Medical Microbiology I - Lecture2

MEDICAL MICROBIOLOGY I

LECTURE 2LECTURE 2

Culture of Bacteria and

Bacterial Growth

Culture Media

Microbiology depends on the ability to GROWand MAINTAIN microorganisms in the laboratory - use of suitable culture media

A culture medium is a solid or liquid preparation A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms.

They must contain water and sources of nitrogen, carbon, mineral salts and essential vitamins.

Some bacteria may require additional specific substances which may be added to the medium.

Culture Media

Two categories of culture medium:

1. Chemically defined media

Concentration of each ingredient is known

Made of highly purified inorganic salts and Made of highly purified inorganic salts and simple organic compounds such as glucose or purified amino acids

Little or no difference in composition between batches

Expensive and not for routine use

Used to determine specific growth requirements of bacteria

Culture Media

2. Complex Media

Media prepared using natural products such

as meat extract or vegetable infusions.

Natural products contain essential bacterial Natural products contain essential bacterial

nutrients

Exact concentration of nutrients are unknown

Used routinely

Easy to prepare, relatively cheap and able to

support the growth of many bacteria

Types of Media

1. Liquid media or broth

Bacteria can move freely in them

Growth in liquid medium is shown by turbidity

Some organism show surface growth Some organism show surface growth

Used for biochemical testing, blood culture,

testing for motility and as enrichment broth

Disadvantage: purity of growth cannot be

guaranteed

Types of Media

2. Solid Media

Microorganism grown on solid media will grow and

multiply at the site of inoculation and form visible

colonies

Liquid agar is made solid by adding a solidifying agent Liquid agar is made solid by adding a solidifying agent

which does not alter the nutritional content of the

medium - agar

Agar is an inert carbohydrate extract obtained from a

type of seaweed found in Japan, New Zealand, and

California.

Types of Media

Properties of agar:

1. Melts at 98C,

2. Sets at around 40C,

3. Easily soluble 3. Easily soluble

4. Remains clear

5. Concentration of 1% to be gel.

6. It is solid at 37C

7. Once solidified, it can be remelted

8. Bacteriologically inert (will not be degraded by

most bacteria)

Types of Media

3. Basal Media

Simple media that will support the growth of most

microorganisms that do not need special

nutritional requirementsnutritional requirements

Contain basic nutrients: peptone, mineral salts

and water

Normally called nutrient broth

2 types: infusion broth and digest broth

Types of Media

4. Enriched media

Culture media that are enriched with whole or

lysed blood, serum, special extracts or nutrients

Support growth of bacteria that cannot grow on Support growth of bacteria that cannot grow on

basal media

Nutrient broth + agar = nutrient agar

Nutrient agar + blood = blood agar

Blood agar + heat = chocolate agar

Types of Media

5. Selective media

Solid media has substances that prevent, slow

down or inhibit the growth of microorganisms

other than those for which the media are other than those for which the media are

devised e.g.

tellurite medium for diptheria organism

Deoxycholate citrate agar (DCA) for Salmonella

and Shigella groups

Types of Media

6. Enrichment media

Liquid media, similar in function to selective

media

Difference: selective media is broth Difference: selective media is broth

e.g. selenite F broth for the isolation of

Salmonella group

Types of Media

7. Differential media

Contain substances or indicators that will

differentiate one organism from another

e.g. MacConkey agar - differentiate lactose e.g. MacConkey agar - differentiate lactose

fermenting bacteria from non-lactose fermenting

bacteria; blood agar - differentiate haemolytic

bacteria from non-haemolytic ones.

Types of Media

8. Transport media

Usually semi-solid

Transportation of clinical specimens containing

delicate microorganisms, if there is to be a delay in

their delivery to the laboratory or in processingtheir delivery to the laboratory or in processing

Contain substances that can prevent the

overgrowth of commensals and prevent bacteria

from dying as a result of pH change or enzyme

action, e.g.

Amies transport medium for Neisseria gonorrhoeae

Stuarts transport medium for delicate organisms

including anaerobes

Isolation of Pure Cultures

In their natural habitats, microorganisms

usually grow in complex, mixed populations

with many other species.

