Microbiology

Microbial Growth

Bacterial Growth - increase in the #

of cells
Binary Fission
Generation Time (Doubling Time)
time required for a cell to divide
most about 1 Hr. To 3 Hrs.
E. coli - 20 minutes
Mycobacterium tuberculosis - 24 Hrs.

Binary Fission - unchecked

E. coli - generation time of 20 min.
20 generations (about 7 hrs.)

1 million cells
30 generations ( about 10 hrs.)

1 billion cells
72 generations ( about 24 hrs.)

1 x 1021
1,000,000,000,000,000,000,000 cells

Phases of Growth

1. Lag Phase
2. Log Phase
3. Stationary Phase
4. Death Phase

1. Lag Phase
- no immediate increase in cell number and mass occurs
- cells adapt to new medium and factors
- evtl. new enzymes and metabolic pathways are induced
- temperature adaptation
- length of lag phase dependent on different factors:
e.g. age of cell culture, type of medium, temperature,
presence or absence of important co-factors or supplements

2. Log Phase
Rapid cell growth (exponential growth)
bacteria are dividing and doubling in number at constant
rate
microbes are sensitive to adverse conditions
antibiotics
anti-microbial agents
-

2. Log Phase
the cell count N of a cell population with a number of N0 cells (at
the beginning of the inoculation) and after n cell divisions can be
calculated using following formula:
N(n) = N0 2n
Number of cell divisions n:
n = lgN - lgN0 / lg2
Division rate or mean growth rate constant k:
k = n / t = lgN - lgN0 / lg2 (t - t0)
--> number of generations per unit of time
Mean generation time or Doubling time g:
g = 1/ k

3. Stationary Phase
population growth ceases and growth curve becomes
horizontal
Death rate = rate of reproduction
bacterial population density reaches maximum number: usually 109 cells / ml
cells begin to encounter environmental stress
lack of nutrients, water, carbon source (e.g. glucose, amino acids)
Energy-consuming production of starvation proteins, e.g. increased
peptidoglucan cross-linking, DNA-protecting proteins (Dps), chaperons,
stress-response proteins
not enough space
metabolic wastes
Oxygen down
pH down (becomes acid)

Endospores would form now

4. Death Phase
Death rate > rate of reproduction
Due to limiting factors in the environment
Lack of food, water or nutrients
space
accumulation of metabolic wastes
lack of oxygen
changes in pH
temperature
decline in number of viable cells due to build-up of toxic
metabolic end products
cells loose ability to reproduce and die off; some produce
endospores death rate may be exponential

Microbial Growth - refers to the # of

cells, not the size of the cells
Requirements for Growth
Physical (Temperature, pH, Osmotic pressure)
Chemical (nutrient/elements, oxygen)

Physical Requirements
Temperature
psychrophiles (cold loving microbes )
range

0 C - 20 C

mesophiles (moderate temp. loving microbes)

range

thermophiles
range

20 C - 40 C

(heat loving microbes)

40 C - 100 C

pH
Most bacteria grow between pH 6.5 - pH 7.5
Very few can grow at below pH 4.0
many foods, such as sauerkraut, pickles, and cheeses
are preserved from spoilage by acids produced during
fermentation

Osmotic Pressure
Microbes obtain almost all their nutrients in solution from
surrounding water
Tonicity
isotonic
hypertonic
hypotonic

Chemical Requirements
Macro elements (there are ten):
C, O, H, N, S, P, K, Ca, Mg and Fe,
micro- or trace-elements f(or optimum growth):
Mn, Mo, Zn, Co, Cu, Ni, V, B, Cl, Na, Se, Si and W,

Oxygen
Bacteria can be classified base on their oxygen requirements

Obligate
Aerobes,

Obligate
Anaerobes,

Facultative
Anaerobes,

Microaerophilic,

Oxygen is lethal to some organisms

All organisms produce superoxide ( O2-)
Superoxide is toxic to cells (steals electrons)
Superoxide must be neutralized
Superoxide dismutase:
O2- + O2- + 2 H+ -------> H2O2 + O2
Catalase
2 H2O2 --------> 2 H2O + O2

Obligate Anaerobes lack:

Superoxide dismutase ( SOD )
Catalase

Growth Media
Synthetic media : solution which is comprised of defined amounts
chemical ingrediences and compounds
- a minimum medium is a solution which contains just enough of the
chemicals and compounds to allow good growth of the specific
microorganism
Complex media: nutrient solution which contains defined amounts
of chemically not defined extracts, e.g. extracts from yeast,
peptone, tryptone or meat
- they are often the choice or only option if the exact growth
conditions and requirements of a given microorganism are not
known
- commonly used complex media in microbiology are:
1. Tryptic soy broth
2. Nutrient broth
3. Peptone yeast extract broth
4. Luria Bertani (LB) broth
5. MacConkey agar

Selective Media
Inhibits the growth of some bacteria while selecting for
the growth of others
Example:
Brilliant Green Agar
dyes inhibit the growth of Gram (+) bacteria
selects for Gram (-) bacteria
Most G.I. Tract infections are caused by Gram (-)
bacteria

Selective Media
EMB (Eosin Methylene Blue)
dyes inhibit Gram (+) bacteria
selects for Gram (-) bacteria
G.I. Tract infections caused by Gram (-) bacteria

Differential Media
Differentiates between different organisms growing on the
same plate
Example:
Blood Agar Plates (TSA with 5% sheep blood)
used to differentiate different types of Streptococci

Beta Hemolytic
Streptococci

Selective and Differential Media

Mannitol Salt Agar
used to identify Staphylococcus aureus
High salt conc. (7.5%) inhibits most bacteria
Contains sugar mannitol
pH Indicator (Turns Yellow when acid)
MacConkeys Agar
used to identify Salmonella
Bile salts and crystal violet (inhibits Gram (+) bacteria)
Contains lactose
pH Indicator

Thank you!

Culture Media
1. Chemically Defined
the exact chemical composition is known
used to grow fastidious organisms

2. Complex Media
exact chemical composition is not known
most bacteria and fungi are grown with this

Special Culture Techniques

1. Anaerobic Bacteria

a. Reducing Media
b. Anaerobic Container
c. Agar Stab
d. Agar Shake

Special Culture Techniques

2. Microaerophilic Bacteria
grow best under reduced O2 levels and increased CO2 levels
Normal Atmosphere

21 % O2, 0.3 to 0.03 % CO2

Microaerophilic Bacteria

A. Candle Jar

16 % O2
4%

CO2

Microaerophilic Bacteria
B. CO2 Generating Packet

Enumeration of Bacteria
Turbid culture - 10 million bacterial cells per ml
Serial Dilution

 1. Petroff-Hauser Counting Chamber

2.