Syllabus Outline till Christmas

Week Date /Time Event Reading

9 26/9 Lecture 1 Unit Introduction/History of Chapter 1
Microbiology
10 3/10 Lecture 2 Microscopes and Microbial Cell Chapter 1 and 2
Structure
11 10/10 Lecture 3 Cell structure and function Chapter 2

All dates 12 14/10 Lecture 4 Microbial nutrition Chapter 3 (15

and times further reading)
12 17/10 (9-10, 11-12, Laboratory introduction and training Online Lab
are 2-3,) Manual
13 24/10 (9-11,12-2, 3- Laboratory 1 Lab Manual
subject to 5,)
25/10 Chapter 5
13 Lecture 5 Microbial Growth and Control
change-
15 7/11 (9-11, 12-2, 3-5,) Laboratory 2 Lab Manual
please
check your 16 14/11 (9-11,12-2, 3-
5,)
Laboratory 3 Lab Manual

portal! 18 28/11 (9-11,

3-5)
12-2, LAB EXAM Lab Manual

20 9/12 Lecture 6 Viruses Chapter 8

Formative MCQ assessment online

Sporulation
• Draw the process of endospore formation
Microbial Nutrition and
Growth
 Reproduction in Microbes
• The reproductive strategies of eukaryotic
microbes
– asexual and sexual, haploid or diploid
• Binary fission
• Budding
• Conidiospores (actinomycetes)
• Fragmentation of filaments
Growth
• May result in:
– increase in cell number
– increase in cell size
• Growth refers to
population growth
rather than growth
of individual cells
Microbial Growth
• Microbial - growth is an
increase in cell number. Cell elongation

One generation
• Microbial cell division
Septum
occurs by binary fission. Septum formation
1 cell divides into two, two
into four, four into eight. Completion
of septum;
formation of
walls; cell
separation
 Bacterial Cell Cycle
• Cell cycle sequence of events from formation of new cell
through the next cell division
• Two pathways function during cycle
– DNA replication and partition
– cytokinesis
 Growth and Determination of Cell Shape
• Determined by peptidoglycan synthesis in bacteria
– penicillin binding proteins (PBPs) – link peptidoglycan strands
and catalyze controlled degradation for new growth
The Population Growth Curve
• Observed when microorganisms are cultivated
in batch culture
• Usually plotted as logarithm of cell number
versus time
• Has four distinct
phases
Lag Phase
• Cell synthesizing new components
– e.g., to replenish spent materials
– e.g., to adapt to new medium or other
conditions
• Varies in length
– in some cases can be very short or even absent
 Exponential Phase
• Also called log phase
• Rate of growth is constant
and maximal
• Population is most uniform in
terms of chemical and
physical properties
• During log phase, cells exhibit
balanced growth
– cellular constituents
manufactured at constant rates
relative to each other
The Mathematics of Growth
• Generation (doubling) time
– time required for the population to double in size
– varies depending on microorganism and
environmental conditions
Stationary Phase
• Closed system population growth eventually
ceases, total number of viable cells remains
constant
– active cells stop reproducing or reproductive rate is
balanced by death rate
• Nutrient limitation
• Limited oxygen availability
• Toxic waste accumulation
• Critical population density reached
Stationary Phase and Starvation Response
• Entry into stationary phase due to starvation
and other stressful conditions activates survival
strategy
– morphological changes
• e.g., endospore formation
• Production of starvation proteins
• Cells are called persister cells
 Senescence and Death Phase
• Two alternative hypotheses
– cells are Viable But Not Culturable (VBNC)
– Programmed cell death -fraction of the population
genetically programmed to die (commit suicide)
• Bacterial population continually evolves
Nature Reviews Microbiology vol 17, 679–690 (2019) 10.1038/s41579-019-0253-y
Measuring Growth
 Measurement of Cell Mass
• Dry weight
• Quantity of a particular cell
constituent
– e.g., protein, DNA, ATP,
or chlorophyll
• Turbidometric measures
(light scattering)
– quick, easy, and sensitive
Direct Measurement of Cell Numbers
• Direct cell counts
– counting chambers
– electronic counters – flow cytometry
– on membrane filters
Membrane filter technique
– bacteria from aquatic samples are trapped on
membranes
– membrane soaked in culture media
– colonies grow on membrane
– colony count determines # of bacteria in sample
 Direct Counts on Membrane Filters
• Cells filtered through special membrane that provides
dark background for observing cells
• Cells are stained with fluorescent dyes
• With certain dyes, can distinguish living from dead cells
Viable Counting Methods
• Spread and pour plate techniques
– sample of bacteria is spread over solid agar
surface or mixed with agar and poured into Petri
plate
– after incubation the numbers of organisms are
determined by counting the number of colonies
multiplied by the dilution factor
– results expressed as colony forming units (CFU)
The Spread Plate and Pour Plate
• Both may be used to determine the number of
viable microorganisms in an original sample
Spread-plate method
Surface
colonies

