Organism # 6
Molecular Biosciences 101
Section #
Purpose
Materials and Methods
Gram Staining: Three circles were marked at the bottom of a clean glass slide and they were labeled as gram(+), gram(-) and unknown respectively. The three labeled circles were then inoculated with microorganism 6 using an inoculating loop. The bacterial smears were then air dried and heat fixed over a gentle flame. The slide was then flooded with primary stain, crystal violet for a minute by placing it on a staining rack. Excess crystal violet was washed off with a gentle stream of water. Thereafter, gram iodine was added and left for 90 seconds on the microbial cells. The excess iodine solution was then rinsed off gently with water. The bacterial cells were then treated drop by drop with decolorizing solution of ethanol: acetone and washed off with water after 6 seconds. Bacterial cells were then counterstained with safranin for about 40 seconds and excess was rinsed with water carefully. The slides were examined under the microscope to detect the gram(+) and (-) cells on the basis of presence or absence peptidoglycan cell wall. Gram (+) bacterial cells are supposed to stain purple due to retention of primary stain in their thick peptidoglycan cell wall whereas, gram (-) stains red as their cell wall is thin peptidoglycan and LPS and hence not able to retain crystal violet.
Carbohydrate Fermentation: Two tubes were prepared with nutrient broth, phenol red as pH indicator and an inverted Durham tube in both the tubes. One nutrient tube had sucrose and the other tube had lactose as primary carbohydrate sources. The unknown microorganism 6 was inoculated into each culture tube using distinct sterilized inoculating loops at 37oC. The color change in the medium was observed after 48 hours in each tube. Phenol red’s color change from red to yellow is indicative of medium turning acidic from basic due to anaerobic sugar fermentation by bacterial cells. The inverted Durham tube indicates any gas released during the process. Color change to deep pink or red or no gas formation is negative interpretations.
Sulfide Production: The microorganism 6 was aseptically inoculated in a Sulphite Indole Motility (SIM) medium tube by stabbing the medium. Casein digests with animal tissues (amino acid sources) and sodium thiosulfate (inorganic sulfur) in the SIM medium were sulfur sources to the bacterium, that can be decomposed or reduced by bacterial enzymes to H2S on inoculation at 37oC for 48 hours. If positive, then a black precipitate (ferric sulfate) will be observed due to reaction of H2S with ferrous sulfate.
Tryptophan Hydrolysis: Microorganism 6 was inoculated using sterilized loop in a tube containing 1% tryptophan broth. Few drops of Kovac’s reagent were added and shaken once the tube was inoculated at 37oC for 48 hours. After ten minutes, the tube was examined for appearance of a red ring at the top of the tube to detect the presence of indole produced by bacterial breakdown of tryptophan through enzyme tryptophanase.
Motility: The motility was observed using SIM medium tube along with the H2S production. Unknown microbial cells were inoculated in the SIM medium tube using sterilized loop via stabbing the medium. The tube was then incubated at 37oC for 48 hours. Growth pattern of bacterial cells was observed for motility and non-motility. The diffused cloudy growth away from the stabbed line mainly at the top and bottom of the line indicated motile bacteria whereas along the line indicated non-motile bacteria.
Urea Hydrolysis: A urea broth tube was prepared using urea, yeast extract and phenol red. Microorganism 6 was aseptically inoculated in the urea tube and incubated at 37oC for 48 hours. Positive result was the color change of pH indicator, phenol red from orange to pink due to alkalinity of the medium after ammonia accumulation. It results from urea hydrolysis by bacterial enzyme urease.
Starch Hydrolysis: A starch agar plate was inoculated using sterilized loop with microorganism 6 in a straight line. It was then incubated at 37oC for 48 hours. Iodine was then added along the inoculated growth line and observed for a color change. Presence of a clear zone indicated positive result due to starch hydrolysis by bacterial amylase enzyme and a color change to blue-black indicated negative result.
Casein Hydrolysis: Using nutrient agar and skim milk, culture plates were prepared. The skim milk agar plate was inoculated with unknown microorganism in a straight line. It was then inverted and incubated at 37oC for 48 hours. Caseinase enzyme, if present, will hydrolyze casein indicating as a halo around the inoculated growth line.
Catalase Activity: The unknown microorganism 6 was streaked on a nutrient agar plate and incubated in an inverted position at 37oC for 48 hours. 3% Hydrogen peroxide was then added to a bacterial colony and if the enzyme catalase is present in the bacterial cells, hydrogen peroxide would break into oxygen gas and water. The oxygen gas will appear as bubbles indicating a positive result.
Results
Gram stain: This differential staining test demonstrated microorganism 6 to be Gram (+) as the third circle on the slide was purplish-blue in color. The bacterial cells were spherical in shape suggesting the bacteria to be coccus.
