lecture+07+microbial+growth+ +cultivation+note+form

7/27/2019 Lecture+07+Microbial+Growth+ +Cultivation+Note+Form

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MICR570/ZMR/F12 7-1

MICROBIAL GROWTH & MULTIPLICATIONLecture 7

LEARNING OBJECTIVES

At the end of this lecture, you should be able to:1. Define bacterial replication and describe the various stages of the bacterial growth

cycle, recognizing the limiting factors that affect each stage in a closed environment.

2. List the physical and chemical factors required for or affecting the growth of bacteria &

fungi and differentiate/classify the bacteria on the basis of their requirements.

3. Explain the importance of these factors with respect to isolation and identification of

bacteria & fungi.

4. Differentiate between the various media types used for isolation and identification of

bacteria & fungi.

INTRODUCTIONMicrobes that are provided with nutrients and required environmental factors becomemetabolically active and grow. Growth occurs on two levels; a cell synthesizes new cellcomponents and increases its size and the number of cells in a population increases. Thecapacity for multiplication has tremendous importance in microbial control, infectious diseaseand biotechnology.

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BACTERIAL GROWTHIs the coordination of chemical & physical processes. Requires metabolism, regulation &division. CHARACTERIZED: Continuous macromolecular synthesis; Cell elongation occursalong with genome replication

BACTERIAL CELL CYCLE Refer to: Murray et al. P31 Fig3-10: Bacterial Cell Division.SIMPLE: The division of a mother cell into 2 daughter cells THEN 1 daughter cell divides into2 more daughter cells.ASEXUAL PROCESS Known as BINARY FISSION. Division is geometrical (the population

doubles).DOUBLING TIME: (formerly known as Generation time) is the time required for a single cell todivide into 2 daughter cells.

GROWTH RATE: time for cell to reproduceDOUBLING TIME: Time required for a complete fission cycle i.e., 1 parent cell → 2 new daughter cells (1st Generation)2nd = 4 cells3rd = 8 cells and so on & on & on



MICR570/ZMR/F12 7-2

Examples (Under Optimal Conditions)Bacillus stearothermophilus 11 mins Escherichia coli 20 minsStaphylococcus aureus 28 minsLactobacillus acidophilus 60-80 minsMycobacterium tuberculosis 360 minsTreponema pallidum 1980 mins

QUANTITATIVE ASSEMENTENUMERATION: Measurement of viable bacteria

• Serial dilution: used for concentrated samples and enumeration of viable bacterialcells. The stock suspension is diluted 10-folds and viable bacteria from each dilutionare determined by the plate count method.

• Plate count: used for enumeration of viable bacteria (CFU – colony forming units). Afraction (0.1ml) of each dilution is plated on an appropriate agar plate and followingincubation the total number of colonies formed, are counted. Assuming that eachcolony is formed by one bacterial cell, the total number of viable bacteria is equal tothe total number of colonies. Therefore total viable count is determined by multiplying

the number of colonies by the sample size (0.1ml) and the dilution factor.

GROWTH CURVEIn a closed system: nutrients and space finite; no removal of waste products

A LAG PHASE1. Newly inoculated cells need to adjust to new environment (nutrient, temp, pH, etc)2. NO cell division during this phase3. Population is sparse or dilute

B EXPONENTIAL (LOG) PHASE1. Cell adjustment completed2. Population growth is at geometric/logarithmic rate2. Cells reach maximum rate of cell divisionThis continues as long as nutrients and environment is favorableFACTORS INFLUENCING LOG PHASE LENGTH = microbial species, temperature & nutrientavailabilityE.g., E. coli @ 30oC g = 1hr, @ 37oC g = 30mins

Time (hrs)

Log10

viable cell #’s

A Lag

B Log

C Stationary

D Death



MICR570/ZMR/F12 7-3

C STATIONARY PHASE1. Population reaches maximum numbers2. Rate of cell inhibition (death) = Rate of multiplicationFACTORS INFLUENCING STATIONARY PHASE = nutrient depletion, pH changes,accumulation of waste, reduced O2. (This can induce sporulation - Bacillus, Clostridium sp.)

