Upon observing he growth on this plate, it is fairly obvious that one of the organisms is Serrated mercenaries. However, it is necessary to perform the requisite tests in order to be certain that this is accurate. The growth on this streak plate is bright red and white. With the color contrast, the separation of organisms is certain and pure cultures can be grown. Each organism was used to inoculate a liquid nutrient broth which was then incubated at 37-C for 18-24 hours. The red growth is labeled #1 and the white growth Subsequent tests and procedures are described elsewhere in this report.
Procedures Gram Stain: The organisms must first be identified as gram-negative or gram- positive. This is determined using a Gram Stain process. (Cappuccino & Sherman, 2008, p. 73) Using the fresh pure cultures grown in nutrient broth, a sample is placed on a slide and allowed to dry completely before heat setting. The staining process is accomplished using first Crystal Violet, rinsing, then Gram’s Iodine for a mordant, rinsing with alcohol, then water, and finally staining with Seafaring and rinsing with water.
After blotting carefully, the slide is placed on a microscope and focused on the organism at the lowest signification, progressing to the highest magnification with oil immersion. Using this process, the results were inconclusive for specimen #1, but #2 is definitely gram-positive rods. Specimen #1 initially appeared to be gram-positive diplomacy. Further testing is needed. Penitently alcohol agar (PEA): PEA is a selective medium which is used to identify gram-positive bacterium. Both organisms were inoculated onto opposite sides of the PEA plate and incubated at ICC for 18-24 hours.
Observation shows that for #1, there is no growth and no change in the appearance of the medium, while #2 does have growth, but tit no change in the appearance of the medium. This is further indication that #2 is gram-positive, while #1 is most likely gram-negative. McCracken agar: McCracken agar is both a differential and selective medium used to select for gram-negative bacteria. It also differentiates lactose-fermenting bacteria from non-lactose-fermenting bacteria. Only #1 was used to inoculate this medium. After inoculation, the plate was incubated at ICC for 18-24 hours.
Observation shows there is a bright red growth but no change in the appearance of the medium. This indicates no lactose fermentation, however since there is Roth, this means the bacterium in gram-negative. Imitation salt agar: This medium was inoculated with both specimens and incubated at ICC for 18-24 hours. There being no growth and no change in appearance of the medium for either organism, this indicates there was no imitation fermentation and they do not tolerate salt. Methyl Red/Vogues-Proteases tests: The Methyl Red-Vogues Proteases medium was used to perform two tests.
The methyl red test is used to identify enteric bacteria based on glucose metabolism. The Vogues-Proteases test is used to determine if an enteric bacterium produces acidic or neutral end reduces. First, the medium was inoculated with isolate #1, and incubated at ICC for 48 hours. After incubation, 2. 5 ml of the medium was transferred to another tube to be used for the Vogues-Proteases test. For the methyl red test, five drops of pH indicator methyl red is added to one of the tubes of medium. After being gently mixed by rolling the tube between the palms of the hands, the results are observed.
The MR..-UP broth has changed to a yellow-orange color. This is a negative reaction and indicates that pyrrhic acid was metabolize to neutral end products. For the Vogues-Proteases test, 10 drops of Barite’s reagent A is added to the second tube of medium. The culture was shaken, then immediately 10 drops of Barite’s reagent B was added and it was shaken again. For the next 15 minutes, the culture was shaken every 3-4 minutes. Observations were made at 15 minutes. There was no change in the medium which indicates a negative reaction and also indicates that does not ferment glucose.
This is another contradiction of expected results and is contradicted in the next test. Triple Sugar-Iron (ITS) agar test: This test was used for both isolates, #1 ND #2, and is designed to differentiate based on differences in carbohydrate fermentation patterns and hydrogen sulfide production. The medium used is a ITS slant. Each isolate was inoculated into a ITS slant by using a sterile needle with a stab-and-streak technique. After inoculation, the slant tubes were incubated at ICC for 18-24 hours. Observation showed #1 was red on the slant but yellow in the butt of the tube.
In addition, the stab pathway revealed some motility of the organism. This indicates the organism is a motile, anaerobe that ferments glucose with no gas. Upon observing #2, the entire medium was yellow with a small amount of liquid at the bottom of the slant, and slight motility in the stab pathway. This indicates the organism is a lactose/ sucrose/glucose fermented, an anaerobe and has motility. Catalane test: This test is to determine whether the isolates can produce catalane in order to degrade toxic hydrogen peroxide. To do this, a few drops of 3% hydrogen peroxide were placed on a smear of each of the isolates.
As expected, isolate #1 bubbled nicely, indicating the presence of catalane and allowing the conclusion that #1 is a facultative anaerobe instead of a strict anaerobe. Isolate #2 had no reaction so the result is, it does not produce catalane. Oxides test: This test is designed to distinguish among groups of bacteria based on stockroom oxides activity. Oxides enzymes play an important role in electron transport during aerobic respiration. Both isolates were tested for oxides by adding p-anodic-antihistamine oxalate to colonies grown on a medium, and neither had a reaction, indicating that they do not produce stockroom oxides.
