Growth & Culture of Bacteria

Chapter 4, 5

2

Key Words

Obligate aerobe

Obligate anaerobe

Facultative anaerobe

Microaerophilic

Mesophile

Thermophile

Psychrophile

Generation time

Growth curve

Growth of bacteria

Bacterial Multiplication - Binary Division

1 to 2 to 4 to 8 to N

Log Growth ?

Chapter 4

20’-30’ /generation

For most bacteria

Phase Growth Rate

Lag Zero

Exponential Constant

Maximum stationary Zero

Decline Negative (death)

Chapter 4

Fig. 4-2

Phases of bacteria growth (in vitro)

Growth curve

• Lag

– Adapt to nutrients

• Log

– Active growth

• Stationary

– Death = Growth rate

• Death

– Nutrients consumed

– pH too low (why?)

• Optimize curves in production

Note the drop-off as you get closer to the Optimum!

Is it the same as in human body ? Why?

Bacterial Growth Curve

Nutritional Factors

• Carbon Source

– Hetero- or Auto-

– Organic compounds or CO2

• Nitrogen Source

– NO3- ==> NO2 ==> NH3 to Amino Acids ==> Proteins

• Sulfur

• Phosphorus

• Trace Elements

– Fe, some required for growth or virulent

– Host sideophores bind Fe

All pathogenic bacteria are heterotrophic

-obtain energy by oxidizing organic molecules

(Carbohydrates, lipids and protein)

Metabolism- yield ATP as an energy source

-Aerobic respiration, 1 glucose produce 38 ATP -

enzymes

-Anaerobic respiration, 1 glucose produce 2 ATP

(fermentation) -enzymes

Requirement for oxygen in respiration

-obligate

-facultative

Chapter 5

p.657

Cultivation of bacteria

Requirements for bacterial growth

Nutrients

H2O, C-source(organic), N-source (organic)

Inorganic salts

Growth factors

Temperature

pH

Gas

Incubator Temperature, gas

Culture medium Nutrients, pH

Pathogenic bacteria

culture condition?

Moisture

Salt concentration 0.9% Nacl…PBS(Phosphate

buffer saline)=?

Osmotic pressure

Pseudomonas grows in distilled water

Jelly- Jams (high sugar)

Salt pork (high salt)

Isotonic

Temperature Requirements

• Mesophile

37℃ (Pathogenic bacteria)

• Thermophiles

– Hot spring, hot tubs-public

– Heat Resistant Enzymes in PCR

(Tag DNA polymerase)

• Psychrophiles

– Cold food (growth in transit)

– Listeria grows in hot dogs

South pole

What effect would have a fever on Mesophiles?

Most pathogenic bacteria

Chapter 6

Hydrogen Ion concentration (pH)

• Neutralophiles (pH 6.0-8.0)

• Acidophiles (< pH 3.0) (Helicobacter pylori)

• Alkaliphiles (pH10.5) (bacteria from salina,

Vibrio. cholera)

Microorganisms regulate their internal pH

over a wide range of external pH values by

pumping protons in or out of the cell

Oxygen Requirements

• Classification

– Aerobic

– Anaerobic

– Falcultative Anaerobic

– Microaerophilic

O2 is reactive-toxic (binds e-’s)

Oxidization of compounds

Loss of energy from compounds

DNA molecule breaks

Why can some organisms grow in the presence of oxygen?

Toxic forms of oxygen need to be neutralized by enzymes

-Superoxide dismutase

-Catalase

-Peroxidase

If microbe does not produce these enzymes must have

anaerobic conditions Aero-

tolerant

Microaerophile

Obligate

aerobe

Obligate

anaerobe

Facultative

anaerobe

Culture medium

-the mixture of various nutrients that is suitable for

the growth of microorganisms

Types of Culture Media

-based on the function and chemical components

-based on the physical state

.

• Culture medium

A liquid or gel with mixture of various nutrients that is suitable for the growth of microorganisms

Classification of culture medium with based on the function and chemical components

components：

Basic medium

Enrichment medium (Basic medium + nutrients

Selective medium（medium + inhibitor）

Differential medium (medium + indicator)

Anaerobic medium

Liquid medium (nutrient broths or LB medium)

Solid medium (liquid medium + agar from seaweed)

Semi- solid culture

19

Based on the function and the chemical components

• Basic Medium

-contains the basic nutrients for the most bacterial growth;

-the base of other kind of media. e.g. broth.

Nutrient Medium/Enriched Medium

Additional or special nutrients (e.g., serum, growth

factors, trace elements) are added to support some

fastidious bacterial growth. e.g. blood agar.

20

• Selective Medium

the medium that can prevent the certain bacterial growth while permitting others.

e.g. SS agar

Differential Medium

Some special substrates and indicators are added into the media in order to produce a visual differentiation when several bacteria grow on the same kind of medium.

e.g. EMB agar (Eosin-methylene blue agar).

Growth of bacteria in the liquid medium

Surface turbid precipitate

No single bacterial cell

Liquid medium

Solid medium（agar 15g/L ）

Semi-solid medium（ agar 3~5g/L）

Solid medium

Richard Petri Petri dish

Semi-solid

medium

Angelina Fannie Eilshemius Hesse

Agar-used for jam、jelly、jelly drops…

1. Chemical: agarobiose

2. Not digested by bacteria，no nutrient；

3. Melted at 85 ℃； solidified at 32-40 ℃

Isolation：

• Streaking on the surface of

solid medium in Petri dish

Colony：

• A single cell bacteria grow to as

a visible cluster on the surface

of solid medium

-pure bacterial strain

Bacterial growth on solid

medium

• solid medium：

Plates (colony) Slant（bacterial lawn)

Bacterial growth in semi-

solid medium

Flagella bacteria

Grow Diffusion,

turbid around

No flagella bacteria

Growth along the stab line,

clear around

No motility Motility

Review questions (chapter 2)

1. p41:1, 2, 3, 5, 6, 7, 8, 9 and think about explainations.

2. Summary the differences between the cell wall of

G+bacteria and G-bacteria (related with clinical practice)

3. Why penicillin and lysozyme have less effect on G-

bacteria?

4. Structures of bacteria associated with clinical practice.

Explain which bacterial structures can be as an

antibiotics target.

Review questions(chapter 4 & 5)

1. In p65: question 1, is it right? Why?

2. In p65: question 2, 4, 5

3. Why a doctor should know the growth curve of

bacteria?

4. In p76: 1, 3, 5, 10

Ref. “Medical microbiology” Published by Mosby

ed. MIMS, PLAYFAIR, ROITT WAKELIN, WILLIAMS

Review of Medical Microbiology and Immunology

-AccessMedicine: www.accessmedicine.com