NUTRITION, ISOLATION, CULTIVATION AND COUNTING OF

BACTERIA

Dr. Jigar ShahIPNU

Chemical RequirementsCarbon: One of the most important requirement for growth Half the dry weight of a typical bacteria cell is carbon Chemoheterotrophs get most of their carbon from source of energy –

organic materials like proteins, carbohydrates, and lipids Chemoautotrophs and photoautotrophs derive from carbon dioxide

Nitrogen, Sulfur, and Phosphorus: Protein synthesis requires considerable amounts of nitrogen as well as

some sulfur Synthesis of DNA and RNA require nitrogen and some phosphorus Nitrogen makes up about 14% of the dry weight of a bacterial cell Sulfur and phosphorus together constitute about 4% Organisms use nitrogen primarily to form the amino group of amino

acids of proteins

Chemical Requirements…

Many bacteria meet this requirement by decomposing protein-containing material and reincorporating the amino acids into newly synthesized proteins and other nitrogen containing compounds.

Other bacteria use nitrogen from ammonium ions (NH4+), nitrates,

gaseous nitrogen (photosynthesizing cyanobacteria) from atmosphere. This process is called Nitrogen Fixation. The nitrogen fixed in the symbiosis is used by both the plant and the bacterium.

Sulfur is used to synthesize sulfur-containing amino acids and vitamins such as thiamine and biotin.

Phosphorus is essential for the synthesis of nucleic acids and the phospholipids of cell membranes.

An important source of phosphorus is the phosphate ion (PO4-3).

Potassium, magnesium, calcium, iron, copper, molybdenum, and zinc are also elements or trace elements that microorganisms require often as cofactors for functions of enzymes.

Chemical Requirements…Oxygen: Organisms that require oxygen to live are called obligate

aerobes. Many of the aerobic bacteria have developed, but retained ability to

continue growing in the absence of oxygen. Such organisms are called facultative anaerobes.

Means these organisms can use oxygen when it is present but are able to continue growth by using fermentation, or anaerobic respiration when oxygen is not available. E.g. E. coli.

However, their efficiency in producing energy decreases in absence of oxygen.

Obligate anaerobes are unable to use molecular oxygen for energy-yielding reaction, but are harmed by it. E.g. Clostridium titani.

Aerotolerant anaerobes can’t use oxygen for growth, but they tolerate it fairly well.

These bacteria characteristically ferment carbohydrates to lactic acid. As lactic acid accumulates, it inhibits the growth of aerobic competitors and established a favorable ecological environment for lactic acid producers.

Chemical Requirements… E.g. Lactobacilli – used in production of many acidic fermented

foods, such as pickles and cheese. These bacteria can tolerate oxygen because they possess Superoxide

dismutase (SOD) or an equivalent system that neutralizes the toxic forms of oxygen.

Microaerophiles are aerobic, require oxygen. They grow only in oxygen concentrations lower than those in air.

In a test tube of solid nutrient media, they grow only at a depth where small amounts of oxygen were diffused into the medium.

They do not grow near the oxygen – rich surface or below the narrow zone of adequate oxygen.

This limited tolerance is probably due to their sensitivity to superoxide free radicals and peroxides, which they produce in lethal concentrations under oxygen rich conditions.

Toxic forms of Oxygen Singlet oxygen is normal molecular oxygen (O2), extremely reactive. It is present in phagocytic cells, which play an important part in the

body’s defenses against pathogens such as bacteria. Once the phagocytic cell ingests bacteria, they are killed by exposure

to singlet oxygen. Superoxide free radicals (O2

-) formed in small amounts during normal respiration, are toxic to cellular component, organisms attempting to grow in atmospheric oxygen must produce an enzyme, superoxide dismutase (SOD), to neutralize them.

SOD convert superoxide free radical into molecular oxygen (O2) and hydrogen peroxide (H2O2).

E.g. Obligate aerobes, Facultative anaerobes, aerotolerant anaerobes. Hydrogen peroxide produced contains toxic peroxide anion (O2

-2). Microbes have developed enzymes catalase and peroxidase to

neutralize hydrogen peroxide.