Incubation

Sample is pipetted onto Sample is spread evenly over Typical spread-plate results
surface of agar plate surface of agar using sterile
(0.1 ml or less) glass spreader

Pour-plate method
Surface
colonies

Solidification
Subsurface
and incubation
colonies
Sample is pipetted into Sterile medium is added and Typical pour-plate results
sterile plate mixed well with inoculum
Fig. 6-16

Sample to
be counted
1 ml

1 ml 1 ml 1 ml 1 ml 1 ml

9-ml
broth

Total 1/10 1/100 1/103 1/104 1/105 1/106

dilution (10–1) (10–2) (10–3) (10–4) (10–5) (10–6)
Plate 1-ml samples

159 17 2 0
Too many colonies colonies colonies colonies colonies
to count

159  103 = 1.59  105

Plate Dilution Cells (colony-forming
count factor units) per milliliter of
original sample
 Culture Media
• Culture medium: Sterile
• Inoculum: Introduction of microbes into medium
• Culture: Microbes growing in/on culture medium
• Agar: Complex polysaccharide -not metabolized
by microbes- Fannie Hesse
• plates, slants, and deeps
• Liquefies at 100°C
• Solidifies ~40°C
• Must contain all the nutrients required by the organism
for growth
• Chemically defined media: Exact chemical composition
is known
• Complex media: Extracts and digests of yeasts, meat, or
plants
– Nutrient broth
– Nutrient agar
Isolation of Pure Cultures
• Population of cells arising from a single cell -
Robert Koch
• Allows for the study of single type of
microorganism in mixed culture
• Each cell can reproduce to form a separate colony
(visible growth or cluster of microorganisms)
Defined or
Synthetic
Media

Complex Media
 Types of Media
• Supportive or general purpose media (e.g. TSA)
– support the growth of many microorganisms
• Enriched media (e.g. blood agar)
– general purpose media supplemented by blood or
other special nutrients
• Selective
• Differential
 Selective Media
• Suppress unwanted
microbes and
encourage desired
microbes.
• e.g., MacConkey agar
– selects for gram-
negative bacteria

Figure 6.9b–c
 Differential Media
• Distinguish between different groups of
microorganisms based on their biological
characteristics
• e.g., blood agar
– haemolytic versus
non-haemolytic bacteria
• e.g., MacConkey agar
– lactose fermenters versus non-fermenters
 Enrichment Media
• Encourages growth of desired microbe
• PCB
 Biofilms
• Most microbes grow attached to surfaces (sessile)
rather than free floating (planktonic)
• These attached microbes are members of complex,
slime enclosed communities called a biofilm
• Biofilms are ubiquitous in nature in water
• Can be formed on any conditioned surface
Factors affecting Growth

Chemical
Physical
Physical Factors affecting Growth

•Temperature
•Acidity/ alkalinity (pH)
•Pressure
•Salt concentration
Temperature
•All organisms have a optimal temperature for
growth- Cardinal temperature

•The range is usually about 30 ºC- no one m/o can

grow at all temperatures

•Optimum slightly below their maximum growth

temperature.

•Minimum- membranes still have to be fluid

•Maximum- proteins have to be able to function

 Enzymatic reactions occurring
at maximal possible rate

Optimum
Growth rate

Enzymatic reactions occurring

at increasingly rapid rates

Minimum
Maximum

Temperature

Membrane gelling; transport Protein denaturation; collapse

processes so slow that growth of the cytoplasmic membrane;
cannot occur thermal lysis
 Classification based on optimal growth
temperature
Thermophile
Example: Hyperthermophile Hyperthermophile
Geobacillus Example: Example:
Mesophile stearothermophilus Thermococcus celer Pyrolobus fumarii
Example:
Escherichia 60°
coli 88° 106°
Growth rate

Psychrophile 39°
Example:
Polaromonas
vacuolata

4°

0 10 20 30 40 50 60 70 80 90 100 110 120

Temperature (°C)
Tolerance of environmental stress
•Microorganisms have colonised many extreme
habitats on earth
•Boiling hot water, Antarctic ice, pH 0 and pH 14
and high pressure
•Some microorganisms are just tolerant
•Some actually require these conditions-
extremophiles
 Cold adaptation

