3: Endospore Stain, Glucose Fermentation, and MSA Tests of Unknown Bacteria

Endospore Stain, Glucose Fermentation, and MSA Tests of Unknown Bacteria
Throughout my experimental process of identifying an unknown bacteria, I have so far observed that my unknown bacteria has a positive cell wall and bacillus cell morphology. To further test my unknown bacteria in hopes of identifying the Genus species, I performed an Endospore Stain, followed by a Glucose Fermentation test when the unknown bacteria tested positive for endospores. After the Glucose Fermentation test revealed that the unknown bacteria had a pH level< 7, I performed a MSA (Mannitol Salt Agar) test. The MSA test was positive. Using the Identification Flow Chart, I was able to identify the unknown bacteria as Bacillus subtilis. See below for Experimental Procedures and Results.






Smear Preparation and Endospore Stain Method Procedure:
1. Prepared two slides by drawing a nickel-sized circle on each slide and labeled slides “1” and
“2”.
2. Used micropipette to place one drop of water within each circle of the slides.
3. Placed inoculating loop in bacti-cinerator for 5 seconds (used stopwatch to keep time).
4. After, used the sterilized inoculating loop and aseptic technique to obtain a sample of the
    unknown bacteria and smeared it into the water droplets of each slide.
5. Let bacteria smear dry on each slide.
6. After each bacteria smear dried, they were heat-fixed for 5 seconds each (used stopwatch to
    keep time).
7. Brought beaker full of water to a boil using hot plate.
8. Placed slide 1 over beaker of boiling water using rack tool and covered the slide with a
    paper towel.
9. Soaked the paper towel in Malachite Green solution and timed it for five minutes using
    stopwatch. Added more Malachite Green solution as needed when paper towel dried.
10. After five minutes, rinsed slide for 40seconds with water.
11. Added Safranin Stain and timed it for 75 seconds. Blotted dry with bibulous paper.
12. Repeated steps 8-11 for slide 2.
13. Prepared both slides for oil immersion under microscope.
14. Placed coverslip on slides and focus objective to 40x lens.
15. Rotated 40x lens halfway to 100x lens and placed one drop of immersion oil on each slide.
16. Viewed slides 1 and 2 through 10x and 40x lens.
17. Unknown bacteria was positive for endospores.
Experiment errors:
Initially placed wrong coverslip on slides.
Timing for second slide was 30 seconds instead of 40.
Could not focus to 100x lens.
Glucose Test Fermentation Procedure:
1. Used Aseptic Technique and took inoculation loop and retrieved sample of unknown bacteria.
2. With the inoculating tool, placed sample of unknown bacteria in test tube.
3. Incubated test tube at 37°C for approximately 16 hours.
Results: The Glucose Fermentation test revealed a yellow liquid color indicating a positive result for a pH <7.
MSA Test Procedure:
1. Used Aseptic Technique and took inoculation loop and performed isolation streak of the microbe onto the surface of the MSA plate.
4. Incubated MSA plate at 37°C.
5. Left inoculated MSA plate in incubator for approximately 16 hours.
Results: The isolation streak revealed a yellow color indicating a positive result