Reichert 1

Endospore Staining to Differentiate Endospore features of

Bacillus megaterium from Escherichia coli

Allyson Reichert

Medical Microbiology Lab

SBL 223 02

15 October 2018
 Reichert 2

TITLE:

Endospore staining to differentiate endospore features of Bacillus megaterium from Escherichia

coli

INTRODUCTION:

The purpose of this experiment was to perform an endospore stain with malachite green

in order to observe which species form spores, Escherichia coli or Bacillus megaterium.

Endospore staining is an example of a structural stain, meaning that it is designed to visualize

particular structures on or in a bacterial cell. The target structures viewed were endospores.

Endospores are formed by bacteria when environmental conditions become unfavorable.

Endospores are very resistant to desiccation, heat, radiation, and a variety of toxic chemicals,

thus making them extremely difficult to kill. The importance of studying these structures on

these particular species is that they can cause human disease. In order to prevent these diseases,

techniques must be developed to manipulate the environment in order to prevent germination of

or to kill endospores (Keating 2016). In this experiment, it was determined that Escherichia coli

was not a spore former due to the overall pink appearance of the cell. In contrast, Bacillus

megaterium stained a pink color but had green areas within, indicating that there were spores

present, making it a spore former.

MATERIALS AND METHODS:

Materials used in this lab included disposable gloves, one (1) slide holder, one (1)

inoculating loop, two (2) clean glass microscope slides, paper towels, Bibulous paper, two (2)

empty, plastic petri dishes, one (1) staining tray, one (1) compound microscope with 100x oil

immersion lens, immersion oil, malachite green, safranin, one (1) squirt bottle containing water,
 Reichert 3

overnight cultures of Bacillus megaterium and Escherichia coli, one (1) Bunsen burner, one (1)

striker, incubator, labelling tape, and pen.

Bunsen burner was ignited using a striker to create a sterile field. Glass microscope slides

were labelled with tape and a pen to designate one as Escherichia coli and one as Bacillus

megaterium. One loopful of water was added to the center of each slide using the inoculating

loop. The inoculating loop was flame sterilized and cooled. A loopful of cells from each culture

was placed on their corresponding slides, making sure that the loop was flame sterilized between

cultures. Using the inoculating loop, the bacteria were mixed with the water, and spread thin onto

the slide. The slides were set aside to dry before proceeding. Once the slides were completely

dried, the cells were heat fixed by running back and forth through the flame a total of three times

using a slide holder. Next, each slide was placed into an empty plastic petri dish. The smear of

cells on each slide were covered completely using a piece of paper towel cut to fit the slide. It

was important that the paper towel did not hang over the edge of the slide. The slides and paper

towels were flooded completely with malachite green stain making sure that the paper towel

remained over the cells on the slide. The petri dishes holding the slides were placed into an

incubator set to 55 degrees Celsius, taking extra caution to ensure that the pool of stain remained

on the slide. The slides were kept in the incubator for 30 minutes, and checked every 8 minutes

to make sure that the paper towel remained saturated with malachite green. After 30 minutes had

passed, the petri dishes were removed from the incubator. The paper towel was removed from

the slide and discarded. The slides were rinsed with water until the malachite green was

removed. The slides were blotted dry using Bibulous paper and placed on the staining platform.

Each slide was flooded with safranin and allowed to sit for 60 seconds. Each slide was rinsed

with water until the safranin was removed. The slides were gently blotted dry with Bibulous
 Reichert 4

paper. Once the slides were completely dried, they were observed under a microscope. The slide

containing Bacillus megaterium was observed using 400x total magnification. Escherichia coli

was viewed under 1000x total magnification using immersion oil. Observations were recorded

and workplace was cleaned.

RESULTS:

Figures 1 and 2 featured below show the results of the endospore staining viewed under a

compound microscope. Corresponding magnifications are listed.

Figure 1: Escherichia coli viewed under 1000x total magnification with immersion oil.
 Reichert 5

RESULTS, CONT.:

Figure 2: Bacillus megaterium viewed at 400x total magnification.

DISCUSSION:

In a typical endospore stain, malachite green is used as the primary stain. It normally

binds relatively weakly to cell structures. When staining with malachite green, heat is a key

element. If the stain is applied to spores or to endospore containing cells for a small amount of

time at room temperature, then the stain will not be able to infiltrate the endospore coat and thus

they will not stain. The heat drives the malachite green into the spores, making them appear

bright green. The decolorizer in this process is simply water since the malachite green binds so

weakly to non-spore structures. Additionally, once the stain has penetrated the spore, it is

difficult to remove, so the water won’t remove the malachite green from the spore. The
 Reichert 6

counterstain used in this lab is safranin. The safranin does not stain over the malachite green, but

will stain any colorless cell parts, making them appear pink (Keating 2016). In the slide

containing Escherichia coli, there was no evidence of malachite green on the cells and only had

cells stained with safranin; therefore, it can be concluded that Escherichia coli is a non-spore

forming bacteria. The few spots of malachite green that are visible in the picture may have been

particles stained by the malachite green that were not rinsed off properly, which would be a step

to perfect in future labs. Furthermore, in the slide containing Bacillus megaterium there are

spots of malachite green surrounded by pink areas of safranin. It can be concluded from this that

Bacillus megaterium is a spore forming bacteria. In an article from the Journal of Bacteriology,

scientists used Bacillus megaterium to regulate carotenoid production, which are thought to

provide health benefits in decreasing risk of disease (Takano et all 2015). This article

emphasizes the fact that Bacillus megaterium is a spore forming bacteria which is why it is

useful in their field of study. Therefore, the results of this experiment can be supported from this

article in that Bacillus megaterium is, indeed, an endospore former. Moreover, in an article

published by LWT- Food Science and Technology, scientists performed experiments that used

UV-C treatment of soymilk in coiled tube UV reactors to inhibit Escherichia coli cells and

Bacillus cereus endospores (Srinivasarao 2012). This article relates to the findings of this lab in

that it confirms that Escherichia coli does not have endospores. In the article, if Escherichia coli

had endospores then they would have been inhibited like the ones in Bacillus cereus. Since

Escherichia coli cells were completely inactivated, they had no spores to protect them. The

information in these articles solidifies the fact that the experiment was successful in

distinguishing between endospore features in Escherichia coli and Bacillus megaterium.

 Reichert 7

REFERENCES:

Keating, Steve. 2016. Microbiology: The Laboratory Experience. New York (NY): W.W.

Norton and Company 109 p.

Srinivasarao, Bandla, et all. 2012. UV-C treatment of soymilk in coiled tube UV reactors for

inactivation of Escherichia coli W1485 and Bacillus cereus endospores. LWT- Science

and Technology. 46(1):71-76.

Takano, Hideaki, et all. 2015. Role and Function of LitR, an Adenosyl B12-Bound Light-

Sensitive Regulator of Bacillus megaterium QM B1551, in Regulation of Carotenoid

Production. Journal of Bacteriology. 197(14):2301-2315.