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1. 1
Microbial Growth
Chapter 6

2. 2
Microbial Growth
• Increase in number of cells
• Very short time
• Understanding the condition
necessary for the microbial
growth.
In this chapter:
• Examine physical and chemical
requirements
• The various kinds of culture media
• Bacterial division
• The phases of bacterial growth
• Methods of measuring microbial
growth.

3. 3
Factors Affecting Bacterial Growth
Physical and chemical factors
Physical requirements: Temperature, PH, and osmotic pressure
Chemical requirements: Source of carbon, nitrogen, sulfur, phosphorous, trace
elements, oxygen, and organic growth factors.
Physical Requirements
Temperature:
Most microorganism grow well at temperature favored by human.
However, certain bacteria are capable at growing at extremes of
temperature.
– Each bacteria grows at particular minimum, optimum, and maximum
temperature.
• Minimum growth temperature (lowest T at which the species will grow).
• Optimum growth temperature (T at which the species grows best).
• Maximum growth temperature (highest T at which growth is possible).

4. 4
• Microorganisms are classified into groups depending on temperature:
– Psychrophiles:
One group grow 15-20 ºC (optimum); 0 ºC; cold lovers; found in the ocean’s
depth, such organism seldom cause problem in food preservation.
Other group grow 20-30 ºC (optimum); 0 ºC; mold on fruit in fridge.
The term psychrotrophs is favored by food microbiologists for this group
of spoilage microorganisms.
– Mesophiles: 25-40ºC; most bacteria; Optimum temperature of human
pathogens 37 ºC, and incubators for culturing these microorganisms are
set at this temperature; include common spoilage and disease organisms.
– Thermophiles: capable of growth at high temperature > 45 ºC ; 50 –60 ºC;
heat lovers; boiling springs.
Typical growth rates of
different types of
microorganisms in
response to
temperature

5. 5
The term Psychrotrophs is used by food microbiologists
• Refrigeration is the most
common method of preserving
household food supplies. It is
based on the principle that
microbial reproductive rates
decrease at low temperatures,
as a results there will be a
decline in number.
• Organism that are capable to
grow between 0°C and 30°C.
• Cause food spoilage
(slowly degrade food, form
molds on food surfaces, off
tastes, off colors in food).
When large amounts of food
must be refrigerated, it is
important to keep in mind the
slow cooling rate of a large
quantity of warm food.
Food spoilage temperatures.

6. 6
Hyperthermophiles organisms
• Members of Archea
• Optimum growth temperature of 80°C or higher.
• Live in hot springs associated with volcanic activity.
Sulfur is usually important in their metabolic activity.
• The known record for bacterial growth at high
temperature is about 110 °C near deep-sea
hydrothermal vents.

7. 7
pH (refers to the acidity or alkalinity of the solution).
Optimum pH: 6.5-7.5 for most of the bacteria.
- Very few can grow at an acidic pH. This is why a number of foods such as
pickles, and many cheeses, are preserved from spoilage by acids produced by
bacterial fermentation.
- Alkalinity also inhibits microbial growth but is rarely used to preserve
foods.
• Acidophiles bacteria: 1 – 5.4 ; Lactobacillus; Bacteria which found in the
drainage water from coal mines and oxidizes sulfur to form sulfuric acid.
Example. Helicobacter pylori is a bacteria that has been associated with
stomach ulcer.
Most bacteria can’t tolerate acidity of the stomach (PH<2).
• Neutrophiles bacteria: 5.4 – 8.5; cause human disease
• Alkalophiles bacteria: 7.0 – 11.5; Many soil bacteria live at these higher pH
enviroment.
• In the laboratory when bacteria are cultured, they often produce acids that
eventually interfere with their own growth (Lactic acid & pyruvic acid -
products of fermentation – inhibit growth)
To maintain proper pH, chemical buffers are included in the growth medium.
The peptones and amino acids acts as buffers, and many media also contain
phosphate salts. Phosphate salts are non toxic and they provide
phosphorous, an essential nutrient.

8. 8
Osmotic Pressure
Microorganisms obtained almost all their nutrients in solution from the
surrounding water. Thus, they require water for growth and are made up of
80-90% water.
• Hyperosmotic environment: cells lose water (higher conc. of solutes than in
the cell) causes plasmolysis – cell shrinks.
– The importance of this phenomena is that the growth of cell is inhibited as the
plasma membrane pulls away from the cell wall.
– The addition of salts (or other solutes) to a solution, and the resulting increase
in osmotic pressure, can be used to preserve foods. Use of salts –pickles; sugars
–jams & jellies causes high O.P. and kills or inhibits bacterial growth; require no
refrigeration due to high O.P.
• Halophiles – salt lovers organisms; Example. Oceans, Great Salt lake, Dead
Sea (30% salt);
• Most microorganisms must be grown in medium that is nearly all water. For
example, the concentration of Agar (complex of polysaccharides isolated
from marine algae) used to solidify microbial growth media usually about
1.5%. IF higher concentrations are used, the increased osmotic pressure
can inhibit the growth of some bacteria.
• Hypoosmotic environment – cell gains water (low conc. of solutes than in the
cell) ; cell becomes rigid or lysed depends of the cell wall.