Pure culture - a population of cells arising from Pure culture - a population of cells arising from

a single cell to characterise an individual species

Approaches:

1. Spread plate and streak plate

2. Pour plate

3. Microbial growth on agar surfaces

Mixed Bacterial Growth


1. Spread plate and streak plate

Mixture of cells is spread out on agar surface at a

relatively low density.

Every cell grows into a completely separate Every cell grows into a completely separate

colony, a macroscopically visible growth of

clusters of microorganism on a solid medium.


I. Spread plate

Small volume of dilute microbial mixture

containing around 30 - 300 cells is transferred to

the center of agar plate and spread evenly over the center of agar plate and spread evenly over

the surface with a sterile bent-glass rod.

Dispersed cells develop into isolated colonies

The number of colonies should equal the

number of viable organism in the sample.

Spread plates can be used for colony count.


II. Streak plate

The microbial mixture is transferred to the edge of

an agar plate with an inoculating loop or swab and

then streaked out over the surface in one of several

pattern.pattern.

After the first quarter is streaked, the inoculating

loop is sterilised and an inoculum for the second

quarter is obtained from the first quarter.

Similar process is repeated for the subsequent

quarters.

This is essentially a dilution process to separate

individual colonies.

Streak Plate


2. Pour plate

After the agar solidifies, each cell is fixed in place

and forms an individual colony

Plates containing 30 -300 colonies are counted Plates containing 30 -300 colonies are counted

The colonies equal the number of viable

microorganism in the sample that are capable of

growing in the medium used

Colonies growing on the surface also can be used

to inoculate fresh medium and prepare pure

cultures

Pour Plate


3. Bacterial growth on agar surfaces

Colony development on agar surfaces aids

microbiologists in identifying microorganisms

because individual species often form colonies of because individual species often form colonies of

characteristic size and appearance

In nature, microorganisms often grow on

surfaces in biofilms - slime-encased aggregations

of microbes

Sometimes they form discrete colonies


Generally the most rapid cell growth occurs at

the colony edge

Growth is much slower in the center, and cell

autolysis takes place in the older central autolysis takes place in the older central

portions of some colonies.

These differences in growth are due to

gradients of oxygen, nutrients, and toxic

products within the colony.


At the colony edge, oxygen and nutrients are

plentiful

The colony center is much thicker than the

edgeedge

Oxygen and nutrients do not diffuse readily

into the center, toxic metabolic products

cannot be quickly eliminated, and growth in

the colony center is slowed or stopped


Cells on the periphery can be growing at

maximum rate while cells in the center are

dying

The bacteria growing on solid surfaces vary The bacteria growing on solid surfaces vary

with nutrient diffusion and availability, the

hardness of the agar surface, bacterial

chemotaxis, and the presence of liquid on the

surface

Isolation of Pure Culture

Tube culture methods

Used for specific identification tests

Only small volumes of media are required for

the teststhe tests

Not used for colonial morphology study

e.g. are:

1. Slope cultures

2. Deep cultures

3. Stab cultures


Slope (slant) cultures are tubes containing

small quantity of medium that has been

allowed to set in sloped position.

Aka slopes or slants Aka slopes or slants

Used for maintenance of isolated bacteria or for

performing biochemical tests

e.g. Loeffler's serum agar, Dorset egg medium and

Lowestein-Jensen medium


Deep culture media are prepared in tubes of

about 150 x 20 mm to a depth of about 60-70

mm.

Aka shake

Cultivation of anaerobic bacteria and can be used for Cultivation of anaerobic bacteria and can be used for

viable count

Stab cultures refer to the method of inoculation,

where the inoculating needle is stabbed through

the center of the medium.

e.g. Kohns II medium, motility medium, indole urea

medium


Inoculation into broth

Carried out using inoculating loop, inoculating

needle or Pasteur pipette

Depends on whether the inoculum is colonial Depends on whether the inoculum is colonial

growth, liquid culture or specimen

Pick colonies with sterile inoculating loop, hold

the tube at an angle, and rub the loop against the

inner side of the container below the level of the

broth

Sub-culturing

Sub-culturing means to transfer

microorganism from one medium to another.

Basically, it is starting new bacterial cultures

from the old cultures.from the old cultures.

