Visualizing microbes

Basic Microscopy and Staining

 Brightfield Microscopy
• Used to observe objects (microbes)
too small to be seen with the
unaided eye

• Here are the parts of the

microscope labeled
 Microscopy-Background
• Total Magnification=Ocular Mag x Objective Mag

• Resolving Power- minimum distance that two objects can be seen as

separate
• RP= λ/(2*NA) NA=numerical aperture
 Microscopy-Background
• Parfocal-remains in focus as you change objectives

• Parcentric- Center of the field of view remains in center of field of

field for all magnifications
 Microscopy-Background
• Working Distance=the distance between the objective lens and slide
when in focus.
• As the magnification of the lens increases, the working distance decreases.

• Immersion Oil- used with the 100x objective only

• Minimized refraction of light away from lenses to improve
resolving power
Microscopy- Background
• Microorganism
shapes that we will
commonly see

• Rod is also referred

to as bacillus
Smear Preparation and
Simple Stains
Why stain microbes?
• Cells are predominately made up of water

• Hard to get good contrast between the microbe and the

background

• Add stains to increase that contrast

• Can be simple or differential stains
 Bacterial Smear
• Technique to adhere cells onto the surface of a slide to allow for
staining and microscopic visualization, required for any staining
procedure where the stain is washed off

• Cells are placed on to surface of slide within a target circle(s) with a

little water

• Once the water dries completely and cells are passed in front of
incinerator to gently heat the cells and denature the protein making
them stick to slide surface

• Avoid overheating which will distort cells

 Simple Staining: Basic Staining
• Technique used to stain
microorganisms to increase the Blue cocci shaped cells

contrast
• Cells are dyed using a single stain
• Today using a Basic(cationic) stain
which has a positively charged
chromogen
• Due to charge it adheres to the
negative charge of the cell wall
and nucleic acids
• Stain used: methylene blue
Simple Stain: Basic Staining
 Simple staining: Acidic/Negative Staining
• Acidic stain so the chromogen is
anionic (negatively charged)
• How will this interact with the cell?
• No heating fixing is used so able
to see the true size and shape of
the cells since heat can distort.
• Shown with Bacillus megaterium
following protocol in manual
using Nigrosin stain
• At right B.megaterium at
1000x+oil
Procedure of Negative Staining
1.Add a drop of stain to the edge of a clean slide
2.Use the sterile loop to pick up a small amount of culture
3.Mix the culture directly into the stain using the loop
4.Sterilize the loop
5.Use a second slide to drag the stain across the slide surface
6.Allow stain/microbe mixture to dry completely before viewing
• This procedure gives you a gradient of stain and when viewing you would
look for the best balance of a dark background to the transparent cells . You
only need to put a small drop of nigrosin onto the slide otherwise the
nigrosin is too thick on the slide and starts cracking as it dries.
• These are done with same stain. Why do you think there is a difference in color?
What would cause the image on the right to look like this?
Differential Stains
Differential Stains
• Allow you to determine a characteristic of the organism (differentiate)
• Multiple stains and steps required
• Have to prepare smear first to allow for multiple washing steps
Gram Staining
• Stain determines the thickness of
the peptidoglycan layer, Gram
type
• Gram-Positive: thick layer
• Gram negative: thin layer
• Following the procedure carefully
is necessary to obtain correct
results
• Important in clinical settings to
determine type of antibiotics and
also if LPS will be an issue
• Crystal Violet- Primary Stain

• Iodine- Mordant

• Alcohol (ethanol or acetone)- Decolorize

• Safranin- Counterstain
Identify the Gram Type and Cell
shape
Bacterial Spore Staining

• Spores produced by Clostridia, Desulfotomaculum and

Bacillus
• Used to survive in harsh environmental conditions, heat,
chemicals, UV
• DNA and ribosomes are protected within the endospore
• Have to use moist heat to open up strong protein coat and
get the malachite green stain inside the spore
• Use Safranin (a basic stain) as a counterstain to stain the
vegetative portion of the cell
Staining Procedure
• Prepare a thin smear of the organism
• Malachite green the primary stain
• Moist heat
• Steam bath or 60°C incubator
• Heat required for stain to enter spore
• Spores must already be formed to stain
• Heat acts as a mordant
• Rinse excess stain with water
• Decolorizer, washes stain out of
vegetative cells, remains trapped in
spores
• Apply Safranin
• counterstain to stain the vegetative
portion of the cell
(Cappuccino and Welsh 2017)
Vegetative cell

Sporulating cell

Free endospore

• Endospore stain at 1000x + additional optical magnification.

