Chapter 6

Microbial Growth

• Exercise 9: Aseptic Technique

• Exercise 10: Pure Culture Technique

• Turn in cultures from home for incubation• Finish microscope worksheet if necessary

Lab 2 Goals and Objectives :

Lecture: Chapter 6 (Microbial Growth)Learn aseptic technique and pure culture isolation

• Microbiology is the study of life and organisms that are too small to be seen with the naked eye.

• Microbial growth - an increase in the number of cells in a population , not increase in size.

1. Culture Medium: Nutrients prepared for microbial growth

•Sterile: No living microbes

2. Inoculation: Introduction of microbes into medium

Inoculum: The material used in an inoculation

3. Culture: Microbes growing in/on culture medium

Growing microorganisms

• Nutrient broth- liquid form • Nutrient agar- solid form

– Agar - complex polysaccharide

• Used as solidifying agent for culture media in Petri plates, slants, and deeps

• Generally not metabolized by microbes

• Liquefies at 100°C

• Solidifies ~40°C

Culture Media

Reproduction of Prokaryotes - Binary fission

Bacterial growth in culture• Bacteria multiply by binary fission• The population grows in geometric progression• 12• 2 4• 3 8• 4 16• 5 32• 6 64• 7 128• 8 256• 9 518• 10 1036• 11 2064• 12 4128• 13 8256• ………

-Lag phase: initial period of little to no cell division as bacteriaacclimate to new media-Log phase: period of exponential growth with a constant generationtime-Stationary phase: cell growth is equal to cell death-Death phase: cell death exceeds cell growth

Number of generation

Generation time

• Generation time - the time required for a cell to divide, to undergo one round of binary fission– If 100 cells growing for 5 hours produced 1,720,320 cells:

• E. coli has a generation time of 20 min• Common bacterial generation times range 1-3 hrs

Quantifying Microbial Growth

• Direct Measurements– Plate Counts– Filtration– Most Probable Number (MPN)– Direct Microscopic Count

• Indirect Estimations– Turbidity– Metabolic Activity– Dry Weight

Microbial Growth• The requirements for microbial growth

• Physical – Temperature– pH– Osmotic pressure

• Chemical• Oxygen• Carbon• Nitrogen• Sulfur• Phosphorus• Trace elements• Organic factors

Physical Requirements -Temperature

– Minimum growth temperature • the lowest temperature at which the species will grow

– Optimum growth temperature• species grow best

– Maximum growth temperature• the highest temperature at which growth is possible.

Physical Requirements - pH

• Most bacteria grow between pH 6.5 and 7.5

• Molds and yeasts grow between pH 5 and 6

• Acidophiles grow in acidic environments

Physical Requirements - Osmotic Pressure• Osmotic pressure is the hydrostatic pressure produced by a difference in

concentration between solutions on the two sides of a surface such as a semipermeable membrane.

• An isotonic environment for a cell is created when the solution outside of the cell is isotonic (having equal accent )with the cytoplasm of the cell.

• Hypotonic environment - the solution outside of the cell is hypotonic in comparison to the cytoplasm of the cell. – cause cell lyses for some cells and nothing to bacteria, because of cell wall .

• Hypertonic environments - hypertonic solution has more dissolved solute than the cytoplasm of the cell ( increase salt or sugar) – cause plasmolysis ( shrink the cells )

– Facultative halophiles tolerate high osmotic pressure – Extreme or obligate halophiles require high osmotic pressure

Figure 4.18 - Overview

• Carbon– Structural organic molecules, energy source– Autotrophs use CO2

– Chemoheterotrophs use organic carbon sources• Nitrogen

– In amino acids, proteins– Most bacteria decompose proteins– Some bacteria use NH4

+ or NO3

– A few bacteria use N2 in nitrogen fixation• Sulfur

– In amino acids, thiamine, biotin– Most bacteria decompose proteins– Some bacteria use SO4

2 or H2S• Phosphorus

– In DNA, RNA, ATP, and membranes– PO4

3 is a source of phosphorus• Inorganic elements required in small amounts- potassium, magnesium and

calcium• Trace Elements - iron, copper, molybdenum and zinc - Usually as enzyme

cofactors

Chemical Requirements

• Organic Growth Factors

– Essential organic compounds an organism is unable to synthesize

– Organic compounds obtained from the environment

– Vitamins, amino acids, purines, pyrimidines

Chemical Requirements

Chemical Requirements - Oxygen (O2)

• Singlet oxygen: O2 boosted to a higher-energy state• Superoxide free radicals: O2

• Peroxide anion: O22

• Hydroxyl radical (OH)

Toxic Forms of Oxygen

Clumps of bacteria adhering to surface

Surface

Water currents

Migrating clump of bacteria

Biofilms• Microbial communities

• Form slime or hydrogels

– Bacteria attracted by chemicals via quorum sensing

Culture Media

• Complex Media:

Extracts and digests of yeasts, meat, or plants

• Chemically Defined Media:

Exact chemical composition is known

• Differential Media:

– Make it easy to distinguish colonies of different microbes.

• Enrichment Media

Encourages growth of desired microbe

• Reducing media for anaerobic culture,

Contain chemicals (thioglycollate or oxyrase) that combine O2

Heated to drive off O2

Figure 6.9b

Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.

Lid with O-ring gasket

Envelope containing sodium bicarbonate and sodium borohydride

Anaerobic indicator (methylene blue)

Petri plates

Clamp with clamp screw

Palladium catalyst pellets

• A pure culture contains only one species or strain

• 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)

• The streak plate method is used to isolate pure cultures

Obtaining Pure Cultures

• Exercise 9: Aseptic Technique• Each person make 3 inoculations:

1. Broth to broth - Escherichia coli - 37°C 2. Slant to slant - E. coli - 30°C3. Plate to slant - Serratia marcescens - 30°C (changed)

• Exercise 10: Pure Culture Technique– Each person make 2 streak plates: Quadrant Streak Method B,

page 82.– Incubate at 25°C

• Turn in cultures from home for incubation• Finish microscope worksheet if necessary

Streak Plate

Figure 6.10a, b

Flame the loop, cool it

Flame the loop,cool it

Flame the loop,cool it

• Exercise 9: Aseptic Technique• Each person make 3 inoculations:

1. Broth to broth - E. coli - 37°C 2. Slant to slant - E. coli - 37°C3. Plate to slant - S. marcescens -

30°C (changed)

• Exercise 10: Pure Culture Technique– Each person make 2 streak

plates– Quadrant Streak Method B,

page 85.– Incubate plates at 25°C

Each pair will need:1 broth culture Escherichia coli, 1 slant culture Escherichia coli1 plate culture Serratia marcescensEach person will need:1 Nutrient Broth/BHI tubes, 2 Nutrient Agar/BHIA slants

Each pair will need:1 mixed culture (which contains: Escherichia coli, Serratiamarcescens and Micrococcus luteus)Each person will need:2 Nutrient Agar/BHIA plates

Get help now, today, if you arehaving any difficulty with oilimmersion lens use orspecimen measurements usingthe ocular micrometer!!!