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PowerPoint Lecture Slide Presentation

B.E Pruitt & Jane J. Stein

Chapter 05Microbial Growth

REFERENCESREFERENCES

Tortora GJ, Funke BR, Case CL, 2007, Microbiologyan Introduction, 9th edition, Benjamin Cummings, SanFrancisco, CA 94111, USA

Madigan MT, Martinko JM, 2006, Brock Biology ofMicroorganisms, 11th edition, Pearson Education Inc.,USA

Doorne H, 2008, Seminar ofCourse on CurrentPharmaceutical Microbiology: Methods,Harmonization and Technology, Faculty of Pharmacy

UBAYA, Indonesia

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Microbial Growth

Microbial growth: increase in number of cells,not the cell size

The requirement for growth:

Physical

Temperature, pH, osmotic pressure

Chemical C, N, S, P, trace element, O2, organic

growth factor

Temperature

Most microorganisms grow well at the temperaturesfavored by human

Temperature (cardinal temperature)

Minimum growth temperature

Optimum growth temperature

Maximum growth temperature

Typical range of any given microorganism is 30-40degrees

Physical requirement

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Physical requirement

Temperature

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Psychrophiles: capable of growing at 0C Grup 1: grow at 0C but has optimum growth at

15C; found in oceans depths

Grup 2 (psychrotrophs / moderate psychrophiles /facultative psychrophiles): grow at 0C, hasoptimum growth at 20-30C and cannot growabove 40C; cause refrigerator food spoilage

Mesophiles: optimum growth at 25-40C

Thermophiles: optimum growth at 50-60C

Hyperthermophiles/ extreme thermophiles: optimumgrowth at 80C; members of archaea

Temperature

Food spoilage temperature

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pH (extracellular environment) Most organisms show a growth pH range of 2-3

units

Most natural environments have pH values 5 and 9

Only few species can grow at pH value < 2 or > 9

Most bacteria grow between pH 6.5 and 7.5

Molds and yeasts grow between pH 5 and 6

Acidophiles: grow in acidic environments (pH < 5.5);

stability of cytoplasmic membrane, e.g. archaea Alkalophiles(pH > 9), neutrophiles(pH 6-8)

Physical requirement

pH

The intracellular pH usually must remain relativelyclose to neutral in order to prevent destruction ofacid- or alkali-labile macromolecules in the cell

Buffer: peptones and amino acid, KH2PO4 (pH 6-

7.5) Buffering system for one to another organisms

may be considerably different

Physical requirement

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Osmotic effect

Water activity (aw): ratio of the vapor pressure of theair in equilibrium with a substance or solution to thevapor pressure of pure water

The values ofawvary between 0 and 1

When a cell is in environment of low aw, there is atendency for water to flow out the cell

Hypertonic environments, increase salt or sugar,cause plasmolysis

Hypotonic environments, decrease salt or sugar,

cause osmotic lysis (plasmoptysis)

Extreme / obligate halophiles(30% salt) andfacultative halophiles(2-15% salt)

Physical requirement

Osmotic effect

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Water activity in pharmaceutical preparation

LowHighNoneMultiple0.25Aerosol inhalants

Moderate to lowModerate tolow

LowSingle0.30Vaginalsuppositories

Low to very lowLowVery lowSingle0.30Rectal

suppositories

Very low to noneVery lowVery lowSingle0.36Compressedtablets

Moderate to lowModerate tolow

Moderate to highMultiple0.90Oral liquids (aq.)

Moderate to lowModerateLow to moderateMultiple0.97Topicals(lotions/creams)

LowHighModerate to highSingle andmultuple

0.97Ophthalmicliquids

Moderate to lowModerateModerate to highMultiple0.99Nasal sprays

HighHighHighMultiple0.99Inhalationsolutions

lowVery highModerate to highSingle andmultiple

0.99Parenterals

Potential forpatient infection

Invasivenessof the route of

administration

Potential tosupport

microbial growth

Single useMultiple use

AwDosage form

Carbon

Structural backbone, energy source

Half of the dry weight is carbon

Chemoheterotrophs, autotroph

Nitrogen

Synthesis amino acids (a.a.), DNA, RNA 14% of the dry weight of bacterial cell

Most bacteria decompose proteins andreincorporating a.a. and other nitrogen compound(e.g. NH4

+ or NO3) into newly synthesized proteins

Chemical requirement

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Sulfur

Synthesis amino acids and vitamin (thiamine, biotin)

Important sources: SO42, H2S, sulfur-containing

amino acid

Phosphorus

In DNA, RNA, ATP, and phospholipids membranes

PO43 is a source of phosphorus

S and P together constitute about 4% P, Mg, Ca are also used as cofactor for enzymes

Chemical requirement

Trace Elements

Inorganic elements required in small amounts

Usually as enzyme cofactors

Naturally present in tap water or even distilled water

Organic Growth Factors

Essential organic compounds that is unable to besynthesized by organisms

Obtained from the environment

Vitamins, amino acids, purines, pyrimidines

Non fastidious and fastidious bacteria

Chemical requirement

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Trace element

Trace element

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Organic growth factor

Chemical requirement

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O2

is a powerful oxidant and the best electron receptorfor respiration

O2 is not the poison but certain O2 derivates that toxicto microorganisms

Toxic Forms of Oxygen

Enzymes that destroy toxic O2

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Culture media

a nutrient material prepared for the

growth of microorganisms in a

laboratory

Inoculum

microbes are introduced into a

culture medium to initiate growth

Culture

the microbes that grow and

multiply in or on a culture medium

Culture media

It must content the right nutrient for

the specific microorganisms

Have a sufficient osmotic balance,

adjusted pH and suitable level of O2

Sterile, it must initially contain no

living microorganisms Growing culture should be incubated

at proper temperature

Criteria must the culture medium meet

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Culture media

Agar: complex polysaccharide

derived from marine alga

Used as solidifying agent for

culture media in petri plates

Generally not metabolized by

microbes

Liquefies at 100C

Solidifies at 40C

Chemically Defined Media: exact

chemical composition is known Complex Media: extracts and

digests of yeasts, meat, or plants

Culture Media

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Culture Media

Reducing media

Contain chemicals (thioglycollate) that combine O2

Heated to drive off O2

Special anaerobic jar

Contain sodium bicarbonate and borohydride

Added with H2O H and CO2

Palladium catalyst combines with H and O2 H2O

Oxyrase enzyme (reduces O2 H2O)

