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This study aimed to identify and differentiate two unknown organisms obtained from a nutrient broth. It was known that one of the organisms was gram-positive, and the other was gram-negative, both sourced from vomit. The identification process involved Gram staining, selective media, biochemical tests, and growth observations. The results revealed that the gram-negative organism is Serratia marcescens, while the gram-positive organism is Bacillus cereus. The epidemiological implications of these findings were discussed.
The objective of this study was to distinguish and identify two unidentified organisms derived from a nutrient broth sample.
The only information available about these organisms was that one was gram-positive and the other was gram-negative, both originating from vomit. To achieve this differentiation, various microbiological techniques and tests were employed. The identification process began with Gram staining to categorize the bacteria as either gram-positive or gram-negative. Subsequent tests, including phenotypic and biochemical assays, were conducted to further characterize and differentiate these organisms.
The Gram staining procedure was used to differentiate the organisms based on their cell wall structure.
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Fresh pure cultures grown in nutrient broth were used for this test. A sample was placed on a slide, heat-fixed, and stained with Crystal Violet, Gram's Iodine, alcohol, and Safranin. The stained slide was then examined under a microscope, starting from the lowest magnification and progressing to oil immersion.
PEA is a selective medium for identifying gram-positive bacteria. Both organisms were inoculated onto opposite sides of a PEA plate and incubated at 37°C for 18-24 hours.
The appearance of the medium after incubation was observed.
MacConkey agar is both a selective and differential medium for identifying gram-negative bacteria and differentiating lactose-fermenting from non-lactose-fermenting bacteria. Only the gram-negative organism was inoculated onto this medium. After incubation at 37°C for 18-24 hours, the growth and appearance of the medium were examined.
Mannitol Salt Agar is used to identify organisms based on their ability to ferment mannitol and tolerate salt. Both organisms were inoculated onto this medium and incubated at 37°C for 18-24 hours. The growth and appearance of the medium were observed.
The Methyl Red and Voges-Proskauer tests were performed to assess glucose metabolism and end product formation by the organisms. The medium was inoculated with isolate #1 and incubated at 37°C for 48 hours. After incubation, the results for both tests were observed.
The TSI agar test was used to differentiate bacteria based on carbohydrate fermentation and hydrogen sulfide production. Both isolates, #1 and #2, were inoculated into TSI slants and incubated at 37°C for 18-24 hours. The appearance of the medium and gas production were noted.
The catalase test was performed to determine if the organisms could produce catalase, which helps degrade hydrogen peroxide. A few drops of 3% hydrogen peroxide were added to smears of each isolate, and the presence or absence of bubbling was observed.
The oxidase test was conducted to determine if the organisms produce cytochrome oxidase. Colonies grown on a medium were tested using p-aminodi-methlyaniline oxalate, and the reaction was observed.
Chocolate agar was used to cultivate colonies of isolate #2 for the oxidase test. After inoculation, the plate was incubated at 37°C for 18-24 hours.
Blood agar was used to assess the hemolytic properties of isolate #2. After inoculation, the plate was incubated at 37°C for 24 hours, and the presence of hemolysis was observed.
The starch hydrolysis test was performed to detect amylase production by the organisms. Starch agar plates were inoculated with isolates #1 and #2 and incubated at 37°C for 24 hours. The plates were flooded with Gram's iodine, and the presence or absence of a blue-black halo around the colonies was observed.
The experimental procedures were conducted as follows:
Slides were prepared with fresh cultures, and the Gram staining process was carried out. The appearance of the stained organisms was observed under a microscope.
Both organisms were inoculated onto PEA plates and incubated. The plates were examined for growth and changes in medium appearance.
Only the gram-negative organism was inoculated onto MacConkey agar plates and incubated. The plates were observed for growth and changes in medium appearance.
Both organisms were inoculated onto Mannitol Salt Agar plates and incubated. The plates were examined for growth and changes in medium appearance.
The methyl red and Voges-Proskauer tests were performed for isolate #1, and the results were observed.
