Identifying the Unknown Bacteria: Bacillus cereus
Coastline Community College
Bacillus cereus is a Gram-positive, facultative aerobic, spore-forming, rod-shaped bacterium that is widely distributed among the environment. Introduction
The reason for identification of unknown bacteria was to help students recognize different bacteria through different biochemical tests and characteristics. This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also, knowledge of different bacteria helped others make antibiotics that are used today. Bacillus cereus, a commonly occurring pathogen which can survive in remarkably hostile conditions, is typically found in soil. B. cereus has been recognized as an agent of food poisoning since 1955. The natural environmental reservoir for B. cereus consists of decaying organic matter, fresh and marine waters, vegetables and fomites, and the intestinal tract of invertebrates, from which soil and food products may become contaminated, leading to the transient colonization of the human intestine. Illness associated with B. cereus can occur when heat-resistant B. cereus endospores survive cooking. If the food is then inadequately refrigerated or held for extended periods at improper temperatures the endospores can germinate and multiply. Once the spores germinate, the vegetative cells can multiply and produce illness causing enterotoxins. Bacillus cereus is known to cause two distinctly different types of food-borne illness. The first type of illness, referred to as the Rapid-onset (Emetic) Vomiting-type, is characterized by nausea and vomiting. The incubation period ranges from 1 to 6 hours. Both the symptomology and incubation period mirror those of Staphylococcus aureus. The second type of illness, generally referred to as the Slow-onset Diarrheal-type, is characterized by diarrhea and abdominal pain. The diarrheal-type illness has an incubation period ranging from 6 to 24 hours. The symptoms include watery diarrhea, abdominal cramps and pain.
This type generally mimics the symptoms of Clostridium perfringens. It is the cause of “Fried Rice Syndrome,” named by Luke Fisher, as the bacteria is classically contracted from fried rice dishes that have been sitting at room temperature for hours (such as at a buffet). Because of the ever-present circulation of B. cereus in food products, the bacterium is ingested in small numbers and becomes part of the transitory human intestinal flora. Regardless of type, symptoms typically persist for less than 24 hours. Generally, gastroenteritis symptoms from B. cereus resolve by themselves. In very rare cases, however, some degree of medical intervention may be required. Some antibiotics work by attaching to the cell walls of the bacteria, breaking them down, and killing the microbes. When a bacteria is resistant to an antibiotic, like B. cereus are to many antibiotics, it is because the cell walls have changed slightly over time making it more difficult for the antibiotics to attach to the cell walls. B. cereus is very resistant to Ampicillin. In one study, almost 97% of the B. cereus bacterial population was resistant to the antibiotic. It is also resistant to Penicillin, Ampicillin, Tetracycline, and Streptomycin. Because B. cereus has cell walls that are Gram-positive is makes it susceptible to Gentamycin, Vancomycin, Ciprofloxacin, and Choramphenicol. Outside its notoriety in association with food poisoning and severe eye infections, this bacterium has been incriminated in a multitude of other clinical conditions such as anthrax-like progressive pneumonia, fulminant sepsis, and devastating central nervous system infections, particularly in immunosuppressed individuals, intravenous drug abusers, and neonates. Its role in nosocomial acquired bacteremia and wound infections in postsurgical patients has also been well defined, especially when intravascular devices such as catheters are inserted. Primary cutaneous infections mimicking clostridial gas gangrene induced subsequent to trauma have also been well documented. Results
There are many reasons for identifying an unknown bacterium. The purpose of this exercise was to identify an unknown bacterium from a liquid culture. We chose our unknown bacteria from a rack of test tubes with several different species of bacteria inside. I wanted to pick an unknown bacteria with a number easy to remember so I pick the test tube labeled “745”. Procedures were followed as stated in the lab manual written by Dr. Pedro J.A.