• If you fail to heat the slide how will that affect the stain outcome?

• If you forgot to add the safranin how would that affect the outcome?
What can we determine
about this culture from this
stain?
Media Basics
Growth Conditions
• What parameters do you think influences growth of organisms?
Focus on nutrients supplied in the medium
Media Background
• Media Basics
• Types
• Rich/Complex Medium- has all the nutrients for most organisms to
grow, not necessary quantifiable amounts, e.g. Nutrient, Trypticase soy,
Yeast Extract (Buffet of food)
• Minimal/defined- Has set amounts of all nutrients, usually used to grow
specific types of organisms
• Media state
• Broth- liquid that lacks solidifying agent
• Solid- contains the solidifying agent agar (makes it hard like jello) that
comes from seaweed extract usually in a plate
• Semi-solid- lower concentration of agar so softer consistency
Media Background Cont.
• Plates- solid surface used to visualize individual colonies of
the inoculum (sample that is being used)
• Label the bottom side with the agar attached
• Why?
• Test Tubes- hold the liquid broth
• Label on the glass below the lid
• Always carry by the glass tube, not the lid
• Labels should typically include:
• Initials, date, medium type, inoculum source
Labeling Example
Figure 1: Nutrient Agar plate inoculated
with finger. Plate was exposed to my
finger and then incubated for 1 day at 37
degrees C. Several different colony
morphologies were observed. Colonies A,
B and D, were off white, round with
slightly raised elevations. Colony c was
black with sprawling edges. Etc.…Continue
descriptions for remaining isolates
Aseptic Technique and Pure
Cultures/Isolation of
Bacteria
Aseptic Technique
 Aseptic Technique
• Process to minimize the contamination of yourself and your sample with
unwanted microbes

• Requires keeping the work environment as sterile as possible

• Cleaning surfaces
• Working near a flame/incinerator
• Sterilizing tools and other supplies before use
• Minimizing the time plates/tubes are opened

• Practice Safe Microbiology

• Important when:
• Initially isolating the organism
• Transferring from one culture to another

• Want to make sure you work with desired organisms and not
everything floating around
Isolation of Bacteria
Pure Culture Technique
 Isolation of Pure Cultures
• Technique used to take a sample containing a mixture and
separate out the individual organisms

• You dilute the culture as you pull it around the surface of the plate

• Ideally each colony comes from a single cell(or species) that

replicated repeatedly to form a visible colony to forming a “pure”
culture
 Pure Culture
• Examples of the alternative streak method
• Only pull streaks in one direction
• Sterilize loop when moving between quadrants 12, 23 to dilute cells
• Do not touch the fourth quadrant into the first
• Always drag and dilute the cells from higher to lower concentration areas in one direction
Streak plate- Demonstration (Basic)
Streak plate- Demonstration (Advanced)
Colony Morphology (undulate,wavy, convex,
flat)
 Streak plate- Optional At home Practice
• Each person can try performing their
own streak plate on Jello
• Use a tiny dot of yellow mustard as
your “inoculation” source
• You will dilute that inoculations as
you move around the surface
• Avoid digging into the surface as
growth should occur on top not
imbedded
• Discard or eat after practice DO NOT
INCUBATE
Successfully performed quadrant streak
• The first quadrant is evenly
spread out
• Colonies are clearly isolated
• No gauging into the medium
surface
• There is a decrease in the
concentration of organisms
from the first quadrant to the
rest
• Streak only overlaps the
overlaps two quadrants
Types of errors in on
this streak plate
• 1st quadrant streaks extend
outside of the quadrant
• Concentration does not decrease
steadily from Quad 14
• Colonies not clearly separated in
the 3 and 4th quadrants
• Streaks overlapped with more
than just the one previous
quadrant
Types of errors on
this streak plate
• Loop pressed too hard on
to agar plate surface
• Colonies not clearly
separated in the 3 and 4th
quadrants
• Loop was probably too hot
when streaking
Summary
• The quadrant streak technique useful lets you isolate
and/or determine the purity of a culture in a quick and
easy technique and then observing the morphology