The TSI agar test was conducted for both isolates, #1 and #2, and the results were observed.
The catalase test was performed for both isolates, and the presence or absence of bubbling was noted.
The oxidase test was conducted for both isolates, and the reaction was observed.
Isolate #2 was inoculated onto chocolate agar plates and incubated. The plates were examined for growth.
Isolate #2 was inoculated onto blood agar plates and incubated. The plates were observed for growth and hemolysis.
The starch hydrolysis test was performed for both isolates, and the presence or absence of a blue-black halo was observed.
The results of the various physiological and biochemical tests are summarized in the following tables:
| Test | Reagent or Media | Temperature | Observations | Results | Interpretations |
|---|---|---|---|---|---|
| Gram Stain | Crystal violet, iodine, alcohol, safranin | 37°C | Reddish-purple small rods | Gram-negative | Slightly inconclusive; appears as coccobacilli |
| PEA | N/A | 37°C | No growth, no change in medium | Gram-positive | Indicative of gram-positivity |
| MacConkey Agar | N/A | 37°C | Bright red growth, no change in medium | Gram-negative | No lactose fermentation; gram-negative |
| Mannitol Salt Agar | N/A | 37°C | No growth, no change in medium | Gram-negative | No mannitol fermentation; salt-intolerant |
| Methyl Red | Methyl red | 37°C | Yellow-orange color | Negative | Pyruvic acid metabolized to neutral end products |
| Voges-Proskauer | Barritt’s reagent A and B | 37°C | No change in medium | Negative | Does not ferment glucose |
| TSI Agar | N/A | 37°C | Red slant, yellow butt; slight motility | Motile, ferments glucose with no gas | |
| Catalase | 3% hydrogen peroxide | 37°C | Bubbling | Positive | Facultative anaerobe |
| Oxidase | p-aminodi-methlyaniline oxalate | 37°C | No reaction | Negative | Does not produce cytochrome oxidase |
| Test | Reagent or Media | Temperature | Observations | Results | Interpretations |
|---|---|---|---|---|---|
| Gram Stain | Crystal violet, iodine, alcohol, safranin | 37°C | Gram-positive rods | Gram-positive | Gram-positive rods |
| PEA | N/A | 37°C | No growth, no change in medium | Gram-positive | Gram-positive |
| MacConkey Agar | N/A | 37°C | N/A | N/A | Not tested |
| Mannitol Salt Agar | N/A | 37°C | No growth, no change in medium | Gram-positive | No mannitol fermentation; salt-intolerant |
| Methyl Red | Methyl red | 37°C | No change in medium | Negative | Does not ferment glucose |
| Voges-Proskauer | Barritt’s reagent A and B | 37°C | No change in medium | Negative | Does not produce cytochrome oxidase |
| TSI Agar | N/A | 37°C | Yellow slant, yellow butt; slight motility | Lactose/sucrose/glucose fermenter, anaerobe, motile | |
| Starch Hydrolysis Test | N/A | 37°C | Growth with a clear ring around colonies | Positive for starch hydrolysis and amylase production |
The initial Gram staining of bacteria #1 showed a reddish-purple coloration, which appeared as small rods. Initially, this observation was suggestive of gram-positive diplococci, but further testing was required to confirm the identity.
Subsequent tests, including inoculation on PEA and MacConkey agar, provided additional information. PEA showed no growth and no change in medium appearance, indicating that the organism did not belong to the group of gram-positive bacteria that PEA selects for. MacConkey agar displayed bright red growth with no change in medium appearance, which suggested that the organism was gram-negative and did not ferment lactose.
The Methyl Red test results were negative, indicating that the organism did not ferment glucose to produce acidic end products. Additionally, the Voges-Proskauer test was also negative, indicating the absence of acetoin production. These results further supported the conclusion that bacteria #1 was not an enteric bacterium that ferments glucose.
The Triple Sugar-Iron (TSI) agar test revealed a red slant and yellow butt, along with slight motility. This indicated that the organism was motile, an anaerobe, and able to ferment glucose without gas production.
