Microbial Growth pp Cell wall Partially formed cross wall DNA (nuclear area) Fig Part B 1

Physical and chemical requirements for growth Various types of culture media Bacterial cell division Phases of bacterial growth Ch 6 Overview 2

Distribution of Microbes Growth of a population – Increase in the number of cells – Not size Each ‘spot’ on the agar is a colony When visible – Cells number in the millions Bacterial colonies 3

Requirements for Growth Physical and Chemical Reasons for understanding the conditions necessary for growth: 4 – To control growth of pathogens – To encourage growth of helpful microbes

Physical requirements: Temperature pH Osmotic pressure Carbon, Nitrogen, sulfur, phosphorus, trace elements, organic growth factors Oxygen Chemical requirements: 5

Temperature Classified in 3 groups 1.Psychrophiles (cold-loving) 2.Mesophiles (moderate-temp-loving) 3.Thermophiles (heat-loving) Psychrophiles MesophilesThermophiles Room T 6

Minimum, Optimum, Maximum Growth Temperatures Minimum: the lowest T that a species will grow Optimum: best growth T Maximum: highest T that growth is possible 7

Most grow in a limited range ‒ Maximum & minimum growth T about 30 o C apart ‒ Grow poorly at high & low T in their range Fig

Optimum Growth Rate ‒ Fastest reproduction = peak of curve ‒ At either T extreme = rate drops ‒ At T just above optimum = fastest drop Fig

Psychrophile – Can grow at O o C – Optimal growth at ~15 o C – But, very sensitive to higher T – Will not grow at ~25 o C – Habitat: deep ocean – Pathogenesis: rarely causes food spoilage 10

Psychrotroph A slight ‘variation’ of a psychrophile – Can grow at O o C – Slightly higher optimal growth T ~ o C – Also, very sensitive to higher T – Will not grow above ~40 o C – Much more common than psychrophiles – Pathogenesis: frequent cause of food spoilage 11

Most common food preservation method – (pathogenic) bacteria grow poorly At the high and low T in their range 12 (even though) psychotrophs grow at ~20-30 o C – They grow slowly ‒ And are able to slowly degrade food Refrigeration

1.To slow growth of – Most spoilage microbes 2.To prevent growth of – Most pathogenic species O F O C Fig Set T in Refrigerator at a Low Setting

– Optimal growth ~ 37 o C – Most common bacteria – Aerobic – Can be pathogenic – But also, can do work of industrial importance Mesophiles 14

Produced by – Propionibacteria freudenreichii – Grows at 24 o C – Ferments lactate to acetate, propionate & CO 2 – Acetate, propionate give the cheese its flavor 15 ‘Swiss’ Cheese

‒ Growth: ~50-60 o C, and higher ‒ Habitat: hot springs ‒ H 2 S provides source of energy Thermophiles Boulder Spring Lower Geyser Basin Yellowstone Nat’l Park 16

Describes acidity or alkalinity of a solution Most bacteria grow in a neutral environment – Between ~6.5 and 7.5 Example: – Escherichia coli pH 17 Fig. 2.7

ph below ~4 is acidic Preserves foods..sauerkraut, pickles 18 Bacterial fermentation produces acids Called acidophiles Helicobacter pylori Fig Fig. 2.7

Solute = [NaCl] 19 Osmotic Pressure Fig. 6.4 The pressure [NaCl] is equal across the cell membrane

Refers to the concentration of NaCl 20 Osmotic Pressure Solute = [NaCl] Chapter 4, p. 93-4, Fig Inside a structure 2.And in the solution surrounding the structure

Osmotic pressure [NaCl] concentration is equal across the membrane 21 Isotonic Pressure Fig. 6.4

Osmotic pressure [NaCl] concentration is: 22 Hypertonic Pressure 1.Higher in the solution surrounding the bacterium 2.Than inside the bacterium 3.Causes death of the bacterium

Osmotic pressure [NaCl] concentration is: 23 Hypotonic Pressure Chapter 4, p. 93-4, Fig 4.18d 1.Lower in the solution surrounding the bacterium 2.Than inside the bacterium 3.Also causes death

1.Water leaves the cell 2.Enters the high solute [NaCl] 3.Causes shrinkage of the cell cytoplasm 4.Cell death 24 Plasmolysis -Loss of Water-  High salt used to preserve foods (or sugar)

Microbes must acquire nutrients from its environment to grow & reproduce Chemical Requirements 25 Water, carbon, nitrogen, minerals (S, P, Ca, Mg, Na, etc) In the lab, we provide nutritional needs – In culture media – Media must contain

Water (80-85% weight of cells) Carbon (50% wt/cell) – To get C, use organic molecules (proteins, carbohydrates, lipids) or CO 2 Nitrogen (14% wt/cell) – Digest protein into amino acids – Use N for protein, NA synthesis 26