•Half of the earths surface at 5ºC or below

•Vast polar regions that are permanently cold
•Psychrophiles produce enzymes with lower temperature optima.
They often denature at room temperatures.
•Unsaturated fatty acids in membrane lipids, keeps membranes
fluid at lower temperatures.
 Psychrotrophs
• Grow between 0°C and 20-30°C
• Cause food spoilage
Heat adaptation
•Few environments reach
high temperatures
•Enzymes and Ribosomes
that are heat stable.
•More salt bridges in
proteins.
•Membranes have many
long-chain saturated fatty
acids.
•Histone-like proteins
stabilize DNA
Table 6-1
 pH
• Most microbes maintain an internal pH near neutrality
– the plasma membrane is impermeable to proton
– exchange potassium for protons
• Acidic tolerance response
– pump protons out of the cell
– some synthesize acid and heat shock proteins that
protect proteins
• Many microorganisms change the pH of their habitat
by producing acidic or basic waste products
Classification based on optimal pH
•Acidophiles - grow best when pH pH Example Moles per liter of:
H+ OH–
is below 5.4
0
Example: Thiobacillus thiooxidans Volcanic soils, waters
1 Gastric fluids
has a pH optimum of 2.5. Lemon juice
2 Acid mine drainage
It produces sulfuric acid that Vinegar

Acidophiles
Rhubarb
lowers the pH. Increasing
acidity
3
Peaches
4 Acid soil
Tomatoes
•Neutrophiles 5 American cheese
Cabbage
optimal pH between 5.4 and 8.5 6 Peas
Corn, Salmon, Shrimp
Most disease causing bacteria are Neutrality 7 Pure water
neutrophiles 8 Seawater
Very alkaline
9 natural soil
•Alkaliphiles Alkaline lakes
Alkaliphiles

10
pH optimum above 8.5 for growth Increasing
alkalinity
Soap solutions
Household ammonia
11
Many Bacillus species like alkaline Extremely alkaline
12 soda lakes
soils. Lime (saturated solution)
13

14
Salt concentration
– Hypertonic environments -cause plasmolysis
– Halophiles grow optimally in the presence of NaCl or
other salts at a concentration above about 0.2M
– Extreme halophiles = 2M to 6.2M, cell wall, proteins,
and plasma membrane require high salt to maintain
stability and activity
– Facultative halophiles tolerate high osmotic pressure

Haloquadratum walsbyi
 Chemical Requirements
• Carbon
– Structural organic molecules, energy source
• Microbes can be i) Heterotrophic
• ii) Autotrophic
Heterotrophs - require 1 or more organic compounds as a C source
Autotrophs – CO2 is the source of C (primary producers)
Synthesize organic compounds from CO2
Chemoheterotrophs use organic carbon sources
 Terminology
• Terms denoting the source of Energy that m/o use
– PHOTOTROPHIC = light energy
– CHEMOTROPHIC = chemical energy (organic or inorganic)

• Terms denoting the source of Reducing Power

– LITHOTROPHIC = use of reducing inorganic compounds (e.g.,
hydrogen, reduced sulfur compounds)
– ORGANOTROPHIC = use of organic compounds

• Terms denoting the source of Elements

– AUTOTROPHIC = use of inorganic compounds (e.g., CO2, nitrate,
ammonium, phosphate, sulfate, trace elements)
– HETEROTROPHIC = use of organic compounds (e.g., carbohydrates,
proteins, lipids, organic acids like acetate, methane)
• Nitrogen
– Use NH4+ or NO3–
– Nitrogen fixation
• Sulphur
– Use SO42– or H2S
• Phosphorus

 Trace elements
 Organic growth factors
Oxygen
aerobes organisms = can use oxygen to grow
anaerobic organisms = cannot use oxygen

Classes:
Obligate aerobes -- grow only when oxygen is present
Obligate anaerobes -- die in presence of oxygen
Facultative anaerobes -- grow with or without oxygen, grow better in
oxygen (respire)
Aerotolerant anaerobes -- ignore oxygen, grow equally well with or
without
Microaerophiles -- won't grow at normal atmospheric oxygen (20%),
but require some oxygen for growth (2-10%)
Capnophilic – low oxygen/high carbon dioxide
The Requirements for Growth:
Chemical Requirements
• Oxygen (O2)

Table 6.1
Obligate anaerobes
Oxygen can easily generate toxic byproducts
•superoxide (O2-)
•peroxide (H2O2 )
•hydroxyl radical (OH.)

•strong oxidizing agents that react indiscriminately with any

organic molecules, including DNA, proteins, etc

•Cells must have enzymes to get rid of these radicals.

•Superoxide dismutase and catalase are two crucial enzymes

for growth in oxygen
 Toxic Forms of Oxygen
• Singlet oxygen: O2 boosted to a higher-energy
state
• Superoxide free radicals: O2–

• Peroxide anion: O22–

• Hydroxyl radical (OH)

 Anaerobic culture techniques

Anaerobes: use media with

reducing agent (combines with
oxygen chemically)

Coy- pump out air, flush with

pure nitrogen gas or mixture

GasPak jar
Capnophiles Require High CO2
• Candle jar

• CO2-packet

Figure 6.7