Microbial Growth. By Dr. Carmen Rexach Microbiology Mt San Antonio College

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1 Microbial Growth By Dr. Carmen Rexach Microbiology Mt San Antonio College

2 Microbial growth Definition Increase in the number of cells Increase in microbial mass Requires specific physical and chemical conditions

3 Physical requirements Temperature ph Osmotic pressure Minimum, optimum, maximum

4 Classifications by temperature Psychrophiles Psychrotrophs Mesophiles Thermophiles Extreme thermophiles

5 Cold temperature Psychrophiles [-10 to -18 o C (optimum = -15) Example: ocean floor Psychrotrophs 0 to 30 o C (optimum = 20) Example: grow in refrigerator freezing stops microbial growth, does not necessarily kill bacteria

6 Moderate temperature Mesophiles o C (optimum 25-40) Examples: human pathogens, spoilage, disease organism

7 Hot temperature Thermophiles o C (optimum 50-60) Example: hot springs Extreme thermophiles o C Example: hydrothermal vents Taq polymerase = enzymes from Thermus aquaticus

8 ph Most organisms require for optimum growth Acidophils Bacteria tolerant to acidity Optimum ph in lab Maintained by adding buffers (peptones, phosphate salts, etc)

9 Osmotic pressure Bacteria 80-90% water Hypertonic solutions crenation Hypotonic solutions Lysis of membrane Prevented by cell wall Halophiles Require salt for growth (Dead Sea) Facultative halophiles Can grow in salt, not required

10 Halophiles Salt lake

11 Chemical requirements Carbon Nitrogen, Sulfur, Phosphorus Trace Elements Oxygen

12 Carbon All organisms require carbon source By definition: all organic molecules contain carbon and hydrogen Chemoheterotrophs Utilize organic carbon sources Chemoautotrophsand photoautotrophs Utilize inorganic carbon = CO 2

13 Nitrogen, sulfur, phosphorus N amino acid formation Some bacteria fix inorganic nitrogen Others use organic nitrogen = symbiosis S amino acid synthesis and some vitamins P synthesis of nucleic acids and phospholipids

14 Trace elements Small amount of mineral elements Usually needed by enzymes as cofactors Include Fe Ca ++ Mg ++

15 Oxygen All organisms using oxygen require special enzymes to protect cells from toxic effects of oxygen Superoxide dismutase Catalase or peroxidase Two categories based on oxygen requirements Aerobes Anaerobes

16 Aerobes Aerobes = use oxygen obligate aerobes = require oxygen How much? Microaerophiles = grow in oxygen concentrations lower than air Facultative anaerobes = aerobes with the ability to grow in absence of oxygen Intestinal bacteria, yeasts

17 Anaerobes Aerotolerant anaerobes Tolerate oxygen Can t use it for growth Many ferment carbohydrates to lactic acid Ex: Lactobacillus Obligate anaerobes Killed by oxygen Do not contain enzymes necessary to detoxify

18 Culture medium Any material prepared for growth of bacteria in a lab Culture = microbes growing in or on culture medium Agar used to solidify culture

19 Types of culture media (start with sterile media) Chemically defined Complex media Anaerobic growth media/methods Special techniques Selective and differential media Enrichment culture Pure culture

20 Culture media Chemically defined Know exact chemical composition Complex Several components Varies from batch to batch Ex) nutrient agar, nutrient broth Special techniques Ex) culturing certain bacteria or parasites on living tissues or cells

21 Anaerobic growth media and methods Special reducing media Thioglycolate broth Ties up oxygen BBL jars Removes oxygen chemically Contains anaerobic indicator (methylene blue) Other options: anaerobic glove box

22 Anaerobic methods

23 Culture media Selective and differential media Encourages growth of selected microorganisms Ex) sabdex Helps to identify certain organisms Ex) CCFA Blood agar = α, β, γ hemolysis Enrichment culture Mixed culture and want to encourage preferential growth of one organism

24 Pure culture Techniques work on basis that each colony arises from division of single bacterial cell Two techniques Pour plate Streak plate Goal: to grow isolated colonies

25 Bacterial division Binary fission Budding Spore formation Actinomycetes (at tip of filaments) Fragmentation Filamentous bacteria

26 Binary fission Bacterial cell elongates. Bacterial DNA replicates. Cell wall and plasma membrane grow inward from both sides, forming a septum. Cross wall forms and two cells are produced identical to the parent cell.

27 Generation time Amount of time it takes for cell to divide or for population to double Growth rate Population growth = change in the number of cells or cell mass per unit of time Bacterial cells multiply logarithmically Rate varies among different organisms and under different nutritional and genetic influences

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29 Phases of growth

30 Lag phase Getting used to new environment Lots of metabolism No lag phase if inoculate from exponentially growing culture Lag phase If inoculate from stationary phase due to depletion of certain nutrients If move from richer culture to poorer culture

31 Log phase & stationary Log phase phase Exponential growth Greatest metabolic activity Increased susceptibility to adverse conditions Stationary phase Number of microbial deaths equal to new cells produced Nutrient depletion, increased wastes, changes in ph

32 Death or logarithmic decline Deaths exceed new cell formation Exponential function but slower than log phase

33 How to measure microbial growth Plate counts Serial dilutions to keep number of colonies to be counted low Other methods Direct count under microscope using slides with counting chambers Electronic cell counters, etc. Indirect methods Turbidity = indicator of increased number Measure amount of metabolic activity Amount of CO 2 produced Dessicate and weigh

34 Bacterial plate count