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Nutritional Requirements for the Cultivation of Bacteria

Background and Introduction

Media used to grow bacteria in the laboratory must meet their nutritional requirements.  All living organisms have basic requirements for the major element, Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorous, and Sulfur (acronym CHNOPS).  Other major elements include potassium, magnesium, calcium and iron.  As a group, bacteria have a remarkably broad range of metabolic abilities.  Many bacteria are chemoheterotrophs that, like us, utilize organic carbon as an energy and carbon source.  However, there are a good number of bacteria that are photosynthetic, obtaining energy from light, and even some that use inorganic compounds, such as sulfate, nitrate, and ammonia, for energy. 

Carbon & Energy Sources Table

As shown in the table below, all living organisms can be categorized based on the carbon and energy sources they utilize.  

Category

Energy Source

Carbon Source

Example

photoautotrophs

light

CO2

purple and green sulfur bacteria, algae, and green plants

photoheterotrophs

light

Simple organics

purple and green non-sulfur bacteria

chemoautotrophs (chemolithoautotrophs)

inorganic chemicals

CO2

Thiobacillus (S), Nitrobacter (NO2-1), and Nitrosomonas (NH3)

chemoheterotrophs (chemoorganoheterotrophs

organic source eg:  glucose 

organic compounds

Most bacteria, all fungi, humans and other animals.

Types of Media

Background

Many different types of culture media have been developed for a number of different purposes.  In general, media can be divided into two categories, defined and undefined (complex) media.  A Defined medium is a relatively simple medium that is made up of specific chemicals at known concentrations.  An Undefined medium is composed of mixtures of yeast cell extracts or enzymatic digests of protein; the exact amount and kinds of nutrients present in the medium are not known.  An Undefined medium tends to support the growth of bacteria better than a defined medium because it contains more preformed nutrients/growth factors (amino acids, nucleotides, and vitamins).  Consequently, organisms do not have to expend valuable energy and materials to synthesize the "preformed" compounds supplied in the undefined medium.  

Microbiologists have also developed special media that can be used to select for the growth of certain organisms or differentiate between bacterial isolates based on physiological characteristics.  These media are referred to as selective or differential media.  Selective media contain at least one ingredient that inhibits the growth of unwanted organisms, but permits growth of the desired bacteria.  Therefore, these types of media allow for the isolation of a certain bacteria even if they constitute a small percentage of the population in a sample.  Differential media are formulated to distinguish different microorganisms growing in the medium.  These media usually contain a chemical that is utilized or altered by some organisms but not by others.  By observing the appearance of the medium or colonies growing on differential media one can identify different organisms exhibiting different physiological traits. 

Today you will inoculate three different types of media with four different bacterial species.  The objective of this exercise is to illustrate the different growth characteristics of organisms on defined, undefined (rich), selective and differential media. 

Glucose Salts Agar (GSA): is a simple, defined medium.  Only organisms that can make all their cellular components from glucose and inorganic salts are able to grow on this medium.

 

 

 

 

 

Tryptic Soy Agar (TSA): is a rich, undefined medium containing products of an enzymatic digest of protein and soy product.  Organisms that require vitamins or other growth factors are able to grow on TSA. 

 

 

 

 

 

Eosin Methylene Blue (EMB) Agar: is a selective AND a differential medium.  The selective components are the eosin and methylene blue dyes that inhibit the growth of Gram-positive bacteria and permit the growth of Gram-negative enteric rods.  The differential component of the medium is lactose, which is a sugar that some organisms can ferment as an energy source.  Organisms that ferment lactose produce dark purple (sometimes shiny) colonies and those that cannot, produce white or very light pink colonies. 

GSA plate with recipe

TSA plate with recipe

EMB plate with recipe

Procedure

Procedure: (work in pairs) Collect 1 GSA plate, 1 TSA plate, and 1 EMB plate. 
Organisms that you will be working with: 
Escherichia coli (G-) 
Staphylococcus epidermidis (G+) 
Pseudomonas aeruginosa (G-) 
Enterobacter aerogenes (G-) 
1. With a marker, divide the bottom of all three Petri plates into quadrants.  Label the plates with your name, lab section, and the date.  Label each quadrant with one of the four organisms as shown in the diagram below. 

plate diagram

2. Follow the procedure for Inoculating organisms into four quadrants. 
3. Repeat the procedure for all four organisms.  Streak them in their appropriately labeled quadrants. 
4. Repeat the inoculation procedure, as described in the video, for the remaining 2 plates.  
5. Invert the plates and incubate them at 37°C for 24-48 hours. 

Results

GSA plate results

Observer the growth of each organism on the GSA plate.

GSA plate with all four organismsGSA plate with Enterobacter aerogenesGSA plate with E. coliGSA plate with Pseudomonas aeruginosaGSA plate with Staphylococcus epidermidis

TSA results

Observe the growth on the TSA plate. Compare growth here to that on the GSA plate.

TSA plate with all organismsTSA plate with Enterobacter aerogenesTSA plate with E. coliTSA plate with Pseudomonas aeruginosaTSA plate with Staphylococcus epidermidis

EMB results

Remember that EMB is both selective and differential. Observe the EMB plate for growth and the ability to ferment lactose

EMB plate with all organismsEMB plate with Enterobacter aerogenesEMB plate with E. coliEMB plate with E. coli with metallic sheenEMB plate with E. coli with metallic sheenEMB plate with Pseudomonas aeruginoasEMB plate with no growth for S. epidermidis

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