Toxic forms of Oxygen…1) O2

- + O2- + 2H+ H2O2 + O2

2) 2 H2O2 2H2O + O2

3) H2O2 + 2H+ 2H2O Hydroxyl radical (OH.) is another intermediate form of oxygen and

probably the most reactive. It is formed in the cellular cytoplasm by ionizing radiation. Most

aerobic respiration produces traces of hydroxyl radicals but they are transient.

Chemical Requirements… Organic Growth Factors: Essential organic compounds, an organism is unable to synthesize are

know as organic growth factors. One group of organic growth factors is vitamins. Most vitamins function as coenzymes, the organic cofactors required

by certain enzymes in order to function. Many bacteria can synthesize all their own vitamins and are not

dependent on outside sources. Some bacteria lack the enzymes needed for the synthesis of certain

vitamins, and for them those vitamins are organic growth factors. Other organic growth factors required by some bacteria are amino

acids, purines, and pyrimidines.

CULTURE MEDIA A nutrient material prepared for the growth of microorganisms in a

laboratory is called a culture medium. When microbes are introduced into a culture medium to initiate

growth, they are called an inoculum. The microbes that grow and multiply in or on a culture medium are

referred to as a culture. Criteria for the culture medium to meet for particular specimen:1) Must contain the right nutrients for the specific microorganism,2) Contain sufficient moisture, properly adjusted pH, suitable level of O2

3) Medium must be sterile – contain no living microorganisms4) Growing culture should be incubated at the proper temperature. Media are constantly being developed or revised for use in the

isolation and identification of bacteria that are of interest to researchers in field such as food, water, and clinical microbiology.

CULTIVATIONChemically Defined Media: To support microbial growth, a medium must provide an energy

source, as well as sources of carbon, nitrogen, sulfur, phosphorus, and any organic growth factors the organism is unable to synthesize.

A chemically defined medium is one whose exact chemical composition is known.

For a chemoheterotroph, the chemically defined medium must contain organic growth factors that serve as a source of carbon and energy.

For example, glucose is included in the medium for growing chemoheterotroph E. coli.

Constituent Amount Constituent AmountGlucose 5.0 g Magnesium sulfate 0.2 g

Ammo phosphate 1.0 g Potassium phosphate 1.0 gSodium chloride 5.0 g Water 1 liter

Cultivation… Many organic growth factors must be required in the chemically

defined medium to cultivate a species of Neisseria. Organisms that require many growth factors are described as

“fastidious”. Organisms of this type, such as Lactobacillus are sometimes used in

tests (microbiological assay) that determine the concentration of a particular vitamin in a substance.

This media are usually reserved for the growth of autotrophic bacteria.

Complex Media: Most heterotrophic bacteria and fungi are routinely grown on complex

media, made up of nutrients such as extracts from yeasts, meat, or plants, or digests of proteins from these and other sources.

In complex media, the energy, carbon, nitrogen, and sulfur requirements of the growing microorganisms are primarily provided by protein.

Cultivation… Composition of Nutrient Agar, a complex medium for the Growth of

Heterotrophic Bacteria

Constituent AmountPeptone (partially digested protein) 5.0 gBeef extract 3.0 gSodium Chloride 8.0 gAgar 15.0 gWater 1 liter

Protein is a large, relatively insoluble molecule that a minority of m.organisms can utilize directly, but a partial digestion by acids or enzymes reduces protein to shorter chains of amino acids called peptones.

Vitamins and other organic growth factors are provided by meat extracts. Yeast extracts are particularly rich in the B vitamins.

Cultivation… If a complex medium is in liquid form, it is called nutrient broth.

When agar is added, it is called nutrient agar. Anaerobic Growth Media and Methods: Special media called reducing media must be used to protect

anaerobes from exposure to oxygen. These media contain ingredients, such as sodium thioglycolate, that

chemically combine with dissolved oxygen and deplete the oxygen in the culture medium.

When the culture must be grown in Petri plates to observe individual colonies, special anaerobic jars are used.

