The Case of the Mystery Bacteria: Part I

For my first research project I have been tasked with identifying an unknown bacteria.  After discussing safety protocols for correctly using the equipment I would need, I was presented with a test tube with a yellowish liquid a microbiology lab book and encouraged to get at it.

The first thing I learned about was Aseptic Technique.  Aseptic technique is an approach to handing materials in the lab such that one can isolate and transfer living microbes from without contamination.  This includes cleaning of one's work space before use to proper sterilization of equipment to the thoughtful and careful handling of cultures and tools.

With this in mind it was time to culture some microbes.

I  have no background in microbiology and prior to the Anatomy and Physiology class I took last semester the last biology class I took was in high school (17 years ago).  As such, I took detailed notes of the procedures I would need to perform to prepare the bacteria for further testing.  The work I would be doing would give me a basic microbiology introduction through the culturing of microbes.

The Five I's of Culturing Microbes¹

1. Inoculation:  Inoculation involves the transfer of a microbe from a patient, surface or container into a tube or plate containing nutrient media (food).
2. Incubation:  During incubation, the microbe is placed in an incubator set at the preferred temperature for growth.  For most microbes this ranges between 25°C and 37°C.  When you want to slow the growth of microbes, cultures should be placed at cooler temperatures, for example in a refrigerator set at 4°C.
3. Isolation:  In order to isolate microbes for a sample, the microbes must be separated into district colonies or groups of cells. 
4. Inspection:  The colonies or broth cultures are observed for macroscopic and microscopic characteristics
5. Identification:  The major purpose of culturing microbes is to identify the specific type of microbe.  This can be done by examining the physical and biochemical characteristics of the microbe.  

First was inoculation.  This process is quite simple.  It takes advantage of the clonal reproductive process of bacteria (that they divide to produce identical copies of each other, clones).  The reason for doing so is to not only get some idea of what the bacteria is due to its colony morphology (what the colony looks like as it grows), but to create a greater volume of the sample bacteria that we can test.  Culturing is necessary when there is just a small amount of a sample.  This is very important in areas like medicine where you want to take as small a sample from a patient as possible, but still have enough to perform the needed tests.

I inoculated my unknown bacteria with a plate using two techniques.  A plate is what many would call a petri dish, except that this petri dish has a solid media spread across the bottom, in this case trypticase soy agar (TSA).  Therefore it called a TSA plate.  The TSA media is food for the bacteria so that it can grow and reproduce, giving us evidence of its identity through its cell/colony morphology and providing more bacteria to test.

I performed two inoculation techniques.  A lawn culture and an isolation streak.  The lawn culture is intended to produce a plate that is blanketed with microbes and does so by a particular pattern of swabbing the plate:

 The isolation streak (also known as a 3-quadrant streak plate) is used to isolate individual types of bacteria from a mixed culture (a sample containing two or more microbes), the goal being to isolate single colonies independently.  This is done by drawing a "T" on the bottom of the plate and using these "boundaries/zones" to perform a series of inoculating swipes across these zones with minimal overlap:

After the plates had been prepare it was time for incubation.  I taped them, making sure they were labeled properly, and they were dropped off in the incubator set at 36.4°C

The condensation seen is due to the TSA plates being stored in the refrigerator prior to my using them.  For this reason they were placed in the incubator bottom side up.  

We will see what grows tomorrow.

References:

¹Biosciences Department. (2017). BIO 205 Laboratory Manual. Phoenix, AZ: Phoenix College.