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7/27/2019 Lecture+07+Slides+for+Posting

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Microbial Growth & Cultivation

MICR570/ZMR/F12 7


Bacterial & Fun al

Lecture 7

OBJECTIVES LECTURE 7

• Describe stages of bacterial growth (reproduction)

• Describe fungal growth (reproduction)

• Describe physical & chemical factors required for

(& affecting growth)

• Discuss the cultivation of bacteria/fungi - media

BACTERIAL GROWTH

• Coordinated chemical & physical processes

• Requires metabolism, regulation & division

• Knowledge Important: Clinical & Investigative purposes

• CHARACTERIZED:Continuous macromolecular synthesis

Cell elongation occurs along with genome replication

• BACTERIAL CELL CYCLE: SIMPLE

Time of division of mother cell into 2 daughter cells THEN 1daughter cell then divides into 2 more daughter cells

nown as

Division is GEOMETRICAL (Population Doubles)




MICR570/ZMR/F12 7

BACTERIAL GROWTH• GROWTH RATE: time for cell to reproduce

• GENERATION TIME: Time for a complete fission cycle

i.e., 1 parent cell Æ 2 new daughter cells (1st Generation)

=

3rd = 8 cells

4th = 16 cells

5th = 32 cells

AND SO ON & ON & ON….

http://www.youtube.com/watch?v=gEwzDydciWc

• GENERATION TIMEFormation of each new bacterial cell, its growth and eventualdivision into 2 cells

E.g. (Optimal conditions)Bacillus stearothermophilus 11 mins

Escherichia coli 20 mins

Staphylococcus aureus 28 mins

Lactobacillus acidophilus 60-80 mins

Mycobacterium tuberculosis 360 mins

Treponema pallidum 1980 mins

Log 10

• QUANTITATIVE ASSEMENT

Expressed as 21, 22, 23, 24 …..2n

Power value increases by 1 each generation

(the number of the generation) Termed EXPONENTIAL

og10

No' of CellsCell No's

Time

Logrithmic graphs preferred -

more accurate cell no’s during early growth




MICR570/ZMR/F12 7

ENUMERATION

1st Step: SERIAL DILUTIONS

Step 2: PLATE OUT

• GROWTH CURVEIn a closed system: nutrients and space finite

no removal of waste products

Log10

CStationary

Time (hrs)

cells

A

D

Lag

Log Death

FUNGAL GROWTHN.B Moulds: Hyphae Æ Mycelium

• Extension at tip (Apical Growth)

• Continuous protoplasm movement into tip

• Up to 40μm/min

• Provides penetrating power (fresh nutrients)

Movies Taken from http://www.icmb.ed.ac.uk/research/read/movies.html




MICR570/ZMR/F12 7

FUNGAL REPRODUCTION• MOULDS: Spores (Sexual & Asexual)

• Ascomycotina

– Sexual spores (internal sac: ascus)

– Asexual spores: borne externally as conidia

N.B Yeasts: Single cellsÆ Daughter cells

Budding (Asexual process)

Saccharomyces cerevisae

Cryptococcus neoformans

Pictures Taken from http://helios.bto.ed.ac.uk/bto/microbes/yeast.htm

PHYSICAL & CHEMICAL

FACTORS AFFECTING

GROWTH




MICR570/ZMR/F12 7

3 Cardinal Temperatures

• MINIMUM

Lowest temp, permits microbial growth + metabolism

• MAXIMUM

TEMPERATURE

Highest temp permits growth + metabolism

• OPTIMUM

Small range of temp’s, (promotes fastest growth &

metabolism rates)

Extremes of MIN & MAX beyond which growth is inhibited

TEMPERATURE RANGES (OPTIMA GROWTH) OF SOME BACTERIA

Bacterium Growth Temperature oC

Min Max Optimum

1. Pseudomonas fluorescens 2-4 36-38 25-30

2. Pseu. aeruginosa 10-15 41-44 c. 37

3. Escherichia coli 15-20 45 37

4. Bacillus polymyxa 5-10 35-45 30-32

5. B. stearothermophilus 30-45 65-75 c. 55

6. Thermus sp. 40 79 70-72

TEMPERATURE ADAPTATIONS

• PSYCHROPHILE: Psychrophilic

Opt temp below 15oC

Capable of growth at 0oC

Cannot grow above 20oC

Found: SNOW FIELDS, POLAR ICE, DEEP OCEAN

EXAMPLES: Flavobacterium, Alcaligenes & Achromobacter sp.

