By Dr Norshuhaila Mohamed Sunar

AIM:

Ability to define, describe and utilize microbial quantification and growth system in biotechnology and biological process

name the various phases of growth that occur in closed culture systems and describe what is occurring in each phase

describe the number of generations, specific growth rate constant, mean generation time that describe microbial growth

compare and contrast the various methods for measuring microbial growth

describe the various types of continuous culture systems and explain the differences in their function

describe the influence of various environmental factors (water availability, pH, temperature, oxygen concentration, pressure, radiation) on the growth of microorganisms

categorize microorganisms according to the environmental factors that are conducive to optimal growth of the organism

Measuring microbial growth Direct measurement Indirect measurement Cell kinetic growth and metabolism Cell Death in Culture System

Growth requirement Sources of carbons, energy and electrons

Environmental effects on microbial growth Temperature pH, osmolarity and oxygen

Outline

The interval for the formation of two cells from one is called a generation

The time required for this to occur is called the generation time.

Generation time is the time required for the cell population to double (the cell mass doubles during this period as well).

Because of this, the generation time is also called the doubling time.

In nature, microbial doubling times may be much longer than those obtained in laboratory culture.

This is because in nature, ideal growth conditions for a given organism may exist only intermittently.

Depending on resource availability, physiochemical conditions (temperature, pH, and the like), moisture availability, and seasonal changes, bacterial populations in nature double only once every few weeks, or even longer.

observed when microorganisms are cultivated in batch culture culture incubated in a closed vessel with a

single batch of medium

usually plotted as logarithm of cell number versus time

usually has four distinct phases

Lag Adapt to nutrients

Log Active growth

Stationary Death = Growth rate

Death Nutrients consumed pH too low

Optimize curves in production

Log Growth

The ideal growth curve for cells in culture

the cells are adjusting to their new environment

most cells do not reproduce immediately, but instead actively synthesize enzymes to utilize novel nutrients in the medium.

bacteria inoculated from a medium containing glucose as a carbon source into a medium containing lactose must synthesize two types of proteins:

membrane proteins to transport lactose into the cell

the enzyme lactase to catabolize the lactose.

bacteria synthesize the necessary chemicals for conducting metabolism in their new environment, and they then enter a phase of rapid chromosome replication, growth, and reproduction.

population increases logarithmically

reproductive rate reaches a constant as DNA protein syntheses are maximized.

preferred for Gram staining because most cells' walls are intact – an important characteristic for correct staining.

the metabolic rate of individual cells is at a maximum during log phase

this phase is sometimes preferred for industrial and laboratory purposes.

If bacterial growth continued at the exponential rate of the log phase, bacteria would soon overwhelm the earth.

does not occur because as nutrients are depleted and wastes accumulate, the rate of reproduction decreases.

the number of dying cells equals the number of cells being produced, and the size of the population becomes stationary

During this phase the metabolic rate of surviving cells declines.

The onset of the stationary phase can be postponed indefinitely by a special apparatus called a chemostat, which continually removes wastes (along with old medium and some cells) and adds fresh medium.

Chemostats are used in industrial fermentation processes.

total number of viable cells remains constant may occur because metabolically active

cells stop reproducing may occur because reproductive rate is

balanced by death rate

nutrient limitation limited oxygen availability toxic waste accumulationcritical population density reached

If nutrients are not added and wastes are not removed, a population reaches a point at which cells die at a faster rate than they are produced.

Such a culture has entered the death phase (or decline phase).

during the death phase, some cells remain alive and continue metabolizing and reproducing, but the number of dying cells exceeds the number of new cells produced, so that eventually the population decreases to a fraction of its previous abundance.

In some cases, all the cells die, while in others a few survivors may remain indefinitely. The latter case is especially true for cultures of bacteria that can develop resting structures called endospore

two alternative hypotheses Cells are Viable But Not Culturable

(VBNC)▪ Cells alive, but dormant

programmed cell death Fraction of the population genetically

programmed to die (commit suicide)

A batch culture is a fixed volume of culture medium that is continually being altered by the metabolic activities of growing organisms and is therefore a closed system. A continuous culture is an open system of constant volume which fresh medium is added continuously and spent culture medium removed continuously, both at a constant rate.

