Unformatted text preview: Cultivating and Identifying Microbes
In this lecture, we will answer the questions of WHY would one want to cultivate and
identify microorganisms and HOW does one cultivate and identify microorganisms.
The first step in cultivating and identifying a microorganism is identifying an
environmental sample in which you believe the microorganism of interest to be present
(e.g. the spinal fluid of a human or the intestinal fluid of a cow). In the next step, you
form a hypothesis about the kind of medium that you can use to grow the
microorganism you want and that will also minimize the growth of unwanted
microorganisms that might be present. This hypothesis forms the basis of an
enrichment culture. Enrichment cultures demonstrate Darwin’s concept of natural
Textbook Reading Page 142-143, “Origins of Enrichment Culture Methodology” What you should get out of this text:
Be able to explain the basic principle behind an enrichment culture. How does this
demonstrate the Darwinian concept of natural selection? Choice of growth medium
There are many choices of growth media that can be used to grow microorganisms.
This textbook reading will explain the difference between two major classes of growth
media – complex and defined. It will also present some of the major environmental
conditions that need to be considered when growing microorganisms.
Textbook Reading Page 138-139, “A Culture Medium” &
“Incubation Conditions” What you should get out of this text:
1. Be able to compare and contrast complex medium and defined
2. Know that growth media can be liquid or solid.
3. Know that temperature and oxygen are the two environmental
factors that must be considered when designing a growth medium.
How would one decide whether to use a complex or a defined medium? Complex
media can be useful when the nonessential nutrients required for growth of an organism
are unknown. Nonessential nutrients will be those factors that most organisms need to
growth, for example an amino acid like alanine. Some microorganisms are able to
synthesize alanine de novo and thus do not required that alanine be added to the
growth medium. Other microorganisms cannot synthesize alanine and it must be added
to the growth medium of these organisms. Alanine can either be added to a defined
medium or obtained via a complex medium, such as one with yeast extract. However,
there are times when a complex growth medium can be detrimental to the growth of a
desired microorganism. For example, the metals found in complex media, such as 1 yeast extract, typically inhibit the growth of bacteria that live in the open ocean where
the metal concentration is low. Liquid or Solid Enrichment Culture
After deciding on the growth medium and the environmental conditions to use, the next
step is to decide whether to grow the environmental sample on a liquid or solid medium.
The textbook explains the pro’s and con’s of each technique.
Textbook Reading Page 144, “Aerobic Enrichment” What you should get out of this text:
Be able to explain why a greater diversity of microorganisms can be isolated from
enrichment on solid media than liquid media.
Be able to explain why if you had a growth medium that was very selective for
your desired microorganism, you might chose to use a liquid medium rather than a
solid medium. Pure culture and Streak Purification
After enriching for your desired microorganism, whether on liquid or solid, the next step is to
isolate your organism in pure culture. The most common method to accomplish this is streak
Textbook Reading Page 139-140, “Streak Plate Procedure” What you should get out of this text:
1. Know what is the goal of the streak plate procedure.
2. Know how a colony arises.
3. Know how the streak plate procedure can be used to assess the purity of a
culture. Identifying the Isolated Microorganism
Often a first step in identifying a bacterium is to simply ask is it a Gram+ or Grambacterium.
Textbook Reading Page 81, Box 4.1 “The Gram Stain” What you should get out of this text:
1. Know that the Gram strain is a differential strain that uses two different stains.
2. Know that cells of a different type stain differently.
(Note: The physiological basis of the differential staining will be explained in a
Gram strains yield a very rough identification of a microbe. As you will have learned in
Lecture 2, a standard way to identify a microorganism is by the sequence of the gene
for 16S rRNA. But, this technique will not identify different strains of a bacterium. When
this level of detailed information is need, DNA fingerprinting is commonly used. There
are currently several different methods for DNA fingerprinting; they all have the goal of
2 distinguishing different entities of DNA based on base pair differences. The method that
will be presented in class involves isolating bacterial chromosomal DNA and then
digesting it with a restriction enzyme. The fragments of DNA are then separating by gel
electrophoresis. A specialized form of gel electrophoresis that is better at separating
large fragments of DNA, called pulsed-field gel electrophoresis (PFGE), is used. I do not
expect you to know the patterns of current in PFGE or why it is better at separating
large DNA fragments. The following link has a nice short explanation of the technique
and an animated cartoon of PFGE
http://www.bio.davidson.edu/Courses/genomics/method/pulse_field.html). You will also
see that after electrophoresis the DNA is made visible due to staining with ethidium
bromide. You will also see how different sequences of DNA yield different patterns on
the gel. Think about and be able to answer why different strains of a bacterium would
yield different patterns of DNA. Estimating Microbial recovery
The procedure outlined above is for isolating a particular microorganism in pure culture
from an environmental sample. But another goal one may have is to identify all
microorganisms present in an environmental sample. To date we have isolated and
grown in pure culture only a small fraction of the microorganisms that are present in the
environment. How can we know many microorganisms are present in the environment
relative to the number we can culture in the lab? The textbook describes two different
methods that are commonly used to estimate the microbial cells present in an
environment – a direct microscopic count and a viable cell count on solid growth
medium. Be able to explain why the direct microscopic count would yield far greater
numbers of cells than the viable cell count.
Textbook Reading Page 161-162, “Direct Count of Cells” What you should get out of this text:
1. Know that the number of cells per milliliter of sample can be determined
using a microscope and a special glass slide with a grid.
2. Know that this method cannot distinguish between dead and living cells
and, sometimes, even debris can look like a bacterial cell.
Textbook Reading Page 162, “Viability Count-Living Cells” What you should get out of this text:
1. Understand how the number of viable cells per milliliter of sample can be
determined by diluting and plating the sample.
2. Understand why the viability count of a sample can yield much lower
numbers of cells that the direct count of cells. 3 ...
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