Lab 8

Endospore Stain and Gram Stain

Alyssa Ference

7 October 2019

SBL 223 02
I. Title: Performing an Endospore stain and Gram stain with Escherichia coli and Bacillus

megaterium.

II. Materials and Methods:

To begin the experiment, three clean, glass slides were collected and labeled, one

B.megaterium, one E.coli, and another one E.coli for the gram stain. The Bunsen burner was set

up and the inoculating loop was sterilized in the flame. The slides were placed on the staining

tray and the inoculating loop was used to transfer one loopful of water to the center of each slide.

The inoculating loop was sterilized by running it through the Bunsen burner flame. Then, cells

from a given species were transferred to the drop of water on the slide with the matching label.

The cells were mixed and spread on the plate with the inoculating loop and the slides were left to

air dry. Once the slides were completely dry, they were picked up with a clothespin, and passed

through the Bunsen burner flame three times in order to heat fix the cells onto the slides. To

perform the endospore stain, one of the slides with E.coli and one of the slides with

B.megaterium were both placed in separate petri dishes. A fitted paper towel was placed on each

of the slides in the petri dishes. Malachite green dye was then used to stain the slides in the petri

dishes by flooding the placed paper towels with the dye. The petri dishes were placed in the

incubator for 17 minutes at 55-60 ° C. While the endospore plates were in the incubator, the

gram stain was completed with the labeled E.coli plate. Once finished, the gram stained E.coli

plate was observed under the microscope. After 17 minutes, the endospore stained plates were

collected from the incubator and the paper towels were removed. The slides were rinsed with

distilled water and stained with Safranin for 60 seconds. Again, the slides were rinsed with

distilled water after the 60 seconds and dried with bibulous paper. Once dried, the slides were

observed under the microscope to identify the presence of endospores.

The purpose of this experiment was to identify the formation of endospores through the

performance of an endospore stain on Bacillus megaterium and Escherichia coli with Malachite

green. The staining technique used was the Schaeffer-Dulton Technique, which includes staining

with Malachite green, rinsing with distilled water, and restaining with Safranin. The different

results of these two organisms helped identify the nature of each one. Endospore stains help

identify bacteria as Gram-positive or Gram-negative by the performance of a gram stain in the

beginning, then helps identify them as endospore forming or non-endospore forming organisms

by staining them with Malachite green.

An endospore stain is a differential stain used to help visually represent bacteria cells that

can form endospores (Welcome to Microbugz, 2019). One of the bacteria that form endospores is

Bacillus. The formation of spores helps certain bacteria survive hostile conditions and the spores

can be formed in only 6-8 hours after being exposed to the hostile conditions (Welcome to

Microbugz, 2019). When you view the endospore stained bacteria under a microscope, the

normal cells seen are referred to as the vegetative cell. The endospores can form within different

areas of the cell and once the cell returns back to ideal conditions, the endospores germinate into

vegetative cells (Welcome to Microbugz, 2019). There are three different types of endospores,

based on their location in the cell; central endospores, terminal endospores, and sub-terminal

endospores (Welcome to Microbugz, 2019). Often times, the shape and size of the endospore can

determine the organism being viewed. Because of this, endospore stains are used in the medical

field to help identify what bacteria is invading a patient’s immune system or body (Endospore

stain, 2019). Spores are resistance to normal staining procedures, like direct and indirect staining,

because they have tough protein coats made of keratin. By incubating the cells after they have
been stained with Malachite green, the stain is driven into the endospores (Keating, pg 113).

Malachite green is water-soluble, which allows it to be rinsed easily and for the vegetative cells

to be stained by the counterstain, Safranin (Welcome to Microbugz, 2019). It is important to

understand which bacteria are endospore-forming because spores increase a bacterium’s ability

to resist antibiotics. When an antibiotic is introduced to a bacterium in an attempt to destroy it,

the bacteria is placed in hostile conditions (Reay). If the bacteria can produce endospores, it

makes the bacteria harder to reach by the antibiotic, due to the touch keratin layer protecting the

bacteria cell (Reay). Because of this, knowledge of endospores and their formation is important

in the medical field in order to develop proper treatment to infections and diseases.

This experiment confirmed Bacillus megaterium as an endospore-forming bacterium after

the Schaeffer-Dulton Technique endospore stain was performed. The vegetative cells remained

red while the spores formed in central, terminal, and sub-terminal areas appeared green and

circular. The process was also completed successfully because Escherichia coli showed no

formation of endospores, but the cells remained the pink color expected from a Gram-negative

bacterium.

IV. Results:
Figure 1: Gram stain of Escherichia coli.

Figure 1 shows the E.coli gram stained cells as pink and rod shaped.
Figure 2: Endospore stain of Escherichia coli.

The endospore stain of E.coli showed rod-shaped, pink cells with no stained endospores,

as expected and represented in Figure 2. E.coli does not form endospores, therefore there was no

green endospore presence on the cells.

Figure 3 represents the B.megaterium endospore stain results that showed rod-shaped,

dark pink/light purple cells with endospores on the insides, outsides, and around the cells.

V. Discussion:

The endospore stain and gram stain were completed successfully. Individual rod-shaped

cells were distinguished for the gram stain of E.coli. All of the cells were pink, which proved the

Gram-negative nature expected from E.coli. By determining E.coli cells as Gram-negative, it

showed that E.coli has a cell wall composed of a thin layer of peptidoglycan. Because E.coli is
gram-negative and has a thin layer of peptidoglycan, it will be easier to distinguish between what

antibiotics would work best to destroy E.coli in its parasitic bacteria form. The endospore stain

was also successful because endospores were identified on the B.megaterium cells. The

individual cells of B.megaterium were a dark pink, rather than purple. This was not expected

because B.megaterium is a gram-positive bacterium. However, because the cells were stained

with Safranin, in an endospore, B.megaterium cells are expected to be redder than purple, which

proves the experiment was successful (Todar, 2012). The endospores formed in B.megaterium

attached to the Malachite green stain, which caused them to turn green and appear under the

microscope. No endospores were found in the E.coli endospore stain, as expected, because E.coli

does not form endospores due to the fact that it is a Gram-negative bacterium. Therefore, the

endospore and gram stain were performed properly.

VI. References:

“Bacillus and Related Endospore-Forming Bacteria.” Textbookofbacteriology.Net, 2012,

textbookofbacteriology.net/Bacillus.html.

“Endospore Stain - Understanding Definitions, Techniques and Procedures.” MicroscopeMaster,

2019, www.microscopemaster.com/endospore-stain.html.

“Endospore Staining: Principle, Procedure and Results - Microbeonline.” Microbeonline, 29

June 2016, microbeonline.com/endospore-staining-principle-procedure-results/.

Keating, Steven. Microbiology: The Laboratory Experience. 2016. W.W. Norton &

Company.

“Welcome to Microbugz - Endospore Stain.” Austincc.Edu, 2019,

www.austincc.edu/microbugz/endospore_stain.php. Accessed 8 Oct. 2019.