1. Ch 6 & 7 - Microbial Growth and Control
Stephanie Lanoue

2. Requirements for Growth
Learning Objectives 6-1 Classify microbes into five groups on the basis of preferred temperature range. 6-2 Identify how and why the pH of culture media is controlled. 6-3 Explain the importance of osmotic pressure to microbial growth.

3. Environmental factors For Growth (Overview)
Physical requirements
__________________
_____
Osmotic pressure
Chemical Requirements
CHONPKS
Trace elements-Fe, Cu, Zn, Mb
Organic growth factors

4. Physical Requirements, Temperature
Minimum growth temperature
The _________ temperature at which the species will grow
Optimum growth temperature
Species grow _______
Maximum growth temperature
The __________ temperature at which growth is possible

5. Physical Requirements, Temperature
Temperature (optimal enzyme operation)
Psychrophiles, _____-loving microbes (-10oC to 20o C)
Mesophiles, __________ temperature-loving microbes (10o C to 50o C)
Pathogens
Thermophiles, _____-loving microbes, (40o C to 70o C)
Extreme thermophiles (65o C to 110o C)

6. Physical Requirements, pH
Most bacteria grow between pH ______ and 7.5 (best pH for most bacteria)
Molds and yeasts grow between pH ____ and 6
Acidophiles grow in ______ environments pH
Ex. Lactobacillus acidophilus, H. pylori
Alkalophiles pH
Ex. Bacillus alcalophilus

7. Physical Requirements, Osmotic Pressure (OP)
Bacteria are better adapted to _____ OP
High OP remove water from a cell
Environment is hypertonic
High ______ or sugar concentration in environment (solute)
Plasmolysis occurs at high OP
Shrinkage of cells
Figure 6.4

8. Cell in isotonic solution. Under these
Figure 6.4 Plasmolysis.
Plasma
membrane
Plasma
membrane
Cell wall
H2O
Cytoplasm
Cytoplasm
NaCl 0.85%
NaCl 10%
Cell in isotonic solution. Under these
Plasmolyzed cell in hypertonic solution.
conditions, the solute concentration in the
cell is equivalent to a solute concentration
of 0.85% sodium chloride (NaCl).
If the concentration of solutes such as NaCl
is higher in the surrounding medium than in
the cell (the environment is hypertonic), water
tends to leave the cell. Growth of the cell
is inhibited.

9. Requirements for Growth
Learning Objectives 6-4 Name a use for each of the four elements (carbon, nitrogen, sulfur, and phosphorus) needed in large amounts for microbial growth. 6-5 Explain how microbes are classified on the basis of oxygen requirements. 6-6 Identify ways in which aerobes avoid damage by toxic forms of oxygen.

10. Chemical Requirements
Carbon
Structure of ________ molecules
Most important requirements for microbial growth
Nitrogen, sulfur, phosphorous
To synthesize cellular material such as _____ and protein
trace elements, small amount of other chemical
such as, zinc
Function of enzymes as cofactors
We have talked about cofactors before in a previous chapter. What
is a cofactor?

11. Chemical Requirements, Oxygen
Obligate aerobes—_______ oxygen
Facultative anaerobes—grow via fermentation or anaerobic respiration when oxygen is available
Obligate anaerobes—grow occurs where there is ___ oxygen

12. Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

13. Chemical Requirements, Organic Growth Factors
Organic compounds obtained from the ____________
Vitamins, amino acids, purines, and pyrimidines

14. Culture Media
Learning Objectives 6-8 Distinguish chemically defined and complex media. 6-9 Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective and differential media, enrichment medium Differentiate biosafety levels 1, 2, 3, and 4.

15. Culture Media Agar __________ polysaccharide
Used as a solidifying agent for culture media in Petri plates, slants, and deeps
Generally ____ metabolized by microbes
Liquefies at 100C
Solidifies at ~40C

16. Culture Media

17. Culture Media
Culture medium: nutrients prepared for microbial __________
Sterile: ___ living microbes
Inoculum: ___________ of microbes into a medium
Culture: microbes growing in or on a culture medium

18. Culture Media
Chemically defined media: exact chemical composition is ________
Complex media: extracts and digests of yeasts, meat, or plants; chemical composition varies batch to batch
Nutrient _______
Nutrient agar

19. Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli

20. Special Culture Techniques
Biosafety levels
BSL-1: __ special precautions; basic teaching labs
BSL-2: lab coat, gloves, eye protection
BSL-3: biosafety cabinets to prevent airborne transmission
BSL-4: sealed, negative pressure; "hot zone"
Exhaust air is filtered twice through HEPA filters