• FACULTATIVE PSYCHROPHILE: Psychrotroph

Grow slowly in cold conditions

BUT have opt temp above 20oC

EXAMPLES: Staphylococcus aureus, L. monocytogenesCONCERN: Contaminants of food/dairy products

• MESOPHILE: Mesophilic

Opt temp 20-40oC

Capable of growth 10-50oC

Group containing HUMAN PATHOGENS (30-37oC)

EXAMPLE: E. coli

• THERMOPHILE: Thermophilic

Opt temp >45oC

Capable of growth 45-85oC

Incapable of growth at usual body temp

(NOT INVOLVED in HUMAN INFECTIONS)

Found: VOLCANO, DIRECT EXPOSURE TO SUN




MICR570/ZMR/F12 7

N.B. Dimorphism in Fungi & Temp – Depends on chemical & physical factors

• FREE-LIVING STATE

– Mycelial or hyphal form

– At sub-physiological temperature (25oC)

– s nc sexua orms are sp aye

• PARASITIC STATE

– Yeast form

– Due mainly to physiologic temperature (37oC)

– Oval morphology (Nothing distinct)

Histoplasma capsulatum Pictures Taken from:

http://www.monkeytime.com/sciencemaster/galleries/fungi/05.php; www.doctorfungus.org

GAS REQUIREMENTS

Oxygen important- terminal electron acceptor (Respiration)

N.B. Oxygen - limited solubility in water ∴ limiting factor

Enzymes are requiredWhy?

Reduce Oxygen to water and toxic products

(hydrogen peroxide + superoxide)

Microbes convert toxic products to molec Oxygen by:

1. CATALASE

H2O2 H2O + O2

2. SUPEROXIDE DISMUTASE

2O2- + 2 H+ H2O2 + O2

Peroxide is metabolized by Catalase (as above)

BASED ON OXYGEN REQUIREMENTS

microbes divided into 4 groups

• (OBLIGATE) AEROBES

Totally dependant on O2 for growth

Requirement of 1 atmosphere (20%)

Produce H O and O - but ossess catalase and su eroxide

dismutase - can tolerate high [O2]

• MICROAEROPHILES

Grow in presence of O2 BUT tolerate only 4%

Possess enzymes BUT if toxic products ↑, enzyme systems

overload INHIBITING GROWTH




MICR570/ZMR/F12 7

• (OBLIGATE) ANAEROBESGrow ONLY in ABSENCE of O2

Effect of presence: LETHAL

LETHAL - org’s lack enzymes to remove toxic products

Brief exposure will KILL

• FACULTATIVE ANAEROBESGrow in presence or absence of O2

Presence: Aerobic respiration

Absence: Fermentation for energy prodn

Grow best under AEROBIC CONDITIONS

E.g. Enterobacteriacea

Bacterial Enzymes that Protect the Cell

Against Toxic Forms of Oxygen

Microorganism Catalase Superoxidedismutase

Aerobe + +

Facultative anaerobe + +

Microaerophile - +

Obligate anaerobe - -

MICROBIAL GROUPS

• Bacteria: All 4 groups

• Fungi (Moulds): Normally aerobic

• Yeasts: Facultative anaerobes

WATER ACTIVITY (Aw)

ALL ORGANISMS require water (growth & reproduction)

Essential solvent, biochemical reactions

Aw = index amount of water free to react

= atmospheric measure (Relative Humidity)

RH = 1.00 Aw. Therefore, 90% RH = 0.90 Aw

Absorption & Solution factors reduce availability (↓ Aw)

Pure distilled water (Aw =1)

E.g., Saturated soln NaCl (Aw = 0.8)

Seawater [NaCl] ≈3% (Aw = 0.98)




MICR570/ZMR/F12 7

Most microbes - grow opt Aw = 1.0

↓ Aw = slow growth rate

Below Aw 0.9 Bacteria unable to grow

XEROTOLERANT: lower Aw

Fungi able to grow Aw 0.60

Yeasts (conc sugar soln’s Aw = 0.60)

Salt-tolerant Bacteria - Halophiles (High [Solute], low Aw)

Effect of Aw on growth of

Staphylococcus aureus

The Interrelationships of Aw of various

foods & susceptibility to microbial spoilageEFFECTS OF pH

pH - degree of acidity/alkalinity of a soln related to [H+]

pH = -log H+ (1/log H+)

Neutral Solutions (pH 7)

Alkaline (Basic) Soln (pH >7)

<

GROWTH RATES INFLUENCED BY pH VALUES

(NATURE OF PROTEIN)