Gutierrez. Lab Day 1: After receiving my unknown bacteria, I streaked a TSA plate and incubated at 37°C for 48 hours. I then picked a single colony from the plate with my sterile loop and streaked a TSA slant and labeled it “Working Stock”. I did the same with another TSA slant and label the second one “Back-up Stock”. This would be the samples I used to complete the following procedures through the next four weeks to determine my unknown bacteria. Lab Day 2: The first procedure that was done was a simple stain to identify the bacterial shape. Bacteria tends to be transparent, so a stain must be use to color the cells of the bacteria so it can be viewed under a compound microscope. After heat-fixing three separate loopfuls of my unknown bacteria onto a slide, I used Methylene blue, Safranin, and Crystal violet to stain the three different samples. After rinsing the slide and observing my finding under the microscope, I determine that the shape of my unknown bacteria is bacilli (rod-shaped), and arranged with single bacilli and streptobacilli.
Figure 1 Bacillus cereus simple stain
Next I performed a negative stain on my unknown sample. The negative stain is an extension of the simple stain, but uses an anionic chromophore (acidic) instead of a cationic (basic) one. Therefore the background will stain but the cell will not. No heat fixing is used in this technique so the cells remain alive on the slide. The sizes of the cells are more accurate with negative staining because the cells do not shrink as they do when they are heat fixed.
Figure 2 Negative stain on B. cereus
Lab Day 3: In order to identify the cell wall type, I performed a Gram Stain. Gram positive cell walls are composed of a thick layer of peptidoglycan and teichoic acids, while Gram negative cell walls have two lipid bilayers that sandwich a thin layer of peptidoglycan. After observing my slide under the microscope I determine that my unknown bacteria #745 is a Gram positive bacteria because the cells were a purple color. This happens because when the decolorizer (Ethanol or acetone) is added to Gram positive cells, the thick layer of peptidoglycan is dehydrated and becomes more compact. This ends up trapping the Crystal violet stain in the cell and
therefore the cell remains purple.
Figure 3 an example of B. cereus that has been Gram stained
Lab Day 4: To determine whether my unknown bacteria produced endospores I needed to perform an endospore stain. An actively growing and metabolizing bacterium is called a vegetative cell. If these actively growing cells encounter adverse environmental conditions, such as significant depletion of nutrients, the usual result is cell death. Yet some bacterial genera posses a survival mechanism which allows them to package their DNA into an extremely resistant, dormant cell type called a spore. After sporulation, the spore remains inside the cell until cell lysis. When the spore is inside a dying bacterial cell, it is called an endospore. After performing my endospore stain I examine under the microscope my findings. I can see little green dots, which confirm that my unknown bacteria produce endospores giving me a positive result.
Figure 4 an example of an endospore stain of B. cereus. Since malachite green is water-soluble and does not adhere well to the cell, and since the vegetative cells have been disrupted by heat, the malachite green rinses easily from the vegetative cells, allowing them to readily take up the counterstain, making the vegetative cells pink and the endospores green.
After completing the endospore stain, I inoculated my unknown bacteria on selective and differential media, and incubated for 48 hours at 37°C. This included Eosin Methylene Blue Agar (EMB), Phenylethyl Alcohol agar (PEA), Mannitol Salt Agar (MSA), and Blood Agar (BA).
Lab Day 5: I obtain my selective and differential medias out of incubation, and my results are as followed. EMB can inhibit the growth of gram-positive and color the colonies present due to the differential components of EMB (lactose, eosin-y and methylene blue). These inhibiting agents combine to form a precipitate at acid pH thus serving as indicators of acid production. Strong lactose fermenters will be dark purple/black with a metallic sheen due to the update and fermentation of lactose, slow or weak lactose fermenters will be purple, and lactose nonfermenters will be pink. I had
very little growth on the surface of my EMB plate and negative (pink colonies) for lactose fermentation.
Figure 5 On the left is a non-lactose fermenting bacteria and on the right side is a bacteria that ferments lactose producing dark purple colonies.
