1. Bacterial Growth drh. Maxs U.E. Sanam, M.Sc.
Fakultas Kedokteran Hewan UNDANA

2. Growth of Bacteria How do bacteria grow?
                
Growth of Bacteria
How do bacteria grow?
Measured as increase in cell number not size.
Transverse binary fission or
Binary Fission

3. Goals for Chapter What bacterial cells need to grow?
How do we measure cell growth?
What are the stages of cell growth?
What is happening in cells during cell growth?
What are the effects of environmental conditions on cell growth?

4. Cell division What is happening? Figure: 06-01 Caption:
The general process of binary fission in a rod-shaped prokaryote. For simplicity, the nucleoid is depicted as a single circle in green.

5. Microbial Growth Physical Requirements of Microbes
Temperature (optimal enzyme operation)
Psychrophiles, mesophiles, thermophiles
pH (optimal enzyme operation)
Using buffers in media
Molds & yeasts versus bacteria
Chemical Requirements
Carbon source in medium
Nitrogen, sulfur, phosphorous, trace elements
Oxygen requirements
Obligate aerobes, anaerobes, facultative anaerobes
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes

6. Culture Media for Microbes
Chemically defined vs. complex media
Anaerobes: reducing media/Brewer jar
Other: animals, eggs, tissue culture, CO2
Media types
Selective, Differential, Enrichment
Bacterial Population Growth
Growth Curve: Lag, Log, Stationary, Death
Quantifying Growth

7. Characterizing Microbes By Optimal Growth Temperature
Figure 6.1

8. Temperature Growth Ranges and Food Safety
“ ”
If > 2 hrs at oF, don’t eat it!
Figure 6.2

9. Effects of Temperature on Growth
Most of our plates are incubated at 37oC (98.6oF).
Conversion C to F = 1.8 x C + 32
From the Virtual Microbiology Classroom on ScienceProfOnline.com

10. Physical Requirements: pH
Acidophiles
Neutrophiles
Alkalophiles
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6

11. Physical Requirements: Osmotic Pressure
Hypertonic environments, increase salt or sugar, cause plasmolysis
Extreme or obligate halophiles require high osmotic pressure
Facultative halophiles tolerate high osmotic pressure

12. Microbial Growth Physical Requirements of Microbes
Temperature (optimal enzyme operation)
Psychrophiles, mesophiles, thermophiles
pH (optimal enzyme operation)
Using buffers in media
Molds & yeasts versus bacteria
Chemical Requirements
Carbon source in medium
Nitrogen, sulfur, phosphorous, trace elements
Oxygen requirements
Obligate aerobes, anaerobes, facultative anaerobes
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes

13. Culture Media for Microbes
Chemically defined vs. complex media
Anaerobes: reducing media/Brewer jar
Other: animals, eggs, tissue culture, CO2
Media types
Selective, Differential, Enrichment
Bacterial Population Growth
Growth Curve: Lag, Log, Stationary, Death
Quantifying Growth

14. The Requirements for Growth: Chemical Requirements
Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs use CO2

15. The Requirements for Growth: Chemical Requirements
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
Trace Elements
Inorganic elements required in small amounts
Usually as enzyme cofactors

16. Microbial Growth Physical Requirements of Microbes
Temperature (optimal enzyme operation)
Psychrophiles, mesophiles, thermophiles
pH (optimal enzyme operation)
Using buffers in media
Molds & yeasts versus bacteria
Chemical Requirements
Carbon source in medium
Nitrogen, sulfur, phosphorous, trace elements
Oxygen requirements
Obligate aerobes, anaerobes, facultative anaerobes
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes

17. Culture Media for Microbes
Chemically defined vs. complex media
Anaerobes: reducing media/Brewer jar
Other: animals, eggs, tissue culture, CO2
Media types
Selective, Differential, Enrichment
Bacterial Population Growth
Growth Curve: Lag, Log, Stationary, Death
Quantifying Growth