The culture plates are placed in the jar, a packet of chemicals (sodium bicarbonate and sodium borohydride) in the jar is moistened with a few mls of water and jar is sealed.

H2 and CO2 are produced by the reaction of the chemicals with the H2O.

Cultivation… A palladium catalyst in the jar combines the oxygen with the

hydrogen, and water is formed. As a result, oxygen quickly disappears CO2 produced aids the growth of many anaerobic bacteria. A relatively new tech. to provide an anaerobic environment makes use

of enzyme, oxyrase, that reduces oxygen to water. Oxyrase is a respiratory enzyme derived from the plasma membranes

of certain bacteria. When it is added to growth media, it transforms the Petri plate,

OxyPlate, into a self contained anaerobic chamber. Researchers regularly working with anaerobes use transparent

anaerobic chambers equipped with air locks and filled with inert gases.

Technicians can manipulate equipment by inserting their hands into airtight rubber gloves called glove ports, which are fitted to wall of chamber.

Cultivation… Oxygen can be removed from anaerobes growth media by several

ways: 1) Special anaerobic media containing reducing agents such as

thioglycollate or cysteine may be used. Boiling of the media drives off oxygen, and reducing agents will eliminate any dissolved O2 remaining with medium.

2) O2 can be removed by eliminating air with a vacuum pump and flushing out residual O2 with nitrogen gas.

3) Gas pack jar4) Plastic bags or pouches make convenient containers for anaerobic

growth. Plastic bags or pouches contain catalyst, calcium carbonate (to produce an anaerobic, carbon – dioxide- rich atmosphere), special solution in reagent compartment. Petri plates are placed in pouch, clamped shut and placed in an incubator.

Cultivation…Special Culture Techniques: Many clinical laboratories have special carbon dioxide incubators in

which aerobic bacteria that require concentrations of CO2 higher or lower than the atmosphere will grow.

High CO2 levels are also obtained with simple candle jars. Cultures are placed in a large sealed jar containing a lighted candle,

which consumes oxygen – stops burning when air in jar has lowered concentration of oxygen and elevated concentration of CO2 is present.

Microbes that grow better at high CO2 concentrations are called capnophiles.

The low oxygen, high CO2 conditions resemble those found in the intestinal tract, respiratory tract, and other body tissues where pathogenic bacteria grow.

Sometimes, commercially available chemical packets are used to generate CO2 atmospheres in containers.

Cultivation… When only one or two Petri plates of cultures are to be incubated, use

of small plastic bags with self-contained chemical gas generators that are activated by crushing the packet or moistening it with a few milliliters of water.

These packets are sometimes specially designed to provide precise concentrations of carbon dioxide and oxygen, for culturing organisms such as microaerophilic campylobacter bacteria.

Selective and Differential Media: To detect the presence of specific microorganisms associated with

disease or poor sanitation, selective and differential media are used. Selective media are designed to suppress the growth of unwanted

bacteria and encourage growth of desired microbes. E.g. bismuth sulfite agar and brilliant green agar are two medium used

to isolate the typhoid bacterium, the gram negative Salmonella typhi from feces.

Cultivation…Bismuth sulfite and brilliant green inhibit gram-positive bacteria

and most gram-negative intestinal bactreia.Bile salts or dyes like basic fuchsin and crystal violet favor the

growth of gram negative bacteriaEndo agar, eosin methylene blue agar and MacConkey agar used

for detection of E. coli.

Differential Media: It is used to distinguish colonies of the desired organism from

other colonies growing on the same plate.Pure cultures of microorganisms have identifiable reactions with

differential media in tubes or plates. Microbiologists used blood agar (contains red blood cells) to

identify bacterial species Streptococcus pyogenes which destroy red blood cells (beta – hemolysis), show a clear ring around their colonies.

Cultivation… Sometimes, selective and differential characteristics are combined in a

single medium. E.g. Staphylococcus aureus has a tolerance for high concentrations of

sodium Chloride; can also ferment the carbohydrate mannitol to form acid.

