Microbial physiology. Microbial metabolism. Enzymes. Nutrition. Bioenergetics. Bacterial growth and multiplication.

Growth and multiplication

mode: Binary fissionmode: Binary fission

Bacterial Cell Division

1. Replication of chromosome

2. Cell wall extension

3. Septum formation

4. Membrane attachment of DNA pulls into a new cell.

It is an increase in all the cell components, which ends in multiplication of cell leading to an increase in population.

It involves - an increase in the size of the cell & an increase in the number of individual cells.

Bacteria divide by binary fission.

Growth

Generation time

Interval of time between two cell divisions

OR The time required for a bacterium to

give rise to 2 daughter cells under optimum conditions

Also called population doubling time.

Generation time

Coliform bacilli like E.coli & other medically important bacteria – 20 mins

Staphylococcus aureus- 27-30 mins

Mycobacterium tuberculosis - 792-932 mins

Treponema pallidum -1980 mins

Colony – formed by bacteria growing on solid media. (20-30 cell divisions)

Each bacterial colony represents a clone of cells derived from a single parent cell.

Turbidity – liquid media - 107-109 cells/ml

Biofilm formation – thin spread over an inert surface.

Growth form in Laboratory

Solid medium

Colony

Liquid medium

Cell Counts ... many ways

2 methods – Total cell count - Viable cell count

Bacterial counts

Total Count

Total number of cells in the sample = living + dead.

Can be obtained by : Direct counting under microscope using

counting chambers.

Counting in an electronic device – Coulter counter.

Counting chambers

Over method

Direct counting using stained smears - by spreading a known volume of culture over a measured area of slide.

Opacity measurements using an absorptiometer/ nephalometer.

Chemical assays of cell components.

Turbidity- a spectrophotometer measures how much light gets

through 

Viable Cell Count

Measures the number of living cells.

Methods – Surface colony count Dilution method Plating method

Number of colonies that develop after incubation gives an estimate of the viable count.

Plate counts

When a bacterium is added to a suitable liquid medium and incubated, its growth follows a definite course.

If bacteria counts are made at intervals after inoculation & plotted in relation to time, a growth curve is obtained.

Shows 4 phases : Lag, Log or Exponential, Stationary Decline.

Bacterial Growth Curve

Phases of Growth Curve

1. Lag phase – No increase in number but there may be an increase in the size of the cell.

2. Log OR Exponential phase – cells start dividing and their number increases exponentially.

3. Stationary phase – cell division stops due to depletion of nutrients & accumulation of toxic products.

- equilibrium exists between dying cells and the newly formed cells, so viable count remains stationary

4. Phase of Decline – population decreases due to the death of cells – autolytic enzymes.

Phases of Growth Curve

Morphological & Physiological alterations during growth Lag phase – maximum cell size towards the

end of lag phase.

Log phase – smaller cells, stain uniformly

Stationary phase – irregular staining, sporulation and production of exotoxins & antibiotics

Phase of Decline –involution forms(with ageing)

Availability of Nutrients & H2O Temperature Atmosphere – O2 & CO2 H-ion concentration Moisture & drying Osmotic effects Radiation Mechanical & sonic stress.

Factors Affecting Bacterial Growth

Bacterial Nutrition

Water constitutes 80% of the total weight of bacterial cells.

Proteins, polysaccharides, lipids, nucleic acids, mucopeptides & low molecular weight compounds make up the remaining 20%.

Moisture & Drying

Water – essential ingredient of bacterial protoplasm. Hence drying is lethal to cells.

Effect of drying varies : T. pallidum – highly sensitive Staphylococci sp– stand for months

Spores – resistant to desiccation, may survive for several decades.

Nutrients

Functions– Generation of energy– Synthesis of cellular materials

– Essential nutrients (basic bioelements needed for bacterial cell growth)

– H2O: universal solvent; hydrolyzing agent– Carbon: food & E* source; in form of prot., sugar, lipid– Nitrogen: for prot. syn; nucleic acid syn (purines &

pyrimidines)– Sulfur (sulfate): AA syn (i.e., Cystine)– Phosphate: key component of DNA & RNA, ATP, and

inner & outer membrane phospholipids– Minerals: assoc’d w/ PRO (i.e., Fe:PRO); common

component of enzymes.