PEA contains an alcohol which helps denature the outer membrane of Gram negative cell walls. This allows Gram positive bacteria to grow, while the growth of Gram positive. The color of the agar is the same as that of a TSA plate. My result for my unknown bacteria was a beige-yellow colored growth where I streaked on the plate, confirming I have a Gram positive bacteria.
Figure 6 shows a PEA plate streaked with different bacteria. Quadrant one is a negative result while Quadrant two and three have a positive growth.
MSA contain 7.5% NaCl. Therefore MSA selects for salt tolerant bacteria. It also contains mannitol and phenol red, a pH indicator. If the bacteria ferment mannitol they produce acid that turns the phenol red to yellow. My results were growth of a bacteria although it did not produce a yellow halo around the streak meaning that it does not ferment mannitol.
Figure 7 shows an MSA plate. The left half shows negative for mannitol fermentation, and the right half shows a positive reaction for mannitol fermentation.
Lastly BA contains 5% sheep blood added to normal TSA plates. The blood provides proteins and hormones that help the bacterial cells grow. Certain bacterial strains can lyse red blood cells to varying degrees. The result of my unknown bacteria was a formation of a clear halo around the streak, making it beta hemolytic, meaning that it can completely lyse red blood cells.
Figure 8 is a blood agar plate streaked with B. cereus, showing that it is beta hemolytic. After analyzing and documenting my results, I inoculated a Brain Heart Infusion (BHI) molten agar shake with my unknown bacteria to determine its oxygen requirements. After rolling the inoculated tube in my palms 15 times I quickly placed the tube in the ice bath for 5 minutes until the agar solidified. I remove from the ice bag and noticed a growth pattern determining that Unknown #745 is a facultative anaerobe, which means it grows best where oxygen is present but can still grow where oxygen is not present. Next I inoculated my unknown bacteria into four TSB tubes and stored them for 48 hours in four different temperature of 0°C, 25°C (room temp.), 35°C, and 55°C.
Lab Day 6: I retrieve my four inoculated TSB tubes and analyze and record my findings. The tubes labeled 0°C, 25°C (room temp.), and 55°C showed no turbidity, while the 35°C TSB tube showed slight turbidity. As the temperature increases, substrates and enzymes collide more frequently and the rate of enzymatic reactions increase. This continues until increased temperature begins to denature the three-dimensional conformation of the enzymes. Once enzymes unfold, they are rendered inactive. Next, I inoculate a phenol red broth test tube with Unknown #745 to test for carbohydrate fermentation and I also inoculate my unknown into a tryptone broth and MR-VP tube, and I streak a loopful of my unknown on a citrate slant. I incubate everything at 35°C for 48 hours.
Lab Day 7: I perform a catalase test to determine whether my bacteria produce catalase (turns hydrogen peroxide into water and oxygen). I place a loopful of my unknown on a microscope slide and add 2-3 drops of hydrogen peroxide, and I have a positive reaction. I know it is positive because bubbles appeared immediately when the hydrogen peroxide was added. After this test I inoculate a gelatin deep with my inoculating needle four times and incubate at 35°C for 48 hours. I then pick up my test tubes and slant that I inoculate from the last lab session and record my results. The carbohydrate test showed that Unknown #745 turned the phenol red broth yellow meaning that it ferments glucose and fructose acid, but not lactose and an absence of an air bubble proved that Unknown #745 did not produce gas from glucose, lactose, or fructose.
