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Unknown Lab Report in Microbiology

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Unknown #124

Amie Booth

December 3, 2013


BIO: 203.604, Fall



When unknown microorganisms are present, it is necessary to identify them in a laboratory setting.  Microbes can be identified by their cellular structure and function.  Different organisms also behave differently in various environmental conditions.  Determination of pathogenicity is possible after correct identification.  The ability of bacteria to cause infection and disease requires identification in order to determine treatment options for the host.  Laboratory microbe identification can also be useful to determine if bacteria are part of the normal flora of animals and humans.  Bacteria have been identified that have beneficial qualities such as necessary organic matter decomposition and use in food, chemical and antibiotic drug production.  The identification of the unknown bacteria in this study was completed by using methods learned in the microbiology lab class for BIO203.


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An unknown labeled as number 124 was obtained from the lab instructor.  The unknown mixed culture contained a Gram positive bacterium and a Gram negative bacterium as indicated by the instructor.  The first procedure that needed to be done was to create an isolation streak from the mixed culture on a nutrient agar plate.  After the plates were incubated at 37°C for 48 hours, individual colonies were observed.  Gram stains were performed on individual colonies to determine if isolation had been achieved.  The isolation streak and inoculation procedures were repeated four times resulting in the observation of mixed colonies.  Gram stains were performed in order to verify the mixed colony results. Separate bacterial colonies were observed on a sixth isolation streak, and gram stains were performed to verify the results.   Isolation of the Gram negative unknown was confirmed.

In order to encourage growth of the Gram positive unknown, a streak was created on a Mannitol Salt Agar plate from the original mixed culture.  The plate was incubated at 37°C for 48 hours.  No growth was observed.  Without another method available to isolate the Gram positive unknown, the lab instructor provided a pure culture of the unknown Gram positive bacterium.   A Gram stain was performed confirming the presence of Gram positive bacteria.  After determining the gram reaction of both unknowns, biochemical tests chosen from the lab manual were performed.  All tests were performed by the methods listed in the Lab Manual for General Microbiology by McDonald et al. (1).   Results were compared to the key provided by the lab instructor.

All of the following tests were performed on this unknown:

1. Maltose fermentation

2. Lactose fermentation

3. Casein hydrolysis

4. Methyl Red test

5. Oxidase test

The organisms were inoculated into maltose and lactose broth tubes by transferring a sample from each isolated unknown with a sterile inoculating loop into a separate tube.  Each type of broth contains peptone, phenol red (a pH indicator) and a single kind of carbohydrate (1).   A sample of the Gram negative unknown was spread on a milk agar plate to test for casein hydrolysis. The glucose fermentation tubes and milk agar plate were incubated at 37°C for 48 hours. The tubes were observed for the indication of carbohydrate fermentation.  The milk agar plate was observed for clearing around the bacterial growth to indicate the production of the enzyme casease

To prepare the methyl red test, the bacteria were inoculated into broth tubes containing glucose, peptone, and a pH buffer.  These tubes along with a control tube were incubated at 37°C for 48 hours.  The pH indicator methyl red was then added to the tubes to test for the fermentation of the glucose. A sterile wood applicator was used to spread a sample of each isolated culture onto filter paper.  Oxidase reagent was added, and the sample was observed for color change indicating a positive result.



The morphology observed in growth on a nutrient agar plate of unknown #124A was circular, opaque, dome-shaped colonies with smooth glistening surfaces.  The Gram stain performed on #124A showed the presence of pink rods when examined microscopically indicating that it was a Gram negative bacterium.  The unknown tested negative for lactose and maltose fermentation.  No clearing around the colony growth on a milk agar plate indicated a negative result for casein hydrolysis.  The methyl red test produced a positive result and the oxidase test provided a negative result.  The unknown microbe #124A was determined to be Proteus vulgaris.

Unknown #124B had the following morphology on a nutrient agar plate: larger dry, flat, off-white, opaque colonies with irregular margins.  This unknown was determined to be Gram positive as indicated by the presence of purple rods.  This unknown tested negative for lactose and maltose fermentation.  Clearing around the colony growth on a milk agar plate indicated the presence of casease and a positive test result.   The methyl red test produced a negative result and the oxidase test provided no color change indicating a negative result.  The unknown microbe #124B was determined to be Bacillus subtilis. Table 1 summarizes all of the biochemical tests and results.  The results are also presented in a flow chart form.


TABLE 1: Unknown #124 Biochemical Tests






Gram Stain To determine the gram reaction of the bacterium Crystal violet,  Iodine, Alcohol, Safranin Pink Rods

Gram-negative rodsPurple Rods

Gram positive rodsMaltose

FermentationTo determine the ability of a bacterium to ferment a specific carbohydratePeptone, Phenol red, MaltoseNo color change


Negative maltose fermenterNo color change

Negative maltose fermenterLactose

FermentationTo determine the ability of a bacterium to ferment a specific carbohydratePeptone, Phenol red, MaltoseNo color change

Negative lactose fermenter color change

Negative lactose fermenterCasein hydrolysis to determine the ability of a bacterium to hydrolyze a specific protein milk agar plate clearing around bacteria growth

Negative cases production, casein not hydrolyzedClearing around bacteria growth

Positive cease production, casein hydrolyzedMethyl RedTo determine if the ability of a bacterium to produce acid as a result of glucose fermentation glucose, Peptone, pH buffer

The methyl red pH indicator color changes from light yellow to red

Positive methyl red test, acid production as a result of glucose fermentation color change

Negative methyl red test, no acid production as a result of glucose fermentationOxidase TestTo determine the presence of cytochrome filter paper, Oxidase reagent color change

Negative oxidase testNo color change

Negative oxidase test




Gram Stain

Gram negative Rod

Maltose Fermentation (negative)



