Identification of the Unknown Microorganisms
Unknown #1
Unknown #2
1. The identification of bacterial strains is a common practice in clinical and medical fields. The ubiquitously present microorganisms can be both, beneficial or harmful to the host they attack or inhabit. Since times, several biochemical characteristics have been identified for many bacteria that commonly are related to humans. These characteristics and morphology have been arranged in a systematic way to identify the targeted unknown microorganism along with ruling out the ones giving negative results for the characteristic tests. This way the search was narrowed down for each of the two unknown microorganisms. The Gram staining differentiates bacteria broadly into gram positive and gram negative based on the stain retained by bacterial cell walls. Along with this, Gram staining also gives information about the morphology that broadly classifies the gram (+) or (-) bacterium into rod shaped (Bacillus), spherical (Coccus) or spirillium. Further, on the basis of this differentiation, biochemical tests are performed.
Both the unknowns under investigation were found to be Gram negative bacteria and were rod shaped when observed under the microscope. This ruled out all the gram positives and gram negative coccus bacteria on the list leaving genus Proteus, Enterobacter, Serratia, Escherichia and Pseudomonas to be tested further. The next step was to decide upon the biochemical tests for further characterization of these bacteria. The carbohydrate fermentation tests for sugars glucose, sucrose and lactose were performed that were positive for glucose and sucrose in both the cases. The sugars when added to the medium were fermented by the bacterial cells that releases acids making the medium acidic from alkaline which is detected by pH indicator phenol red. Also Durham tube was added to the sugar containing medium that trapped any gas released while fermentation proceeded. Unknown #1 released gas in both glucose and sucrose tubes turning the medium acidic from alkaline i.e. color change from red to yellow. No color change was observed in the lactose containing tube. Unknown #2 did change the color from red to yellow in the glucose and sucrose tubes without any gas released and no change was seen in the lactose tube. This ruled out E. coli and Enterobacter as they are mandatorily lactose fermenters along with Pseudomonas that do not ferment sucrose. Thus, only Proteus and Serratia were left for examination.
Next test performed was to test the presence of tryptophanase enzyme in the tested bacterium. It was done through indole test. The enzyme dissociates tryptophan amino acid into indole and pyruvic acid that is detected in the SIM medium. The Kovac’s reagent present in the medium detects indole presence by the appearance of red ring on the top of the medium in the tube. Unknown#1 was detected positive and unknown#2 was detected negative for this test. Further, methyl red test was performed to detect mixed acid formation upon glucose fermentation that reduces pH below 4.4. Only large amount of acid produced changes the medium color from yellow to red. Unknown#1 was positive for MR test and unknown#2 was negative. The interpretation of both these tests defined unknown#1 as Proteus vulgaris and unknown#2 as Serratia marcescens.
The ultimate determining test was urease test that detects urease enzyme presence in bacterial cells which hydrolyzes urea into ammonia and carbon di oxide. Ammonia presence increases alkalinity and is detected by phenol red changing the medium color to pink. Genus Proteus readily gives a positive urease test and is at times used to identify enterobacteriaceae family members from other bacteria. Unknown#1 & unknown#2 both gave positive results for urease test, hence confirming the interpretations.
2. The Gram staining test for both the unknown microorganisms here gave red color under the microscope due to retention of safranin counterstain. Thus, both were identified as Gram negative bacteria. Further observation under the microscope revealed the shape of these bacteria as rod shaped i.e. bacilli. Though, slight difference was observed in arrangement of bacilli i.e. unknown#1 was at times seen in chains but unknown#2 was never in chains but as diplococcus at few instances. But, Gram staining results for both the unknowns were almost similar and hence Gram staining results alone were not sufficient to identify the unknowns.
3. The different bacterial strains ferment different carbohydrate sources to derive energy for carrying out cellular processes in anaerobic conditions. Here, Proteus vulgaris and Serratia marcescens could ferment the monosaccharide simple sugars glucose as well as sucrose to do so. Along with this, it is noticeable that MR test was positive for Proteus which means that this organism is able to ferment glucose explicitly to produce a mix of acids (Delost, 2014). Thus, a medium containing added glucose and sucrose will enhance microbial growth for both when added along with the organic molecules.
Moreover, it is worth mentioning that these sugars are added along with other medium components to differentiate between specific sugar fermenters and the original medium ingredients are sufficient in raising the microbe. It happens because such sugars are basically utilized by the microbe for energy production under anaerobic conditions.
4. Disinfectants are used on inanimate objects like equipment, beds, table tops, surfaces, counter tops to kill the pathogenic bacterial strains so as to make the objects safe for usage. It is a regular practice in medicinal field so as to prevent the infection from spreading (ASM, 2016).
P. vulgaris usually inhabits human gastrointestinal gut but does cause infections in urinary tracts. S. marcescens is the most mastitis causing bacterial strain that spreads majorly via food materials. Both are mostly acquired in hospitals and are classified as a nosocomial infection and measures should be taken to prevent it by applying disinfectants. They have been found to be resistant to most antibiotics (Proteus against cephalosprins, aminoglycosides and imipenem; Serratia against beta-lactams and amino glycosides) and certain disinfectants like chlorhexidine-gluconate (Public Health Agency of Canada, 2011; NMC, 2011). Thus, a broad spectrum accelerated 0.5% hydrogen peroxide is shown to be very effective in controlling the spread if applied for 30 seconds and more. 1% Sodium hypochlorite, phenolics, 70% ethanol, 5% formaldehyde, gluteraldehyde, iodine and quaternary ammonium compounds could also be used as effective disinfectants against gram negative bacteria (Public Health Agency of Canada, 2011).
Hydrogen peroxide will be the choice to control contamination by both the organisms as it has proven to be highly effective, if used at proper concentrations, against gram negative bacteria.
References
ASM. (2016). Outbreaks associated with Contaminated Antiseptics and Disinfectants. Accessed at http://aac.asm.org/content/51/12/4217.full on 10 May 2016.
Delost, D, M. (2014). Introduction to Diagnostic Microbiology for the Laboratory Sciences. Jones & Bartlett Learning. Ch.3:43-48.
NMC. (2011). Serratia species: A Practical Summary for Controlling Mastitis. Udder Topics. 34(4&5):5.
Public Health Agency of Canada. (2011). Proteus Spp. Accessed at http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/proteus-eng.php on 10 May 2016.
