che 315: microbiology - wordpress.com 315: microbiology microbial nutrition and growth •growth...

CHE 315: Microbiology

Microbial Nutrition and Growth • Growth requirements and classification

• Physical parameters that effect growth and classification based on growth patterns

• Chemical parameters that effect growth and classification based on growth patterns

• Population growth -- growth curve

• Population growth -- Methods

1 9/16/2015 Microbiology CHE 315 AP Wacoo

Environmental Effects on Bacterial Growth

• Temperature

• pH

• Osmotic pressure

• Oxygen classes


Temperature and Microbial Growth

• Cardinal temperatures

– minimum

– optimum

– maximum

• Temperature is a major environmental factor controlling microbial growth.


Temperature

• Minimum Temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow.

• Optimum Temperature: Temperature at which its growth rate is the fastest.

• Maximum Temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow.


Classification of Microorganisms by Temperature Requirements


Temperature Classes of Organisms • Mesophiles ( 20 – 45C)

– Midrange temperature optima – Found in warm-blooded animals and in terrestrial and

aquatic environments in temperate and tropical latitudes • Psychrophiles ( 0-20C)

– Cold temperature optima – Most extreme representatives inhabit permanently cold

environments • Thermophiles ( 50- 80C)

– Growth temperature optima between 45ºC and 80ºC • Hyperthermophiles

– Optima greater than 80°C – These organisms inhabit hot environments including boiling

hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC


pH and Microbial Growth

• The (measure of [H+]) acidity or alkalinity of an environment

can greatly affect microbial growth. Internal pH regulated

by BUFFERS and near neutral adjusted with ion pumps

• Most organisms grow best between pH 6 and 8, but some

organisms have evolved to grow best at low or high pH. The

internal pH of a cell must stay relatively close to neutral even

though the external pH is highly acidic or basic.

– Acidophiles (optimum in pH range 1-4) : organisms that

grow best at low pH( Helicobacter pylori, Thiobacillus

thiooxidans, lactic acid bacteria – 4-7 )

– Alkaliphiles (optimum in pH range 8.5-11): organismsa

that grow best at high pH ( Vibrio cholera)

– Most of pathogenic bacteria are neutrophiles 7 9/16/2015 Microbiology CHE 315 AP Wacoo

Osmotic Effects on Microbial Growth

• Osmotic pressure depends on the surrounding solute concentration and

water availability

• Water availability is generally expressed in physical terms such as water

activity (aw)

• Water activity is the ratio of the vapor pressure of the air in equilibrium

with a substance or solution to the vapor pressure of pure water ( aw

1.00).

aw

= P solu

P water


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Environmental factors and growth

1. Osmotic Effect and water activity

organisms which thrive in high solute – osmophiles

organisms which tolerate high solute – osmotolerant

organisms which thrive in high salt – halophiles

organisms which tolerate high salt – halotolerant

organisms which thrive in high pressure – barophiles

organisms which tolerate high pressure – barotolerant

9/16/2015 Microbiology CHE 315 AP Wacoo

Microbial Nutrition

The hundreds of chemical compounds present inside a living cell are formed from nutrients.

• Macronutrients : elements required in fairly large amounts

• Micronutrients : metals and organic compounds needed in very small amounts


Main Macronutrients

Carbon:

(C, 50% of dry weight) and nitrogen (N, 12% of dry

weight)

• Virtually all chemical substances in microorganisms contain carbon in some form, whether they are proteins, carbohydrates, or lipids. 50% of a bacterium's dry weight is carbon. Carbon can be obtained from organic materials in the environment, or it may be derived from carbon dioxide.

• Autotrophs are able to build all of their cellular organic

molecules from carbon dioxide


Classification of organisms based on sources of C and energy used


Nitrogen:

• is used for the synthesis of proteins, amino acids, DNA, and RNA. Bacteria that obtain nitrogen directly from the atmosphere are called nitrogen-fixing bacteria. They include species of Rhizobium and Azotobacter, both found in the soil.

• Most Bacteria can use Ammonia -NH3 and many can

also use NO3-

• Nitrogen fixers can utilize atmospheric nitrogen (N2)


Classification of organisms based on O2 utilization

• Utilization of O2 during metabolism yields toxic by-products including O2

-, singlet oxygen (1O2) and/or H2O2.

• Toxic O2 products can be converted to harmless substances if the organism has catalase (or peroxidase) and superoxide dismutase (SOD)

• SOD converts O2- into H2O2 and O2

• Catalase breaks down H2O2 into H2O and O2

• Any organism that can live in or requires O2 has SOD and catalase (peroxidase)


Classification of organisms based on O2 utilization

• Obligate (strict) aerobes require O2 in order to grow

• Obligate (strict) anaerobes cannot survive in O2

• Facultative anaerobes grow better in O2

• Aerotolerant organisms don’t care about O2

• Microaerophiles require low levels of O2 17 9/16/2015 Microbiology CHE 315 AP Wacoo

Toxic Forms of Oxygen and Detoxifying Enzymes

18

Hydrogen

peroxide

Superoxide


19

Environmental factors and growth

4. Oxygen

anaerobes lack superoxide dismutase and/or catalase

anaerobes need high -, something to remove O2

chemical: thioglycollate; pyrogallol + NaOH

H2 generator + catalyst

physical: removal/replacement


20

Special Culture Techniques

Candle Jar


21

Special

Culture

Techniques

Gas Pack

Jar Is Used

for

Anaerobic

Growth 9/16/2015 Microbiology CHE 315 AP Wacoo

Other Macronutrients

• Phosphate (P), sulfur (S), potassium (K), magnesium

(Mg), calcium (Ca), sodium (Na), iron (Fe)

• Iron plays a major role in cellular respiration, being a

key component of cytochromes and iron-sulfur

proteins involved in electron transport.

