Microbiology

1. PHYSIOLOGY OF
MICROORGANISMS

2. OBJECTIVES
 Growth and Multiplication
 Metabolism
 Cultivation
 Culture medium

3. BACTERIAL GROWTH
 Increase in number of bacterial cells rather than
increase in size of individual bacteria
Bacterial species only maintained if
population continues to grow
Growth depends on temperature, pH,
osmotic pressure, oxygen, and
nutrients

4. BACTERIAL DIVISION
Bacteria and archaea reproduce
asexually through binary fission
Eukaryotic microbes can engage in
either sexual or asexual
reproduction.
Other, less common processes can
include multiple fission, budding,
and the production of spores.

5. BINARY FISSION
1. Prokaryote cells`
Replication is by
binary fission, the
splitting of one cell
into two
2. Therefore, bacterial
populations
increase by a factor
of two (double)
every generation
time.

6. GENERATION TIME
The time required to for a
population to double (doubling
time) in number.
 Escherichia coli (E. coli) double
every 20 minutes
 Mycobacterium tuberculosis
double every 12 to 24 hours
4-6

7. CONSEQUENCES OF BINARY FISSION
Very large number of cells very fast
Mathematical progressions
 arithmetic (2>4>6>8>10>12>14>16)
 geometric(1>2>4>8>16)
 For example, 1 E. coli organism will
produce
over 1000 progeny in about 3 hours
and
over 1 million in about 7 hours.

8. Bacteria growing in the culture
produce a growth curve with up to
four distinct phases.
1. The first is the lag phase, during
which vigorous metabolic activity
occurs but cells do not divide. This
can last for a few minutes up to many
hours.
 For example, with a nutritionally poor
medium, several anabolic pathways
need to be turned on, resulting in a lag
before active growth begins.
Growth in Batch Culture

9. 2. The log (logarithmic) phase is when rapid cell
division occurs.
 β-Lactam drugs, such as penicillin, act during this
phase because the drugs are effective when
cells are making peptidoglycan (i.e., when they
are dividing).
The log phase is also known as the exponential
exponential phase. The doubling time varies not
varies not only with the species, but also with the
the amount of nutrients, the temperature, the
the pH, and other environmental factors.
Growth in Batch Culture

10. 3. Due to nutrient consuming (expenditure) and/or
accumulation of toxic end products, replication
stops and cells enter a stationary phase where
there is no net change in cell number.
4. The final phase is the death phase, which is
marked by a decline in the number of viable
bacteria.
Growth in Batch Culture

12. FACTORS INFLUENCING LAG PHASE
 Age of culture inoculum
 old culture -> long lag
 young culture-> short lag

13. METABOLISM
Definitions
 Metabolism: The processes of catabolism and
anabolism
Catabolism: The processes by which a
living organism obtains its energy and raw
materials from nutrients
Anabolism: The processes by which
energy and raw materials are used to build
macromolecules and cellular structures
(biosynthesis)

14. METABOLISM RELATIONSHIPS

15. VARIOUS TYPES OF PROKARYOTIC
ENERGY PRODUCTION PROCESSES
 Aerobic Respiration
 Fermentation: Anaerobic Respiration
 Lithotrophy
 Photoheterotrophy
 Anoxygenic photosynthesis
 Methanogenesis

16. CATABOLISM:
 Fermentation
 Transfer of electrons to organic
substrate
 Respiration
 Transfer of electrons to inorganic
acceptor

17. GLYCOLYSIS: EMBDEN-MEYERHOFF
 Glycolytic
 In the Cytoplasm
 Anaerobic
 End products
 C6H12O6 + 6O2  2
Pyruvic acids
2 ATP are used
4 ATP are produced
 4-2 = 2 net ATP by
substrate level
phosphorylation
 2 NADH are produced

18. MICROBIAL METABOLISM
Fermentation
A) incomplete oxidation of glucose
B) does not require O2
C) follows glycolysis when O2 is absent
Pyruvate is converted to either an acid or alcohol and
NADH is converted back to NAD
E) 2 types
1) alcoholic fermentation: a) results in 2 ATP, CO2, and an
alcohol (usually ethanol)
2) acidic fermentation: a) results in 2 ATP plus an acid such as
lactic acid and butyric acid

19. FERMENTATION
Figure 5.18b

20. RESPIRATION
 The series of chemical reactions that
accomplish complete oxidation is called
the Krebs Cycle in the cell membrane.
a) the complete oxidation of glucose
b) C6H12O6 + 6O2  6CO2 + 6H2O + 38
ATP
Krebs Cycle

22. TEMPERATURE
 The range of enzyme activity determines
the range for growth of specific bacteria,
analogously leading to a value for optimal
growth rate.
 In the case of temperature, bacteria are
divided into categories based on the
temperature range
where they can grow and the temperature
that provides optimal growth.