Usually performed to maintain continuous

growth of microbes

Measurement of Microbial Growth

To determine growth rates and generation

times

Either population number or mass may be

followed because growth leads to increases in followed because growth leads to increases in

both

No single technique is always best, the most

appropriate approach will depend on the

experimental situation


Classified into 2 methods:

1. Cell number

i. Direct counting

ii. Coulter counter and flow cytometerii. Coulter counter and flow cytometer

iii. Membrane filter technique

iv. Plating methods (visible counting methods, spread

plate and pour plate methods)

2. Cell mass

i. Microbial dry weight

ii. Spectrophotometry


1. Direct counting

Using counting chamber e.g. Petroff-Hausser

counting chamber, haemocytometers

Prokaryotes are more easily counted when stained,

or if phase contrast or flourescence microscope is or if phase contrast or flourescence microscope is

used

Advantage: easy, inexpensive, and relatively quick; it

gives information about the size and morphology of

organisms

Disadvantage: the microbial population must be

fairly large for accuracy because only a small volume

is sampled

Petroff-Hausser Counter


2. Coulter chamber and flow cytometer

Large microorganisms like protists and yeasts can

be directly counted with electronic counters,

Coulter chamber and flow cytometer

The microbial suspension is forced through a small

hole or orifice in the Coulter counter

An electrical current flows through the hole, and

electrodes placed on both sides of the orifices

measure its electrical resistance

Coulter Counter


Every time a microbial cell passes through the orifice,

electrical resistance increase (or conductivity drops)

and the cell is counted

Advantage: accurate with large cells and extensively

used

Disadvantage: interference by small particles, the

formation of filaments and other problems


3. Membrane filter techniques

The number of bacteria in aquatic samples is

frequently determined from direct counts after

the bacteria have been trapped on special the bacteria have been trapped on special

membrane filters

Sample is first filtered through a black

polycarbonate membrane filter.

Then, the bacteria are stained with a fluorescent

dye and observed microscopically


Stained cells are easily observed against the black

background of the membrane filter and can be

counted when viewed with an epifluorescence

microscope

Traditional counting methods do not distinguish

dead cells from live cells

New methods (e.g. commercial kits) make this

possible

First traps bacteria in aquatic samples on a

membrane filter


The filter is then placed on an agar medium or on

a pad soaked with liquid media and incubated

until each cell forms a separated colony

A colony count gives the number of

microorganisms in the filtered sample, and special

media can be used for specific microorganisms

Specially useful technique in analysing water

purity


4. Plating methods

To determine the number of viable microbes

Viable counting methods - count only those cells

that are alive and able to reproducethat are alive and able to reproduce

Pour plate and spread plate methods - diluted

samples of bacteria or other microorganisms is

dispersed over a solid agar surface

Simple, sensitive, and widely used for viable

counts


Low counts will result if clumps of cells are not

broken up and microorganisms not well dispersed

Results expressed in colony forming units (CFU) -

30 - 300 colonies for most accurate counting


5. Microbial dry weight

Cells growing in liquid medium are collected by

centrifugation, washed, dried in an oven and

weighedweighed

Especially useful technique for measuring the

growth of filamentous fungi

Time-consuming and not very sensitive

Bacteria weigh so little, it may be necessary to

centrifuge several hundred millimeters of culture

to collect a sufficient quantity


6. Spectrophotometry

More rapid and sensitive

Depend on the fact that microbial cells scatter

light that strikes themlight that strikes them

Microbial cells in a population are of roughly

constant size

The amount of scattering is directly proportional

to the biomass of cells present and indirectly

related to cell number


When the concentration of bacteria reaches

about 107/mL, the medium appears slightly

cloudy or turbid

The population growth can be easily measured

as long as the population is high enough to give as long as the population is high enough to give

detectable turbidity


If the amount of a substance in each cell is constant,

the total quantity of that cell constituent is directly

related to the total microbial cell mass

Example, a sample of washed cells collected from a Example, a sample of washed cells collected from a

known volume of medium can be analysed for total

protein or nitrogen

An increase in the microbial population will be

reflected in higher total protein levels.

ATP can be used to estimate the amount of living

microbial mass

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Medical Microbiology I - Lecture2