• Many different versions of the technique but end goal for

all is to dilute the culture around the surface of the plate

• Allows you to get “pure” cultures if completed correctly

when you see well separated isolated colonies
Growth and Enumeration
of Bacteria
Cell Growth and Binary Fission
• Microbial growth involves an increase in
the number of cells. Growth of most
microorganisms occurs by the process of
binary fission.
 Exponential Growth
• Microbial populations show a
characteristic type of growth pattern
called exponential growth, which is best
seen by plotting the number of cells over
time on a semilogarithmic graph.
 Figure 9.4

• The parental cell divides and gives rise to two daughter cells. Each of the daughter cells, in turn, divides, giving
a total of four cells in the second generation and eight cells in the third generation. Each division doubles the
number of cells.
 Figure 9.6

• Both graphs illustrate population growth during the log phase for a bacterial sample with an
initial population of one cell and a doubling time of 1 hour.
(a)When plotted on an arithmetic scale, the growth rate resembles a curve.
(b)When plotted on a semilogarithmic scale (meaning the values on the y-axis are
logarithmic), the growth rate appears linear.
Figure 9.5

• The growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a
function of time. The graph can be divided into four phases according to the slope, each of which matches events
in the cell. The four phases are lag, log, stationary, and death.
• Microorganisms show a characteristic growth pattern when inoculated into a fresh culture medium in a closed
system
• There is usually a lag phase, then exponential
growth commences. As essential nutrients are
depleted or toxic products build up, growth ceases,
and the population enters the stationary phase. If
incubation continues, cells may begin to die (the death
phase).
Figure 9.6

• Both graphs illustrate population growth during the log phase for a bacterial sample with an initial
population of one cell and a doubling time of 1 hour.
(a) When plotted on an arithmetic scale, the growth rate resembles a curve.
(b) When plotted on a semilogarithmic scale (meaning the values on the y-axis are logarithmic), the
growth rate appears linear.
Examples for Comparison
• E. coli in the lab – 20 minute doubling time
• E. coli in the gut – 12 hour doubling time
• Heterotrophs in pond water – 2-10 hour doubling time
• Heterotrophs in the deep subsurface – 100 years
doubling time
 Figure 9.7

• A chemostat is a culture vessel fitted

with an opening to add nutrients (feed)
and an outlet to remove contents
(effluent), effectively diluting toxic
wastes and dead cells. The addition
and removal of fluids is adjusted to
maintain the culture in the logarithmic
phase of growth. If aerobic bacteria are
grown, suitable oxygen levels are
maintained.
Measuring Microbial Growth
 Direct Measurements of Microbial
Growth: Total and Viable Counts
• Growth is measured by the change in the number of
cells over time.

•Cell counts done microscopically measure the total

number of cells in a population, whereas viable cell
counts (plate counts) measure only the living,
reproducing population.
Figure 9.8- Direct Counts

(a) A Petroff-Hausser chamber is a special slide designed for counting the bacterial cells in a measured volume of
a sample. A grid is etched on the slide to facilitate precision in counting.
(b) This diagram illustrates the grid of a Petroff-Hausser chamber, which is made up of squares of known areas.
The enlarged view shows the square within which bacteria (red cells) are counted. If the coverslip is 0.2 mm
above the grid and the square has an area of 0.04 mm 2, then the volume is 0.008 mm3, or 0.000008 mL. Since
there are 10 cells inside the square, the density of bacteria is 10 cells/0.000008 mL, which equates to
1,250,000 cells/mL. (credit a: modification of work by Jeffrey M. Vinocur)
 Direct Counts
(Petroff-Hausser Counter)
Figure 9.10

• A Coulter counter is an electronic device that counts cells. It measures the change in resistance in an
electrolyte solution that takes place when a cell passes through a small opening in the inside
container wall. A detector automatically counts the number of cells passing through the opening.
(credit b: modification of work by National Institutes of Health)
 Viable Counting
(Spread Plating or
Plate Counting)

To calculate the number of cells per ml

 The Great Plate Count Anomaly
• Plate counts can be highly unreliable when used to assess total cell numbers
of natural samples such as soil and water. Direct microscopic counts of natural
samples typically reveal far more organisms than are recoverable on plates of
any given culture medium.

•This is referred to as "the great plate count anomaly," and it occurs because
microscopic methods count dead cells whereas viable methods do not

•In addition, different organisms may have vastly different requirements for
resources and conditions in laboratory culture.
 Indirect Measurement of
Microbial Growth: Turbidity

• Turbidity measurements are an indirect but very rapid and

useful method of measuring microbial growth.

