MICROBIAL GROWTH & GENETICS

GROWTH

Increase in cellular constituents

 Increase in size

or

 Increase in cell number

REPRODUCTION OF MICROBES
Asexual

Modes: Binary fission budding Fragmentation Formation of conidiospores / sporangiospores

REPRODUCTION OF MICROBES
Binary fission

Bacteria

REPRODUCTION OF MICROBES
Binary fission

Paramecium

REPRODUCTION OF MICROBES
Binary fission

Giardia

REPRODUCTION OF MICROBES

REPRODUCTION OF MICROBES
Binary fission

REPRODUCTION OF MICROBES

REPRODUCTION OF MICROBES
Budding most commonly in yeasts small bud develops @ one end of cell

bud develops into new cell

REPRODUCTION OF MICROBES
Budding

REPRODUCTION OF MICROBES
Fragmentation
occurs in filamentous species one filament small cells

REPRODUCTION OF MICROBES
Fragmentation

REPRODUCTION OF MICROBES
Conidiospores / sporangiospores filamentous species

REPRODUCTION OF MICROBES
Conidiospores / sporangiospores

hypha

sporangia

REPRODUCTION OF MICROBES
Conidiospores / sporangiospores

Spores are enclosed in a sheath

POPULATION GROWTH
Means of characterizing specie determine generation time different species have different generation times Establish growth curve

How do we do this?

POPULATION GROWTH
Establish growth curve

Why are we able to do this? Cell numbers increase by geometric progression i.e 1 2122232n (n = no of generations)

POPULATION GROWTH
Closed system (batch culture) Culture of microbes without removal of waste or replenishment of nutrients

Determination of generation time is done using a batch culture

Growth curve

POPULATION GROWTH

POPULATION GROWTH
Lag phase: length varies age of inoculum type of medium
Log cell no. Old inoculum Time in min Young inoculum

POPULATION GROWTH
Log phase / exponential: inc @constant rate

POPULATION GROWTH
stationary phase: constant no of cells max cell no. 109 / ml

POPULATION GROWTH
death phase: cell no. nutrients waste products

GENERATION TIME
aka doubling time = g This is the length of time it takes for a population of cells to double in number. Need to determine 2 parameters before we can determine g number of generations within a specific time period the rate of growth (k)

GENERATION TIME
Use exponential phase

GENERATION TIME

Need 2 time points on the exponential part of the graph T0 = 8 hours = 480 min Tt= 12 hours = 720 min

GENERATION TIME
How many generations are produced between the 2 time points (i.e tt-t0)? Let No = initial cell no. @ T0 = 10 000 Let Nt = cell no. @ Tt = 10 000 000 Nt = N0 x 2n Log Nt = log N0 + n log 2 n= log Nt - log N0

log 2

GENERATION TIME
n= log Nt - log N0 log 2

n= 74

log 10 000 000 log 10 000 log 2 3 = 9.97 generations

n=

0.301

0.301

GENERATION TIME
Next need to determine growth rate constant (k) i.e no of generations / unit time n t 9.97 generations 720 - 480 min 9.97 generations 240 min

k=

k = 0.0415 generations / min

GENERATION TIME
Finally the generation time (g) g=
1 k

1 0.0415 generations / min

g = 24.09 min / generation

MEASURING GROWTH
Determine cell number or cell mass or turbidity

CELL NUMBER
Determine either
total count (live and dead bacteria) or viable count (live bacteria only)

TOTAL COUNT
for total count (live and dead bacteria) can use a counting chamber electronic counter

TOTAL COUNT
a counting chamber
Improved neubauer

Modified fuchsin

TOTAL COUNT
a counting chamber

TOTAL COUNT
a counting chamber

TOTAL COUNT
a counting chamber

TOTAL COUNT

Area: 0.04 mm2

Area: 1 mm2

TOTAL COUNT
Formula: Cells / ml =

counted cells x dilution factor

area counted x chamber depth

TOTAL COUNT
a counting chamber Advantages: Quick Cheap Easy Disadvantages: Inaccurate small volume sampled

TOTAL COUNT

Electronic counter Sample taken up via a probe Electrodes on either side of probe Electrical resistance increases when a cell passes electrodes

TOTAL COUNT
Electronic counter Advantages: Quick Easy Disadvantages: probe clogged Expensive

VIABLE COUNT
Can use Spread / pour plates

membrane filter counts

VIABLE COUNT
Spread / pour plates make dilutions

VIABLE COUNT
Spread / pour plates make dilutions

VIABLE COUNT
Spread plate add dilution to ready made agar plate

VIABLE COUNT
Spread plate spread dilution across the agar surface incubate

VIABLE COUNT
Pour plate add dilution to empty Petri dish

VIABLE COUNT
Pour plate add molten agar to the dilution & mix

VIABLE COUNT
Pour plate allow agar to solidify

VIABLE COUNT
Pour plate incubate

VIABLE COUNT
Spread / Pour plate After incubation

Pour plate

Spread plate

VIABLE COUNT
Spread / Pour plate After incubation, count the colonies

VIABLE COUNT
Spread / Pour plate
To calculate the original cell concentration
In 100 there are cell count 70 colonies in 1 m there are 70 x 10 colonies = 700 colonies dilution factor : 109 original cell concentration = 7.00 x 1011 / ml

VIABLE COUNT
Spread / Pour plate
To calculate the original cell concentration
In 1 m there are cell count 590 colonies Dilution factor : 109 original cell concentration = 5.90 x 1011 / ml