Nutrients

2 types1. Macronutrients – needed in large quantities

for cellular metabolism & basic cell structure C, H, O, N

2. Micronutrients – needed in small quantities; more specialized (enzyme & pigment structure & function)

Mn, Zn

– Fastidious Bacteria: microbes that require other complex - nutrients/growth factors ( i.e., Vitamins or AAs)

Vary in the temperature requirements.

Temperature range – growth does not occur above the maximum or below the minimum.

Optimum Temperature – growth occurs best, 37ºC for most pathogenic bacteria.

Temperature

Uptake of nutrients by bacteria

o Passive diffusiono simple diffusiono facilitated diffusion

o Active transport

Psychrophiles: -10 to 20C Psychrotrophs: 0 to 30 C Mesophiles: 10 to 48Ce.g. most bacterial pathogens Thermophiles: 40 to 72C Hyperthermophile: 65 to 110C

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Some pathogens can multiply in the refrigerator: Listeria monocytogenes

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Neutral or slightly alkaline pH (7.2 – 7.6) – majority of pathogenic bacteria grow best.

acidic pH – Lactobacilli

alkaline pH -Vibrio cholerae

H-ion Concentration

Osmotic Pressure or Osmolarity

Most bacteria require an isotonic environment or a hypotonic environment for optimum growth.

Osmotolerant - organisms that can grow at relatively high salt concentration (up tp 10%).

Halophiles - bacteria that require relatively high salt concentrations for growth, like some of the Archea that require sodium chloride concentrations of 20 % or higher.

34Similar effect with sugars

Radiation X rays & gamma rays exposure – lethal

Mechanical & Sonic Stress May be ruptured by mechanical stress.

Radiation, stress

Some bacteria require certain organic compounds in minute quantities – Growth Factors OR Bacterial Vitamins.

It can be : Essential – when growth does not

occur in their absence. Accessory – when they enhance

growth, without being absolutely necessary for it.

Growth Factors

Identical with eukaryotic nutrition Vitamin B complex – thiamine riboflavine nicotinic acid pyridoxine folic acid & Vit.B 12

Growth Factors

Primary gases = O2, N2, & CO2

O2 - greatest impact on microbial growth (even if the microorganism does not require it)

Aerobic respiration – terminal electron acceptor is oxygen.

Anaerobic respiration – terminal electron acceptor is an inorganic molecule other than oxygen (e.g. nitrogen).

Presence or Absence of Gases

Strict (Obligate) Aerobes – O2 present, require O2 for growth e.g. Pseudomonas aeruginosa

Obligate aerobe – 20% O2: only grows with O2 Microaerophile – 4% O2: best growth with small amount O2

e.g. Campylobacter spp, Helicobacter spp

Strict (Obligate) Anaerobes – O2 depleted, grow in the absence of O2 & may even die on exposure to O2 e.g. Bacteroides fragilis

Obligate anaerobe: only grows in absence of O2 Aerotolerant anaerobe: anaerobes that “tolerate” +/or survive in O2, but do NOT

utilize O2 during E* metabolism e.g. Clostridium perfringens

Facultative Anaerobe – grows both in presence & absence of O2; but grows BEST under Aerobic conditions; considered to be aerobic organism; O2 present – aerobic respiration for E*; O2 absent – anaerobic pathways (fermentation)

e.g. Staphylococcus spps

Capnophilic organism – requires high CO2 levels eg Neisseria spps

Depending on the O2 requirement

Oxygen-related growth zones in a standing test tube

Oxygen is readily converted into radicals (singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical)

Most important detoxifying enzymes are superoxide dismutase and catalase

Cells differ in their content of detoxifying enzymes and hence, ability to grow in the presence of oxygen

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Classification of gram-positive cocci Staphylococci are catalase + Streptococci are catalase -

Staphylococci

Streptococci

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pH Majority of bacteria grow BEST at neutral or

slightly alkaline pH• pH 7.0 – 7.4 => this is near most normal body

fluids

Acidophiles: grow BEST at low pH (acid: pH 0 – 1.0)

T.B. - pH 6.5-6.8

Alkalophiles: grow BEST at high pH (alkaline: pH 10.0)

V. cholerae - pH 8.4-9.2