1. Ch 6 Microbial Growth

2. Student Learning Outcomes:
Classify microbes into five groups on the basis of preferred temperature range.
 Explain the importance of osmotic pressure to microbial growth.
 Provide a use for each of the four elements (C, N, S, P) needed in large amounts for microbial growth.
Explain how microbes are classified on the basis of O2 needs.
Identify 2 ways in which aerobes avoid damage by toxic forms of O2.
Explain biofilms, describe their formation and their potential for causing infection.
Distinguish between chemically defined and complex media.
Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective and differential media.
Define colony and CFUs and describe how pure cultures can be isolated with streak plates.
Explain how microbes are preserved by deep-freezing and lyophilization.
Distinguish between binary fission and budding.
Define generation time and explain the bacterial growth curve.
Review some direct and indirect methods of measuring bacterial cell growth. 1

3. SLOs cont.: Check Your Understanding
How would one determine whether a microbe is a strict anaerobe?
If bacterial cells were given a sulfur source containing radioactive sulfur (35S) in their culture media, in what molecules would the 35S be found in the cells?

4. Microbial Growth
Microbial growth: Increase in cell number, not cell size!
Physical Requirements for Growth: Temperature
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature
Five groups based on optimum growth temperature
Psychrophiles
Psychrotrophs
Mesophiles
Thermophiles
Hyperthermophiles
Fig 6.1

5. Figs 6.2/3: Food preservation temperatures and Effect of amount of food on its cooling rate

6. Physical Requirements for Growth: pH and Osmotic Pressure
Most bacteria, the so-called ______________ , grow best between pH 6.5 and 7.5.
Some bacteria prefer a pH range 1 to 5. These are the __________________ What bacterium lives in the stomach?
Some bacteria do well in hypertonic (?) environments:
Obligate vs. facultative __________________

7. What happens if bacterium is not salt tolerant?
Fig 6.4

8. Chemical Requirements for Growth: C, N, S, P, etc.
Carbon
 Half of dry weight
Chemoheterotrophs use organic carbon sources
Nitrogen and Sulfur
Found in ?
Needed for ?
S also in thiamine and biotin
Phosphorus: Phosphate ions (PO43–)
Also needed K, Mg, Ca, trace elements (as cofactors), and organic growth factors
Vit B1
Vit B7

9. Chemical Requirements for Growth: Oxygen
O2 requirements vary greatly
Table 6.1: The Effects of Oxygen on the Growth of Various Types of Bacteria

10. Toxic Forms of Oxygen 2 · ·
Superoxide free radicals (also known as superoxide anions)
Peroxide anion: O22– 2

11. Fig 6.5
Biofilms
Microbial communities form slime or hydrogels and share nutrients
1st step: Attachment of planctonic bacteria to surface structures.
Bacteria in biofilm are sheltered from harmful factors (disinfectants etc.)
Cause of most nosocomial infections, i.e.: : Indwelling catheters
Bacteria communicate by chemicals via quorum sensing “The secret social lives of bacteria” TED talk by Bonnie Bassler

12. Culture Media Culture medium: Nutrients prepared for microbial growth
Have to be sterile (?)
Inoculum: Introduction of microbes into medium
Culture: Microbes growing in/on culture medium
Chemically defined media: Exact chemical compo- sition is known (for research purposes only)
Complex media: Extracts and digests of yeasts, meat, or plants, e.g.:
Nutrient broth
Nutrient agar
Blood agar

13. Agar Complex polysaccharide
Used as solidifying agent for culture media in Petri plates, slants, and deeps
Generally not metabo- lized by microbes
Liquefies at 100°C
Solidifies ~40°C

14. Anaerobic Culture Methods
Reducing media contain chemicals (e.g.: thioglycollate) that combine with O2 . Keep in tightly capped test tubes.
Novel method in clinical labs for Petri plates: Add oxyrase to growth media  OxyPlate
Anaerobic jars
Anaerobic chambers
Figs 6.6 & 6.7

15. Capnophiles
Aerobic bacteria grow better in high ____________ and low ______________
In human body, these conditions found in
__________________
E.g: Campylobacter jejuni
Use candle jar, CO2- generator packets, or CO2 incubators
Candle jar

16. Biosafety Levels Biosafety Level 3
Biosafety Level 4

17. Selective and Differential Media
Selective media: Additives suppress unwanted and encourage desired microbes – e.g. EMB and mannitol salt agar etc.
Differential media: changed in recognizable manner by some bacteria  Make it easy to distinguish colonies of different microbes.
MacConkey agar
EMB
Mannitol salt agar
 and  hemolysis on blood agar
Compare to Fig 6.9

18. EMB
MacConkey

19. MSA
Compare to Fig 6.10

20. Pure Cultures Contain only one species or strain.
Most patient specimens and environmental samples contain several different kinds of bacteria
Streak-plate method is commonly used. More details in lab.
Colony formation: A population of cells arising from a single cell or spore or from a group of attached cells (also referred to as CFU).
Only ~1% of all bacteria can be successfully cultured
Aseptic technique critical!
Compare to Fig. 6.11

21. Preserving Bacterial Cultures
Deep-freezing: Rapid cooling of pure culture in suspension liquid to –50°to –95°C. Good for several years.
Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum. Good for many years.

22. The Growth of Microbial Cultures
Binary fission – exponential growth
Budding
Generation time – time required for cell to divide (also known as doubling time)
Ranges from 20 min (________) to > 24h (_____________)
Fig 6.12a

23. Consider reproductive potential of E. coli!
Fig 6.13
Type of curve?
Fig 6.14

24. Bacterial Growth Curve
Foundation Fig 6.15
Illustrates the dynamics of growth
Phases of growth
________ phase
Exponential or logarithmic (_____) phase
_______________ phase
_____________ phase
Compare growth in liquid and on solid media

25. Direct Measurements of Microbial Growth
Viable cell counts: Plate counts: Serial dilutions put on plates CFUs form colonies
Fig 6.16

26. Fig 6.17
Figure 6.15, step 1

27. Additional Direct Counts
Direct microscopic count: Counting chambers (slides) for microscope
Filtration method of choice for low counts
Fig 6.18

28. Indirect Count: Spectrophotometry
Fig 6.21
Measures turbidity
OD (Absorbance) is function of cell number

29. Clinical Case: Glowing in the Dark
In the Clinic
H2O2 and wound care
Clinical Case: Glowing in the Dark
The End