VIABLE COUNT
Membrane filter counts: samples with few bacteria eg water 0.22 m filter

VIABLE COUNT
Membrane filter counts: a known volume is filtered

VIABLE COUNT
Membrane filter counts: filter is removed placed on an agar plate incubated

VIABLE COUNT
Membrane filter counts: colonies counted
If 100 ml of water was filtered And 45 colonies grew on the membrane Then cell concentration is. 45 cells / 100 ml

MULTIPLE TUBE TEST

qualitative test

MULTIPLE TUBE TEST

Presumptive test
Coliforms? MPN lactose fermentation
Dilute water sample 1:2 1:10 1:100

MULTIPLE TUBE TEST

Presumptive test

Acid & gas production

MULTIPLE TUBE TEST

Presumptive test

Determine most probable number (MPN)

MULTIPLE TUBE TEST

MPN No of tubes positive in Set 1 Set 2 Set 3 No of tubes positive in Set 1 Set 2 Set 3 MPN No of tubes positive in Set 1 Set 2 Set 3 MPN No of tubes positive in Set 1 Set 2 Set 3 MPN

0 0 0

0 0 0

0 1 2

3 3 6

1 1 1

0 0 0

0 1 2

36 72 11

2 2 2

0 0 0

0 1 2

9.1 14 20

3 3 3

0 0 0

0 1 2

25 39 61

0
0 0 0 0 0 0 0 0 0 0 0 0

0
1 1 1 1 2 2 2 2 3 3 3 3

3
0 1 2 3 0 1 2 3 0 1 2 3

9
3 6.1 9.2 12 6.2 9.3 12 16 9.4 13 16 19

1
1 1 1 1 1 1 1 1 1 1 1 1

0
1 1 1 1 2 2 2 2 3 3 3 3

3
0 1 2 3 0 1 2 3 0 1 2 3

15
7.3 11 15 19 11 15 20 24 16 20 24 29

2
2 2 2 2 2 2 2 2 2 2 2 2

0
1 1 1 1 2 2 2 2 3 3 3 3

3
0 1 2 3 0 1 2 3 0 1 2 3

26
15 20 27 34 21 28 35 42 29 36 44 53

3
3 3 3 3 3 3 3 3 3 3 3 3

0
1 1 1 1 2 2 2 2 3 3 3 3

3
0 1 2 3 0 1 2 3 0 1 2 3

95
43 75 120 160 93 150 210 290 240 460 1100 2400

CELL MASS
Wet weight scrape cells off agar weigh Dry weight use a broth culture wash cells in distilled water Dry cells in an oven and weigh.

TURBIDITY
Spectrophotometer is used to measure absorbance of light in a tube of bacteria Concentration of bacteria > 107/ml All bacteria of the same specie have the same size Bacteria scatter light The greater the absorbance, the greater the cell no.

TURBIDITY
OD 550 600 nm First need to establish a std curve

Cell number X 10 6 (Obtained from spread/pour plate) absorbance (obtained from spec)

METABOLIC ACTIVITY
Cell concentration may also be measured by o ATP

o ATPase activity
o pH

o Oxygen production or consumption

o Carbon dioxide production or consumption

McFarlands Standards
McFarland 0.5 standard number Volume of 1 % 0.05 Barium chloride Volume of 1% 9.95 sulphuric acid Approximate cell density 1.5 1x108CFU/ml % Transmittance 74.3 0.13 Absorbance 2 1 0.1 2 0.2 3 0.3 4 0.4

9.9
3 55.6 0.257

9.8
6 35.6 0.451

9.7
9 26.4 0.582

9.6
12 21.5 0.669

ENVIRONMENTAL FACTORS
Water activity pH Temperature Oxygen Pressure

TEMPERATURE
Affects enzyme & protein activity temp disrupts membrane

Cardinal temp range

TEMPERATURE
Used as a means of killing

TEMPERATURE
Different species of bacteria grow @ different temperatures

OXYGEN

OXYGEN

OXYGEN

OXYGEN

OXYGEN

OXYGEN

pH
acidophile

neutrophile

alkalophile

pH
Most bacteria grow at neutral pH

pH

pH
Enzyme activity is pH dependent

pH
Adverse effects of great change in pH damage to plasma membrane, enzymes & proteases Internal pH kept neutral in neutrophiles by exchanging K+ for H+ extreme alkalophiles by exchanging Na+ (int) for H+ ions (ext)

pH
Microbes also contribute to change in pH produce waste products Adapt to change in environmental pH by using K & Na ion gradients other mechanisms acidic tolerance response chaperone proteins

WATER ACTIVITY
aw availability of free water in the microbes habitat inversely related to osmotic Pa = Psolution vapour Pvapour pure water

WATER ACTIVITY
Hypertonic environment has low aw Would expect plasmolysis to occur Bacteria avoid this by internal osmotic conc Done by [compatible solute]

WATER ACTIVITY
Most bacteria grow @ aw = 0.98

WATER ACTIVITY
Osmotolerant microbes: grow over a wide range of aw

S. aureus

Halophiles: live in high salt conc (2.8 6.2 M) modify protein & membrane structure

WATER ACTIVITY
Extreme halophiles: increase their uptake of K+ to 4 7 M stabilise enzymes, ribosomes & permeases Na+ stabilise cell wall, plasma membrane