Petri (OxyPlate); self-contained anaerobic chamber

Anaerobic Culture Methods

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Anaerobic Culture Methods

Canophiles

Special culture technique

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A pure culture contains only one species or strain

A colony is a population of cells arising from a singlecell or spore or from a group of attached cells

A colony is often called a colony-forming unit (CFU)

Obtaining pure culture

Streak Plate

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Deep-freezing: pure culture is placed in asuspending liquid and quick-frozen at -50to -95C

Lyophilization (freeze-drying): frozen (-54 to-72C) and dehydrated in a vacuum (sublimation)

The microorganisms can be revived at any time byhydration with a suitable liquid nutrient medium

Preserving bacteria cultures

Binary fission, budding, conidiospores(actinomycetes), fragmentation of filaments

Generation time: the time required for a cell to divide(and its population to double)

Most of bacteria have a generation time of 1-3 hours,

others require more than 24 hours per generation E. colihave a generation time of 20 minutes

The Growth of Bacterial Culture

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Binary Fission

Binary Fission

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Growth curve

If 100 cells growing for 5 hours produced 1,720,320 cells:

Generation time calculation

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Phases of Growth

Lag Phase

It can last for 1 hour or several days, the cells arenot dormant

Period of intense metabolic activity (synthesis ofenzymes and various molecules)

Log or Exponential Growth Phase

Generation time reaches a constant minimum

Metabolically active and preferred for industrialpurposes

Sensitive to adverse conditions (e.g. radiation andantimicrobial drugs)

Phases of Growth

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Stationary Phase or period of equilibrium

Cryptic growth

Metabolic activities of surviving cells are slowing

Exhaustion of nutrients, accumulation of wasteproducts, changes in pH may all play a role

Chemostatapparatus; continuous culture

Death or Logarithmic Decline Phase

Involution: their morphology changes dramatically,making them difficult to be identified

Some species pass through all the phases in few days;others retain some surviving cells almost indefinitely

Phases of Growth

Chemostat apparatus

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Measuring Microbial Growth

Weightof some component of cell mass (protein,nucleic acid or dry weight of the cells)

Populations total mass (numberof the cells in amilliliter liquid or in a gram solid material)

Methods:

Direct measurement

Direct microscopic count, viable count,filtration, MPN

Indirect measurement Turbidity, metabolic activity and dry weight

Direct measurements of microbial growth Direct Microscopic Count

Petroff-Hausser cell counter

Advantage:

No incubation is required (fast)

Disadvantages:

It is needed special attention to count the cells

Dead cells are not distinguished from living cells Precision is difficult to achieve

Motile bacteria are difficult to be counted

A rather high concentration of cells is required tobe countable (e.g. 106 bacteria / milliliter)

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Direct microscopic count

Viable count

Measures the number of viable cells

Plate count or colony count

Assume: each viable cell can grow and divide toyield one colony

Unit: Colony-forming unit(CFU)

Serial dilution

Pour plate (0.1-1.0 ml) and spread plate (0.1 ml orless)

Direct measurement of microbial growth

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Serial dilution

After incubation, count

colonies on plates thathave 30-300 colonies

Plate count

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Advantages:

Only living cells is counted

Some species can be countable all at once

Can be used for isolation and identification

Disadvantages:

The result does not reflect the actual value; somecells can closely growth resulting one colony

Different time, medium and incubation condition will

result a different value The microbial must be grown in/on the solid medium;

show dense, distinct and not spreading colonies

Plate count

Filtration method

Used for very small quantity bacteria

Usually using 100 ml of sample

Applied frequently to detection and enumerationof coliform bacteria

Direct measurements of microbial growth

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Direct Measurements of Microbial Growth

Most Probable Number (MPN)

Useful when the microbes being counted will notgrow on solid media or when the growth ofbacteria in a liquid differential medium is used toidentify the microbes

The greater the number bacteria in a sample, themore dilution is needed to reduce the density tothe point at which no bacteria are left to grow inthe tubes in a dilution series

The MPN is only the statement that there is a

95% chance that the bacterial population fallswithin a certain range and that the MPN isstatistically the most probable number

Most probable number

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Multiple tube

MPN test Count

positivetubes andcompare tostatisticalMPN table

Most probable number

Turbidity measurement

As bacterial multiply in a liquid medium, the mediumbecomes turbid or cloudy with cells

Spectrophotometerorcolorimeter

Parameter: absorbanceor sometimes called opticaldensity(OD)

About 106 107 cells / milliliter are needed to makesuspension turbid enough to read on aspectrophotometer

Indirect measurements of microbial growth

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Turbidity measurement

Metabolic activity

The amount of a certain metabolic product (acid,CO2) is proportional to the number of bacterialpresent

Vitamin bioassay

Dry weight

Used for filamentous bacteria or molds

The molds is removed from the growth medium,filtered to remove extraneous material, dried in adesiccator and then weighed

Indirect measurements of microbial growth