Chocolate agar: This medium was used to grow colonies of isolate #2 to be used in the oxides test. After inoculation, the plate was incubated at CZ for 18-24 hours, before the test. There was heavy gray growth on this enriched medium. Blood agar: Blood agar can be used to cultivate fastidious organisms and also shows the hemolytic properties of some microbes. Bacterium #2 was inoculated onto a blood agar plate using a sterile loop. The plate was incubated at ICC for 24 hours. It was then observed that there was heavy growth on the plate with a clear ring around the colonies.
This indicates lysine of red blood cells occurred, which is beta-hemolytic. Starch hydrolysis test: Starch agar is used to determine if bacteria produce the enzyme amylase which breaks down starch, causing hydrolysis. The starch agar plate was inoculated with isolates #1 and #2 using a sterile loop. After inoculation, the plate was incubated at ICC for 24 hours. The plate was then flooded with Gram’s iodine which reacts with starch to turn purple-blue. Both bacteria had growth on the starch, however, only isolate #2 had a ring around the colonies. This indicates that the bacterium is positive for starch hydrolysis and the presence on amylase. 1 was negative for starch hydrolysis, not producing amylase. The following tables show the physiological and biochemical tests performed for this study, as well as the student’s observations and interpretations. Table 1: Physiological & Biochemical Test Results – #1 Serrated mercenaries Test Reagent or Media Temperature Observations Results Interpretations Gram Stain Crystal violet, iodine, alcohol, seafaring Reddish-purple small rods Gram negative Slightly inconclusive. Looks like sociability. Selective media Penitently alcohol agar ICC No growth No change in medium As a selective medium, PEA identifies this as a gram-negative bacterium.
Differential/ Selective media McCracken agar Red growth Indicates this is a gram-negative bacterium No lactose fermentation Imitation salt agar No imitation fermentation Does not tolerate salt Methyl Red MR..-UP broth Methyl red indicator Yellow-orange color in broth Negative Indicates that pyrrhic acid was metabolize to neutral end products Vogues- Proteases Barite’s reagents A & B Negative-should be positive Does not ferment glucose (contradicted below) Triple Sugar-Iron agar ITS slant Red color on slant, yellow in bottom, movement away from stab site Motile Anaerobic Glucose fermentation, no gas
Catalane 3% hydrogen peroxide Bubbles present Positive for catalane Indicates presence of catalane; facultative anaerobe Oxides p-amino-dimensionality oxalate Does not produce stockroom oxides Starch hydrolysis Starch agar Gram’s iodine Growth Does not produce amylase Table 2: Physiological & Biochemical Test Results -#2 Bacillus cereus Test Purple rods Gram positive Definitely gram positive rods PEA selects for gram positive organisms Enriched agar Chocolate agar Heavy gray growth Grows well on this medium Blood agar Growth with clear area around colonies Positive for beta-hemolytic
An expected result for B. Cereus Medium turned yellow, small amount of liquid, motile Positive for lactose/sucrose/ glucose fermentation Fermentation occurred creating an acidic pH in the medium No reaction Negative for catalane No catalane is present; anaerobic paminodimethylaniline oxalate Oxides negative Growth with ring around colonies Positive for starch hydrolysis Indicates the presence of amylase Discussion The identification of bacteria #1 was very easy since there are few organisms which have a red growth, and Serrated mercenaries is the only one that is on our unknowns list.
When the Gram stain was inconclusive for this bacterium, it was sensible to use other tests to positively identify it. By inoculating PEA and McCracken agar, it was possible to conclusively identify #1 as a gram-negative organism. Observation of the bacterium with oil immersion, seemed to indicate a duplicitous, however after completing the aforementioned physiological and biochemical tests, it must be concluded that isolate #1 is probably a short rod. Once it is determined to be a gram-negative rod, the oxides test being negative leads to doing a methyl red test.
This test is also negative. The McCracken agar indicates there is no lactose fermentation, leading to the final conclusion that this organism is Serrated mercenaries. Organism #2 was obviously a gram- positive rod from the very first Gram stain. A spore stain was not done, however positive result for the starch hydrolysis test leads to the final conclusion that this unknown is Bacillus cereus. Several other tests were done, such as for detection of hemolytic and glucose fermentation.
Each result was consistent with expected results for B. Cereus. Epidemiology The patient presenting with vomiting likely has food poisoning due to the intimidation of a starchy dish such as pasta salad or rice. Bacillus cereus can be characterized by nausea and vomiting within h – 6 hours after ingesting the contaminated food. Other symptoms may include abdominal pain and headache. There is also a diarrhea type of food poisoning due to B. Cereus, however in this instance the illness presents only with vomiting.
The second organism, Serrated mercenaries, might be present in the vomit due to the same contaminated food; however it could also have been in the intestines of the patient, without causing a problem. (General Background, 2009) B. Cereus food singsong is usually self limiting and not very severe. It has a very quick onset and usually resolves within several hours. Because of this, the illness often goes unreported. This emetic food poisoning is common year round and can affect everyone.