9. 9
The Requirements for Growth: Physical Requirements
Normal cell in isotonic solution.
Under these conditions, the
osmotic pressure in the cell is
equivalent to a solute
concentration of 0.85% sodium
chloride.
Plasmolyzed cell in hypertonic solution.
If the concentration of the solutes
such as NaCl is higher in the
surrounding medium than in the cell
(hypertonic), water tends to leave the
cell. Growth of the cell is inhibited.

10. 10
Chemical Requirements
Carbon
It is needed for all the organic compound that make up a living cell.
• Chemoheterotrophs organisms use organic molecules as a source of
carbon and energy (such as proteins, carbohydrates, lipids).
• Chemoautotrophs (use inorganic chemical as energy source) and
photoautotrophs (use light as energy source) organisms use CO2 as
carbon source.
Factors Affecting Bacterial Growth
Nitrogen
• In addition to carbon, other elements are needed by microorganisms
for the synthesis of cellular material.
For example,
– protein synthesis requires considerable amounts of nitrogen as well as
some sulfur;
– DNA and RNA synthesis, requires N and some phosphorus;
– the synthesis of ATP molecule, which is important for the storage and
transfer of chemical energy within the cell.

11. 11
Protein: Amino acids are joined to
form unbranched polypeptides by a
peptide bond. Covalent bond
between the carboxyl group of one
amino acid and amino group of the
next amino acid.
Organisms use nitrogen to form amino groups of the amino acids of proteins.
• Many bacteria meet this requirement by decomposing proteins and
incorporating the amino acids into newly synthesized proteins and other N
containing compounds.
• Some bacteria use nitrogen from ammonium ions (NH4
+) or nitrate ions
(NO3
).
• A few bacteria including many of the photosynthesizing cyanobacterium, use
gaseous nitrogen (N2) from the atmosphere. This process is called nitrogen
fixation.
• Some organisms that can use this method are free-living, mostly in the
soil,
• but others live cooperatively in symbiosis with the roots of legumes
such as clover, alfalfa, beans and peas. The nitrogen fixed in the
symbiosis is used by both the plant and the bacterium.

12. 12
DNA STRUCTURE
chemical structure of the 4 nucleotides in DNA

13. 13
Sulfur
Is used to synthesis sulfur-containing amino acids and vitamins, such as
thiamine, biotin.
–Most bacteria decompose proteins
–Some bacteria use SO4
2 or H2S
Phosphorus
Phosphorus is essential for the synthesis of nucleic acids and the
phospholipids of the cell membranes. Also found in the energy bonds of
ATP.
–PO4
3 is an important source of phosphorus
Potassium, magnesium, and calcium are also elements that microorganisms
require, often as cofactors for enzymes (help catalyze a reaction by
forming a bridge between the enzyme and a substrate).
Trace Elements
–Other mineral elements required in small amounts such as iron, copper and
zinc.
–Most are essential for the functions of certain enzymes, usually as
cofactors. Most of these naturally present in tap water.

14. 14
obligate
aerobes
Facultative
anaerobes
Obligate
anaerobes
Aerotolerant
anaerobes
Microaerophiles
Oxygen
We accustomed to thinking of molecular oxygen (O2) as a necessity of life, but
it was found that large number of bacterial species live in the absence of O2.
• Obligate aerobes: Organisms that require oxygen to live. EX. Pseudomonas*
• Obligate anaerobes – no oxygen; oxygen kills; bottom of broth; improperly
canned foods; Ex. Clostridium spp.
• Facultative anaerobes – Organisms that have the ability to continue growing
with or without oxygen; Ex. E. coli, Staphylococci.
Can any bacteria live in a reduced oxygen atmosphere?
Microaerophiles bacteria – need little (low concentration) of oxygen; turbid just
below surface in broth; found in Urinary and digestive tracts in humans and
some species can cause infection in these systems. EX. Campylobacter
• Many of the Microaerophiles are dependent on carbon dioxide for their
metabolism. These organisms are called Capnophiles – CO2 lovers
Aertolerant anaerobes:
can’t use O2 for
growth, but tolerate it
fairly well.

15. 15
1. Singlet oxygen: is normal O2 boosted to a higher-energy state and is extremely
reactive. It is present in phagocytic cells, which play an important role in the
body’s defenses against pathogens such as bacteria.
2. Superoxide free radicals: O2

are formed in small amounts during the normal respiration of organisms that use
O2 as final electron acceptor, forming water.
3. The H2O2 produced in this reaction contains peroxide anion O2
2 and is also
toxic.
4. Hydroxyl radical (OH) is another intermediate form of O2 and probably the
most reactive. Most aerobic respiration produces traces of hydroxyl radicals
but they are transient.
Obligate anaerobes usually produce neither SOD nor catalase. Because aerobic
conditions lead to an accumulation of superoxide free radicals in their
cytoplasm, obligate anaerobic extremely sensitive to O2 .
In Order to understand how organisms can be harmed by oxygen we
require some knowledge of the toxic forms of oxygen
H+
2