Once such a system is in equilibrium, the chemostat volume, cell number, and nutrient status main constant, and the system is said to be in steady state.

The chemostat controls both the growth rate and the population density of the culture simultaneously

Two factors are important in such control: the dilution rate and the concentration of a limit

nutrient, such as a carbon or nitrogen source.

nutrient concentration can affect both the growth rate and the growth yield of a culture

growth rate and growth yield can be controlled independently of each other

Growth rate by adjusting the dilution rate and the growth yield by varying the concentration of a nutrient present in a limiting amount.

Applications such as :

the study of a particular enzyme

enzyme activities may be quite lower in stationary phase cells than in exponential phase cells and thus chemostat-grown cultures are ideal.

in microbial ecology

enrichment and isolation of bacteria

constant supply of cells in exponential phase growing at a known rate

study of microbial growth at very low nutrient concentrations, close to those present in natural environment

study of interactions of microbes under conditions resembling those in aquatic environments

food and industrial microbiology

“Continuous culture devices (chemostats) are a means of maintaining cell

populations in exponential growth for long periods. In a chemostat, the rate, and the population size is governed by the concentration of the growth-limiting

nutrient entering the vessel”

Differentiate the definition of batch cultures and continuous culture

What are the advantages of chemostat compare to batch culture.

A batch culture is a fixed volume of culture medium that is continually being altered by the metabolic activities of growing organism and is therefore a closed system. In the early stage of exponential growth in batch culture, conditions may remain relatively constant, but in later stages when cell numbers become quite large, drastic changes in the chemical composition of the culture medium occur.

A continuous culture is an open system of contrast volume to which fresh medium added continuously and spent culture medium is added continuously both at a constant rate . Once such a system is in equilibrium, the chemostate volume, cell number, and nutrient status remain constant, and the system is said to be in steady state.

The chemostat controls both the growth rate and the population density of the culture simultaneously . Two factors are important in such control: the dilution rate and the concentration of a limiting nutrient, such as a carbon or nitrogen source.

In a batch culture, nutrient concentration can affect both rate and the growth yield of a culture. At very low concentrations of a given nutrient, the growth rate is reduced, probably because the nutrient cannot be transported into the cell fast enough to satisfy metabolic demand. At moderate or higher nutrient levels, the growth rate may not be affected while the cell yield continues to increase.

In contrast to a batch culture, in a chemostat, growth rate and growth yield can be controlled independently of each other, the former by adjusting the dilution rate and the latter by varying the concentration of a nutrient present in a limiting amount. A practical advantage to the chemostat is that a population may be maintained in the exponential growth phase for long periods, thus, experiments can be planned in detail and then performed whenever most convenient.

viable plate countsmembrane filtrationmicroscopic counts the use of electronic counters the most probable number

method.

spread and pour plate techniques diluted 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)

What if the number of cells in even a very small sample is still too great to count?

for example, a 1-milliliter sample of milk containing 20,000 bacterial cells per ml were plated on a Petri plate, there would be too many colonies to count.

In such cases, we make a series of dilutions and count the number of colonies resulting on a spread or pour plate from each dilution.

We count the colonies on plates with 25-250 colonies and multiply the number by the reciprocal of the dilution to estimate the number of bacteria per ml of the original culture.

This method is called a viable plate count

The accuracy of a viable plate count is also dependent on the homogeneity of the dilutions, the ability of the bacteria to grow on the

medium used, the number of cell deaths, and the growth phase of the sample population.

Thoroughly mixing each dilution, inoculating multiple plates per dilution, and using log-phase cultures minimize errors.

In this method, a large sample (perhaps as large as several liters) is poured (or drawn under a vacuum) through a membrane filter with pores small enough to trap the cells.

