1. Microbiology: Principles and Explorations
Jacquelyn G. Black
Microbiology: Principles and Explorations
Sixth Edition
Chapter 6:
Growth and Culturing of Bacteria
Copyright © 2005 by John Wiley & Sons, Inc.

2. Growth and Cell Division
Microbial Growth Defined:
Mother or parent cell doubles in size (asexual)
Divides into two daughter cells
if the cell prevented from division (by some types of antimicrobial agent) >>> static cell >>> autolysis
Microbial growth is defined not in
terms of cell size BUT as the increase in the number of cells, which occurs by cell division.
Growth in human differ from growth in m.o; in human increase in the size of the cell, but in m.o increase in the number of cells.

3. In fungi >>> sexual and asexual
yeast >>> budding (random, not organized division)

4. Cell Division
Binary fission –in bacteria- (equal cell division): A cell duplicates its components and divides into two cells. 1m.o give 2, 2m.o give 4, 4m.o give 8 (exponential growth).
Septum: is a partition that grows between the two daughter cells and they separate at this location. they may separate completely or not completely (if separation not completely that give the phenomena of arrangement; staphylococci, Streptococci…).
Budding التبرعم –in yeast- (unequal cell division): A small, new cell develops from surface of existing cell and subsequently separates from parent cell

5. Binary Fission

6. Thin section of the bacterium Staphylococcus, undergoing binary fission

8. Budding in Yeast

9. Phases of Growth
Consider a population of organisms –in the lab- introduced into a fresh, nutrient medium (nutrient is one of the needs of growth; source of carbon and nitrogen…) environmental and nutrition conditions must be optimum (PH, temp, oxygen) to grow.
Such organisms display four major phases of growth (typically)
The lag phase
The logarithmic phase
The stationary phase
The death phase

10. Practically, bring flask –sterile one- with growth media, then put in it bacteria –from pure culture- this process is (inoculation).
inoculum size: the initial no. of bacteria.
Inoculation: process include taking bacteria by inoculating loop from pure culture –kown by streaking- and putting it in water and nutrient (broth).
First the water will be clear but after a while there will be turbidity; particles in water (evidence of the presence of bacterial growth), (solution >>> suspension).
Solid media (agar) >>> colonies of bacteria
Liquid media (broth) >>> turbidity (bacterial suspension), diff. smell ,color change.

11. Growth curve

12. To know and identify the bacteria its important to draw a growth curve.
Growth curve: taking samples of bacteria -in specific points of time - to define (تحديد) the no. of bacteria –by special method- and plotting the time with no. on semi log paper; no. of bacteria on the log y-axis and the time on normal x-axis.

13. The Lag Phase Organisms do not increase significantly in number
They are metabolically active because they are adapting the exist environment.
Grow in size, synthesize enzymes, and incorporate molecules from medium
Produce large quantities of energy in the form of ATP

14. The Log Phase Organisms have adapted to a growth medium
The highest rate of growth. from this phase we calculate the growth rate or doubling time (specific for m.o). Usually minutes but in mycobacterium because of the waxy layer it has long doubling time (PCR are used instead of agar to diagnose it).
Growth (doubling or binary fission) occurs at an exponential (log) rate. (exponential phase)
The organisms divide at their most rapid rate
During this phase the m.o is consuming nutrient and give waste –it’s accumulating if there isn’t sink conditions- so accumulating of toxins .
But if there is sink condition >> no accumulating of toxins >> continues growth.
a regular, genetically determined interval (generation time)

15. Synchronous growth: A hypothetical situation in which the number of cells in a culture would increase in a stair-step pattern, dividing together at the same rate (not available in the nature, but exist in theory)
Nonsynchronous growth: A natural situation in which an actual culture has cell dividing at one rate and other cells dividing at a slightly slower rate (naturally happened)

16. Microbes growing continuously in a chemostat

17. Stationary Phase:
Cell division decreases to a point that new cells are produced at same rate as old cell die.
The number of live cells stays constant. number of new bacteria is the same of which die.
Decline (Death) Phase:
Condition in the medium become less and less
supportive of cell division
Cell lose their ability to divide and thus die
Number of live cells decreases at a logarithmic rate