18. How Toxic Forms of Oxygen Are Handled
Singlet oxygen: O2 boosted to a higher-energy state
Handling superoxide free radicals: O2 2O2- + 2O2- + 8H  4H2O2
oxygen radicals hydrogen peroxide
Superoxide Dismutase (SODS)
Handling peroxide anion: O22
2H2O  H2O O2
hydrogen peroxide water oxygen gas
Catalase (Peroxidase)
Catalase Test: Bacteria + H2O2 bubbles

19. Growth in thioglycollate broth reveals oxygen preferences
Obligate aerobes
Faultative anaerobes
Obligate anaerobes
Aerotolerant anaerobes
Microaerophiles
Oxygen gradient
Resazurin dye is red in the presence of oxygen
Thyoglycollate binds molecular oxygen, reducing it and removing it: R-SH + O2  R-SO2

20. Microbial Growth Physical Requirements of Microbes
Temperature (optimal enzyme operation)
Psychrophiles, mesophiles, thermophiles
pH (optimal enzyme operation)
Using buffers in media
Molds & yeasts versus bacteria
Chemical Requirements
Carbon source in medium
Nitrogen, sulfur, phosphorous, trace elements
Oxygen requirements
Obligate aerobes, anaerobes, facultative anaerobes
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes

21. Culture Media for Microbes
Chemically defined vs. complex media
Anaerobes: reducing media/Brewer jar
Other: animals, eggs, tissue culture, CO2
Media types
Selective, Differential, Enrichment
Bacterial Population Growth
Growth Curve: Lag, Log, Stationary, Death
Quantifying Growth

22. Culture Media: Chemically Defined or Complex
Table 6.2 & 6.4

23. Anaerobic and Low O2 Culture Methods
Candle jar
Brewer or anaerobic jar
CO2 packet

24. Unusual Culture Methods
Grows only in certain cell types: using armadillos to culture M. leprae
Grows only inside live cells: eggs as culture vessels for influenza virus
Grows only in certain cell types: using tissue culture with low O2, enriched CO2 incubators

25. Microbial Growth Physical Requirements of Microbes
Temperature (optimal enzyme operation)
Psychrophiles, mesophiles, thermophiles
pH (optimal enzyme operation)
Using buffers in media
Molds & yeasts versus bacteria
Chemical Requirements
Carbon source in medium
Nitrogen, sulfur, phosphorous, trace elements
Oxygen requirements
Obligate aerobes, anaerobes, facultative anaerobes
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
Chemically defined vs. complex media
Anaerobes: reducing media/Brewer jar
Other: animals, eggs, tissue culture, CO2
Media types
Selective, Differential, Enrichment
Bacterial Population Growth
Growth Curve: Lag, Log, Stationary, Death
Quantifying Growth

26. Nutrienth broth
Nutrienth agar

27. Selective Media
Goal: To chemically (or physically) suppress unwanted microbes and encourage desired microbes.
MSA
Mannitol salt agar : selective for halophiles with 7% salt (osmotic challenge) and differential for mannitol fermenters: good for skin bacterial cultures.
EMB Agar: kills gram positives with eosin and methylene blue, selective for gram negatives. Differential for lactose fermenters. Good for growing enterics.
McConkey Agar: supresses gram positives with crystal violet and bile salts; also differential for
EMB MA
Figure 6.9b, c

28. Differential Media
Distinguish between different species based on a metabolic ability.
Blood agar
(sheep’s blood) reveals if hemolytic
Mannitol salt agar contains the pH sensitive dye phenol red (yellow when acidic)
Se Sa
Figure 6.9a

29. Hemolyses of Streptococcus spp. (left) α-hemolysis (S
Hemolyses of Streptococcus spp. (left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes); (right) γ-hemolysis (= non-hemolytic, S. salivarius)