The membrane is then transferred onto a solid medium, and the colonies present after incubation are counted. In this case, the number of colonies is equal to the number of CFUs in the original large sample.

simple and sensitivewidely used for viable counts of

microorganisms in food, water, and soil

inaccurate results obtained if cells clump together

easy, inexpensive, and quick

useful for counting both eucaryotes and procaryotes

cannot distinguish living from dead cells

Specialized chamber with etched grid used to count the number of cells in a sample.

use of trypan blue allows differentiation between living and dead cells

Diagram represent cell count using hemocytometer.

Remove the hemacytometer and coverslip (carefully) in solution and dry thoroughly with a kimwipe.

Center coverslip on hemacytometer

Barely fill the grid under the coverslip via the divet with your cell suspension.

Count cells in ten squares (5 on each side) by following diagram at station.

Bright refractile “spheres” are living cells,

Blue cells about the same size as the other cells are dead.

Keep a differential count of blue vs. clear for viability determination.

Sometimes there will be serum debris, and this will look red or blue and stringy粘性的 or gloppy--don’t count it!

These are blood cells,You will not have this many

Count 10 squaresAny 10 will do but we will follow convention

Watch for stringy, reddishmaterial—those aren’t cells!

serum

Top group

Count cells thattouch top and left lines

DO NOTCount cells thattouch bottom and right lines

useful for large microorganisms and blood cells, but not procaryotes

microbial suspension forced through small orifice

movement of microbe through orifice impacts electric current that flows through orifice

instances of disruption of current are counted

it is less useful for bacterial counts because of debris in the media and the presence of filaments and clumps of cells.

Cellometer lets you: • View cell morphology, for visual confirmation after cell counting • Take advantage of 300+ cell types and easy, wizard-based parameter set-up • Save sample images with results securely on your computer, plus autosave results on the network for added convenience and data protection

is a variation of counting with a Coulter counter.

A cytometer uses a light-sensitive detector to record changes in light transmission through the tube as cells pass.

a statistical estimation technique based on the fact that the more bacteria in a sample, the more dilutions are required to reduce their number to zero.

Metabolic Activity Dry Weight Turbidity

Under standard temperature conditions, the rate at which a population of cells utilizes nutrients and produces wastes is dependent on their number.

Once they establish the metabolic rate of a microorganism, scientists can indirectly estimate the number of cells in a culture by measuring changes in such things as nutrient utilization, waste production, or pH.

The abundance of some microorganisms, particularly filamentous 丝状 microorganisms, is difficult to measure by direct methods.

Instead, these organisms are filtered from their culture medium, dried, and weighed.

The dry weight method is suitable for broth cultures, but growth can not be followed over time because the organisms are killed during the process

As bacteria reproduce in a broth culture, the broth often becomes turbid (cloudy)

An indirect method for estimating the growth of a microbial population involves measuring changes in turbidity using a device called a spectrophotometer

more cells

more lightscattered 分散

less lightdetected

dry weight time consuming and not very sensitive

quantity of a particular cell constituent e.g., protein, DNA, ATP, or chlorophyll useful if amount of substance in each

cell is constant

turbidometric measures (light scattering) quick, easy, and sensitive

Sources of carbon, energy and electrons

- From inorganic source (i.e CO2) that called autotrophs.

- From organic molecules (proteins, carbohydrates) they acquire from other organisms.We called as heterotrophs.

- From redox reactions involving inorganic and organic chemicals that called chemotrophs.

- From light source that called as phototrophs.

-Therefore based on carbon and energy sources, microbes an be categorized into one of four basic groups:

i. Photoautotrophsii. Chemoautotrophsiii. Photoheterotrophsiv. Chemoheterotrophs

- From same organic molecules that provide C and energy are called organotrophs.

- From inorganic sources (H2, NO2-, H2S) are called lithotrophs.

•Temperature• pH•water availability•oxygen

minimum temperature below which growth no longer occurs,

an optimal temperature at which growth is most rapid, and

maximum temperature above which growth is not possible

cardinal temperatures

an characteristic of each organism but are not completely: fixed entities, as they can be modified slightly by other factors of the environment, in particular, by the composition of the growth medium.

