1. Chapter 6
Microbial Growth

2. Lab 2 Goals and Objectives :
Lecture: Chapter 6 (Microbial Growth)
Learn aseptic technique and pure culture isolation
Exercise 9: Aseptic Technique
Exercise 10: Pure Culture Technique
Turn in cultures from home for incubation
Finish microscope worksheet if necessary

3. 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.

4. Growing microorganisms
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

5. Culture Media 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

6. Reproduction of Prokaryotes - Binary fission

7. Bacterial growth in culture
Bacteria multiply by binary fission
The population grows in geometric progression
12
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 bacteria
acclimate to new media
-Log phase: period of exponential growth with a constant generation
time
-Stationary phase: cell growth is equal to cell death
-Death phase: cell death exceeds cell growth

8. Number of generation

9. 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

10. Quantifying Microbial Growth
Direct Measurements
Plate Counts
Filtration
Most Probable Number (MPN)
Direct Microscopic Count
Indirect Estimations
Turbidity
Metabolic Activity
Dry Weight

11. Microbial Growth The requirements for microbial growth Physical
Temperature pH
Osmotic pressure
Chemical
Oxygen
Carbon
Nitrogen
Sulfur
Phosphorus
Trace elements
Organic factors

12. 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.

13. 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

14. 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

15. Chemical Requirements
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
Some bacteria use SO42 or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43 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

16. 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

17. Chemical Requirements - Oxygen (O2)

18. Toxic Forms of Oxygen
Singlet oxygen: O2 boosted to a higher-energy state
Superoxide free radicals: O2
Peroxide anion: O22
Hydroxyl radical (OH)

19. Biofilms Microbial communities Form slime or hydrogels
Bacteria attracted by chemicals via quorum sensing
Clumps of bacteria adhering to surface
Migrating clump of bacteria
Surface
Water currents

20. 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

21. Envelope containing sodium bicarbonate and sodium borohydride
Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Clamp with clamp screw
Lid with
O-ring gasket
Envelope containing sodium bicarbonate and sodium borohydride
Palladium catalyst pellets
Anaerobic indicator (methylene blue)
Petri plates

22. Obtaining Pure Cultures
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

23. Exercise 9: Aseptic Technique
Each person make 3 inoculations:
Broth to broth - Escherichia coli - 37°C
Slant to slant - E. coli - 30°C
3. 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

24. Streak Plate Flame the loop, cool it Flame the loop, cool it
Figure 6.10a, b

25. Exercise 9: Aseptic Technique Each person make 3 inoculations:
Broth to broth - E. coli - 37°C
Slant to slant - E. coli - 37°C
3. 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 coli
1 plate culture Serratia marcescens
Each person will need:
1 Nutrient Broth/BHI tubes,
2 Nutrient Agar/BHIA slants
Each pair will need:
1 mixed culture (which contains: Escherichia coli, Serratia
marcescens and Micrococcus luteus)
Each person will need:
2 Nutrient Agar/BHIA plates
Get help now, today, if you are
having any difficulty with oil
immersion lens use or
specimen measurements using
the ocular micrometer!!!