The citrate slant determines whether a bacterial strain can use citrate as its sole carbon source. A positive reaction would turn the green to blue agar producing an alkaline product that raise the pH. Unknown #745 stayed its green color meaning it does not use citrate as its sole carbon source. The Methyl red test detects mixed acid fermentation. After methyl red was added to my unknown MR-VP tube and it turns a deep red color meaning that the pH level is below 6.4, therefore it produces mixed acids. The Voges-Proskaeur tests for the production of a non-acid byproduct of glucose metabolism called acetoin. After adding the VP reactants and waiting 30 minutes I notice that the broth color does not turn a pink/red color which means it does not produce acetoin. I now have enough information to map out a dichotomous key to determine what is Unknown #745. Discussion
Figure 9 a Gram positive dichotomous key used to identify Unknown #745 Refer to Figure 9, which is the dichotomous key I used to determine my unknown bacteria. I have confirmed that B. cereus is Gram positive and bacilli (rod shaped). This leads me to answer whether I have a positive or negative catalase test. The catalase test proved positive, so I scroll to the next choice of positive or negative results for endospore formation. Unknown #745 did produce endospores. Next did the unknown produce acid from mannitol.
The answer was negative it did not produce acid from mannitol, which lead me to the conclusion that Unknown #745 is in fact B. cereus. Although I correctly identified my unknown bacteria, I did have some bumps in the road along the way. For starters, when I did my Gram stain the first time out I had rod-shaped bacilli but it was a mix of purple and pink colors so I wasn’t quite sure if I had a Gram negative or Gram positive bacteria. These results occurred because I overcooked the bacteria on the slide when heat fixing, killing the bacteria which turned some of the bacteria from purple to pink. I restained my bacteria and waited for my smear to air dry before heat-fixing and my results were clear as day. I have a Gram-positive bacteria. Another limitation of the experiment was I was not able to directly autoclave my tubes the lab assistants were responsible for that task.
Another thing that could possibly cause errors would be incubation times. Some of the procedures said to incubate for 48hours, but if we inoculated our specimens on a Wednesday lab class we were not able to get our results until the following Monday when we came back to class. Bacara is a chromogenic selective and differential agar that promotes the growth and identification of B. cereus, but inhibits the growth of background flora. The chromogenic agar has been suggested for the enumeration of B. cereus group as a substitute for MYP (Mannitol Egg Yolk Polymyxin Agar). Also, if a budget isn’t an issue there is always genetic testing that can be done to determine the accurate strain of the unknown is, in fact, Bacillus cereus.
Bottone, E.J., Bacillus cereus, a Volatile Human Pathogen. (2010) US National Library of Medicine National Institutes of Health. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863360/ Gutierrez, P.J.A., BIOL C210:General Microbiology: Microbiology Exercises., 3rd Edition. (2012) Coastline Community College. McCarthy, A.L., Stevens, S.K., & Weber, R.A. Bacillus Cereus Fact Sheet (2013) Food Safety Counsel. Retrieved from http://www.foodsafetycounsel.com/food-safety-law/common-food-borne-pathogens/bacillus-cereus/ Todar, K. The Microbial World: Bacillus cereus food poisoning. (2009) Department of Bacteriology University of Wisconsin-Madison. Retrieved from http://textbookofbacteriology.net/themicrobialworld/homepage.html Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins Handbook Bacillus cereus and other Bacillus spp.. (2013) FDA U.S. Food and Drug Administration Website. Retrieved from http://www.fda.gov/Food/FoodborneIllnessContaminants/CausesOfIllnessBadBugBook/ucm296005.htm Microbiology: Bacterial Sensitivity on Antibiotics (2009) Healthmad Web site. Retrieved from http://healthmad.com/medicine/microbiology-bacterial-sensitivity-on-antibiotics/
B. cereus negative staining. Digital image. Gelose.com. N.p., n.d. Web. 3 Nov. 2013. Dichotomous Key, Coastline Community College Microbiology Lab. Personal photograph by author. 2013. EMB Positive and Negative results. Digital image. Flickriver.com. Flickriver Viewer Software, 2007. Web. 3 Nov. 2013. An endospore stain of Bacillus cereus using malachite green. Digital image. Academic.missouriwestern.edu. N.p., n.d. Web. 3 Nov. 2013. Hahn,