D DEATH PHASE

1. Decline in growth rate (reverse of Log phase)2. Death occurs in geometric fashionFACTORS INFLUENCING DEATH PHASE = depletion of nutrients, O2, excretion of toxicwaste products, increased density of cells (limited space), release of lytic enzymes

FUNGAL GROWTH (N.B Moulds & Yeasts)Moulds: Hyphae→ Mycelium

Extension at tip (Apical Growth). Continuous protoplasm movement into tip. Up to 40μm/min.Provides penetrating power (fresh nutrients)

FUNGAL REPRODUCTIONMoulds: Sporulation is the means by which fungi reproduce and spread through theenvironment. Spores (Sexual & Asexual): metabolically dormant, released by mycelium inlarge numbers.

Asexual Sporulation Asexual spores (conidia) formed by mitosis in/on specialized hyphae (conidiophores).

Sexual SporulationInitiated when haploid nucleus from compatible strains of same species fuse to form transient

diploid. Sexual spores (ascospores) formed from meiosis of transient diploid.

Fungal Sub Division Sexual Asexual

Zygomycota Thick walled zygospores Internally sporangium

Ascomycotina Internal in ascus As conidia

Basidomycotina External on club basidium Usually none

Deuteromycotina(Fungi Imperfecti)

NOT KNOWN On conidia

Yeasts: Single cells → Daughter cells Refer to: Murray et al. P59 Fig5-2: Fungal CellMorphology

Reproduce by budding (Asexual process)



MICR570/ZMR/F12 7-4

FACTORS AFFECTING GROWTH OF BACTERIA & FUNGIThere are 2 categories of influencing factors. 1. Physical/Environmental & 2. Chemical

1 A. TEMPERATUREThere are 3 Cardinal Temperatures of importance to bacteria

MINIMUM: The lowest temp that permits microbial growth and metabolism MAXIMUM: The highest temp that permits growth and metabolism

OPTIMUM: A narrow range of temp’s that promotes the fastest growth (metabolism rates) Each bacterial species has its own specific min, max and optimal temps.Extremes of MIN & MAX beyond which growth is inhibited

ExampleGrowth Temperature oC

Bacterium Min Max OptimumPseudomonas fluorescens 2-4 36-38 25-30 Pseudomonas aeruginosa 10-15 41-44 c. 37Escherichia co li 15-20 45 37Bacillus polymyxa 5-10 35-45 30-32 B. stearothermophilus 30-45 65-75 c. 55

Thermus sp. 40 79 70-72

BACTERIAL CLASSIFICATION ACCORDING TO TEMPERATUREi. PSYCHROPHILE: PsychrophilicOptimum temp below 15oC; Capable of growth at 0oC; Cannot grow above 20oCFound in snow fields, polar ice, depth of ocean (NOT involved in human infections)E.g., Pseudomonas, Flavobacterium, Alcaligenes & Achromobacter sp.

ii. FACULTATIVE PSYCHROPHILE: PsychrotrophGrow slowly in cold conditions BUT have optimum temp above 20oCE.g., Staphylococcus aureus, L. monocytogenes

This group are of CONCERN: Contaminants of food/dairy products

iii. MESOPHILE: MesophilicOptimum temp 20-40oC; Capable of growth 10-50oCGroup containing HUMAN PATHOGENS (30-37oC) E.g., E. coli

iv. THERMOPHILE: ThermophilicOptimum temp >45oC; Capable of growth 45-85oC; Incapable of growth at usual body tempFound in Volcanos, with direct exposure to sun, hot springs (NOT involved in humaninfections)

N.B. DIMORPHISM IN FUNGI (Depends on chemical & physical factors)FREE-LIVING STATEMycelial or hyphal form. At sub-physiological temperature (25oC). Distinct sexual forms aredisplayed

PARASITIC STATEYeast form. Due mainly to physiologic temperature (37oC). Oval morphology (Nothing distinct)E.g., Candida albicans (Yeast: human tissue, Mycelial form: in culture (Pseudohyphae))Histoplasma capsulatum (Yeast: human tissue, Mycelial form: in nature)



MICR570/ZMR/F12 7-5

1 B. GAS REQUIREMENTSOxygen is important as the terminal electron acceptor (Respiration).

It has limited solubility in water ∴ is considered a limiting factor in bacterial growth.In order to cope with oxygen containing toxic products, enzymes are required to reduceoxygen to water and toxic products (H2O2 + O2

-) superoxide.

