Moon

Bacillus cereus

Sally Moon
BIOL 2325L-2
10 December 2013

Moon 2
The purpose of this experiment was to properly identify unknown organism #11 by utilizing a

variety of different methods learned throughout the course of the microbiology lab. Proper identification of
unknown organisms provides access to study and characterize the specific discovered organism. The
discovered organism, such as pathogenic bacteria, may be harmful to humans or the environment. The
specific characteristics of the microorganisms such as its structure determine how to treat the organism, if
found to be dangerous, disadvantageous, or unfavorable.
Both qualitative and quantitative data were collected via different techniques and tests, which
played an important role in determining the successive steps necessary to further continue the discovery
process to ultimately identify the designated organism. By process of elimination, each step performed
distinguished the available options of which tests to perform for the continuing steps.
To begin the process, it is necessary to obtain a pure culture in order to study and characterize the
organism. Naturally, microorganisms are commonly found to exist as mixed cultures, which is why it is
very important to successfully isolate the microorganism culture provided. One of the most important steps
in any and all procedures used the process is the proper use of aseptic techniques including proper and
thorough hand-washing as well as inoculating sterile materials. These cautious steps aid in obtaining a pure
culture as well as preventing cross contamination. In order to isolate a pure culture from the bacterial
sample on the given agar plate, the method of plate streaking was used.
Plate Streaking
-Inoculate loop via bacti-cinerator before and after each use of inoculating loop
-Allow the loop to cool before use
-Crack lid on agar plate enough to slide loop in
-Obtain small sample of a well-isolated colony
-Place lid back on plate
-Use loop to transfer sample onto the new media
-To streak, crack lid of new media and streak Quadrant I of the surface via zigzag motion
-Close lid and sterilize loop again (perform between each streak)
-Use sterilized loop to cross the streak from Quadrant I into Quadrant II and streak again
-Repeat streaking steps from Quadrant II to III, then III to IV

Moon 3
-When finished, close plate lid and sterilize loop

Once the streaking method was successfully completed, the next step was preparing a bacterial
smear on a microscope slide in order to be able to use staining techniques, which allow for the viewing of
the specimen under a microscope.
Preparing a Bacterial Smear
-Begin with a clean microscope slide
-Label slide
-Place small drop of water on slide
-Use sterile loop to touch the surface of a well-isolated colony
-Spread the cells in the water and spread evenly to the size of a dime (approximately)
-Allow slide to air dry for 2 minutes
-Heat fix slide using provided hot plates
-Allow plate to cool before performing staining techniques

The first test used was a gram stain, which allows for visualization of cells under a microscope
and show differences in cell structure depending on cell wall composition. This test is used to determine
whether the cells are gram positive or gram negative, which is the first differential category in
differentiating an organism.
Gram Stain Procedure:
-Place slide on staining rack placed over the sink
-Flood the smears with the primary stain, crystal violet
-Let stand 1 minute, then rinse off with deionized water
-Flood smear with Grams Iodine
-Let stand 1 minute, then rinse off with deionized water
-Decolorize with 95% ethanol by adding 8-10 drops while holding slide at a 45 degree angle
(Do not over-decolorize)
-Immediately rinse with deionized water

Moon 4
-Flood stain with Safranin (counterstain)
-Let stand 1 minute, then rinse with deionized water
-Use a folded Kimwipe to gently dry the slide in order to use under microscope
Following the gram stain procedure, the specimen was carefully viewed under the microscope

using the oil immersion lens as the final objective lens. The gram stain procedure provided important data.
The cells viewed under the microscope appeared to be purple in color and the shape appeared to be long
rod-shaped with many refractile areas present in the rods. These cells were also in clusters. Judging from
the microscopic view, the following conclusions were made: the cells were gram positive (purple color),
staphylo (in arrangement), and bacilli (rod-shaped) with what seemed to be spores were present.
However, in order verify that the above information were correct, additional steps were taken to
assure that the cells were spore forming and gram positive. The next test was testing if the organism would
grow on PEA agar, which is a selective agar that is specifically selective for gram positive bacteria. This
agar contains phenyl ethyl alcohol that inhibits the growth of gram negative bacteria, so if growth appeared
on this media, it would further confirm that the unknown organism is gram positive. Transferring the
original specimen to the PEA agar followed the same steps as the beginning portion of the streaking
methods. Aseptic techniques were used to simply transfer a sample of the specimen onto the PEA agar. The
results showed that there was, indeed, obvious growth on the PEA agar. Therefore, the conclusion was
made that the bacteria were definitely gram positive. Also, gram positive bacteria are the only type of
bacteria that are capable of forming endospores. Therefore, this step reassured that the refractile areas in the
rods seen under the microscope represented endospores.
At this point, the various charts and lists provided by the instructor for this specific experiment
were reviewed, to narrow down possible organisms. According to the information presented, the specimen
had to fall under one of two categories: Bacillus or Clostridium. However, the list of possible organisms
assigned for the particular assignment did not include any species from Clostridium. Therefore, it was
further narrowed down to one of the following three organisms: Bacillus subtilis, Bacillus cereus, and
Bacillus megaterium. Following the review of the information, the next step was to list some of the main
distinguishing characteristics of the three possible organisms that would aid in selecting the next testing
method.