•However, to relate a direct cell count to a turbidity value, a

standard curve must first be established to find out how many
cells are expected for a given turbidity value.
 Figure 9.15

(a)A spectrophotometer is commonly used to measure the turbidity of a bacterial cell suspension as an indirect measure of cell density.
(b)A spectrophotometer works by splitting white light from a source into a spectrum. The spectrophotometer allows choice of the
wavelength of light to use for the measurement. The optical density (turbidity) of the sample will depend on the wavelength, so once one
wavelength is chosen, it must be used consistently. The filtered light passes through the sample (or a control with only medium) and the
light intensity is measured by a detector. The light passing into a suspension of bacteria is scattered by the cells in such a way that some
fraction of it never reaches the detector. This scattering happens to a far lesser degree in the control tube with only the medium. (credit a:
modification of work by Hwang HS, Kim MS; credit b “test tube photos”: modification of work by Suzanne Wakim)
 Standard Curve and
Generation Time with
Escherichia coli
 Determining Generation Time
• Track growth of organism over a period of time in the
exponential phase to determine the of the organism
• Measuring two ways:
• Directly using dilutions and colony forming units/mL from
plate counts

• Indirectly using turbidity of the culture measured with the

spectrophotometer to determine Optical Density (OD)

• Both measurements must be taken at each time point

Parts of the Pipette

Gilson Pipetman ® P User’s Guide

Setting the Volume

Gilson Pipetman ® P User’s Guide

 Pipetting

Gilson Pipetman ® P User’s Guide

Plate Counts- Even dispersal
• Want to evenly spread dilution
across plate
• Next week will count the number
of colonies
• Each colony represents 1 cell that
replicated over and over from the
original culture
• Calculate the colony forming
units/mL (CFU/mL) the
concentration of cells at that point
Indirect Measurement-Spectrophotometer

• Used to measure optical density

(OD)
• One measurement taken each
time an aliquot is removed for
dilution series
• Calibrated with a blank

• CELLS DO NOT ABSORB LIGHT,

they scatter the light
Optical Density

(Analyzing differences in bacterial Optical Density: Matlock et al.)

 • Semi-log paper has one axis
as a logarithmic scale and
the other axis is linear
• This paper has two log cycles
• Generation Time (GT)
Calculation
• Use trendline to calculate GT
• Pick two points that are
double each other in
concentration
• Change in time between
those points is GT
 Count the Colonies on all Plates
• Can divide the plate into quadrants to make easier to count
• Record colony counts for all time points and dilutions
• Dilutions with 30-300 colonies used for statistical reasons to
calculate the CFU/mL
• CFU/mL=number of colonies/dilution Colony Counts
Time 10-5 10-6 10-7
Duplicate Plates Duplicate Plates Duplicate Plates
0 min

30 min

60 min

90 min
 Colony Counts

Time 10-5 10-6 10-7

Duplicate Plates Duplicate Plates Duplicate Plates
0 min 35 41 3 4 0 0

30 min 76 78 7 8 0 1

60 min 181 184 18 20 2 3

90 min TMTC TMTC 42 38 4 5

• Calculate the CFU/mL at each time point

• Duplicate Plates at each dilution to increase accuracy
• Only use plates that fall between 30-300
 Colony Counts

Time 10-5 10-6 10-7

Duplicate Plates Duplicate Plates Duplicate Plates
0 min 35 41 3 4 0 0

30 min 76 78 7 8 0 1

60 min 181 184 18 20 2 3

90 min TMTC TMTC 42 38 4 5

• At the zero min which plates should be used?

 Colony Counts

Time 10-5 10-6 10-7

Duplicate Plates Duplicate Plates Duplicate Plates
0 min 35 41 3 4 0 0

30 min 76 78 7 8 0 1

60 min 181 184 18 20 2 3

90 min TMTC TMTC 42 38 4 5

• Average the yellow plates

• Determine CFU/mL at the Point
 Colony Counts

Time 10-5 10-6 10-7

Duplicate Plates Duplicate Plates Duplicate Plates
0 min 35 41 3 4 0 0

30 min 76 78 7 8 0 1

60 min 181 184 18 20 2 3

90 min TMTC TMTC 42 38 4 5

• What is the CFU/mL for the 90 minute data point?

