15 Metabolic Activities A: Inoculation of Media

When microbiologists or doctors come across a microbe that may be causing an infection, they need to be able to isolate, characterize, and identify this unknown microbe in order to treat the patient, develop further antimicrobials, or simply learn.

The process of identifying involves analyzing morphology, cultural characteristics, and physiological characteristics. Morphology includes viewing the cell shape, colony color, and results of specific stains, particularly Gram stain. “Cultural characteristics” refers to how the bacteria grows on various liquid and solid media, and how it consumes oxygen (obligate aerobe vs. facultative anaerobe vs. obligate anaerobe and so on). Physiological characteristics are determined through a series of biochemical assays. A biochemical assay is testing for aspects of microbial metabolism, which is the means of a microbe getting its energy and nutrients (carbon source) to live and reproduce. Essentially these assays, or tests, are detecting which enzymes are being producing by the bacterium. Bacteria can be differentiated by which specific enzymes are present or absent.

You will be learning the procedure of how to inoculate and perform the metabolic tests with a “known” bacterium for a two-week period. You will be working in pairs. Afterwards, you will be working individually for the next two-week period identifying an unknown bacterium.

For identifying the unknown bacterial strain, you will perform a Gram stain to determine if the strain is gram positive or gram negative and observe the cell shape. You will also decide what the colony color is by examining the original culture given to you on a TSA plate. These three traits are considered most important when you are using a key to identify a stain. Unfortunately, while the colony color and other visual characteristic are very powerful, they also require extensive experience to use reliably. Only a few colony colors are easily interpreted, but when you have a distinctive color that characteristic should be given a rank just after Gram stain and cell shape. The metabolic tests are a “best-fit” system which works well even if some tests fail. Not every strain of a bacterial species may produce an expected result. A positive result simply means that the metabolism of interest occurred.  Species range from mostly positive to mostly negative results and this helps make identification possible. You have to look at all results as a whole and figure out which bacterium matches closest to a bacterium in the Key, or “Unknown Booklet”. The “Unknown Booklet” is a list of bacteria with morphological and cultural characteristics described and results of the biochemical assays. Your unknown will be one of these bacteria.

MATERIALS

Per pair of students for “Knowns” **
2 Blue racks
2 Inoculating loops
1 Inoculating needle
Wax pencil
2 Bunsen Burners
2 Strikers
9 test tube labels
Coffee can
1 of each type of medium:

**Per student for “Unknowns”:
1 Blue rack
1 Inoculating loop
1 Inoculating needle
Wax pencil
Bunsen Burner
Striker
9 test tube labels
Coffee can
1 of each type of medium listed above

PROCEDURE OF METABOLIC ACTIVITIES: INOCULATION

1. The instructor will distribute one cultural plate per pair of students to share for the learning process (“Knowns”), but for identifying the Unknown Bacteria, students will work individually.
2. Inoculation of plates:
   a.  Label the bottom of the plates with names, date, medium name, and bacterial label (name, letter, or number).
   b.  Using aseptic technique, you will inoculate the medium with a single streak of bacteria on the milk and starch plates. You will inoculate the TSA plate with two streaks of bacteria. See below. When you remove bacteria from streak plate, you take from an isolated colony.                                   
   Milk & Starch Strike Plate                                       TSA Strike Plate
3. Simmon’s Citrate Agar (screw cap):
   a.  Using aseptic technique, you will transfer bacteria from an isolated colony using an inoculating loop from the bacterial plate onto a slant surface.
   b.  Using a test tube label, label the tube “Citrate”.
4. SIM Medium (clear cap):
   a.  Using aseptic technique, you will transfer bacteria from an isolated colony an inoculating needle from the bacterial plate, and then stab the needle all the way to the bottom on the medium.
   b. Using a test tube label, label the tube “SIM”.
5. Inoculation of all other broths (one tube of each):
   a.  Using aseptic technique, you will transfer bacteria from an isolated colony using an inoculating from the bacterial plate into a broth. This only has to be done once per tube.
   b.  The tubes are pre-labeled by their cap-color which indicates the medium:

   MR-VP broth	(black cap broth tube)	Phenol Red (PR) tubes have Durham tubes inside
   Nitrate broth	(yellow cap broth tube)	PR Sucrose	(blue cap broth tube)
   Simmon’s Citrate agar	(screw-top agar slant)	PR Dextrose	(green cap broth tube)
   SIM medium agar	(clear cap agar tube)	PR Mannitol	(red cap broth tube)
   Urea broth	(white cap broth tube)	PR Lactose	(yellow cap broth tube)

6. Place all test tubes into a coffee can. Put lab tape onto the outside of the coffee can. Label the coffee can with your name(s), bacterial label, and date. Place in your lab section’s incubator and correctly labeled shelf.
7. Store your 4 plates (this includes the original bacterial culture plate) in a rack upside down in your lab section’s lab bench area at room temperature. We do not incubate the plates for a week due to dehydration of the medium.

REFERENCES

Brown, A. E. (2009). Benson’s Microbiological Applications: Laboratory Manual in General Microbiology. New York: McGraw Hill.

Chess, B. (2015). Laboratory Applications in Microbiology: A Case Study Approach. New York: McGraw Hill. Cowan, M.K. (2015). Microbiology: A Systems Approach (4th edition). New York: McGraw Hill.