18. Serial Dilution and Standard Plate Counts
Standard plate count: One method of measuring bacterial growth
Agar plate: A petri dish containing a nutrient medium solidified with agar
Serial dilutions are used to dilute the original bacterial culture before you transfer known volume of culture onto agar plate

19. Serial Dilution

20. Calculation of the number of bacteria per milliliter of culture using serial dilution
Pour plate: made by first adding 1.0ml of diluted culture to 9ml of molten agar
Spread plate: made by adding 0.1ml of diluted culture to surface of solid medium

23. Counting colonies using a bacterial colony counter

24. Bacterial colonies viewed through the magnifying glass against a colony-counting grid

25. Countable number of colonies (30 to 300 per plate)
Which of these plates would be the correct one to count? Why?

26. Direct Microscopic Counts
Another way to measure bacterial growth
Petroff-Hausser counting chamber
Bacterial suspension is introduced onto chamber with a calibrated pipette
Microorganisms are counted in specific calibrated areas
Number per unit volume is calculated using an appropriate formula

27. The Petroff-Hausser Counting Chamber

28. Most Probable Number (MPN)
Method to estimate number of cells
Used when samples contain too few organisms to give reliable measures of population size by standard plate count
Series of progressively greater dilutions
Typical MPN test consists of five tubes of each of three volumes (e.g. 10, 1, and 0.1ml)

29. A MPN test: those tubes in which gas bubbles are visible (labeled +) contain organisms

30. Positive carbohydrate fermentation test
+ Gas/+Acid
+ Acid
- No Acid or Gas

31. Turbidity, or a cloudy appearance, is an indicator of bacterial growth in urine in the tube on the left

32. A Spectrophotometer: This instrument can be used to measure bacterial growth by determining the degree of light transmission through the culture

33. Factors Affecting Bacterial Growth
The kinds of organisms found in a given environment and the rates at which they grow can be influenced by a variety of factors, both physical and biochemical
Physical factors include: pH, temperature, oxygen concentration, moisture, hydrostatic pressure, osmotic pressure, and radiation
Nutritional factors include: availability of carbon, nitrogen, sulfur, phosphorus, trace elements and, in some cases, vitamins

34. pH
Optimum pH: the pH at which the microorganism grows best (e.g. pH 7)
According to their tolerance for acidity/alkalinity, bacteria are classified as:
1. Acidophiles (acid-loving): grow best at pH
2. Neutrophiles: grow best at pH 5.4 to 8.0
3. Alkaliphiles (base-loving): grow best at pH

35. Temperature
Obligate: organism must have specified environmental condition
Facultative: organism is able to adjust to and tolerate environmental condition, but can also live in other conditions
According to their growth temperature range, bacteria can be classified as:
psychrophiles: oC
Mesophiles: oC
Thermophiles: 50-60oC

36. Thermophiles: Thermophilic sulfur bacteria can live and grow in the runoff waters from such geysers despite the near-boiling temperatures

37. Growth rates of psychrophilic, mesophilic, and thermophilic bacteria

38. Oxygen Aerobes: require oxygen to grow
Obligate aerobes: must have free oxygen for aerobic respiration (e.g. Pseudomonas)
Anaerobes: do not require oxygen to grow
Obligate anaerobes: killed by free oxygen (e.g. Bacteroides)
Microaerophiles: grow best in presence of small amount of free oxygen
Capnophiles: carbon-dioxide loving organisms that thrive under conditions of low oxygen
Facultative anaerobes: carry on aerobic metabolism when oxygen is present, but shift to anaerobic metabolism when oxygen is absent
Aerotolerant anaerobes: can survive in the presence of oxygen but do not use it in their metabolism

39. Patterns of Oxygen Use

40. Hydrostatic Pressure
Water in oceans and lakes exerts pressure exerted by standing water, in proportion to its depth
Pressure doubles with every 10 meter increase in depth
Barophiles: bacteria that live at high pressures, but die if left in laboratory at standard atmospheric pressure

41. Osmotic Pressure
Environments that contain dissolved substances exert osmotic pressure, and pressure can exceed that exerted by dissolved substances in cells
Hyperosmotic environments: cells lose water and undergo plasmolysis (shrinking of cell)
Hypoosmotic environment: cells gain water and swell and burst