23. FIG. 7.8

24. TEMPERATURE
 Psychrophilic forms grow best at low temperatures (-0–
18°C)
 They are usually found in such environments as the Arctic and
Antarctic regions;
 Psychrotrophs have a temperature optimum between 20°C
and 30°C but grow well at lower temperatures.
 They are an important cause of food spoilage.
 Mesophilic forms grow best at 30–37°C (25-45 °C).
 Most organisms are mesophilic; 30°C is optimal for many free-
living forms.
 The body temperature of the host is optimal for symbionts of
warm-blooded animals or pathogen bacteria.
 Most thermophilic forms grow best at 50–60°C.
 Some organisms are hyperthermophilic and can grow at well
above the temperature of boiling water, which exists under
high pressure in the depths of the ocean.
Bacteria can be stored in stock cultures for years at -20 and -80 ° C.

25. OXYGEN REQUIREMENTS
 Obligate aerobes – require O2
 Facultative anaerobes – can use O2 but
also grow without it
 Obligate anaerobes – die in the presence of
O2
The use of oxygen by bacteria generates toxic
products such as superoxide and hydrogen
peroxide.
Aerobes and facultatives have enzymes, such as
superoxide dismutase and catalase, that detoxify
these products,
but anaerobes do not and are killed in the presence
of oxygen.

26. OXYGEN REQUIREMENTS
 Some bacteria, such as M. tuberculosis,
are obligate aerobes;
 they require oxygen to grow because their ATP-
generating system is dependent on oxygen as
the hydrogen acceptor.
 Other bacteria, such as E. coli, are
facultative anaerobes; they utilize oxygen,
 if it is present, to generate energy by respiration,
but they can use the fermentation pathway to
synthesize ATP in the absence of sufficient
oxygen.

27. OXYGEN REQUIREMENTS
 The third group of bacteria consists of the
obligate anaerobes, such as Clostridium
tetani,
 which cannot grow in the presence of oxygen
because they lack either superoxide dismutase
or catalase, or both.
 Obligate anaerobes vary in their response to
oxygen exposure; some can survive but are not
able to grow (aerotolerant anaerobes), whereas
others are killed rapidly (strict anaerobes).
 Microaerophiles, which require small
amounts of oxygen (2%–10%) for aerobic
respiration (higher concentrations are
inhibitory);

28. OXYGEN
Obligate aerobes
 Only aerobic growth, oxygen required
Facultative anaerobes (most human pathogens)
 Greater growth in presence of oxygen
Obligate anaerobes
 Only anaerobic growth, cease with oxygen
Aerotolerant anaerobes (e.g., C. perfringens)
 Only anaerobic growth, continues with oxygen
Microaerophiles
 Only aerobic growth with little oxygen

29. 29

30. PH AND MICROBIAL GROWTH
 Acidophiles : organisms that grow best at
low pH (Helicobacter pylori, Thiobacillus
thiooxidans )
 Many bacteria and viruses survive low pH of stomach to
infect intestines
 Helicobacter pylori lives in stomach under mucus layer
 Alkaliphiles : organisms that grow best at
high pH (Vibrio choleraea)
 Most of pathogenic bacteria are
neutrophiles

31. OSMOTIC PRESSURE
High osmotic pressure
(hypertonic) removes water
causing plasmolysis – inhibits
growth i.e. salt as preservative
Low osmotic pressures
(hypotonic) cause water to enter
and can cause lysis
Bacteria are more tolerant to
osmotic variations because of the
mechanical strength of the cell
wall
 Organisms requiring high salt
concentrations are called halophilic;
those requiring high osmotic pressures
are called osmophilic.
NaCl 0.85% NaCl 10%
H2O
Plasma membrane
Plasma membrane
Cell wall

32. MOISTURE AND DESICCATION
Moisture is essential - 80% body weight is water
Effect of drying varies by organism
 T pallidum, gonococcus are very susceptible
 Mycobacterium tuberculosis, staphylococci may survive
for weeks
 Bacterial spores survive several years
Lyophilization
 is a process in which water is removed from a product
after it is frozen and placed under a vacuum, allowing the
ice to change directly from solid to vapor without passing
through a liquid phase.