30. On the left is a positive Staphylococcus infection, on the right a positive Streptococcus culture.

31. TSI Agar

32. Enrichment Media
Encourages growth of desired microbe by providing special growth conditions or added growth factors
Thioglycollate
Anaerobic or Brewer Jar
Lysed red blood cells provide unique nutrients in blood/chocolate agar
Glucose Salts Agar (enriches for microbes that can growth only on glucose and some inorganic nutrients

33. Pure Cultures Used To Study Characteristics of a Particular Species
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)

34. Microbial Growth Physical Requirements of Microbes
Temperature (optimal enzyme operation)
Psychrophiles, mesophiles, thermophiles
pH (optimal enzyme operation)
Using buffers in media
Molds & yeasts versus bacteria
Chemical Requirements
Carbon source in medium
Nitrogen, sulfur, phosphorous, trace elements
Oxygen requirements
Obligate aerobes, anaerobes, facultative anaerobes
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes

35. Culture Media for Microbes
Chemically defined vs. complex media
Anaerobes: reducing media/Brewer jar
Other: animals, eggs, tissue culture, CO2
Media types
Selective, Differential, Enrichment
Bacterial Population Growth
Growth Curve: Lag, Log, Stationary, Death
Quantifying Growth

36. Bacterial Growth is Exponential (Logarithmic)
Bacterial “growth” means an increase in the number
of individuals, not an increase in cell size.
Figure 6.12b

37. Generation Cell
Number Count
0 1
1 2
2 4
3 8
4 16
5 32
,024
,048,576

38. Growth Curve for Bacteria (Logarithmic Plot)
Figure 6.14

39. Estimating Bacterial Numbers by Indirect methods
Direct Measures
Plate counts of viable bacterial forming colonies
Counting low viable bacterial numbers by filtration
Counting viable bacteria with Most Probable Number
Counting bacteria per ml in direct microscopy
Indirect Measures
Turbidity/Absorbance with a spectrophotometer
Metabolic activity tracking conversion of colored molecules
Dry weight by weighing a set volume and knowing weight of one cell

40. Plate Assays: Spread Plate or Pour Plate Methods
After incubation, count colonies on plates that have colonies (CFUs)
The dilution in a particular tube = ml of fluid added to tube/total volume after addition; e.g. 1ml/(9ml + 1ml) = 1/10 = 10-2
Figure 6.15

41. Direct Measurements of Microbial Growth
Figure 6.19

42. Direct Measurements of Microbial Growth
Filtration: Good for measuring very dilute samples of bacteria
Figure 6.17a, b

43. Direct Measurements of Microbial Growth
Multiple tube MPN test
Count positive tubes and compare to statistical MPN table
Produces a range of concentrations
Figure 6.18b

44. Estimating Bacterial Numbers by Indirect methods
Direct Measures
Plate counts of viable bacterial forming colonies
Counting low viable bacterial numbers by filtration
Counting viable bacteria with Most Probable Number
Counting bacteria per ml in direct microscopy
Indirect Measures
Turbidity/Absorbance with a spectrophotometer
Metabolic activity tracking conversion of colored molecules/enyzme assay
Dry weight by weighing a set volume and knowing weight of one cell

45. Estimating Bacterial Numbers by Indirect Methods
Turbidity
Figure 620

46. Metabolic Conversion/Enzyme Assay
1 bacterium produces 4.6 x 1012 NADH/sec/cell under idea growth conditions.
In a 1 ml sample of growing cells, 5.2 x 1023 NADH/sec/ml are produced per second (as revealed by a color-based assay of NADH on the sample)
Therefore, (4.6 x 1012 NADH/sec/ml) x (5.2 x 1023 NADH/sec/cell) = 2.3 x 1024 cells/ml

47. Determining dry mass of a fixed volume
An E. coli cell has a dry mass of about 7.0 x mg.
A 1 ml sample with a dry mass of 2 mg therefore has:
2 mg/ml x 1 cell/7 x mg
= 2.8 x 1020 cells/ml