Week 5 Microbiology Lab: Heat Inactivation and Media Techniques

Week 5 Microbiology Lab: Heat Inactivation and Media Techniques

This Week 5 Microbiology Lab focuses on heat inactivation of microbes, specifically E. coli survival at 60°C. Students will explore differential and selective media, including Lactose MacConkey agar and Simmons citrate agar, to identify Enterobacteriaceae. The lab also includes a Pseudomonas project, enriching and isolating Pseudomonas species from soil samples. Designed for microbiology students, this handout provides detailed procedures and key concepts for understanding microbial survival and media differentiation.

Key Points

  • Demonstrates E. coli survival rates at 60°C using absorbance measurements.
  • Explains the principles of differential and selective media for identifying Enterobacteriaceae.
  • Covers the use of Lactose MacConkey agar and Simmons citrate agar in microbiological analysis.
  • Details the enrichment techniques for isolating Pseudomonas species from soil samples.
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  • Niesha Dupree
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Week 5. Heat inactivation of microbes, Differential/selective media, and
Pseudomonas project Day 1
5.1 Heat inactivation of microbes
Heat is a commonly used technique in food and medical industry to inactivate
microbial pathogens. For example, traditional pasteurization is a process of food
preservation in which packaged and unpacked foods are treated with mild heat,
usually to less than 100°C. Ultra-high-temperature (UHT) pasteurization involves
heating milk or cream to 138–150°C (280–302°F) for one or two seconds. High
temperature kills microbes by causing proteins (e.g., enzymes) to denature. In
today’s exercise, we are going to demonstrate the effect high temperature on
bacterial survival determined by both the total viable count and the absorbance.
Exercise: determine E. coli survival at 60°C
Materials (each group)
8 mL of E. coli culture
3 microcentrifuge tubes (2.0 mL)
5 cuvettes
5 mL of TSB for blanking
1 microcentrifuge tube rack
Sterile micropipette tips (1 box of 1000 µL and 1 box of 200 µL)
1 spreader
1 bicker containing 95% (caution: flammable)
4 TSA plates
Determine E. coli cell survival at 60°C
• Gently vortex the E. coli culture and transfer 1.8 mL of the E. coli culture from
the conical tube into 3 microcentrifuge tubes, respectively.
• Label and place all 3 microcentrifuge tubes into the 60°C water bath incubator
to start the heat treatment. Each microcentrifuge tube represents a sampling
time point.
• Once all microcentrifuge tubes are at 60°C, start your timer and retrieve one
microcentrifuge tube every 7 min. The total heat treatment lasts 21 min.
• At each time point, plate 0.1 mL of the E. coli sample onto a TSA plate without
serial dilution. Transfer the rest 1.7 mL of the E. coli cells into a cuvette to
measure absorbance.
• The rest of the E. coli culture in the conical tube is used as time “0 min” sample.
While the heat treatment is underway, plate 0.1 mL of the “0 min” E. coli
sample on TSA plate without serial dilution and transfer 1.7 mL of the E. coli
cells into a cuvette to measure absorbance.
• Incubate all TSA plates at 37°C for 24 h.
Note: After performing the plating, aseptic technique is no longer necessary when
measuring the absorbance.
5.2 Differential/selective media
The term “enterics” refers to organisms belonging to the family Enterobacteriaceae.
This is a family of Gram-negative, oxidase-negative, and facultative anaerobic rods.
Several groups are included in this family. One group includes genera such as
Escherichia (e.g., E. coli), Klebsiella, and Proteus, which are generally found as
normal inhabitants of the human body, but they may also cause diseases under
certain circumstances. Other groups include Salmonella and Shigella which are
usually associated with a disease state in animals.
Because of their importance in human disease, the Enterobacteriaceae have
been studied extensively. Although these bacteria are morphologically alike and in
many ways metabolically similar, laboratory procedures for identification of
enterics are based on biochemical activities. In this exercise, you will use a limited
number of biochemical tests to differentiate between selected members of the
Enterobacteriaceae.
The media used in this exercise are differential and/or selective media. Selective
media contain ingredients that only allow certain organisms to grow. For example,
selective media contain antimicrobial compounds that inhibit some organisms but
allow others to grow. Another type of selective media contains carbon/nitrogen
sources that only support the grow of certain organisms. Differential media contain
chemical compounds that allow different microorganisms to be visually distinguished
by the appearance of the colony or the surrounding media, usually on the basis of
metabolic differences between the organisms. Some media can possess both
characteristics (i.e., selective and differential). Below is the principle of each individual
selective/differential medium used in this exercise.
Lactose MacConkey agar
Lactose MacConkey Agar is a selective-differential medium. In this medium,
pancreatic digest of gelatin and peptones (meat and casein) provides the essential
nutrients, vitamins, and nitrogenous factors required for growth of microorganisms.
Lactose is a fermentable source of carbohydrates. Microbes that can ferment lactose
will lower the pH during growth, resulting in red color around colonies. The selective
action of this medium is attributed to crystal violet and bile salts, which are inhibitory
to most species of Gram-positive bacteria.
Phenol red mannitol broth with Durham tube
This medium is a differential medium. Peptone and beef extract serve as sources for
carbon and nitrogen. Phenol red is the pH indicator, which turns from red to yellow at
acidic pH due to mannitol fermentation. Gas production, if any, is captured in Durham
tubes.
Simmons citrate agar
In the absence of fermentable glucose or lactose, some enterics are capable of using
citrate as a carbon source. Citrate agar contains an ammonium salt as the sole
nitrogen source, citrate as the sole carbon source, and a bromothymol blue indicator,
which is green at pH 6.8 and blue at pH > 7.6. Microbes that can grow on this medium
utilize citrate as a sole carbon source and ammonium salt as the sole nitrogen source.
Metabolism of ammonium salt releases ammonia that raises the pH, changing the
color of the agar from green to deep “Prussian blue”. The ability to use citrate
as a carbon source relies on the presence of a citrate permease that facilitates the
transport of citrate into the cell. Simmons citrate agar is a selective-differential
medium.
MR-VP (Methyl Red and Vogues-Proskauer) broth
Glucose is the major substrate oxidized by enteric bacteria for energy production. The
end products of the oxidation process vary depending on the specific metabolic
pathways. When mixed acids (e.g., lactic, formic, and acetic acid) fermentation takes
place, pH is lowered to approximately 4.5-5.0. Methyl red is a pH indicator that is red
between pH 4.4 and 5.0 and yellow between pH 6 and 7. The addition of methyl red
after 24 h growth results in red color in medium where mixed acid fermentation occurs.
A yellow color indicates a negative for methyl red test
In contrast, some microbes can turn organic acids to non-acidic end products such as
2, 3-butanediol and acetoin. The Vogues-Proskauer test detects the presence of
acetoin, which is oxidized to a diacetyl compound in the presence of oxygen and
potassium hydroxide. When acetoin is produced after 48 h growth, addition of VP test
reagents (i.e., alpha-naphthol and potassium hydroxide) results in a pink color within
30 min.
Urea agar
Urea is the product of amino acid metabolism. Many enterics can turn urea into
ammonia with the enzyme urease. Organisms which possess urease hydrolyze urea
to produce alkaline by-product (i.e., ammonia) which is detected by a change in the
phenol red indicator from yellow-orange to pink.
Exercise: inoculation of differential/selective media
Materials (each group)
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FAQs of Week 5 Microbiology Lab: Heat Inactivation and Media Techniques