• Siderophores : Iron-binding agents that cells produce

to obtain iron from various insoluble minerals.


Microbial growth

• Microbes grow via binary fission, resulting in exponential increases in numbers

• The number of cell arising from a single cell is 2n after n generations

• Generation time is the time it takes for a single cell to grow and divide


24

Binary Fission


25

Rapid Growth of Bacterial Population


Growth curve

• During lag phase, cells are recovering from a period of no growth and are making macromolecules in preparation for growth i.e.

• no apparent cell division occurring but the cells are growing in volume or mass, synthesizing enzymes, proteins, RNA e.t.c and increasing in metabolic activity. The duration of lag phase depends on;

• Inoculum size

• Recovery period of cells from physical damage or shock during transfer to a fresh medium

• Time required for synthesis of new inducible enzymes necessary to metabolize substrates present in a medium


log phase; • cultures are growing maximally • All cells are dividing at a constant rate and the population is

growing by geometric progression • Composition of medium and the conditions of incubation

determine the rate of cell division • Rate of exponential growth of a bacterial culture is expressed as

generation time or doubling time of the bacterial population • Generation time G is defined as the time (t) per generation (n-

number of generations). Hence, G = t/n The Stationary phase; • occurs when nutrients are depleted and wastes accumulate

(Growth rate = death rate) • Exhaustion of available nutrients • Accumulation of inhibitory metabolites or end products • Exhaustion of space (biological space)


In this phase, the rate of cell division = rate of cell death and the bacteria population remains constant. Some bacteria produce secondary metabolites i.e metabolites produced after the active stage of growth (antibiotics and pigments). In spore-forming bacteria, the sporulation process is induced during the stationary phase. Death phase/decline phase:- If the incubation continues after the stationary phase, death phase follows. Viable cell population declines. Turbidimetric measurements or microscopic counts cannot detect death phase in culture since living and dead cells are counted. In death phase, number of viable cells decreases geometrically or exponentially. It is the reverse of growth during the log phase.


Calculation of Generation Time If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide. G (generation time) = (time, in minutes or hours)/n(number of generations) G = t/n t = time interval in hours or minutes B = number of bacteria at the beginning of a time interval b = number of bacteria at the end of the time interval n = number of generations (number of times the cell population doubles during the time interval) b = B x 2n (This equation is an expression of growth by binary fission)


Example:

What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth?


Methods used to measure microbial growth Growth;-- the formation of new protoplasm from available nutrients involves the increase in cell mass and the number. The time interval for a bacterial cell to divide into 2 new cells or for a population of bacterial cells to double is called generation time. Generation time for bacteria growing under natural conditions ranges from a few minutes to several days. In bacteria and other unicellular organisms, growth can be measured in terms of 2 parameters;

a) Changes in cell mass

b) Changes in cell numbers


Measurement of cell mass This may be direct or indirect. • Direct determination of cell dry weight, wet weight or volume of

cells is done after centrifugation. This is a physical method. The dry weight of the bacterial mass is directly proportional to their number.

• Direct determination of a chemical constituent of the cells e.g. total N, total protein or total DNA content. This is a chemical method. Total N can be determined by the micro-Kjeldahl method.

• Indirect measurement of a chemical activity e.g. the rate of respiration using optical oxygen sensors, e.t.c.

• Turbidity measurements involve the use of an instrument such as a spectrophotometer to determine the amount of light scattered by cell suspensions. Cells such as bacteria scatter light in proportion to their numbers. Turbidity of a suspension of cells is directly related to the cell mass/cell number. 32 9/16/2015 Microbiology CHE 315 AP Wacoo

Methods used to measure microbial growth

• Count colonies on plate or filter (counts live cells)

Indirect viable cell counts/plate counts methods. This involves inoculating a known volume of culture on a suitable growth medium in plates. Each viable cell grows into a colony. Colonies are then counted to determine the number of colony forming units (CFUs) and hence the viable number of bacteria/unit volume can be calculated.

• Microscopic counts

• Flow cytometry (FACS)

• Turbitity


Viable counts

• Each colony on plate or filter arises from single live cell

• Only counting live cells


35

Direct Count

Pour Plate


Microscopic counts

37

Direct microscopic counts are possible using special slides known as

counting chambers e.g. Petroff-Hausser counting chambers. The

disadvantage is that both dead and living cells are counted. Only

dense populations can be counted.


Turbitity

• Cells act like large particles that scatter visible light

• A spectrophotometer sends a beam of visible light through a culture and measures how much light is scattered

• Scales read in either absorbance or % transmission

• Measures both live and dead cells


Inoculation

• Sample is placed on sterile medium providing microbes with the appropriate nutrients to sustain growth.

• Selection of the proper medium and sterility of all tools and media is important.

• Some microbes may require a live organism or living tissue as the inoculation medium.


Incubation

• An incubator can be used to adjust the proper growth conditions of a sample.

• Need to adjust for optimum temperature and gas content.

• Incubation produces a culture – the visible growth of the microbe on or in the media


Isolation

• The end result of inoculation and incubation is isolation.

• On solid media we may see separate colonies, and in broth growth may be indicated by turbidity.

• Sub-culturing for further isolation may be required.


Inspection

• Macroscopically observe cultures to note color, texture, size of colonies, etc.

• Microscopically observe stained slides of the culture to assess cell shape, size, and motility.


Identification • Utilize biochemical tests to differentiate the

microbe from similar species and to determine metabolic activities specific to the microbe.


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