The catalase test showed a positive result, indicating the presence of catalase. This suggested that bacteria #1 was a facultative anaerobe, capable of degrading hydrogen peroxide.
The oxidase test yielded a negative result, indicating that the organism did not produce cytochrome oxidase.
Collectively, the test results led to the identification of bacteria #1 as Serratia marcescens, a gram-negative, facultative anaerobic bacterium that ferments glucose without gas production.
Bacteria #2 was initially identified as gram-positive rods based on the Gram stain results. This observation was consistent with the typical morphology of Bacillus species, which are known to be gram-positive, aerobic, spore-forming rods.
The PEA test showed no growth and no change in medium appearance, indicative of gram-positivity. However, the MacConkey agar test was not performed for this organism.
The Mannitol Salt Agar test revealed no growth and no change in medium appearance, suggesting that the organism did not ferment mannitol and was salt-intolerant.
The Methyl Red test yielded no change in medium appearance, indicating that the organism did not ferment glucose to produce acidic end products. Similarly, the Voges-Proskauer test showed no change in medium, suggesting the absence of acetoin production.
In the TSI agar test, bacteria #2 exhibited a yellow slant and yellow butt, along with slight motility. This indicated that the organism was a lactose/sucrose/glucose fermenter, an anaerobe, and possessed motility.
The catalase test produced no bubbling, indicating the absence of catalase production. This implied that bacteria #2 was a strict anaerobe.
The oxidase test results were negative, indicating that the organism did not produce cytochrome oxidase.
Additionally, the starch hydrolysis test showed a clear ring around the colonies, indicating the presence of amylase and positive starch hydrolysis.
Based on the overall test results, bacteria #2 was identified as Bacillus cereus, a gram-positive, spore-forming bacterium capable of starch hydrolysis and lactose/sucrose/glucose fermentation.
The epidemiological implications of identifying these bacteria are essential in understanding potential health risks associated with the organisms. In this context, the organisms were isolated from vomit, and it is crucial to consider their possible roles in foodborne illnesses.
Bacillus cereus, identified as bacteria #2, is known to cause food poisoning characterized by symptoms such as nausea, vomiting, abdominal pain, and headaches. This type of food poisoning typically occurs within ½ to 6 hours after consuming contaminated food, often dishes containing starches like pasta salad or rice. In the case of the identified patient with vomiting, it is likely that the ingestion of contaminated food led to Bacillus cereus food poisoning. It is important to note that this bacterium can survive cooking, so proper food handling and storage are crucial to prevent foodborne illness. Prevention measures include prompt refrigeration of rice products and maintaining good hygiene practices.
On the other hand, Serratia marcescens, identified as bacteria #1, might have been present in the vomit due to the same contaminated food source or could have been part of the patient's intestinal microbiota without causing illness. Serratia marcescens is generally considered an opportunistic pathogen and may cause infections in immunocompromised individuals. In this particular case, its presence in the vomit may not be directly linked to the observed symptoms of vomiting.
In conclusion, while Bacillus cereus was likely responsible for the vomiting episode, Serratia marcescens might have been present as a bystander or part of the normal gut flora. Proper food handling and hygiene practices are essential to prevent foodborne illnesses caused by organisms like Bacillus cereus.
This study successfully identified two unknown organisms obtained from a nutrient broth, which were derived from vomit. Bacteria #1 was identified as Serratia marcescens, a gram-negative, facultative anaerobic bacterium capable of fermenting glucose without gas production. Bacteria #2 was identified as Bacillus cereus, a gram-positive, spore-forming bacterium with the ability to hydrolyze starch and ferment lactose/sucrose/glucose.
Understanding the identities of these bacteria is crucial for epidemiological considerations, especially in cases of foodborne illnesses. Bacillus cereus, in particular, is associated with symptoms of food poisoning, emphasizing the importance of proper food handling and storage practices.
Identification of Unknown Bacteria: Serratia Marcescens & Bacillus Cereus. (2016, Mar 05). Retrieved from https://studymoose.com/document/serratia-marcescens-bacillus-cereus-report
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