21. Media Types Selective, suppress growth of _________ bacteria
Contain inhibitors to suppress growth
Mannitol _______ agar : selective for halophiles with 7% salt (osmotic challenge) and differential for mannitol fermenters. Selects for Gram(+) and inhibits Gram(-) bacteria.
Eosin Methylene Blue Agar (EMB) Agar: kills Gram(+) with eosin and methylene blue, selective for Gram(-). Differential for lactose fermenters.
McConkey Agar: suppresses Gram(+) with crystal violet and bile salts and selects Gram(-). Differential for lactose fermenters.

22. Media Types (cont’d)
Differential - distinguishes between ________ species (different microbes on the same plate)
Blood agar
(sheep’s blood) reveals if hemolytic

23. Media Types (cont’d)
Enrichment - __________ nutrients to favor growth of special bacteria
Lysed red blood cells provide unique nutrients in blood/chocolate agar

24. Obtaining Pure Cultures
Learning Objectives 6-11 Define colony Describe how pure cultures can be isolated by using the streak plate method.

25. Pure Cultures • A pure culture contains only ____ species or strain
• A colony is a ___________ 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

26. The Growth of Bacterial Cultures
Learning Objectives 6-14 Define bacterial growth, including binary fission Compare the phases of microbial growth, and describe their relation to generation time.

27. Bacterial Growth Refers to bacterial __________, not the cell size!
Generation time, time required for cell ________ (1 hr to 3hr, for some 24hr)
Growth Curve: Lag, Log, Stationary, Death
Quantifying Growth
If time 20 min, in 30 generation or 10 hr, population 1 billion

28. Bacterial Division, _______ fission

29. Generation Time Time required for a cell to ______
20 minutes to 24 hours
Binary fission _______ the number of cells each generation
Total number of cells = 2number of generations
Growth curves are represented logarithmically

30. Figure 6.13a Cell division.

31. Figure 6.13b Cell division.

32. Phases of Growth (In order)
Log phase
Stationary phase
Death phase

33. Figure 6.15 Understanding the Bacterial Growth Curve.

34. The Growth of Bacterial Cultures
Learning Objectives 6-16 Explain four direct methods of measuring cell growth Differentiate direct and indirect methods of measuring cell growth.

35. Measuring Bacterial Growth
Direct measurement
Plate _______ of viable bacterial forming colonies
Direct microscopic count

36. Counts are performed on bacteria mixed into a dish with agar (pour plate method) or spread on the surface of a plate (spread plate method)
After incubation, count colonies on plates that have colonies (CFUs)

37. Plate counts Figure 6.17 Methods of preparing plates for plate counts.
The pour plate method
The pour plate method
The spread plate method
The spread plate method
1.0 or 0.1 ml
0.1 ml
Inoculate
empty plate.
Inoculate plate
containing
solid medium.
Bacterial
dilution
Spread inoculum
over surface
evenly.
Add melted
nutrient agar.
Swirl to mix.
Colonies grow
only on surface
of medium.
Colonies
grow on and
in solidified
medium.
Plate counts

38. Direct Microscopic Count
Volume of a bacterial suspension placed on a ________
Average _______ of bacteria per viewing field is calculated
Uses a special Petroff-Hausser cell counter
Number of bacteria/ml =
Number of cells counted
Volume of area counted

39. Grid with 25 large squares
Figure Direct microscopic count of bacteria with a Petroff-Hausser cell counter.
Grid with 25 large squares
Cover glass
Slide
Bacterial suspension is added here
and fills the shallow volume over the
squares by capillary action.
Bacterial
suspension
Microscopic count: All cells in
several large squares are
counted, and the numbers are
averaged. The large square
shown here has 14 bacterial cells.
Cover glass
Slide
Location of squares
The volume of fluid over the
large square is 1/1,250,000
of a milliliter. If it contains 14
cells, as shown here, then
there are 14 × 1,250,000 =
17,500,000 cells in a milliliter.
Cross section of a cell counter.
The depth under the cover glass and the area
of the squares are known, so the volume of the
bacterial suspension over the squares can be
calculated (depth × area).

40. Measuring by Indirect Methods
Example: Turbidity—measurement of ___________ with a spectrophotometer
More turbid = More bacteria
Light source
Spectrophotometer
Light
Blank
Light-sensitive
detector
Scattered light
that does not
reach detector
Bacterial suspension