Moon 5
One of the main differences noted was that Bacillus cereus is motile, and Bacillus subtilis and

Bacillus megaterium are non-motile. Also, to further differentiate between the two non-motile organisms,
Bacillus subtilis bacteria form mannitol, and Bacillus megaterium are citrate positive and appear to be long
rod-like in shape. With this information, the decision was made to perform a SIM medium test. The SIM
medium is used to detect three different characteristics such as sulfur reduction, indole production, and
motility. The SIM medium allows motile bacteria to move in the media because of its low concentration of
agar. Therefore, the one necessary in this experiment at this point was the test for motility.
SIM Medium Test Procedure:
-Obtain SIM medium (deep) and original streaked plate containing pure culture of specimen
-Using inoculating loop, obtain a sample of the specimen on loop
- Stab the deep straight down in order to form a single stab line
-Close cap on deep and sterilize loop again before putting away
The result from the SIM Medium Test was a fuzzy line present in the deep. Instead of a distinct
line, there was a fuzzy area where the loop was stabbed. It was a bit difficult to detect, but with the
reassurance of the instructor, it was concluded that the organism was motile. Because of the discovery of
the organisms motility, it was possible to exclude the other two organisms from the three possibilities, and
finally conclude that the unknown organism is Bacillus cereus.
Bacillus cereus is a type of bacteria that harm to humans by causing diarrhea, nausea, and
vomiting, by producing toxins that may be transferred through food. B. cereus is often associated to
Bacillus anthracis and Bacillus thuringiensis because of its similarity to the other two bacteria. However, it
is distinguishable from the other two because it is the most motile of them all. This type of bacteria can
transfer toxins such as emetic toxins and enterotoxins to humans through food such as plants, eggs, soups,
pastries, vegetables, and dairy products, when they are not properly stored and refrigerated. Leaving food
out without refrigerating for a few hours and then consuming the contaminate food is a common cause of
the food poisoning caused by this organisms toxins. In some cases, B. cereus can also cause various
infections in humans (Bacillus cereus, n.d.).
The food poisoning caused by this microorganism results when the produced toxins reach the
gastrointestinal tract, which can lead to diarrhea or other symptoms such as nausea and vomiting. This type

Moon 6

of food poisoning is common, and it usually is not detrimental. Some of the signs and symptoms of this
particular type of food poisoning are watery diarrhea, abdominal cramps, and pain related to ingestion of
contaminated food, which may occur anywhere from 6 to 15 hours after ingestion. Other symptoms such as
nausea and vomiting may result as early as 30 minutes to 6 hours after ingesting the contaminated food.
These symptoms may last for about 24 hours (Bacillus cereus, n.d.).
The bacteria is not known to directly be the cause of other major complications, but have been
associated with cellulitis, septic meningitis, pyogenic infections, and bovine mastitis (Bacillus cereus,
n.d.). If one suffers from food poisoning caused from B. cereus, it is treatable by some basic food safety
precautions. When preparing food, it is important to thoroughly clean them to attempt to prevent
microorganism growth, especially with regards to fresh produce. Also, food should be properly stored and
refrigerated to lessen the risks of contamination. As far as treatment, advised methods are similar to treating
other food poisoning cases. Drinking sufficient amounts of fluids, especially those that contain electrolytes,
is strongly recommended for those that experience food poisoning. However, if symptoms worsen and
become more severe, it is advised to visit a physician or a hospital for proper examination and diagnosis. If
a severe case of food poisoning does occur, fluid consumption will still be a crucial part in recovery.
Antibiotics may also be advised if seen necessary by a physician (Bacillus cereus, n.d.).
This experiment was extremely beneficial in reviewing very important steps in basic lab
experiences. Review of proper aseptic techniques was absolutely necessary in order to perform any test
successfully. Contamination would have made the entire experiment and its results worthless. Therefore, it
is extremely important to remember the importance of following proper aseptic techniques. Also, caution
and care are necessary when performing tests that may not provide results immediately. After performing
the SIM Medium test, it was difficult to determine whether the result surely represented a fuzzy line or no
line at all. However, after careful examination and multiple reviews, it was assured that a fuzzy line was
present in the deep. Making a careless mistake in any of the beginning parts of the experiment would have
affected all of the remaining steps. Therefore, it is advised to follow all instructions fully and thoroughly
mastitis (Bacillus cereus, n.d.).

Moon 7

Moon 8
References

Bacillus cereus. (n.d.). A guide to Food poisoning. Retrieved December 9, 2013, from

http://www.medic8.com/healthguide/food-poisoning/bacillus-cereus.html

Bacillus cereus. (n.d.). Bacillus cereus. Retrieved December 9, 2013, from

http://textbookofbacteriology.net/B.cereus.html