33. WHY CULTIVATE BACTERIA?
 Identification of bacteria
 Antimicrobial susceptibility testing
 For research
 for example vaccine development

34. GROWTH REQUIREMENTS
Physical
 Temperature
 pH
 Osmotic pressure
 Moisture & desiccation
Chemical
 Carbon source
 Nitrogen, sulfur phosphorus
 Oxygen

35.  Most of the dry weight of microorganisms is
organic matter containing the elements
 carbon, hydrogen, nitrogen, oxygen,
phosphorus, and sulfur.
 In addition, inorganic ions such as
potassium, sodium, iron, magnesium,
calcium, and chloride are required to
facilitate enzymatic catalysis and to
maintain chemical gradients across the cell
membrane.

36. CULTURE MEDIUM
Growth media are those used for microbiological culture,
which are used for growing microorganisms, such
as bacteria or fungi.
A classification of media based on their respective usages:
 Basic medium
 Enrichment medium
 Selective medium
 Differential medium

37. CULTURE MEDIUM
 Basic medium:
supplies only the minimal nutritional requirements of a
particular microorganism. e.g. broth
 Enrichment medium:
Nutrient broth, nutrient agar, peptone water are
commonly used in enrichment media. e.g. blood agar
plate

38. Blood agar Chocolate agar

39.  Selective medium:
Supports the growth of desired bacteria
while inhibiting the growth of many or
most of the unwanted ones,
 either by adding one or more
selective agents which is a "poison" to
the unwanted bacteria but not harmful
to desired bacteria, or
 by including certain nutrients for the
desired ones and deleting certain
nutrients for the unwanted ones.
e.g. Lowenstein Medium medium for M.
tuberculosis

40. Selective media
The inhibitory substance is added to a solid media.
Eg:
Mac Conkey’s medium for gram negative bacteria
TCBS (Thiosulfate Citrate Bile Salts Sucrose) – for
V.cholerae
LJ medium – M.tuberculosis
Wilson and Blair medium – S.typhi
Potassium tellurite medium – Diphtheria bacilli

41. Mac Conkey’s medium TCBS
(Thiosulfate Citrate Bile Salts
Sucrose)

42. Potassium Tellurite media LJ media

43.  This medium allows two or more different bacteria to
grow, but it contains dyes and/or other components upon
which different bacteria act in various ways to produce a
variety of end products or effects (usually by showing
different colors).
 Differential medium

44. Differential media
 A media which has substances incorporated in it enabling
it to distinguish between bacteria.
Eg: Mac Conkey’s medium
 Peptone
 Lactose
 Agar
 Neutral red
 Taurocholate
 Distinguish between lactose fermenters & non lactose
fermenters.

45.  Lactose fermenters – Pink colonies
 Non lactose fermenters – colourless colonies
Mixed bacterial colonies
on MacConkey agar,
Escherichia coli (red) and
Salmonella typhimurium (white)

46. Indicator media
These media contain an indicator which
changes its colour when a bacterium
grows in them.
Eg:
 Blood agar
 Mac Conkey’s medium
 Christensen’s urease medium

48. Urease medium

49. Medium
Classification according to physical condition
(according to the content of solidifying agent):
• liquid medium
• Solid medium
• Semi-solid medium

50. Used to obtain a large number of bacteria, and to
perform drug sensitivity test and bacterial growth assay.
The bacteria grown in liquid medium will display some
certain characteristics of bacteria (alignment and
clustering) that can't be seen easily in solid media.
 liquid medium

51. Solid medium
Used to obtain a large number of bacteria, isolate
identical clones of bacteria (colony), and to perform
drug sensitivity test.
A colony is a bacterial cluster which propagated
(multiplied) from a single initial bacterial cell (So a
colony is a pure bacterial culture).
Colony can be used to determine the original bacterial
numbers by counting colonies and to evaluate viability
of bacteria (colony forming units, CFU).

52.  Agar
•The major solidifying agent used in bacteriological
media.
• An polysaccharide gum that extracted from certain
red algae.
• Agar can be dissolved at 100 C, and solidified at
about 43 C.
•Added 1.5-2.0% of Agar for solid plates or slanted
media, 0.1-0.5% for semisolid media.

53. Semisolid medium
Test the motility of bacteria (a
bacterium has a flagellum or flagella
whether or not )
Positive: bacteria grow into the medium
give cloudiness to the medium.
Negative: bacteria grow in situ.

