microbial growth module 8

7/30/2019 Microbial Growth Module 8

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MICROBIAL GROWTH &

GENETICS


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GROWTH

Increase in cellular constituents


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Increase in size

or


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Increase in cell number


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REPRODUCTION OF

MICROBESAsexual

Modes:Binary fission

budding

FragmentationFormation of conidiospores / sporangiospores


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REPRODUCTION OF

MICROBES Binary fission

Bacteria


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REPRODUCTION OF


Paramecium


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REPRODUCTION OF


Giardia


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REPRODUCTION OF

MICROBES


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REPRODUCTION OF



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REPRODUCTION OF

MICROBES


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REPRODUCTION OF

MICROBES Budding

most commonly in

yeasts

small bud develops@ one end of cell

bud develops intonew cell


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REPRODUCTION OF

MICROBES Budding


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REPRODUCTION OF

MICROBES Fragmentation

occurs in

filamentous speciesone filament small

cells


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REPRODUCTION OF

MICROBES Fragmentation


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REPRODUCTION OF

MICROBES Conidiospores / sporangiospores

filamentous species


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REPRODUCTION OF


sporangiahypha


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REPRODUCTION OF


Spores are enclosedin a sheath


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POPULATION GROWTH

Means of characterizing specie

determine generation time

different species have differentgeneration times

Establish growth curve


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POPULATION GROWTH How do we do this?

Establish growth curve

Why are we able to do this?

Cell numbers increase by geometric progression

i.e 1 21

22

23

2n

(n = no of generations)


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POPULATION GROWTH

Closed system (batch culture)

Culture of microbes without removal of

waste or replenishment of nutrients


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POPULATION GROWTH Determination of generation time is done using

a batch culture

Growth curve


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POPULATION GROWTH Lag phase:

length varies

age of inoculumtype of medium

Time in min

Log cell no.

Young inoculum

Old inoculum


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POPULATION GROWTH Log phase / exponential:

inc @constant rate


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POPULATION GROWTH stationary phase:

constant no of cellsmax cell no. 109 / ml


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POPULATION GROWTH death phase: cell no. nutrients

waste products


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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 wecan determine g

number of generations within a specifictime period

the rate of growth (k)


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GENERATION TIME

Use exponentialphase


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GENERATION TIME

Need 2 time points on the exponential part of the graphT0 = 8 hours = 480 min Tt= 12 hours = 720 min


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GENERATION TIME How many generations are produced between the 2 time

points (i.e tt-t0)?Let No = initial cell no. @ T0 = 10 000Let Nt = cell no. @ Tt = 10 000 000

Nt = N0 x 2n

Log Nt = log N0 + n log 2

log Nt - log N0n =log 2


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GENERATION TIME log Nt - log N0

n =log 2

log 10 000 000 log 10 000n =

log 2

7 4 3n = = = 9.97 generations

0.301 0.301


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GENERATION TIME Next need to determine growth rate constant (k)i.e no of generations / unit time

n 9.97 generations 9.97 generations

k = = =t 720 - 480 min 240 min

k = 0.0415 generations / min


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GENERATION TIME Finally the generation time (g)

1 1g = =

k 0.0415 generations / min

g = 24.09 min / generation


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MEASURING GROWTH

Determine

cell numberor

cell massor

turbidity


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CELL NUMBER

Determine either

total count (live and dead bacteria)or

viable count (live bacteria only)


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TOTAL COUNT

for total count (live and dead bacteria) canuse

a counting chamber electronic counter


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TOTAL COUNT

a counting chamber

Improved neubauer

Modified fuchsin


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TOTAL COUNT

a counting chamber


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TOTAL COUNT

a counting chamber


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TOTAL COUNT

a counting chamber


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TOTAL COUNT

Area: 1 mm2

Area: 0.04 mm2


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TOTAL COUNT

Formula:counted cells x dilution factor

Cells / ml =area counted x chamber depth


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TOTAL COUNT

a counting chamberAdvantages:

QuickCheapEasy

Disadvantages:Inaccurate small volume sampled


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TOTAL COUNT

Electronic counter

Sample taken up via a probe

Electrodes on either side of probeElectrical resistance increases when acell passes electrodes


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TOTAL COUNT

Electronic counterAdvantages:

QuickEasy

Disadvantages:

probe cloggedExpensive


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VIABLE COUNT Can use

Spread / pour plates

membrane filter counts


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VIABLE COUNT Spread / pour plates

make dilutions


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VIABLE COUNT Spread / pour plates

make dilutions


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VIABLE COUNT Spread plate

add dilution to ready made agar plate


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VIABLE COUNT Spread plate

spread dilution across the agar surfaceincubate


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VIABLE COUNT Pour plate

add dilution to empty Petri dish


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add molten agar to the dilution & mix


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allow agar to solidify


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incubate


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VIABLE COUNT Spread / Pour plate

After incubation

Pour plate Spread plate


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After incubation, count the colonies


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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


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To calculate the original cell concentration

In 1 m there are cell count 590 coloniesDilution factor : 109

original cell concentration = 5.90 x 1011 / ml


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VIABLE COUNT Membrane filter counts:

samples with few bacteria eg water0.22 m filter


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a known volume is filtered


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filter is removedplaced on an agar plateincubated


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colonies counted

If 100 ml of water was filteredAnd 45 colonies grew on the membraneThen cell concentration is.