What is the purpose of heat inactivation in microbiology?
Heat inactivation is a critical technique used to eliminate microbial pathogens in food and medical applications. By applying heat, such as in pasteurization, proteins and enzymes within microbes are denatured, leading to cell death. This process is essential for ensuring food safety and preventing infections in clinical settings. The effectiveness of heat treatment can be assessed through methods like total viable count and absorbance measurements.
How does Lactose MacConkey agar differentiate between bacteria?
Lactose MacConkey agar is both selective and differential, primarily used to isolate Gram-negative bacteria. It contains lactose, which some bacteria can ferment, lowering the pH and resulting in red colonies. The presence of crystal violet and bile salts inhibits the growth of Gram-positive bacteria, allowing for the selective growth of Gram-negative organisms. This medium is particularly useful for identifying lactose fermenters like E. coli.
What is the significance of the Durham tube in phenol red mannitol broth?
The Durham tube is an essential component of phenol red mannitol broth, used to detect gas production during fermentation. When bacteria ferment mannitol, they produce acids that can lower the pH, causing the phenol red indicator to change color from red to yellow. If gas is produced, it will be trapped in the Durham tube, indicating successful fermentation. This method is crucial for identifying specific bacterial species based on their metabolic capabilities.
What does a blue color indicate in Simmons citrate agar?
In Simmons citrate agar, a blue color change indicates that the organism can utilize citrate as its sole carbon source. The medium contains bromothymol blue, which shifts from green to blue as the pH increases due to ammonia production from ammonium salts. A negative result, where the medium remains green, suggests that the organism cannot metabolize citrate. This test is vital for differentiating among Enterobacteriaceae.
What are the enrichment techniques used for isolating Pseudomonas species?
Enrichment techniques for isolating Pseudomonas species involve using a mineral base medium that lacks organic carbon and nitrogen sources. By adding specific carbon and nitrogen sources, such as sodium benzoate or ammonium chloride, the growth of Pseudomonas is favored over other soil bacteria. This selective enrichment increases the relative abundance of Pseudomonas, allowing for successful isolation on solid media. Such methods are crucial for studying the ecological roles of Pseudomonas in soil.
What is the expected outcome of the E. coli heat treatment experiment?
The E. coli heat treatment experiment aims to determine the survival rate of E. coli at 60°C over a 21-minute period. By sampling at intervals and measuring absorbance, students can assess how heat affects bacterial viability. The expected outcome is a decrease in both viable counts and absorbance readings as the duration of heat exposure increases, demonstrating the effectiveness of heat in inactivating microbial pathogens.

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