42. Halophiles
Salt-loving organisms which require moderate to large quantities of salt (sodium chloride)
Membrane transport systems actively transport sodium ions out of cells and concentrate potassium ions inside
Why do halophiles require sodium?
Cells need sodium to maintain a high intracellular potassium concentration for enzymatic function
Cells need sodium to maintain the integrity of their cell walls

43. Responses to Salt

44. The Great Salt Lake in Utah

45. Nutritional Factors Carbon sources Nitrogen sources
Sulfur and phosphorus
Trace elements (e.g. copper, iron, zinc, and cobalt)
Vitamins (e.g. folic acid, vitamin B-12, vitamin K)

46. A USDA scientist working on his microbial brew – a mix of some 80 ingredients to support growth of nutritionally fastidious spiroplasmas

47. responsible for hundreds of crop and animal diseases
Spiroplasma spp.
responsible for hundreds of crop and animal diseases

48. Locations of Enzymes
Exoenzymes: production of enzymes that are released through cell or plasma membrane
Extracellular enzymes: usually produced by gram-positive rods, which act in the medium around the organism
Periplasmic enzymes: usually produced by gram-negative organisms, which act in the periplasmic space

49. Sporulation
The formation of endospores, occurs in Bacillus, Clostridium and a few other gram-positive genera
Protective or survival mechanism, not a means of reproduction
As endospore formation begins, DNA is replicated and forms a long, compact, axial nucleoid

50. Core (living part of endospore): most of cell’s RNA and some cytoplasmic protein molecules gather around DNA
Dipicolinic acid: contained in the core along with calcium ions
Endospore septum: grows around the core, enclosing it in a double thickness of membrane
Cortex: laminated layer forms when peptidoglycan is released into space between endospore septum membranes
Spore coat: keratin-like protein, impervious to chemicals is laid down around the cortex
Exosporium: found in some endospores, a lipid-protein membrane formed outside the coat

51. Vegetative and Sporulation Cycles in Bacteria capable of Sporulation

52. Germination
A spore returns to its vegetative state, occurs in three stages:
Activation
Germination proper
Outgrowth

53. Bacterial endospores in two Clostridium species

54. Culturing Bacteria
Culturing of bacteria in the laboratory presents two problems:
A pure culture of a single species is needed to study an organism’s characteristics
A medium must be found that will support growth of the desired organism
Pure culture: a culture that contains only a single species of organism

55. The Streak Plate Method uses agar plates to prepare pure cultures

56. A Streak Plate of Serratia marcescens
A Streak Plate of Serratia marcescens. Note the greatly reduced numbers of growth /colonies in each successive region

57. Types of Culture Media
Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter
Synthetic medium: A medium prepared in the laboratory from material of precise or reasonably well-defined composition
Complex medium: contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)

58. Commonly Used Media Yeast Extract Casein Hydrolysate Serum Blood agar
Chocolate agar

59. Selective, Differential, and Enrichment Media
Selective medium: encourages growth of some organisms but suppresses growth of others
(e.g. antibiotics)
Differential medium: contains a constituent that causes an observable change (e.g. MacConkey agar)
Enrichment medium: contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified

60. Three species of Candida can be differentiated in mixed culture when grown on CHROMagar Candida plates

61. Identification of urinary tract pathogens with
differential media (CHROMagar)

62. Don’t Leave Home Without Your CO2

63. Candle Jar culture of anaerobes and microaerophiles

64. To culture obligate anaerobes, all molecular oxygen must be removed and kept out of medium. Agar plates are incubated in sealed jars containing chemical substances that remove oxygen and generate carbon dioxide or water

65. Anaerobic Transfer

66. Preserved Cultures
To avoid risk of contamination and to reduce mutation rate, stock culture organisms should be kept in a preserved culture, a culture in which organisms are maintained in a dormant state
Lyophilization
Frozen at -70oC
Refrigeration
Reference culture (type culture): a preserved culture that maintains the organisms with characteristics as originally defined

67. Methods of Performing Multiple Diagnostic Tests
The Enterotube System
The Analytical Profile Index (API) System

68. The Enterotube Multitest System

70. The API System: Various species of Enteobacteriaceae are shown here with differences in reactions that enable them to be identified