Carbohydrate fermentation: Phenol red in the sucrose culture tube did not change the color and remained red without any gas release in Durham’s tube indicating a negative result. But, phenol red in the lactose culture tube changed the color from red to yellow but no gas was released indicating a positive result.
Sulfide Production: No black precipitate was observed in the SIM tube indicating a negative result.
Tryptophan Hydrolysis: There was no red ring present at the top of the tryptone broth tube indicating a negative result.
Motility: The bacterial growth was along the stabbed line only and did not diffuse away from the line.
Urea Hydrolysis: There was no color change of phenol red in the urea broth tube giving a negative result.
Starch Hydrolysis: The outcome was negative as the medium color changed from straw colored to blue black on adding iodine.
Casein Hydrolysis: There was no halo observed around the growth line and the medium remained white indicating a negative result.
Catalase Activity: There were no bubbles formed on addition of H2O2 on the microbial cells inferring a negative result.
UK = Unknown, BS = Bacillus subtilis, EA = Enterobacter aerogenes, EC = Escherichia coli, PV = Proteus vulgaris, SA = Staphylococcus aureus, EF = Enterococcus faecalis
Discussion
Microbial testing for identifying the unknown organism in cultures is a very common practice in the research, medical and clinical fields. An accurate identification to the ‘species’ level will only aid the professionals to treat the diseases correctly. There were several tests performed to identify the unknown microorganism#6 as only the visible physical characteristics are not sufficient to identify a bacterium. Biochemical tests need to be performed to evaluate biochemical profiles of enzyme production, gas release or color changes in the medium and characterize accordingly.
Primarily, gram staining, a differential staining test was performed that suggested the unknown being Gram positive coccus bacteria as the observed purplish-blue cells were spherical to oval under microscope. This test was useful in categorizing the unknown into a specific group of gram positives that possess a tougher outer cell wall made up of peptidoglycan. Next was to identify its biochemical aspects through several biochemical metabolic tests. Bacteria utilize carbohydrates for ATP production. Some have ability to oxidize oxygen and others can degrade complex carbohydrates like lactose, sucrose to simpler units that are utilized for energy production. The microorganism 6 fermented only lactose indicating the presence of lactase that anaerobically fermented lactose to lactic acid making the medium acidic, hence color changed from red to yellow.
Next was the identification of the ability of bacteria to produce hydrogen sulfide. Microorganism 6 did not produce H2S gas in the medium confirming that it was not amongst the sulfur producing bacteria. Also, the medium showed no motility for bacterial cells as they did not diffuse away from the stabbed line. This suggests the absence of flagella for locomotion in the bacterial cells which goes along with the fact that coccus bacteria are always non-motile.
Tryptophan test was performed to detect the presence of tryptophanase by identifying indole production in the tryptone broth. It gave a negative result as there was no pink ring observed at the tube top indicating absence of tryptophanase enzyme complex. Urea broth was tested for presence of urease enzyme in the bacterial cells that came out to be negative as phenol red in the medium did not show any color change as the pH of the medium did not change. This happened because ammonia did not accumulate in the medium and hence medium did not become alkaline. Urease test eliminated Proteus sp entirely as this genus is a fast hydrolyzer of urea (Watson, n.d.).
The activities of amylases and caseinases enzymes came out to be negative via the starch hydrolysis and casein hydrolysis tests. These enzymes break down complex carbohydrate, starch and complex protein, casein into their simpler units for utilization to produce energy in the cells. Iodine reacts with starch to form a blue-black colored complex and in the absence of starch, there is no such color observed. In case of casein detection, bacterial growth on skin milk agar plates itself demonstrates the enzyme activity by forming a clear halo region around the streaked line that was absent in the test performed. Catalase activity test came out negative that indicates the absence of catalase enzyme which further suggests the microbe 6 to be obligate anaerobe. All aerobic microorganisms do possess this enzyme to decompose the toxic product hydrogen peroxide produced as a result of aerobic activity.
Conclusion
As per the results obtained and their comparison with the available characteristics for known microorganisms, the biochemical characteristic for unknown microorganism 6 matches with Enterococcus faecalis. All the results obtained matched exactly with the provided data and the available documentations (Manero and Blanch, 1999). Rest other bacteria were not a match and there was no confusion in characterizing the microorganism 6 as Enterococcus faecalis. This genus is ubiquitously found in plants, soil and human gut.
References
Manero, Albert and Blanch, R, Anicet. “Identification of Enterococcus spp. with a Biochemical Key”. Applied and Environmental Microbiology. 1999. 65(10); 4425-4430.
Watson, Rachel. “Summary of Biochemical Tests. General & Medical Microbiology”. N.d. Accessed at http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm on 2 March 2016.
