Microbiology and Parasitology - Bacterial Growth Requirements and Culture Media

Culture Media

  • A culture medium is a nutrient material prepared for the growth of microorganisms inside the laboratory.

History of Culture Media

  • Louis Pasteur used simple broths made up of urine or meat extracts.

  • Robert Koch used potato pieces to grow bacteria

  • Fanny Hesse, wife of Walther Hesse, an assistant of Robert Koch suggested the use of agar to solidify culture media.

Types of Culture Media According to Physical State

  1. Liquid media

    • Broth, milk, infusion

    • Do not solidify

  2. Semi-solid media

    • Clot-like consistency at room temperature

    • Solidifying agent (gelatin /agar)

    • Thickens but not firm

  3. Solid Media

    • Firm surface

    • 2 forms

      1. Liquefiable (reversible)

      2. Non-liquefiable (non-reversible)

    • For culture of bacteria & fungi

  • A LIQUID MEDIUM (tubed medium) is a water- based solution contained in a test tube that does not solidify at temperatures above freezing.

  • Examples: broths, milks or infusion

  • Nutrient broth

  • Methylene blue milk

  • Litmus milk

  • Fluid thioglycolate

  • Growth of bacteria: Dispersed, cloudy or flaky appearance

Type According to Function

  1. General purpose media

    • To grow & support growth broad spectrum microbes

    • Contains basic nutrient : nutrient agar, nutrient broth

  2. Enrichment media

    • For selected/ desired m.o.

    • Uses blood, serum or growth factor

    • Examples: Blood Agar, Chocolate Agar

      • ex. BLD. AGAR

      • Streptococcus pyogenes

      • 5% Sheep blood agar (BAP) w/ 5% bld

      • Blood Agar Plate: Hemolysis

      • Some Gr(+)'s exotoxin cause HEMOLYSIS of RBC

  3. Selective Media

    • GROWTH OF 1 SPC. M.O. ONLY

    • Examples:

      • MAC-CONKEY AGAR

        • Inhibits the growth of Gram positive bacteria

        • Selective for Gram negative bacteria

      • PHENYLETHYL ALCOHOL AGAR

        • Selective for Gram positive bacteria

      • COLISTIN NALIDIXIC AGAR

        • Selective for Gram positive bacteria

  4. Differential Media

    • Allows growth of several m.o.

    • For comparison Ex. Variety of size, color, changes in culture media’s color, gas bubble

    • GR (+)purple or blue; GR(-) RED or PINK

    • Examples:

      • BLOOD AGAR

        • Determines the type of hemolysis that the bacterial isolate produces

      • MSA Mannitol Salt Agar (MSA)

        • used to screen for S. aureus; S. aureus will turn the originally pink medium to yellow with yellow zones. Other coagulase-negative Staphylococci will produce pink to red colonies

Bacterial Growth Requirements

  • GROWTH: Orderly and organized increased in the sum of all the components of organisms. The process involves the replication of cellular structures, organelles and components

  • MICROBIAL GROWTH: This is the increase in the number of cells and not an increase in the size of the organisms

Microbial Growth Requirements

  • Nutritional Requirements

    • Carbon

    • Nitrogen, Sulfur, & Phosphorus

    • Inorganic Ions Growth Factors

  • Physical Requirements

    • Moisture/Water

    • Oxygen

    • Temperature

Nutritional Requirements

A. CARBON

  • Makes up the structural backbone of all organic molecules

  • Based on carbon source, microorganisms can be classified as Autotrophs & Heterotrophs

  • Inorganic compounds like CO_2

  • Sugars and glucos

  • Energy from Light (Photolithotrophs & Photo organotrophs)

  • Energy from Inorganic Substances (Chemolithotrophs

B. NITROGEN, SULFUR, PHOSPHORUS

  • These are necessary for the synthesis of cellular materials such as proteins and nucleic acids

  • Nitrogen and sulfur à Synthesis of proteins

  • Nitrogen and Phosphorus à Synthesis of nucleic acids and ATP

  • Approximately 14% of the weight of bacterial cell is Nitrogen and 4% is sulfur and Phosphorus

C. INORGANIC IONS

  • Magnesium, Potassium, Calcium, iron and trace elements (Manganese, Zinc, Copper and Cobalt)

  • These serves as stabilizers, co factors, integrity and enzymatic activities
    D. GROWTH FACTORS

  • Vitamin B Complex and amino acids

  • Promotes growth and development of the bacterial cells

Physical Requirements

A. MOISTURE AND WATER

  • Bacterial cell is mainly composed of water

  • Serves as medium from which bacteria acquire their nutrients

B. OXYGEN

  • Aerobes vs Anaerobes

  • Obligate vs Facultative

  • Prefixes and suffixes:

    • Bacteria are highly diverse in the types of conditions they can grow in.

    • Optimal or required conditions implied by “-phile” meaning “love”

    • Some bacteria prefer other conditions, but can tolerate extremes

    • Suffix “-tolerant”

    • Note the difference!

  • Relation to Oxygen

    • Aerobes: use oxygen in metabolism; obligate.

    • Microaerophiles: require oxygen (also obligate), but in small amounts.

    • Anaerobes: grow without oxygen

    • Capnophiles: require larger amounts of carbon dioxide than are found normally in air.

C. TEMPERATURE

  • Microbes are classified into 3 groups based on their temperature requirements

    • Thermophiles à Higher than 40c

    • Mesophiles à Optimal 20-40c

    • Psychrophiles à Optimum 10-20c

    • Most medically important bacteria are mesophiles

  • Special cases:

    • Psychrotrophs: bacteria that grow at “normal” (mesophilic) temperatures (e.g. room temperature” but can also grow in the refrigerator; responsible for food spoilage.

    • Thermoduric: more to do with survival than growth; bacteria that can withstand

D. pH

  • Acidity or alkalinity of bacterial requirement

  • Lowest = 0 (very acid); highest = 14 (very basic) Neutral is pH 7.

  • Alkalophiles à grow best in pH 8.4-9.0

  • Neutrophiles à 6.5 -7.5

  • Acidophiles à Less than 6.0

E. Osmotic Conditions

  • Determined by salt conditions

  • Halophiles à Organisms that require high salt concentrations for growth rate

  • Osmophiles à organisms the require high osmotic pressure for optimal growth.

The Bacterial Growth Curve

  • Bacterial growth over time can be graphed as cell number versus time.

  • This is called a growth curve.

  • This curve typically has 4 distinct phases:

    • Lag

    • Exponential (log) phase

    • Stationary phase

    • Death phase

Phases of Bacterial Growth Curve

Lag phase

  • In the first phase.

  • No increase in cell number

  • Cells are actively metabolizing, in preparation for cell division.

  • It may be short or very long, according to the growth medium.

Exponential or log phase

  • In the second phase.

  • called the exponential or log phase.

  • This is the period in which the cells grow most rapidly, doubling at a fairly constant rate.

  • The time it takes the culture to double is called the generation time.

  • The generation time depends on several factors:

    • the organism

    • the growth medium

    • temperature

Stationary phase

  • In third phase

  • metabolism slows

  • cells cease rapid cell division.

  • Why?

    • Due to:

      • high cell density.

      • depletion of nutrients .

      • accumulation of waste products.

Death phase

  • In the final phase .

  • cells quickly lose the ability to divide.

  • exponential death.