• Calculate the CFU/ml for each plate that falls between range 30-300
(Make sure to show your work in the post lab)
• CFU/mL=number of colonies/dilution
• Average CFU/mL to get one value for each time point
• Averaged data will be used for graphing
CFU/mL

0 min 3.8x106

30 min 7.7x106

60 min 1.82x107

90 min 4.0x107
How to Graph by Hand
Label the Axes with units
based on range of data

Time(min) CFU/mL OD

0 2.8x107 0.05

30 6.8x107 0.11

60 1.38x108 0.325

90 2.88x108 0.625
Start Plotting the data

Time(min) CFU/mL OD

0 2.8x107 0.05
30 6.8x107 0.11
60 1.38x108 0.325
90 2.88x108 0.625
Time(min) CFU/mL OD

0 2.8x107 0.05
30 6.8x107 0.11
60 1.38x108 0.325
90 2.88x108 0.625
Time(min) CFU/mL OD

0 2.8x107 0.05
30 6.8x107 0.11
60 1.38x108 0.325
90 2.88x108 0.625
Time(min) CFU/mL OD

0 2.8x107 0.05
30 6.8x107 0.11
60 1.38x108 0.325
90 2.88x108 0.625
Draw a trendline that
follows the overall data

Time(min) CFU/mL OD

0 2.8x107 0.05
30 6.8x107 0.11
60 1.38x108 0.325
90 2.88x108 0.625
• Use trendline to calculate the
generation time by picking two
points that are double each
other
• For example:
• 4x107CFU/mL and 8x107CFU/mL
• Change in time between points
in the generation time
• ~26 minutes
• Set scale based on data points
• Graph data Points
• Draw trendline to calculate the
generation time by picking two
points that are double each
other
Time(min) CFU/mL OD

0 2.8x107 0.05
30 6.8x107 0.11
60 1.38x108 0.325
90 2.88x108 0.625
• Standard Curve
• Uses Linear Graph paper
because log functions cancel
each other out
• Set scale based on data points
• Graph data Points CFU/ml vs OD
• Draw trendline
Time(min) CFU/mL OD
0 2.8x107 0.05
30 6.8x107 0.11
60 1.38x108 0.325
90 2.88x108 0.625
 Using Graphing Software

• Enter data as CFU/ml and OD values

• Change the y-axis to log function
• Use an exponential function to get trendline
• The R2 value is how well your data fits to the line
• Use graph to determine generation time
 Graphing the OD Values
OD vs Time
1

f(x) = 0.0472490786458001 exp( 0.029016196878803 x )

R² = 0.975747457056532
Time Average OD

Optical Denstiy
0 0.040063
30 0.144 0.1
60 0.2714
90 0.5904

0.01
0 10 20 30 Time
40 (minutes)
50 60 70 80 90 100
Calculating Generation Time
OD vs Time
1

f(x) = 0.0472490786458001 exp( 0.029016196878803 x )

R² = 0.975747457056532
Time= 50-26=24min
0.2
Optical Denstiy

Double OD
0.1

0.01
0 10 20 30 40 50
Time (minutes) 60 70 80 90 100
 1.00E+08
CFU/mL vs. Time
CFU/mL

1.00E+07

1.00E+06
0 20 40 60 80 100 120 140
Time (min)
 CFU/mL vs. OD
4.00E+04

3.50E+04

3.00E+04

2.50E+04
CFU/mL

2.00E+04

1.50E+04

1.00E+04

5.00E+03

0.00E+00
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
OD
Growth Conditions
 The Great Plate Count Anomaly
• Plate counts can be highly unreliable when used to assess total cell numbers
of natural samples such as soil and water. Direct microscopic counts of natural
samples typically reveal far more organisms than are recoverable on plates of
any given culture medium.

•This is referred to as "the great plate count anomaly," and it occurs because
microscopic methods count dead cells whereas viable methods do not

•In addition, different organisms may have vastly different requirements for
resources and conditions in laboratory culture.
Understanding Bacterial Growth Factors in the Environment

• What are important environmental parameters/factors for

growth?
Understanding Bacterial Growth Factors in the Environment

• What are important environmental parameters for growth?

• Temperature
• pH
• Salinity
• Oxygen (biostimulation)
• Nutrients (biostimulation)
Temperature
 Temperature
• Temperature is a major environmental factor controlling
microbial growth. The cardinal temperatures are the
minimum, optimum, and maximum temperatures at which
each organism grows.
 Microorganisms can be grouped by
the temperature ranges they require.
• Mesophiles, which have midrange temperature optima, are found
in warm-blooded animals and in terrestrial and aquatic environments
in temperate and tropical latitudes.

