Mystery Microbe
Introduction: Being able to identify a particular bacterial species is important. It is very useful in knowing its risk of toxicity to humans or animals, its resistance or susceptibility to antibiotics, and determining how to control its growth or kill it altogether. The purpose of these procedures is to discovery the identity of an unknown microbe by observing its reactions to a barrage of chemical and physical tests. Different microorganisms react in different ways, due to their function, digestibility, morphology, chemical make-up and other details. By observing the responses to these tests performed in a particular sequence, some can be eliminated as possibilities and others require further investigation.
Materials and Methods: A stock culture, labelled 25, was stored at room temperature (in the lab desk). A transfer loop was sterilized and a sample of the stock was transmitted to a tube of sterile BHI broth (for a working culture) and also to an agar plate and incubated for 24 hours. From the incubated plate, distinct and well isolated waxy, yellowish colonies appeared. Another sample was applied to a glass slide, heat fixed and a Gram stain test was performed. From the list of several possible choices for the unknowns, about half are eliminated. Table 1 indicates the tests that were performed on the unknown sample, all materials required for each test, as well as factors determining a positive or negative result.
Test Performed Media/Reagents Used Positive Test Observations Negative Test Observations
Gram Stain Crystal violet
Gram’s iodine
95% Alcohol
Safranin Blue or violet cells Pink cells
Catalase Glass slide
3% H2O2
Agar plates with colonies
Toothpick Bubbles produced No bubbles produced
OF Test 2 tubes of OF medium (glucose agar with bromthymol blue)
1 tube with sterile oil
1mL pipette
Pipette and pump Colour change from blue to yellow in tube covered with oil (F) Blue/no colour change in tube covered with oil (O)
Materials and Methods: A stock culture, labelled 25, was stored at room temperature (in the lab desk). A transfer loop was sterilized and a sample of the stock was transmitted to a tube of sterile BHI broth (for a working culture) and also to an agar plate and incubated for 24 hours. From the incubated plate, distinct and well isolated waxy, yellowish colonies appeared. Another sample was applied to a glass slide, heat fixed and a Gram stain test was performed. From the list of several possible choices for the unknowns, about half are eliminated. Table 1 indicates the tests that were performed on the unknown sample, all materials required for each test, as well as factors determining a positive or negative result.
Test Performed Media/Reagents Used Positive Test Observations Negative Test Observations
Gram Stain Crystal violet
Gram’s iodine
95% Alcohol
Safranin Blue or violet cells Pink cells
Catalase Glass slide
3% H2O2
Agar plates with colonies
Toothpick Bubbles produced No bubbles produced
OF Test 2 tubes of OF medium (glucose agar with bromthymol blue)
1 tube with sterile oil
1mL pipette
Pipette and pump Colour change from blue to yellow in tube covered with oil (F) Blue/no colour change in tube covered with oil (O)
References: Breed, Robert S., Murray, E.D.G., Smith, Nathan R. et al. 1957. Bergey’s Manual of Determinative Bacteriology, Williams & Wilkins Company, Baltimore. 1094 pages Cowan, S.T Cohn, 1872. (Bacleridium hileum Schroeter, Cohn, z6zd., 153; not Micrococcus Zwfews LehFAMILY VII mann and Neumann, Bakt. Diag., 1 Aufi., S, 1896, 161.) This culture has been retested (September, 1955) and has been found to grow slowly in