45 cells / 100 ml


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MULTIPLE

TUBETEST

qualitative test

MULTIPLE


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MULTIPLETUBE

TEST

Presumptive test

Coliforms?

MPN

lactose fermentation

Dilute water sample

1:2

1:10

1:100

MULTIPLE TUBE TEST


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MULTIPLE TUBE TEST

Acid & gas production

Presumptive test

MULTIPLE TUBE TEST


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MULTIPLE TUBE TEST

3 1 2

Presumptive test

Determine most probable number (MPN)

MULTIPLE TUBE TEST


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MULTIPLE TUBE TESTNo of tubes positive in

MPNNo of tubes positive in



MPN

Set 1 Set 2 Set 3 Set 1 Set 2 Set 3 Set 1 Set 2 Set 3 Set 1 Set 2 Set3

0 0 0 3 1 0 0 36 2 0 0 9.1 3 0 0 25

0 0 1 3 1 0 1 72 2 0 1 14 3 0 1 39

0 0 2 6 1 0 2 11 2 0 2 20 3 0 2 61

0 0 3 9 1 0 3 15 2 0 3 26 3 0 3 95

0 1 0 3 1 1 0 7.3 2 1 0 15 3 1 0 43

0 1 1 6.1 1 1 1 11 2 1 1 20 3 1 1 75

0 1 2 9.2 1 1 2 15 2 1 2 27 3 1 2 120

0 1 3 12 1 1 3 19 2 1 3 34 3 1 3 160

0 2 0 6.2 1 2 0 11 2 2 0 21 3 2 0 93

0 2 1 9.3 1 2 1 15 2 2 1 28 3 2 1 150

0

2

2

12

1

2

2

20

2

2

2

35

3

2

2

210

0 2 3 16 1 2 3 24 2 2 3 42 3 2 3 290

0 3 0 9.4 1 3 0 16 2 3 0 29 3 3 0 240

0 3 1 13 1 3 1 20 2 3 1 36 3 3 1 460

0 3 2 16 1 3 2 24 2 3 2 44 3 3 2 1100

0 3 3 19 1 3 3 29 2 3 3 53 3 3 3 2400


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CELL MASS

Wet weightscrape cells off agarweigh

Dry weightuse a broth culturewash cells in distilled water

Dry cells in an oven and weigh.


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TURBIDITY

Spectrophotometer is used to measureabsorbance of light in a tube of bacteriaConcentration of bacteria > 107/ml

All bacteria of the same specie have thesame sizeBacteria scatter lightThe greater the absorbance, the greater the

cell no.


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TURBIDITY

OD 550 600 nmFirst need to establish a std curve

Cell numberX 10 6

absorbance

(Obtained fromspread/pour plate)

(obtained from spec)


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METABOLIC ACTIVITY

Cell concentration may also be measured by

o ATP

o ATPase activity

o pH

o Oxygen production or consumptiono Carbon dioxide production or consumption


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McFarlands StandardsMcFarland

standard number0.5 1 2 3 4

Volume of 1 %

Barium chloride0.05 0.1 0.2 0.3 0.4

Volume of 1%sulphuric acid

9.95 9.9 9.8 9.7 9.6

Approximate cell

density

1x108CFU/ml

1.5 3 6 9 12

% Transmittance 74.3 55.6 35.6 26.4 21.5

Absorbance0.13

2

0.257 0.451 0.582 0.669


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ENVIRONMENTAL FACTORS

Water activitypHTemperature

OxygenPressure


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TEMPERATURE

Affects enzyme & protein activity temp disrupts membrane

Cardinal temp range


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TEMPERATURE

Used as a means of killing


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TEMPERATURE

Different species of bacteria grow @different temperatures


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OXYGEN


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OXYGEN


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OXYGEN


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OXYGEN


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OXYGEN


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OXYGEN


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pH

acidophile

neutrophile

alkalophile


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pH

Most bacteria grow at neutral pH


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pH


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pH

Enzyme activity is pH dependent


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pH

Adverse effects of great change in pHdamage to plasma membrane, enzymes &proteases

Internal pH kept neutralin neutrophiles by exchanging K+ for H+

extreme alkalophiles by exchanging Na+ (int)

for H+ ions (ext)


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pH

Microbes also contribute to change in pHproduce waste products

Adapt to change in environmental pH byusing K & Na ion gradientsother mechanisms

acidic tolerance response

chaperone proteins


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WATER ACTIVITY

awavailability of free water in themicrobes habitat

inversely related to osmotic Pa

Psolution vapour=

Pvapour pure water


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WATER ACTIVITY

Hypertonic environment has low aw

Would expect plasmolysis to occur

Bacteria avoid this by internal osmotic conc

Done by [compatible solute]


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WATER ACTIVITY

Most bacteria grow @ aw = 0.98


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WATER ACTIVITY

Osmotolerant microbes:grow over a wide range of awS. aureus

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


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WATER ACTIVITY

Extreme halophiles:increase their uptake of K+ to 4 7 M stabilise enzymes, ribosomes & permeases

Na+ stabilise cell wall, plasma membrane

microbial growth module 8

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