Mannitol salt agar contains 7.5% sodium chloride, which will discourage the growth of competing organisms and thus select for S. aureus.

This salty medium also contains a pH indicator that changes color if the mannitol is fermented to acid.

The mannitol fermenting colonies of S. aureus are thus differentiated from colonies of other bacteria that do not ferment mannitol.

MacConkey agar medium contains bile salts, crystal violet (inhibit growth of gram positive bacteria) and lactose. Gram negative bacteria metabolized lactose, produced acid (become red or pink colonies) and differentiated from other bacteria (colorless colonies) that can not.

Cultivation… Assay Media: Media of prescribed compositions are used for the assay of vitamins,

amino acids, and antibiotics. Media of special composition are also available for testing disinfectants.

Media for Enumeration of Bacteria: Specific kinds of media are used for determining the bacterial content

of milk and water.Media for Characterization of Bacteria: A wide variety of media are used to determine type of growth

produced by bacteria and to determine their ability to produce certain chemical changes.

Maintenance Media: Satisfactory maintenance of the viability and physiological

characteristics of a culture over time may require a medium. E.g. Glucose enhances growth, but acid produced by glucose is

harmful to the cells so omission of the glucose is preferable in maintenance medium.

Enrichment Culture This is the culture designed to increase very small numbers of the

desired type of organism to detectable levels especially when other organisms are present in very larger numbers.

The medium for this culture is usually liquid and provides nutrients and environmental conditions that favor the growth of particular microbes but not others.

E.g. To isolate from a soil sample a microbe that can grow on phenol.

Pure Culture Pure culture means culture containing only one type of bacterium. Most bacteriological work requires pure cultures or clones of bacteria. If the bacterial species being sought comprises a suitably high

proportion of the mixed population, it can be isolated in pure culture. The descendents of a single isolation in pure culture comprises a

strain. If a strain is derived from a single parent cell, it is termed as a clone.

Isolation of Pure Culture The isolation method most commonly used to get pure cultures is the

streak plate method. A sterile inoculating loop is dipped into a mixed culture – is streaked

in a pattern over surface of nutrient medium – as pattern is traced, bacteria rubbed off the loop onto the medium – the last cells to be rubbed off the loop are far apart to grow into isolated colonies.

When streaking is properly performed, the bacterial cells will be sufficiently far apart in some areas of the plate to ensure that the colony developing from one cell will not merge with that growing from another.

These colonies can be picked up with an inoculating loop and transferred to a test tube of nutrient medium to form a pure culture.

The streak plate method works well when the organism to be isolated is present in large numbers, if in very small numbers, its numbers must be greatly increased by selective enrichment before isolation.

A modification know as the roll-tube technique is used for the isolation of stringent anaerobes.

Isolation of Pure Culture…Pour Plate Method: The mixed culture is diluted directly in tubes of liquid (cooled) agar

medium. The medium is maintained in a liquid state at a temperature of 45ºC to allow thorough distribution of the inoculum.

The inoculated medium is dispensed into Petri dishes, allowed to solidify, and then incubated. A series of agar plates showing decreasing numbers of colonies resulting from the dilution procedure in pour plate technique.

Spread Plate Method: Here, culture is diluted in a series of tubes containing a sterile liquid,

water or psychological saline. A sample is removed from each tube, placed onto the sufrace of the

agar plate, and spread evenly over the surface by means of a sterile, bent glass rod.

On at least one plate the bacterial will be in numbers sufficiently low as to allow development of well separated colonies.

Isolation of Pure Culture… Micromanipulator Technique: It is used in conjunction with a microscope to pick a single cell from a

mixed culture. The micromanupulator permits the operator to control the movements

of a micropipette or a microprobe so that a single cell can be isolated.

Heat Differentiation: If a mixed culture is heated in a temperature controlled water bath the number of heat-sensitive species in samples will decreases as time progresses.

If temperatures above 70ºC are used only spores will be isolated after about 10 minutes and further heating will separate the sporing species according to their heat tolerance.