Optimum growth temperature classifications Close to upper range of growth temperatures Too high and enzymes cease to function

(denaturation)

4 groups:

Psychrophiles (-5°C to 15°C)▪ Arctic and Antarctic bacteria▪ Spoilage of refrigerated goods▪ Some Pseudomonas sp.

Mesophiles (25°C to 45°C)▪ E. coli and many pathogens▪ Core body temp is 37°C and extremities cooler▪ Mycobacterium leprae (leprosy) attacks extremites but not

main trunk▪ Syphilis treatment was heat shock- induce fever or hot spas

Thermophiles (45°C to 70°C)▪ Hot springs▪ Compost heaps 堆肥▪ Water heaters▪ Lactobacillus delbrueckii (bulgaricus)-

yogurt production

Hyperthermophiles (70°C to 110°C)▪ Many Archaea 古▪ Hydrothermal vents of ocean floors▪ Pyrolobus fumarimii up to 113°C

Solute concentrations in the cell’s exterior environment can cause water to diffuse out of the cell- plasmolysis and cytoplasmic membrane shrinks away from cell walls

Some cells avoid plasmolysis by increasing solute concentration inside cells

Osmotolerant organisms-(to about 10% NaCl) Staphylococcus sp

Halophiles – require NaCl ions (> 20% NaCl) Many Archaea

Develop specialized processes to transport H+ across the cytoplasmic membrane to maintain a nearly neutral internal pH

Like temperature, there are optimum growth conditions

3 major groups: Neutralophiles (optimum pH 7)

▪ Most bacteria Acidophiles (optimum pH below 5.5) Alkalophiles (optimum pH above 8.5)

Acido- or alkalotolerant organisms can survive in the pH environments, however do not grow (multiply) under those conditions

O2 concentration varies 20% of earth’s atmosphere Subsurfaces of soils and some aquatic habitats

have much less Stomach and intestines are devoid of O2

Determining requirement: absolutes and tolerances Shake tubes:

solid medium boiled to drive off O2 cooled but molten agar is inoculated with bacteria

and bacteria distributed throughout medium agar solidifies impeding(hinder) diffusion of O2 into

bottom of tube (stratification of O2) watch for region of bacterial growth

Obligate aerobes Need O2 for aerobic respiration to produce

cellular energy Pseudomonas sp.

Obligate anaerobes Cannot multiply if O2 is present Cellular energy produced by anaerobic

respiration or fermentation Bacterioides sp. and Clostridium sp.

Facultative anaerobes Grow best if O2 is present but also grow without it Use aerobic respiration if O2 is present, otherwise

undergo fermentation or anaerobic respiration Aerobic respiration provides more ATP = better growth E. coli and Saccharomyces cervisiae

Microaerophiles Requires low levels of O2 for aerobic respiration High concentrations of O2 are inhibitory Helicobacter pylori

Aeroltolerant anaerobes Can grow in presence of O2 but do not use it for energy

production Use only fermentative pathways = obligate fermenters Streptococcus pyogenes (strep throat)

Leibig’s law of the minimum total biomass of organism determined by

nutrient present at lowest concentration

Shelford’s law of tolerance above or below certain environmental

limits, a microorganism will not grow, regardless of the nutrient supply

organisms become more competitive in nutrient capture and use of available resources

morphological changes to increase surface area and ability to absorb nutrients

mechanisms to sequester 隔离 certain nutrients

stressed microorganisms can temporarily lose ability to grow using normal cultivation methods

microscopic and isotopic methods for counting viable but nonculturable cells have been developed

difficult to culture organisms from natural environments

previously stressed microbes are very sensitive to secondary stresses and may not grow on media normally used to cultivate them

Postgate Microviability Assay uses change in morphology on agar surface as indication of “life signs”

Extra reading:

http://www.dbkgroup.org/Papers/kell_viability_avl98.pdf