Asignatura: Microbiología

Departamento: Química

Profesores: Pedro F. de Brito Brandão

Carlos Yesid Soto O.

Laboratorio: Bioquímica 330

Practica 5. Factores físicos que afectan el crecimiento microbiano: pH del

ambiente extracelular

Objectives
Once students have completed this experiment, they should be familiar with the
pH requirements of microorganisms.

Introduction
Growth and survival of microorganisms are greatly influenced by the pH of the
environment, and all bacteria and other microorganisms differ as to their requirements.
Based on their pH optima, microorganisms may be classified as acidophilic,
neutralophilic, or alkalophilic (Figure 1). Each species has the ability to grow within a
specific pH range, which may be broad or limited, with the most rapid growth occurring
within a narrow optimum range. These specific pH needs reflect the organisms’
adaptations to their natural environment. For example, enteric bacteria are capable of
survival within a broad pH range, which is characteristic of their natural habitat, the
digestive system. Bacterial blood parasites, on the other hand, can tolerate only a narrow
range; the pH of the circulatory system remains fairly constant at approximately 7.4.

Figure 1 – The effect of pH on the growth of microorganisms

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 Despite this diversity and the fact that certain organisms can grow at extremes of the
pH scale, generalities can be made. The specific range for bacteria is between 4 and 9,
with the optimum being 6.5 to 7.5. Fungi, molds, and yeasts prefer an acidic
environment, with optimum activities at a pH of 4 to 6.

Because a neutral or nearly neutral environment is generally advantageous to the

growth of microorganisms, the pH of the laboratory medium is frequently adjusted to
approximately 7. Metabolic activities of the microorganism will result in the production
of wastes, such as acids from carbohydrate degradation and alkali from protein break-
down, and these will cause shifts in pH that can be detrimental to growth.

To retard this shift, chemical substances that act as buffers are frequently
incorporated when the medium is prepared. A commonly used buffering system
involves the addition of equimolar concentrations of K2HPO4, a salt of a weak base, and
KH2PO4, a salt of a weak acid. In a medium that has become acidic, the K2HPO4
absorbs excess H+ to form a weakly acidic salt and a potassium salt with the anion of
the strong acid.

In a medium that has become alkaline, KH2PO4 releases H+ to form water by combining
with the excess OH-, and the remaining anionic portion of the weakly acidic salt
combines with the cation of the alkali.

Most media contain amino acids, peptones, and proteins, which because of their
amphoteric nature, can act as natural buffers. For example, amino acids are zwitterions,
molecules in which the amino group and the carboxyl group ionize to form dipolar ions.
These behave in the following manner:

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Materials
- saline suspensions of 24-hour TSB cultures, adjusted to an O.D. of 0.05 at a
wavelength of 600 nm, Alcaligenes faecalis, Escherichia coli and Saccharomyces
cerevisiae.
- per group of students: 12 Trypticase soy broth (TSB) tubes, three at each of the
following pH designations: 3, 6, 7, and 9. The pH of the culture media has been
adjusted with 1N NaOH or 1N HCl before autoclaving.
- “mecheros”
- micropipettes
- sterile blue and yellow tips
- spectrophotometer
- test tube rack
- glassware marking pencil
- incubators at 37°C and 25°C

Procedure
1. Using a sterile pipette tip, inoculate a series of the appropriate labelled TSB tubes of
media, pH values 3, 6, 7, and 9, with E. coli by adding 0.1 ml of the saline culture to
each, under sterile conditions.

2. Repeat step 1 for the inoculation of A. faecalis and S. cerevisiae, using a new sterile
pipette tip each time

3. Incubate the A. faecalis and E. coli cultures for 24 to 48 hours at 37°C and the S.
cerevisiae cultures for 48 to 72 hours at 25°C.

Questionnaire
1. Using the spectrophotometer, determine the optical density of all cultures after the
incubation time and record the readings in a table.

2. Summarize your results as to the overall range and optimum pH of each organism
studied in a table.

3. Explain the mechanism by which buffers prevent radical shifts in pH.

4. Explain why it is necessary to incorporate buffers into media in which

microorganisms are grown.

5. Why are proteins and amino acids considered to be natural buffers?

6. Explain why microorganisms differ in their pH requirements.

7. Will all microorganisms grow optimally at neutral pH? Explain.