Minerals – Sulfur (amino acids, thiamine, biotin) – Phosphorus (DNA, RNA, ATP, membranes) – K, Mg, Ca (cofactors for enzymes) – Trace elements (usu enzyme cofactors) Iron, copper, molybdenum, zinc – Organic growth factors for some bacteria p

Oxygen Classification based on oxygen requirements 28 1.Obligate aerobes 2.Facultative anaerobes 3.Obligate anaerobes 4.Aerotolerant anaerobes 5.Microaerophiles

Microbes that use oxygen – Produce more energy from nutrients – Than microbes that do not use oxygen Obligate aerobes require oxygen to live Obligate Aerobes Examples: Bacillus, Serratia, Pseudomonas, Vibrio

Oxygen is poorly soluble in water – Therefore, many bacteria have evolved to live in the absence of water Can use oxygen when it is present But, in absence, use fermentation or anaerobic respiration Produces less energy Facultative Anaerobes Examples: Escherichia coli, Shigella, Salmonella

Microbes unable to use oxygen for energy yielding reactions Most are killed by oxygen Obligate Anaerobes Example: Clostridium (botulism, tetanus)

Cannot use oxygen for growth But, can tolerate it – Posses enzyme SOD which neutralizes oxygen Mostly ferment carbohydrates to lactic acid Aerotolerant Anaerobes Example: Lactobacillus

Microaerophiles are aerobic They require oxygen But, only in LOW [O 2 ]’s Sensitive to superoxide free radicals – They do not have the SOD enzyme Microaerophile Example: Micrococcus

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O 2 and Enzymes Aerobes, facultative anaerobes & aerotolerant anaerobes must have the enzyme: Superoxide dismutase (2O 2 – + 2H+ O 2 + H 2 O 2 ) See Lab experiment Peroxide is toxic to cells

And aerobes & facultative anaerobes must have either: Catalase See Lab experiment (2H 2 O 2 2H 2 O + O 2 ) Peroxidase (H 2 O 2 + 2H+ 2H 2 O)

Called binary fission A single cell divides into 2 cells A few species divide by ‘budding’ Bacterial Cell Division 37 Fig. 6.11

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Pure cultures are obtained by the streak plate method 39 Streak Plate Method Fig. 6.10a,b; Ch 6, p 173-4

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Phases of Bacterial Growth Generation time – Time required for a cell to divide – Or, a population to double Most bacteria have a generation time of – One to three hours – Others require more than 24 hours 41

Bacterial division occurs according to a logarithmic progression 42 Logarithmic Representation of Bacterial Populations Fig b – 2 cells, 4 cells, 8 cells, 16 cells, 32 cells… (y x )

43 Bacterial Growth Curve

Q’s 44 1.Which of these terms describes a microorganism that grows at human body temperature (37 o C)? A.Thermophile B.Psychrophile C.Psychrotroph D.Mesophile E.Hyperthermophile 2.During log phase, bacteria are: A.Dividing at fastest rate B.Preparing to divide C.Dying exponentially D.Dying and dividing in equal numbers

Q’s 45 1.Which of these terms describes microbes that do not use oxygen, but can grow if it is present? 2.Which of these is an example of an organic growth factor? A.Obligate aerobe B.Obligate anaerobe C.Facultative anaerobe D. Microaerophile E. Aerotolerant anaerobe A. Glucose B. Oxygen C. Biotin D. Sodium chloride E. Agar

46 Appendix

47 1.Lag phase: – Little or no change in the number of cells, but metabolic activity is high 2.Log phase: – Bacteria multiply at the fastest rate possible under the conditions provided 3.Stationary phase: – An equilibrium between cell division and death occurs 4.Death phase: – Deaths exceed the number of new cells formed Bacterial Growth Curve - Phases

48 Q’s 1.Shrinking of the plasma membrane in response to osmotic loss of water is called: 2.Bacteria that spoil food in the refrigerator are most likely: A.Thermophiles B.Psychrotrophs C.Mesophiles D.psychrophiles 3.The _____ _____ _____ is the most common technique used to obtain pure cultures of microbes.

49 1.Organisms such as Clostridium lack oxygen- detoxifying enzymes, thus they are: A.Obligate aerobes B.Obligate anaerobes C.Facultative anaerobes D.Microaerophiles E.Aerotolerant anaerobes 2.Which enzyme catalyzes the reaction: H 2 O 2 + 2H + 2H 2 O A.Catalase B.Oxidase C.Peroxidase D.Superoxide dismutase Q’s

50 1.Which growth phase is also called the exponential growth phase? A.Lag phase B.Log phase C.Stationary phase D.Death phase Q’s

51 Which test tube in the figure shows how a bacterial culture of facultative anaerobic bacteria would grow? Q’s

52 During which stage in the growth curve shown in the figure is the number of cells dying greater than the number of cells dividing? A.Lag phase B.Log phase C.Stationary phase D.Death phase Q’s