells, which

were initially hypothesized to be coccus in identification.

 Fig. 1. Simple staining of bacterial isolates A and B.

Microbial cells stained with methylene blue absorbed the dye and appeared blue as the

stain itself is cationic in nature thus attracted to the negatively charged particles of bacteria such

as polyphosphates, DNA and RNA (Kiuchi, 2016). Since the advent of microbiology, bacterial

cells spherical or seed-shaped are named as coccus, thus the identification established for the

cells observed in the microscope (Cabeen & Jacobs-Wagner, 2005). In detail, cocci come in two

classes, those with truly round shapes dividing in either two or three alternating

perpendicularplanes during consecutive division cycles such as micrococci, and those with

elongated ellipsoid shape such as enterococci (Pinho, et. al, 2008), but simple staining method

alone is insufficient to specifically identify the bacterial samples, thus further testing is required

for more correct identification.

Which is why, gram staining is also executed in the experiment to distinguish the strain of

the bacterial isolate being tested. Developed in 1884 by Danish physician Hans Christian Gram,

this method is currently the most widely used differential staining technique in bacteriology.

Differential staining procedures such as gram staining delve on the principle that different

microorganisms show different staining reactions with the dyes exhibited in the colors they
produce (Mehrotra & Sumbali, 2009). Gram stain differentiates bacteria either as gram positive,

which retain the initial crystal violet stain, or gram negative which are decolorized and stained

red (Beveridge, 2009). Gram staining reveals the size, shape, arrangement, and kind of strain of

the isolate, thus is important in identifying whetherthe isolate is a pure culture.

Observation of gram stained specimens indicates that bacterial isolate A is gram positive,

evident in the purple color retained from the crystal violet primary stain, while isolate B is gram

negative due to the red color exhibited. It is further observed that the cell shape and arrangement

are similar to those observed from the simple staining method done, signifying that the bacterial

isolate indeed is a coccus. Furthermore, cells observed are of uniform color and shape, indicating

that the isolate is a pure culture.

Fig. 2. Gram staining results of isolate A and B.

The differences in the color retained by microbial species after gram staining is a result of

the chemical and structural makeup of the cell walls of gram positive and gram-negative strains.

Gram positive microorganisms, such as isolate A, have thick, relatively impermeable wall that

resists decolorization with the alcohol (Beveridge, 2009). Several layers of peptidoglycan
comprise gram positive cell walls which are dehydrated by the introduction of alcohol, causing

the pores to close and preventing the crystal violet-iodine complex from escaping. In

comparison, gram negative strains of bacteria like that of isolate B possess thin peptidoglycan

layers and overlying lipid-protein bilayer referred as outer membrane, which can be disrupted by

decolorization. The alcohol readily penetrates the outer membrane and extracts the crystal violet-

iodine complex, leaving the cell colorless unless counterstained with the second dye safranin

(Madigan et. al, 2009).

It must be noted, however, that the distinction between gram positive and gram-negative

bacteria is not absolute, as a gram-positive strain can give erratic response to the stain, resulting

to erroneous identification. Some old culture gram positive bacteria might lose the ability to

retain the crystal violet dye, thus appearing red after absorbing the counterstain safranin

(Mehrotra & Sumbali, 2009). Such can also be attributed to growth stress brought by unsuitable

nutrients, temperatures, pH levels, or electrolytes, resulting inseveral nonviable, gram-negative

cells in a gram-positiveculture (Beveridge, 1990).

To further characterize the bacterial isolates, endospore staining and capsule staining are

also employed. On the one hand, endospore staining is a method done to recognize the presence

of bacterial endospores in vegetative cells, usually done with a malachite green dye, an

alkalinesubstance able to stain the endospore (Kamal et. al, 2017). Bacterial endospores are

highly differentiated cells extremely resistant to heat, harsh chemicals, and radiation, enabling

the microorganism to thrive and endure environments with extreme temperatures (Madigan et. al,

2009).

The complex structure of endospores consisting of outer layer exosporium, spore coats,

and cortex makes this layer difficult to stain, thus a substance that can penetrate the walls of the
endospores is required. Additionally, heating is also employed in the method for the endospores

to absorb the stain used (Kamal et. al, 2017).

In the experiment, bacterial endospores are not observed in both isolates tested,

signifying that such structure is absent from the said bacterial species. Microbiological

researches establish that very few cocci species produce endospores, and the majority of

endospore forming microorganisms are currently assigned into four genera in the family

Bacillaceae. In this family, the genus Bacillus and Clostridium are the most popularly known

endospore forming microorganisms causing diseases to other organisms (Leuschner, 2003).

Endospore forming coccus belong to the genus Sporosarnina, gram positive strains first isolated

by Beijerinck in 1901 (Priest, 1989).

Fig. 3. Endospore staining results of isolates A and B

On the other hand, negative staining for capsules involves using an acidic stain to observe

the presence of capsules in the bacterial isolate. The repulsion between the negative charges of

the stain and the bacterial surface stains the background instead and leaves the cells colorless,
hence the term negative staining. Negative staining for capsules does not require heat fixation, as

the heat might distort the capsule and result to false results (Breakwell et. al, 2009).

Capsules in bacteria are composed of high molecular weight polysaccharides or

polypeptides and are often associated with virulence and biofilm formation (Breakwell et. al,

2009). In the experiment, negative staining procedure of the isolates show that the specimen does

not contain capsules, evident in the absence of a glowing halo like structure surrounding the

cells. While many gram positive and gram-negative pathogens contain capsules and many early

findings on the structure were made from Streptococcus pneumonae (Wen& Zhang, 2015), the

coccus isolates were negative in capsule formation.

Fig. 4. Negative Staining for Capsule result of isolates A and B

The presence of capsules is commonly associated with bacterial pathogenicity, as certain

gram positive and gram-negative bacteria have evolved capsules significant in the pathogenesis

of infections of plants, animals, and insects. All capsulate bacteria are responsible for causing

diseases, some of which the most serious invasive infections, including septicaemia, meningitis,

pneumonia,osteomyelitis, septic arthritis and pyelonephritis (Kroll & Moxon, 1990).

SUMMARY AND CONCLUSION

Microorganisms are diverse in nature most especially in their distinct characteristics and

structures. Bacterial cells are tiny or minute and are living organisms that are hard to be seen in a

bright field background especially when they are motile. In order to view, examine, and

distinguish their cell structure and characteristics properly different staining techniques are

introduced and accomplished precisely in this experiment. Pure culture were fix in a smear

before proceeding to the different staining techniques namely, simple staining, gram staining,

endospore staining, and negative staining for capsule. The results suggest that bacterial cells have

different characteristics, forms, sizes, shapes, and structures. It can also be concluded that

executing the staining techniques properly and precisely can lead to better view and examination

of the distinct characteristics and structures of the bacterial cells.

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