Motility: If a mixed culture is inoculated at the bottom of a moist slope, which

is then incubated upright, motile organisms can be recovered from the top of the slope which they have reached by swimming in the film of liquid on the surface.

Isolation of Pure Culture…Filtration: When the quantity of bacteria is very small, as in lakes or relatively

pure streams, bacteria can be counted by filtration methods. 100ml of water are passed through a thin membrane filter whose pores

are too small to allow bacteria to pass. Thus, the bacteria are filtered out and retained on the surface of the filter.

The filter is then transferred to a Petri dish containing a pad soaked in liquid nutrient medium, where colonies arise from the bacteria on the filter’s surface.

This method is applied frequently to detection and enumeration of coliform bacteria, which are indicators of fecal pollution of food or water.

Preservation of Bacterial Culture Refrigeration can be used for the short term storage of bacterial

cultures. Two common methods of preserving microbial cultures for long

periods are deep-freezing and lyophilization. Deep freezing is a process in which a pure culture of microbes is

placed in a suspending liquid and quick frozen at temperatures ranging from -50º to -95ºC.

In lyophilization, a suspension of microbes is quickly frozen at temperature ranging from -54º to -72ºC, and the water is removed by a high vacuum (sublimation).

The culture can usually be thawed and cultures even several years later.

The organisms can be revived at any time by hydration with a suitable liquid nutrient media.

Preservation of Bacterial Culture… Strains can be maintained by periodically preparing a fresh stock

culture from previous stock culture. The culture medium, storage temperature and time interval at which transfers are made vary with the species.

Many bacteria can be successfully preserved by covering the growth on an agar slant with sterile mineral oil.

Growth of Bacterial CulturesBacterial Division: Bacterial growth refers to an increase in bacterial numbers. Bacteria normally reproduce by binary fission; by budding, by

producing chains of conidiospores carried externally at the tips of filaments.

Generation Time: The time required for a cell to divide (and its population to double) is

called the generation time. It varies considerably among organisms and with environmental

conditions, such as temperature. Most bacteria have a generation time of 1-3 hours; others require

more than 24hours per generation. If a doubling occurred every 20 minutes (E. coli), after 20 generations

(7 hours) a single initial cell would increase to over 1 million cells. In 30 generations (or 10 hours), population would be 1 billion. In 24 hours it would be a number trailed by 21 zeros.

It is difficult to graph population changes of such enormous magnitude by using arithmetic numbers. So logarithmic scales are generally used to graph bacterial growth.

Growth of Bacterial Cultures…Phases of growth: Bacterial growth curve that shows the growth of cells over time, 4

basic phases of growth: the lag, log, stationary and death phases. The Lag Phase: Initially, number of cells changes very little because the cells do not

immediately reproduce in new medium. This period of little or no cell division is called the lag phase, and it

can last for 1 hour or several days. During this time, microbial population is undergoing a period of

intense metabolic activity involving, synthesis of enzymes and various molecules.

The Log Phase: Eventually, the cells begin to divide and enter a period of growth, or

logarithmic increase, called the log phase, or exponential growth phase.

Cellular reproduction is most active, and generation time reaches a constant minimum and a logarithmic plot of growth is a straight line.

Growth of Bacterial Cultures… The log phase is the time when cells are most active metabolically and

is preferred for industrial purposes. During this phase, m.organisms are particularly sensitive to adverse

conditions, Radiation and many antimicrobial drugs. The Stationary Phase: Eventually, the growth rate slows, the number of microbial deaths

balances the number of new cells, and the population stabilizes. The metabolic activities of individual surviving cells also slow at this stage. This period of equilibrium is called the stationary phase.

The exhaustion of nutrients, accumulation of waste products, and harmful changes in pH may all play a role in stopping of exponential growth.

In a specialized apparatus called a chemostat, a population can be kept in the exponential growth phase indefinitely by draining off spent medium and adding fresh medium. This type of continuous culture is used in industrial fermentations.

Growth of Bacterial Cultures…The Death Phase: The number of deaths eventually exceeds the number of new cells

formed, and the population enters the death phase, or logarithmic decline phase.