Proteus vulgaris

Escherichia coli

Pseudomonas aeruginosa

Klebsiella pneumoniae


Enterobacter aerogenes

Lactose Fermentation (negative)

Proteus vulgaris

                        Pseudomonas aeruginosa


Casein hydrolysis (negative)


Negative                                 Positive

Proteus vulgaris                     Pseudomonas aeruginosa

Methyl Red Test (positive)

Proteus vulgaris


Oxidase Test (negative)

Proteus vulgaris

            Unknown #124A- Proteus vulgaris



Gram Stain

Gram positive Rod



Bacillus cereus

Staphylococcus aureus

Bacillus subtilis

Staphylococcus epidermidis


Enterococcus faecalis

Lactose Fermentation (negative)

Bacillus cereus

                        Bacillus subtilis


Maltose Fermentation (negative)


Negative                                 Positive

Bacillus subtilis                      Bacillus cereus

Methyl Red Test (negative)

Bacillus subtilis


Oxidase Test (negative)

Bacillus subtilis

            Unknown #124B- Bacillus subtilis



When viewed microscopically after Gram stain, Unknown #124A was identified as a Gram negative rod-shaped bacteria.  A negative result for maltose fermentation allowed for the elimination of Escherichia coli, Klebsiella pneumoniae, and Enterobacter aerogenes as possible identities.  A negative result for lactose fermentation confirmed this elimination and presented only Proteus vulgaris or Pseudomonas aeruginosa as possibilities.  The inability of the unknown bacterium to hydrolyze casein eliminated P. aeruginosa as a possibility.  A positive methyl red test indicated the presence of acid as a result of glucose fermentation, and a negative oxidase test indicated the inability of the unknown bacterium to produce oxidase.  These tests confirmed the identity of Unknown#124A as Proteus vulgaris.

The bacterial genus Proteus belongs to the family Enterobacteriaceae of gram-negative bacteria (2).  They are motile by peritrichous flagella and facultatively anaerobic (3).  P. vulgaris is considered to be the representative species of the genus (2).  It occurs in soil, polluted water, and intestines of (healthy) man and animals (4).  This bacterium is an opportunistic pathogen that typically affects only immune-suppressed individuals which is problematic as P. vulgaris is resistant to most antibiotics (2).

Unknown #124B was provided by the lab instructor after isolation on a nutrient agar plate was not attained.  After a Gram stain was performed, Gram positive rod-shaped bacteria were observed microscopically.  This eliminated Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis as possibilities since they present as spherically-shaped cocci.  The inability of the unknown to ferment lactose confirmed the possible identities as Bacillus cereus or Bacillus subtilis.  A negative maltose fermentation test result eliminated B. cereus because it is able to ferment maltose. A casein test performed on Unknown #124B provided an unexpected positive result when compared with the provided identification key.  However, B. subtilis has been shown to hydrolyze casein by the production of casease (5).  This test was not necessary to confirm the identity of the unknown.  A negative methyl red test and negative oxidase test confirmed the identity of Unknown #124B as Bacillus subtilis.

The term bacillus is generally used to describe any rod-shaped bacterial cell (4).  Bacillus is a genus of straight rod-shaped bacteria that are either aerobes or facultative anaerobes.  Common among all species of this genus is the ability to convert to an endospore form in order to withstand extreme conditions.  Bacillus cells possess peritrichous flagella to provide motility (6). The ability of both unknown bacterium to be motile with the use of flagella could possibly account for the difficulty observed during the isolation streak process.

B. subtilis has become the model system for bacterial research.  It produces enzymes that are widely used in the baking and beer brewing areas of food industry resulting in its classification as a GRAS (generally regarded as safe) organism (7). This bacterium has a low degree of virulence to humans and is not considered pathogenic or toxigenic.  It is a common inhabitant of soils, and production of enzymes and proteases enable it to contribute to nutrient cycling in decomposing plant residue leading to its classification as saprophytic.  B. subtilis produces novel antibiotics used against a wide spectrum of aerobic and anaerobic bacteria as well as more common antibiotics such as bacitracin, bacillin, and bacillomycin (8).  This species also produce a variety of extracellular enzymes that are useful in commercial products.  These enzymes digest starches, proteins, and gelatins (6).  These digestive enzymes provide many commercial uses for B. subtilis especially in detergents, washing powders, and other cleaning aids as well as utilization in the leather industry.  Because this bacterium is a natural inhabitant of soil, ecological hazards associated with its use are low.  Its use in an industrial setting should not pose a high level of health or infection risk to workers (8). 




(1)McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero.  Lab Manual for General Microbiology.  St. Louis Community College at Meramec, 2011. Print.

(2)Sankaran, Neeraja. Microbes and People: An A-Z of Microorganisms in Our Lives. Arizona:

The Oryx Press, 2000. Print.

(3)Holt, John G., Noel R. Krieg, Peter H.A. Sneath, James T. Staley, and Stanley T. Williams.

Bergey’s Manual of Determinative Bacteriology. Philadelphia: Lippincott Williams & Wilkins, 1994. Print.

(4)Singleton, Paul and Diana Sainsbury.  Dictionary of Microbiology and Molecular Biology. West Sussex, England: John Wiley & Sons, Ltd., 2006. Print.

St. Louis Community College at Meramec. 16 November 2013.

(6)Maczulak, Anne. Encyclopedia of Microbiology. Ann Arbor: Sheridan Books, Inc., 2011.Print.

(7)Lederberg, Joshua (Ed.). Encyclopedia of Microbiology. San Diego: Academic  Press, 2000.Print.

(8)United States Environmental Protection Agency. Bacillus Subtilis Final Risk Assessment. Washington D.C.: February 1997. Web. 24 November 2013.



Another example of an Unknown Lab Paper



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