Microbes convert toxic products to molec Oxygen by:

1. CATALASE (H2O2 → H2O + O2)2. PEROXIDASE (H2O2 + NADH + H+ → 2H2O + NAD+)

3. SUPEROXIDE DISMUTASE (2O2- + 2 H+ → H2O2 + O2) Peroxide is metabolized by

Catalase (as above)

Microbes are divided in to 4 groups based on oxygen requirementsi. (OBLIGATE) AEROBESThese are totally DEPENDANT on O2 for growth. They require at least 1 atmosphere (20%)oxygen. During growth toxic metabolic products (H2O2 and O2

-) start to accumulate but theseorganisms possess catalase and superoxide dismutase enzymes and can therefore toleratehigh [O2].

ii. MICROAEROPHILESThese can grow in the presence of O2 BUT tolerate only upto 4% oxygen. They do possessenzymes BUT if toxic products increase in concentration, enzyme systems overload inhibitinggrowth.

iii. (OBLIGATE) ANAEROBESThese grow ONLY in ABSENCE of O2 as they do not possess any enzymes to remove toxicproducts of respiration. Presence of low levels of oxygen is LETHAL.CAPNOPHILES: Organisms grow better in high CO2, but can tolerate O2 for small periods of time

iv. FACULTATIVE ANAEROBES

These have the ability to grow in the presence or absence of O2. When oxygen is presentaerobic respiration takes place while in the absence of oxygen, fermentation reactionsproduce energy. They all grow best under AEROBIC CONDITIONS

Bacterial Enzymes that protect the cell against Toxic forms of Oxygen

Microorganism Catalase Superoxide Dismutase

Aerobe + +Facultative anaerobe + +

Microaerophile - +

Obligate anaerobe - -

Bacteria: All 4 groups; Fungi: Normally aerobic; Yeasts: Facultative anaerobes

1 C. WATER ACTIVITY (Aw)ALL ORGANISMS require water for their growth & reproduction. It is an essential solvent,involved in all biochemical reactions and indirectly maintains osmotic pressure.

Aw = index of free water to react = atmospheric measure (Relative Humidity; RH)RH = 100 Aw. Therefore, 90% RH = 0.90 Aw



MICR570/ZMR/F12 7-6

Absorption and Solution factors reduce availability of free water (↓ Aw)Pure distilled water (Aw =1)

E.g., Saturated soln NaCl (Aw = 0.8); Seawater [NaCl] ≈3% (Aw = 0.98)

Most microbes have a growth optimum Aw = 1.0. If the amount of free water declines = slowgrowth rate.

Most bacteria have an active metabolism at Aw>0.9. Below Aw 0.9, they are unable to grow.

EXCEPTIONSXEROTOLERANT: survive & grow at lower AwFungi able to grow Aw 0.60; Yeasts (conc sugar soln’s Aw = 0.60)Salt-tolerant Bacteria: Halophiles (High [Solute], low Aw)

Association: Foods having a high Aw spoil quickly as bacteria utilize the water and multiply,whereas foods which have a low Aw (less free water available) will keep fresh longer

1 D. EFFECTS OF pHpH - 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); Acidic Soln (pH <7)Growth rates are influenced by pH values (N.B. Protein structure)

PHYSICAL FACTORS

CHEMICAL FACTORS2. NUTRIENT CONCENTRATION

MACRONUTRIENTS: Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorus & Sulphur (CHNOPS). All of these elements are building blocks for various compounds essential for microbial structures and metabolism, i.e., CH20’s, Lipids, Protein & Nucleic acids

MINERALS: K+, Ca2+, Mg2+, Fe2+/Fe3+. All of these are cations and are involved in enzymaticreactions. (Potassium - enzyme activity, Calcium - heat resistance, Magnesium - enzymecofactor, Iron - cytochromes, cofactor).

Temperature

Psychrophiles (0-20oC)

Mesophiles (20-40oC)

Most pathogens (30-37oC)

Thermophiles (45-85oC)

Gas (Oxygen)

Aerobes (20% O2)

Microaerophile (4% O2)

Anaerobes (No O2)

Facultative Anaerobes (O2)

Water Aw pH

Xerotolerant <0.6

Halophiles <Aw

Acidophiles <pH7

Neutrophiles pH7

Alkalinophiles >pH7



MICR570/ZMR/F12 7-7

TRACE ELEMENTS: Mn, Zn, Co, Ni, Cu, Mo. These are components of various enzymesand/or are cofactors of store enzymes.