This phase continues until the population is diminished to a tiny fraction of the number of cells in the previous phase, or the population dies out entirely.

Some species pass through the entire series of phases in only a few days.

Counting of Bacteria The total number of organisms living and dead in a preparation is

known as the total count, while the number of living organisms is called the viable count.

Plate counts & Serial Dilutions The Most Probable Number Method: This statistical estimating technique is based on the fact that the

greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series.

The MPN is only a statement that there is a 95% chance that the bacterial population falls within a certain range and that the MPN is statistically the most probable number.

Direct Microscopic Count: A measured volume of bacterial suspension is placed within a defined

area on a microscope slide.

Counting of Bacteria… In the Breed Count Method, a specially designed slide called a

Petroff-Hausser cell counter is used, where a diluted suspension of organisms of known volume is spread carefully and average number of bacteria in each of series of these squares is calculated by the factor that produces the count per milliliter.

Motile bacteria are difficult to count by this method, dead cells are about as likely to be counted as live ones, and a rather high concentration of cells is required.(10 million bacteria per ml)

No incubation time is required, very fast. Many electronic cell counters, coulter counters, which automatically

count the number of cells in a measured volume of liquid. Theses instruments are used in some research laboratories and hospitals.

Wright Method: Equal volumes of suspension and blood (5 x 106 per cm3) are mixed, dried, fixed and stained. As the ratio of blood cells to bacteria on the slide is known, the number of bacteria per cm3 in the suspension can be found by proportionality.

Counting of Bacteria…Counting Chamber: A slide with a recessed area that is ruled in squares. A suitable

dilution of the culture is made and a drop is placed on the recess. If the mean number of organisms per square is assumed to be 10,

number of bacteria per mm3 = 20 x 20 x 50 x 10 = 2 x 105

The original culture contained 2x107 mm3

Viable Counts:These are used to determine number of living organisms in

pharmaceutical products and in foods, and to find number of bacteria surviving after exposure to a lethal agent.

Solid media techniques: 1)Pour plate method, 2) Surface viable method –

a) Drop technique: 0.02 cm3 diluted suspension with 20-80 organisms placed in dropping pipette. Placed pipette at height of 10mm from agar surface, two drops of each dilution are dropped on to each plate up to 6-8 drops per plate.

Counting of Bacteria… After about 20 minutes the drops have been absorbed by medium and

the plates are incubated. Counts can be examined in a shorter time in just 8 hours.

b) Spreading technique: or spread plate technique Advantages: 1) Counting is easier; colonies are approximately same

size, because they are on the surface and having same oxygen tension. 2) Obligate aerobes including fungi can be counted satisfactorily. 3) Colony characteristics on solid media are more pronounced because on surface growth, contaminants are more easily detected.

3) Roll tube method: Dilution of organisms added to a small volume of nutrient agar in a test tube or boiling tube, shaking to mix the contents and rotating almost horizontally, under cold water or in a hollowed block of ice, until the medium had set in a thin film around the inside of tube. ASTELL SPINNING BOTTLE technique. A special illuminator, that facilitates counting, can be obtained. The ideal number of organisms is between 100 and 200 per bottle.

Counting of Bacteria… Counting anaerobes: Solid media methods can be used for anaerobes provided that the

containers are incubated under anaerobic conditions. Oxygen free diluting fluids may be necessary, e.g. solutions containing reducing agents or fluid anaerobic media

Two special methods are- 1) The Miller-Prickett Tube: Tube is flattened oval in section and

has a longish narrow neck. The organism is mixed with agar. The agar suspension fills the body of the tube and neck is sealed with agar containing a reducing agent. Colonies grow throughout the agar.

2) Ingram’s Method: Normal tubes or bottles are used. After introducing the suspension in agar, a thick sterile black rod is placed down the centre of container and the medium solidifies around this. Because the agar layer is relatively thin and the organisms are viewed against a black background, counting is facilitated.

Indirect Methods: These involves comparison with standard suspensions and

readings are made with the naked eye or by means of a photoelectric colorimeter or nephelometer.

Photometric methods: