Microbial Growth and Culturing

Microbial Growth

Refers to increase in NUMBER not Size

• Mother Cell Doubles in Size + Dupes Content

• Divides into 2 Daughter Cells

Cell Division

Occurs by Budding or Binary Fission

• Binary Fission → Most Bacteria

  • Cell Dupe + Divide

  • Daughter becomes Independent when a Septum grows Between Them and they Separate
    
    

• Budding → Mainly in Yeast

  • Small New Cells Develops on Surface and Separates

Binary Fission

Dupes their Chromosome before Division

Chromosome attached to Cell Membrane

Cells Temporarily have 2+ Chromosomes

Cells may not Separate Completely

• Diplo, Strepto

Culturing Bacteria

Must have Culture Medium with Nutrients

Culture under Proper Environmental Conditions

• Temp

• pH

• Aeration

• Solute Concentration / Osmolarity

Growth Curve

Inoculate Sterile Liquid Media in Flask with Bacteria from Pure Culture

Incubate Cells at Good Temp

Remove Small Volumes At Intervals and Plate

Incubate and Count Colonies

Total = Number of Colonies x Dilution Factor

Phases of Bacterial Growth - In Liquid Media

Lag Phase → Bacteria do not Increase in Number, but are Metabolically Active

Exponential Log Phase → Once Adapted, Growth is Exponential

• Generation (Doubling) Time → Time it takes to Double

Stationary Phase → Nutrients are Used up + Metabolic Waste Accumulate = Growth Levels Off, Cell Number Stay Constant

Death Phase → Conditions Worsen, Cells Decrease at Exponential Rate

• Involution → Cells take Weird Shapes

• Pleiomorphic

• Don’t Use for Staining

Importance of Growth Curve

Infection and Disease

Food

Industrial

Environmental

Genetic Engineering

Measuring Bacterial Growth - Cell Count

Serial Dilution → Plate Count

• Count Colonies on Plates with 30 - 300 Colonies (CFUs)

Direct Microscopy Counts → Hemocytometer

Measurement of Turbidity → Spectrophotometer

Dry Weight → Heat Cells to Remove Water and then Weigh

Direct Measurements of Microbial Growth

Hemocytometer → Glass Microscope Slide with Grid in Liquid Suspension

• Combo with Viability Stain to Distinguish Live/Dead Cells

Indirect Measurement

Spectrophotometer → Turbidity

• Measures Optical Density (OD600)

• Amount of Light Transferred Through Liquid

• Cells Scatter Light, Reducing Transfer

• Fast

Problem → Can’t Tell Dead or Alive

New Method for Analyzing Growth

Polymerase Chain Reaction → Quantifies Bacteria in Environmental Isolating and Culturing

Tests that Measure ATP

• Used in Food Practice

• Used for Quick Quantification

Microbial Growth

Colony → A Group of Descendants of Original Cell

• Arise from Single Cell, Spore, or Group

• After Growing Rapidly, Edge Continues to Divide → Middle Die

Types of Media

Defined → Made from Known Chemical Ingredients

• Used when exact nutrients of organism is known

• Ex: Glucose salt mediums

• Glucose, K+, Mg++, NH4, PO4, Vitamins, Amino Acids
  
  

Complex → Unknown Chemical Composition

• Used for General Cultivation → Rich in Nutrients

• Extract of animal, plants, or yeast

• May have ground up cells, tissues

General and Enriched Media

General Purpose Media → Help growth of non-picky cells

• Complex Media with wide range of nutrients
  
  

Enriched Media → Complex Organic Substances (Blood, Growth Factors)

• FASTIDIOUS (picky) bacteria like

• Growth Factor → Specific Vitamins / Amino Acids

Selective and Differential Media

Media can Distinguish

• Designed for Growth of Specific Cells

• Cost Effective
  
  

Selective Media → Only Specific Type can Grow

• 1+ Agent that Inhibits Growth of Unwanted
  
  

Differential Media → Lets you Distinguish from Appearance

• Has pH Indicators that Color Cells

BOTH Selective and Differential Media

SOME ARE BOTH

Mannitol Salt Agar

• 7.5% NaCl → Kills Non-Staphylococcus

• Mannitol → Fermentable Carbohydrate (Differential Agent)

• Phenol Red Dye → pH Indicator → Yellow

MacConkey Agar

• Bile Salt → Detergent

• Crystal Violet → Dye → Inhibits Gram +??

• Neutral Red Dye → pH

• Lactose → Fermentable Differential Agent

  • E. coli Turns it Pink

• Test for Enteric (Intestine) Bacteria

Blood Agar and Hemolysis

Blood Agar → Differential Agar → Based on Ability to Break Blood Cells

• Beta Hemolysis → Completely Break RBC → Clearing Around Cells

• Alpha Hemolysis → Partially Break → Greenish Color Around

• Gamma Hemolysis → No Break → No Change

Chocolate Agar - Thayer Martin

Lysed Blood to Media

• Enriches for N. gonorrhea → Picky

• Grow Well in Human Body

• Needs Free Hemoglobin to Survive

Special Culture Techniques

Some Bacteria Can’t be Cultured on Media

Some Needs to be Cultured Inside Cells or Organisms

• 9 Banded Armadillo → Cool Body Temp

  • Can be Infected with Mycobacterium leprae

Bacteria Needing High CO2 → Placed in Jar

• No Oxygen

• Anaerobic Growth