• Extremophiles have evolved to grow optimally under very hot or

very cold conditions.
•Organisms with cold temperature optima are called psychrophiles, and the
most extreme representatives inhabit permanently cold environments.
•Psychrophiles have evolved biomolecules that function best at cold
temperatures but that can be unusually sensitive to warm temperatures.
Organisms that grow at 0ºC but have optima of 20ºC to 40ºC are called
psychrotolerant.
•Organisms with growth temperature optima between 45ºC and 80ºC are
called thermophiles, and those with optima greater than 80°C are called
hyperthermophiles.
How would you design an experiment to test
an organism’s temperature range?
How would you design an experiment to test
an organism’s temperature range?

• Grow samples at a range of temperatures

• Have replicates at each temperature (3+)
• Monitor growth
• In liquid using a spectrophotometer
• Plates +/- growth
Example Data Set
0.7
20
0.6 25

0.5 30
OD at 625 nm

35
0.4
40
0.3 45

0.2

0.1

0
0 10 20 30 40
Hours

Figure 2.2. B. species WAO growth at six temperatures was determined by monitoring the change in OD at 625 nm in
trypticase soy broth. Temperature ranged from 20-45ºC at 5 ºC increments. Each point represents three replicates with
error bars representing the standard deviation between replicates. (Walczak 2016)
Example Data Set
0.7 • Based on the data
20 set what is the
0.6 25 ideal temperature?
0.5 30
35
A) 20C
OD at 625 nm

0.4
40 B) 25C
0.3 45 C) 30C
0.2 D) 35C
0.1 E) 40C
0
0 10 20 30 40
Hours

Figure 2.2. B. species WAO growth at six temperatures was determined by monitoring the change in OD at 625 nm in
trypticase soy broth. Temperature ranged from 20-45ºC at 5 ºC increments. Each point represents three replicates with
error bars representing the standard deviation between replicates. (Walczak 2016)
Figure 9.28

• A black smoker at the bottom of the ocean belches hot, chemical-rich water, and
heats the surrounding waters. Sea vents provide an extreme environment that is
nonetheless teeming with macroscopic life (the red tubeworms) supported by an
abundant microbial ecosystem. (credit: NOAA)
pH
 pH
• The acidity or alkalinity of an
environment can greatly affect microbial
growth.

• Some organisms have evolved to grow

best at low or high pH, but most
organisms grow best between pH 6 and
8. The internal pH of a cell must stay
relatively close to neutral even though
the external pH is highly acidic or basic.

•Organisms that grow best at low pH are

called acidophiles; those that grow best
at high pH are called alkaliphiles.
 Figure 9.25, pH and Food preservation

• Lactic acid bacteria that ferment milk into yogurt or transform vegetables in pickles thrive
at a pH close to 4.0. Sauerkraut and dishes such as pico de gallo owe their tangy flavor to
their acidity. Acidic foods have been a mainstay of the human diet for centuries, partly
because most microbes that cause food spoilage grow best at a near neutral pH and do not
tolerate acidity well. (credit “yogurt”: modification of work by “nina.jsc”/Flickr; credit
“pickles”: modification of work by Noah Sussman; credit “sauerkraut”: modification of
work by Jesse LaBuff; credit “pico de gallo”: modification of work by “regan76”/Flickr)
Example Data Set
1.8 • Based on the data
pH 4.5-5
1.6 set what is the
1.4
pH 5.5
ideal pH range?
1.2
pH 6.0
A) 5.5
pH 6.5
B) 6.0
OD 625 nm

1
pH 7.0-8.0
0.8 C) 6.5
pH 8.5-9
0.6 D) 7-8
0.4
E) 8.5-9
0.2

0
0 10 20 30 40 50 60 70
Hours

Figure 2.3. B. species WAO growth at ten pH values was determined by monitoring the change in OD at 625 nm in trypticase soy broth. The pH
values ranged from 4.5- 9 pH and increased by 0.5 pH increments. Data was grouped when similar growth response was obtained. Each point
represents a minimum of three replicates per pH concentration with error bars representing the standard deviation between replicates. (Walczak 2016)
Osmolarity
 Salinity and Osmolarity
• Some microorganisms (halophiles) have evolved to
grow best at reduced water potential, and some
(extreme halophiles) even require high levels of salts
for growth.