ALL ARE REQUIRED for energy production & macromolecular biosynthesis

N.B. GROWTH LIMITED BY REQUIRED [NUTRIENT]

CLASSIFICATION OF MICROBES is based on the source of energy utilizedCARBON

• Autotroph Utilize Inorganic Carbon (CO2) as sole/principle C source

• Heterotroph Require reduced/pre-formed Organic molecules produced byother microorganisms

ENERGY

• Phototrophs Energy is derived from sunlight and NOT directly from anyorganic or inorganic source (Photosynthesis)

• Chemotrophs Energy id derived from oxidation of organic or inorganicmacromolecules

HYDROGEN/ELECTRON

• Lithotrophs Depend on reduced inorganic molecules for electron transfer • Organotrophs Depend on reduced organic molecules

MOST PATHOGENS are Chemoorganot rophic heterotrophs

CHEMICAL FACTORS

CULTIVATION ON LABORATORY MEDIAMost culture media are developed to isolate and grow a variety of microbes within a particular

group (i.e., bacteria or fungi). These media can be divided according to their use and specificneeds of the microbe of interest.

BASED ON CHEMICAL CONTENT:

Media where the chemical nature of all the medium components is KNOWN= SYNTHETIC or DEFINED MEDIUM E.g., Basal Salts Medium If the medium contains one or more complex organic compounds whose chemical nature isNOT KNOWN= NON-SYNTHETIC or COMPLEX MEDIUM E.g., Nutrient broth

Carbon

Autotrophs (CO2)

*Heterotrophs (reduced or

preformed organic

compds)

Energy

Phototrophs (Light)

*Chemotrophs (Oxidation

of organic/inorganic

compds)

Hydrogen

Lithotrophs (reduce

inorganic molecules)

*Organotrophs

(reduce organic

molecs)



MICR570/ZMR/F12 7-8

Defined Medium (mg/L) Complex Medium (g/L)Basal Salts Medium Nutrient BrothCaCl2 (15) Peptone (5.0)MgSO4 (120) Beef extract (3.0)(NH4)2SO4 (1200) Sodium chloride (8.0)Na2HPO4 (7000) Water 1000mlNaH2PO4 (200)

Glucose (10,000)

VAROUS AVAILABLE MEDIAAll-purpose Medium: supports growth most micro-organisms E.g., Nutrient agar/broth

Enriched media: basal growth support media + nutritive supplements added E.g., Blood agar

Reduced Medium: Addition of a reducing agent (thioglycolate, cystine or ascorbate) tomedium to remove oxygen (anaerobes can grow)

Transport Medium: preserve microorganisms in transit following 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 tensionE.g., Salmonella-Shigella agar, Mannitol salt agar

Differential: More than one type of organism can grow BUT separation is based upondistinguishes between various genera & sp. (Visible changes in media)E.g., MacConkey agar - Enterobacteriaceae; Eosin-methylene blue (EMB) - E.coli &Enterobacter aerogenes

PLEASE NOTE: Media can be selective and differential or enriched and

differential at the same time

FUNGAL CULTIVATIONPrimary/Selective Isolation of Fungi use Sabouraud’s Agar Made from simple peptone (protein hydrolysate) and agar, the pH is adjusted to 5.6 (inhibitsfaster growing bacteria) and contains various antibiotics (Penicillin, Streptomycin,Tetracycline) which inhibit bacteria.

Secondary for Culture and Species Identification use Corn Meal Agar Incubated at 25oC, several days, this allows the characteristic sexual structures develop

providing ID based on morphology (microscopy).

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Review Questions1. What phase of growth are bacterial cells dividing in a regular and orderly process? Howcould this be maintained?2. Why do bacterial cells enter the stationary phase?3. What roles do oxygen, water and pH play to protect us from bacterial colonization in variousparts of our body?



MICR570/ZMR/F12 7-9

4. In which medium do you think Escherichia coli would grow more quickly and why?5. If you wanted to differentiate between different Streptococci species which medium wouldyou use?

References in Murray, P.R., Rosenthal, K. S. & Pfaller, M.A. (2009) Medical Microbiology, 6 th Edition (Recommended Course Text)

Chapter 3: Bacterial Metabolism & Growth p23-38.

Chapter 5: Fungal Classification, Structure & Reproduction p57-63.

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