16. 16
testing for the presence of catalase
H2O2  2 H2O + O2

17. 17
Organic Growth Factors (OGF)
– Essential organic compounds an organism is unable to synthesize
known as OGF. They must be directly obtained from the
environment.
– Vitamins is an organic compounds obtained from the environment.
Most vitamins functions as coenzymes, the organic cofactors
required by certain enzymes in order to function.
– Many bacteria synthesize their own vitamins and are not dependent
on outside source. However, some bacteria lack the enzymes
needed for the synthesis of certain vitamins, and for them those
vitamins are OGF.
– Other organic growth factors required by some bacteria are amino
acids, purines, pyrimidines

18. 18
Culture Media
• A nutrient material prepared for the growth of microorganisms in a laboratory
is called a culture medium. Some bacteria can grow well on just about any
culture medium; others require special media, and still others cannot grow in
any nonliving media such as Mycobacterium tuberculosis.
• Defined - made from purified chemicals
• Complex - exact chemical composition not known

19. 19
Selective Media
Suppress unwanted microbes and encourage desired microbes.
For example, bismuth sulfite agar is one medium used to isolate the typhoid
bacterium, the gram-negative Salmonella typhi, from feces.
Bismuth sulfite inhibits gram positive bacteria and most gram negative intestinal
bacteria (other than S. typhi) as well.
Differntail Media
Make it easier to distinguish colonies of the desired organism from other
colonies growing on the same plate. Example, EMB, blood agar (Streptococcus
pyogenes which lyse the blood; show a clear ring around their colonies)
In clinical and public health microbiology, it is frequently necessary to
detect the presence of specific microorganism associated with disease.
Enterobacter aerogenes on EMB (eosin
methylene blue) medium showing
characteristics dark-centered colonies.
E. coli on EMB (eosin methylene blue) medium
showing the black -centered colonies
surrounded by characteristic metallic green.

20. 20
• Encourages growth of desired microbe (it is also a selective medium, but it
designed to increase numbers of the desired organism to detectable levels).
• Assume a soil sample contains a few phenol-degrading bacteria and
thousands of other bacteria and we want to isolate the bacteria that can
grow on phenol.
• If the soil sample is placed in a liquid enrichment medium in which phenol is
the only source of carbon and energy, microbes unable to metabolize phenol
will not grow.
• Experiment:
– Inoculate phenol-containing culture medium with the soil and incubate
for a few days.
– Transfer 1 ml to another flask of the phenol medium and incubate for
few days (this is enrichment stage).
– After a series of such transfers only phenol-metabolizing bacteria will
be growing.
Enrichment Media

21. 21
Obtaining a Pure Culture
Why?
If there were two or more species being cultivated together it is difficult
to know which effect was being brought about by which species. Also, if a
diagnosis for disease is desired, it would be difficult to know which
microorganisms were responsible for the disease.
• A pure culture contains only one species or strain. The isolation method
most commonly used to obtain a pure cultures is the Streak Plate Method.
• A colony is a population of cells arising from a single cell or spore or from a
group of attached cells
• A colony is often called a colony-forming unit (CFU)

22. 22

23. 23
• Deep-freezing: is a process in which a pure culture of microbes is
placed in suspending liquid and quick frozen at temperatures ranging
from -50°to -95°C.
The culture can usually thawed and cultured even several years later.
• Lyophilization (freeze-drying): a suspension of microbes is quickly
frozen (-54° to -72°C) and dehydrated in a vacuum.
Preserving Bacteria Cultures

24. 24
Population Growth
• Growth rate = change in cell # / time
• Generation time (g) = time for formation of two cells from one = doubling time
• If generation time remains constant, then exponential growth.
Doubling Time = 30 min Exponential Growth
Some bacteria can complete one
generation in as little as 30 min.
Binary Fission

25. 25
• When the culture must be grown in
Petri plates to observe individual
colonies, special anaerobic jars are
used.
Anaerobic Culture Media and Methods
Reducing media
Contain chemicals (thioglycollate or oxyrase) that combine with the
dissolved O2 and deplete the O2 in the culture medium.
These media are heated shortly before use, to drive off absorbed O2
.

26. 26
Capnophiles require high CO2
Many clinical
laboratories have
special CO2 incubators
in which to grow
aerobic bacteria that
require concentrations
of high or low CO2 than
that found in the
atmosphere.
Candle jar
3 % CO2
CO2-chemical
packet
10 % CO2
Campylobacter

27. 27
Serial Dilutions
Plate Counts:
Perform serial
dilutions of a
sample
Direct Measurements of Microbial Growth

28. 28
• Count positive
tubes and
compare to
statistical MPN
table.
Direct Measurements of Microbial Growth Using
Most probable number (MPN) test

29. 29
Direct Measurements of Microbial Growth
Direct Microscopic Count
Figure 6.19
Counts all cells; cannot distinguish between living and dead

30. 30
• Turbidity
Estimating Bacterial Numbers by Indirect Methods