• Halotolerant organisms can tolerate some reduction

in the water activity of their environment but generally
grow best in the absence of the added solute.
 Figure 3.15

• In cells that lack a cell wall, changes in osmotic pressure can lead to crenation
in hypertonic environments or cell lysis in hypotonic environments.
 Figure 3.16

• In prokaryotic cells, the cell wall provides some protection against changes in osmotic pressure, allowing it to
maintain its shape longer. The cell membrane is typically attached to the cell wall in an isotonic medium (left).
In a hypertonic medium, the cell membrane detaches from the cell wall and contracts (plasmolysis) as water
leaves the cell. In a hypotonic medium (right), the cell wall prevents the cell membrane from expanding to
the point of bursting, although lysis will eventually occur if too much water is absorbed.
Concentration increases
• Water activity becomes limiting to an organism
when the dissolved solute concentration in its
environment increases.

• Xerophiles are able to grow in very dry

environments.
 Figure 9.27

• View from space of Lake Natron in Tanzania. The pink color is due to the pigmentation of the
extreme alkaliphilic and halophilic microbes that colonize the lake. (credit: NASA)
• What type of conditions do these organisms grow in based on their classification?
Table 6.2 shows the water activity (aw) of
several substances.
Example Data Set
2
0% NaCl
• Based on the data
1.8 5% NaCl set what is the
1.6 10% NaCl ideal % added
1.4 15% NaCl
NaCl?
OD at 625nm

1.2 0% NaCl
1 1% NaCl a) 0%
2% NaCl
0.8
3% NaCl b) 1%
0.6
0.4
4% NaCl
c) 2%
0.2 d) 4%
0
0 10 20 30 40 50 60 70
e) 5%
Hours

Figure 2.4. B. species WAO growth at eight salinities was determined by monitoring the change in OD at 625 nm in trypticase soy broth with
additional NaCl from 0% - 15% weight/volume. Concentrations 0% (diamond), 5%, 10%, and 15% were completed with growth only observed in
the control 0% NaCl. A second round of growth curves with concentrations between 0% (circle) through 5% were completed. Each point
represents three replicates with error bars representing the standard deviation between replicates. (Walczak 2016)
Growth Conditions
Oxygen
 Oxygen and Microbial Growth
• Microorganisms require different environmental conditions for growth.
• Grow without Oxygen
• Obligate Anaerobes- Can not tolerate oxygen and use alternative terminal e-
acceptors
• Aerotolerant anaerobes- can grow in the presence of oxygen but do not use it for
growth
• Grow with Oxygen
• Obligate Aerobes- require oxygen for growth and use it as terminal e-acceptor for
respiration
• Microaerophiles- use lower than atmospheric oxygen for growth
• Facultative aerobes/anaerobes- grow with and without oxygen (fermentation)
• Metabolic Enzymes to remove free oxygen radicals (aerobes)
• Catalase- degrades hydrogen peroxide into oxygen and water
 Media and Components, Growth Conditions
• Special Media Components
• Resazurin- indicates if oxygen present(pink) or not(clear)
• Sodium thioglycolate- reduces free oxygen in the medium to water
• Media Types using these components
• Fluid thioglycolate medium- broth that allows growth of anaerobes in
aerobic conditions
• Brewers Agar- used to streak for growth in anaerobic jar
• Anaerobic Jar- Sealed incubation container
• GasPak- added to jar to create an anoxic/anaerobic environment by
generating hydrogen that binds with oxygen present to reduce it to
H2 O
 Special techniques are needed to grow
anaerobic microorganisms.

Anaerobic Jar Anaerobic Culture Anaerobic Chamber

Bottles
Growth in Fluid Thioglycolate
Medium
 Anaerobic Jar and Methylene Blue Strip

• Color of the methylene blue strip indicates if oxygen is present in the jar
 Example Experimental Set up
• 6 example micoorganisms divided into two sets
• Set 1: Escherichia coli, Bacillus megaterium, Clostridium acetobutylicum
• Set 2: Staphylococcus aureus, Clostridium sporogenes, Enterococcus faecalis
• Inoculations:
• 6 FTG tubes, (1/organism)
• Inoculate the FTM culture about a 3 rd of the way down the tube by adding 0.2mL of specific
culture
• 4 brewers plates- 2 per set of organisms (replicate plates)
• Per set one for aerobic environment and one for anaerobic environment
• Single streak across the plate with each
of the organisms done carefully not to
cross-contaminate

• Compare growth in the different oxygen requirements