54. ANAEROBIC GROWTH
Reducing media containing
thioglycolate to deplete oxygen;
cooked meat broth
Anaerobic jar, anaerobic
chamber, anaerobic bags/
pouch
Petri
plates
Anaerobic indicator
(methylene blue)
CO2 H2
Envelope
containg
sodium
bicarbonate
and sodium
borohydride
Lid with O-ring gasket
Palladium catalyst pellets

55. CAPNOPHILES
Capnophiles require high
concentration of CO
e.g. Brucella abortus
Petri plates
Candle
Tubes with
liquid media
Glass jar
Petri plates
Gas generator
Gas generator
A. Glass jar
B. CO2 generating package

56. 56
Special Culture Techniques
Candle Jar

57. 57
Special Culture
Techniques
Gas Pack Jar Is
Used for
Anaerobic
Growth

58. Streaking
Procedure:
1. Flame the loop and streak a loopful of broth culture as at A in
the diagram.
2. Reflame the loop and cool it.
3. Streak as at B to spread the original inoculum over more of
the agar.
4. Reflame the loop and cool it.
5. Streak as at C.
6. Reflame the loop and cool it.
7. Streak as at D.
8. Incubate the plate inverted.

59. Streaking
By spreading a large amount of bacteria over the surface of a plate,
the amount of bacteria is diluted and individual cells are
spread. From these individual cells a single colony arises.
Wire loop
Dish
Colony
(pure culture)

60. GROWTH OF MICROBES
 Increase in number of
cells, not cell size
 One cell becomes
colony of millions of
cells

61. COLONY- (CLONE)
 Colony- A bacterial population derived from
one bacterial cell. The cells within the
colony have identical, genus, species,
genetic and phenotypic characteristics.
 Pure bacteria - derived from a single
colony.
 Selection of a pure colony -most important
for bacterial identification
 R,S, M colonia

63. 63

64. PEARLS:
 Bacteria reproduce by binary fission, whereas
eukaryotic cells reproduce by mitosis.
 The bacterial growth cycle consists of four phases:
 the lag phase, during which vigorous metabolic
activity occurs but cells do not divide.
 the log phase, during which rapid cell division
occurs;
 the stationary phase, during which as many cells
are dying as are being formed; and
 the death phase, during which most of the cells
are dying because nutrients have been
exhausted.

65. PEARLS:
 Some bacteria can grow in the presence of
oxygen (aerobes and facultatives),
 but others die in the presence of oxygen
(anaerobes).
 The use of oxygen by bacteria generates
toxic products such as superoxide and
hydrogen peroxide.
 Aerobes and facultatives have enzymes,
such as superoxide dismutase and
catalase,
 that detoxify these products, but anaerobes do not
and are killed in the presence of oxygen.

66.  a bacterial growth curve divided into
phases a, b, c, and d. In which one of the
phases are antibiotics such as penicillin
most likely to kill bacteria?
(A) Phase a
(B) Phase b
(C) Phase c
(D) Phase d

67.  a bacterial growth curve divided into
phases a, b, c, and d. In which one of the
phases are antibiotics such as penicillin
most likely to kill bacteria?
(A) Phase a
(B) Phase b
(C) Phase c
(D) Phase d

68.  Some bacteria are obligate anaerobes. Which of
the following statements best explains this
phenomenon?
(A) They can produce energy both by
fermentation (i.e., glycolysis) and by respiration
using the Krebs cycle and cytochromes.
(B) They cannot produce their own ATP.
(C) They do not form spores.
(D) They lack superoxide dismutase and
catalase.
(E) They do not have a capsule.

69.  A 23-year-old woman has 10 Escherichia coli
inoculated into her bladder while having sex.
These E coli have a generation time of 20
minutes. After a lag of 20 minutes, the E coli
enter the logarithmic phase of growth. After 3
hours of logarithmic growth, the total number of
cells is
(A) 2560
(B) 5012
(C) 90
(D) 1028
(E) 1,000,000

70.  The growth rate of bacteria during the
exponential phase of growth is
(A) Zero
(B) Increasing
(C) Constant
(D) Decreasing
(E) Negative

71.  The growth rate of bacteria during the
stationary phase of growth is
 (A) Zero
 (B) Increasing
 (C) Constant
 (D) Decreasing
 (E) Negative

72.  Most microorganisms pathogenic for
humans grow best in the laboratory when
cultures are incubated at
 (A) 15–20°C
 (B) 20–30°C
 (C) 30–37°C
 (D) 38–50°C
 (E) 50–55°C

73.  Which of the following is NOT a
mechanism for generating metabolic
energy by microorganisms?
(A) Fermentation
(B) Protein synthesis
(C) Respiration
(D) Photosynthesis
(E) C and D

74.  Which of the following is NOT a
component of peptidoglycan?
(A) N-Acetyl muramic acid
(B) N-Acetyl glucosamine
(C) Lipid A
(D) Pentaglycine
(E) Diaminopimelic acid