Bacteria Lower Optimum Upper

E. coli 4.4 6.0-7.0 9.0

L. acidophilus 4.0-4.6 5.8-6.6 6.8




MICR570/ZMR/F12 7

Environmental Factors

Temperature Gas (Oxygen) Water pH

– °

Mesophiles (10 – 50°C)

Pathogens (30 – 37°C)

Thermophiles (45 – 85°C)

Obligate Aerobes (20% O2)

{catalase+SOD}

Microaerophiles (4% O2)

Obligate Anaerobes (No O2)

Facultative anaerobes (O2)

Xerotolerant < 0.60 Aw

Halophiles < Aw

Neutral (pH 7)

Alkaline (pH >7)

Acidic (pH ≤ 7)

• NUTRIENT CONCENTRATION

CHNOPS - Macronutrients

(components: CH20’s, Lipids, Protein & Nucleic acids)

K+, Ca2+, Mg2+, Fe2+ /Fe3+ (Cations) - Minerals

CHEMICAL FACTORS

- , -

Magnesium - enzyme cofactor, Iron - cytochromes, cofactor

Trace Elements (Mn, Zn, Co, Ni, Cu, Mo)

(components: enzymes or cofactors)

REQUIRED: Energy prodn & macromolecular biosynthesis

GROWTH LIMITED BY REQUIRED [NUTRIENT]

CLASSIFICATION: Carbon, Energy & Hydrogen/Electron

CARBON

Autotroph CO2 sole/principle C source

Heterotroph Reduced/pre-formed Organic molec

Other organisms

ENERGY

Chemotrophs Oxidation (organic or inorganic)

HYDROGEN/ELECTRON

Lithotrophs Reduced inorganic molec

Organotrophs Organic molec

MOST PATHOGENS: Chemoorganotroph ic heterotrophs

Chemical Factors

Carbon Energy Hydrogen

Autotrophs (CO2)

*Heterotrophs (Reduced or

preformed organic compounds)

Phototrophs (light)

*Chemotrophs (oxidation

of organic & inorganiccompounds)

Lithotrophs (reduce inorganic

molecules )

*Organotrophs (reduce organic

molecules)

*Most Pathogens




MICR570/ZMR/F12 7/

CULTIVATION OF

BACTERIA & FUNGI

LABORATORY MEDIA

CHEMICAL CONTENT:

KNOWN: SYNTHETIC or DEFINED MEDIUM

E.g., Basal Salts Medium

NOT KNOWN: NON-SYNTHETIC or COMPLEX

E.g., Nutrient broth

Defined Medium (mg/L)

Basal Salts Medium

CaCl2 (15)

MgSO4 (120)

(NH4)2SO4 (1200)

Na2HPO4 (7000)

NaH2PO4 (200)

Glucose (10,000)

Complex Medium (g/L)

Nutrient Broth

Peptone (5.0)

Beef extract (3.0)

Sodium chloride (8.0)

Water 1000ml

All-purpose Medium: supports growth most microorganisms

E.g., Nutrient agar & broth, trypticase soy broth

Enriched media: basal growth support media + nutritivesupplements added

VAROUS AVAILABLE MEDIA

. .,

Reduced Medium: Addition of a reducing agent (thioglycolate,cystine or ascorbate) to medium to remove oxygen (anaerobescan grow)

Transport Medium: preserve microorganisms in transitfollowing isolation from patient until cultivated

Selective: Allows one species to grow and suppresses others

Addn: specific C or energy source, adjust pH, increase osmotic

pressure, adjust O2 tension

E.g., Salmonella-Shigella agar, Mannitol salt agar

Differential: More than one type of organism can grow BUTseparation is based upon distinguishes between various genera

& sp. (Visible changes in media)

E.g., MacConkey agar - Enterobacteriaceae

Eosin-methylene blue (EMB) - E.coli & Enterobacter aerogenes

Media can be selective and differential or enriched and differential at the same time




MICR570/ZMR/F12 7/

GROWTH ON MAC

E. coli S. typhi P. aeruginosa

All Pictures © Z. Ross 2005

FUNGAL CULTIVATION• Primary: Selective Isolation

• Sabouraud’s Agar

– Peptone (protein hydrolysate)

– pH 5.6 (inhibits faster growing bacteria)

– Antibiotics: Penicillin, Streptomycin, Tetracycline (inhibit

bacteria)

Moulds Yeasts

• Secondary: Culture and Species Identification

• Corn Meal Agar – Incubated at 25oC, several days

– Characteristic sexual structures develop

– ID based on morphology (microscopy)

Pictures taken from www.doctorfungus.org

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