Laboratory Investigations in Microbiology
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Chapter 12: Effect of environmental variables on growth

As we have seen in the last exercise, bacterial growth is rapid and bacterial populations can easily reach into the billions of cells per milliliter. The process of bacterial growth, of course, is the sum of all the biological activities required to build cells. Nutrients are taken in and metabolized, energy is generated, precursor molecules are constructed and assembled into proteins, nucleic acids, carbohydrates, and lipids. These molecules are then assembled into the various cell components (ribosomes, membranes) needed to enlarge a cell and then allow it to divide. Virtually all the steps just described require to use of enzymes - biological catalysts that speed up chemical reactions and link reactions so that energy released from one molecule can be used to construct another molecule. Most of these reactions would never happen spontaneously 9without the help of enzymes). Since enzymes are themselves proteins, their structure and function depend on their environment. In particular, temperature, pH, and salt concentrations can affect protein structure. If the environment of an organism is not in the optimal range, enzyme function is impaired or lost, and biological activities cease.

Every living cell is adapted to a certain range of temperature, pH, and osmolarity. We can classify organisms according to their preference in (or tolerance for) pH, temperature, and osmotic concentration. 

An organism that prefers cold temperatures (0° - 15° C) is called a psychrophile, one that prefers moderate temperatures (15° - 35° C) a mesophile, and one that prefers hot temperatures (> 50° C) a thermophile.

Cells that prefer alkaline (pH > 7) media are known as alkalophiles. Cells that prefer neutral (pH 6 - 8) environments are neutrophiles, and those living in acidic (pH < 7) conditions are acidophiles.

Bacteria also vary in their tolerance of salt. This is for two reasons. First, external salt levels affect the movement of water (osmosis) in and out of cells, and high external salt levels can cause bacteria to shrivel up. Secondly, high salt levels can interfere with protein (enzyme) structure. Bacteria that are tolerant of higher-than-normal salt levels are termed osmotolerant, and those that require high salt levels are known as halophiles. 

The objectives of this exercise are to evaluate the effect of varying temperature, pH, and salt levels on the growth of various microorganisms, and to determine the identity (-phile) of each organism.

Materials & Methods

Materials per lab group

1 set of 8 TSB tubes (pH 3, 4, 5, 6, 7, 8, 9, 10)

1 set of 4 salt agar plates (0%, 5%, 10%, 20%)

1 set of 6 TSB tubes designated 4°C, 15°C, 23°C, 35°C, 50°C, 65°C

Cultures (assigned in class)

• Temperature: 1 culture assigned to each lab group
  • E. coli (EC), B. stearothermophilus (Bst), Actinomyces spp. (AT), S. aureus (SA), Vibrio natriegens (VN), or Pseudomonas fluorescens (PF)
• pH: 1 culture assigned to each lab group
  • E. coli (EC),  A. faecalis (AF), Lactobacillus acidophilus (LA), B. laterosporis (BL), S. aureus (SA) or Saccharomyces (Yeast)
• Salt: 3 cultures for each lab group
  • H. salinarum, S. aureus, and E. coli

Procedure

Temperature

1. Obtain 6 TSB tubes and one broth culture of bacteria (assigned)
2. Label your 5 tubes: Lab group #, Lab section, name of bacteria, temperature
3. Vortex your bacterial broth to mix evenly
4. Inoculate each TSB tube with 100 µl of your bacterial broth or one loopful (loop inoculation)
5. Place your tubes into the appropriate locations:
   • 4°C: Refrigerator (222A)
   • 15°C: Circulating water bath #2
   • 23°C: Front lab bench
   • 35°C: Small incubator
   • 50°C: water bath (silver)
   • 65°C: Circulating water bath #1

1. Remove your tubes from these locations. 
2. Measure growth by turbidity.
   1. Calibrate the spectrophotometer with a TSB blank
   2. Pipette 1 ml of your sample into a plastic cuvette
   3. Record your Absorbance value.
3. Graph your data: Temperature (X) vs. Absorbance (Y)
4. Record your data online.

pH

1. Obtain a set of 8 color-coded pH-adjusted TSB tubes {clear = 3, white = 4, red = 5, yellow = 6, green = 7, blue = 8, metal = 9, small white = 10} and one microbial broth (as assigned to your group)
2. Inoculate each TSB tube with 100 µl of the microbial broth or one loopful (loop inoculation)
3. Label your test tubes: Lab group #, Lab section, microbe name, pH

1. Measure growth in each test tube by turbidity.
2. Graph your data: pH (X) vs. Absorbance (Y)
3. Record your data online

Salt

1. Obtain a set of 4 salt agar plates (0%, 5%, 10%, 20%), broth cultures of Staphylococcus aureus and E. coli and 1 culture (shared) of Halobacterium salinarum (HS)
2. Divide your TSA plates into 3 sections, labeled SA, EC, HS. Streak-inoculate each section of each plate with the appropriate microbe.
3. Bring your plates up front to be incubated

1. Visually compare the amount of growth on all 4 plates. 
2. Rate the growth on each plate as heavy (+++), moderate (++), light (+), or none (-).
3. Record your data on your data sheet.

• Data Sheet & Review Questions

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