Controlling Microbial Growth in Vitro

Factors that affect microbial growth

Temperature
psychrophiles (cold loving microbes )
range range 0 C - 20 C 20 C - 40 C

mesophiles (moderate temp. loving microbes)

thermophiles
range

(heat loving microbes)

40 C - 100 C

Factors that affect microbial growth

Availability of nutrients:
Many nutrients are energy sources Nutrients serve as sources of C, O, N, P, & Sulfur Source for trace elements as Iron, Iodine, Zinc

Factors that affect microbial growth

Moisture
All living organisms need water for normal metabolic processes Moisture necessary for growing and reproduction

pH

Most bacteria grow between pH 6.5 - pH 7.5

Very few can grow at below pH 4.0 Acidophilic microbes: Alkalophilic microbes:

Osmotic Pressure

Microbes obtain almost all their nutrients in solution from surrounding water
Tonicity
isotonic hypertonic hypotonic

Cells

Barometric pressure

Barophiles: prefer to live in a high atmospheric pressure like some archeans

Gaseous atmosphere

Some microbes prefer the same human do (e.g O2 21%, N2 78%, other gases 1%) Other microbes need high concentration of O2 called Microaerophiles

Bacterial Growth - increase in the # of

cells

Binary Fission
Generation Time (Doubling Time)
time required for a cell to divide most about 1 Hr. To 3 Hrs.
E. coli - 20 minutes Mycobacterium tuberculosis - 24 Hrs.

Limiting factors in the environment

Lack of food, water or nutrients space accumulation of metabolic wastes lack of oxygen changes in pH temperature

Phases of Growth

4 Phases
1. Lag Phase 2. Log Phase 3. Stationary Phase 4. Death Phase

1. Lag Phase

Bacteria are first introduced into an environment or media Bacteria are checking out their surroundings cells are very active metabolically # of cells changes very little 1 hour to several days

2. Log Phase

Rapid cell growth (exponential growth) population doubles every generation microbes are sensitive to adverse conditions
antibiotics anti-microbial agents

3. Stationary Phase

Death rate = rate of reproduction cells begin to encounter environmental stress

lack of nutrients lack of water not enough space metabolic wastes oxygen pH

Endospores would form now

4. Death Phase

Death rate > rate of reproduction Due to limiting factors in the environment

Inhibiting the growth of microorganisms in Vitro

Methods to Control Microbial Growth

1. Physical
2. Chemical

Terms used:

Sterilization vs. Disinfection Sterilization

destroying all forms of life

Disinfection
destroying pathogens or unwanted organisms

Disinfectant vs. Antiseptic Disinfectant

antimicrobial agent used on inanimate objects

Antiseptic
antimicrobial agent used on living tissue

Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms)

cidal vs. static

Bactericidal - kills bacteria Bacteristatic - inhibits bacterial growth

Fungicidal Fungistatic Algacidal Algastatic

Factors that effect Antimicrobial Activity

1. Temp 2. Time 3. Concentration of Antimicrobial agent 4. Type of Microbe 5. Activity of Microbe 6. Presence of organic matter

Targets of Antimicrobial Agents

1. Cell membrane
2. Enzymes & Proteins 3. DNA & RNA

Physical Methods of Microbial Control

1. Heat
works by denaturing enzymes and proteins

A. Thermal Death Point (TDP)

lowest temp. at which all microorganism in a liquid culture are killed in 10 minutes

B. Thermal Death Time (TDT)

minimum length of time in which all microorganisms in a liquid culture are killed at a given temperature

Moist Heat

1. Boiling Water
kills vegetative bacterial cells, Fungi and many viruses not effective for endospores and some viruses Some spores may survive boiling water for up

Moist Heat

2. Autoclave (Steam under pressure)

preferred method of sterilization Water boils at 100 C Increasing the pressure raises the Temp. 15 lbs./ per sq. inch (psi) ------> 121 C 121 C for 15 min.

Dry Heat

1. Direct Flaming
Inoculating Loop and Needle 100% effective

2. Incineration
disposable wastes (paper cups, bags, dressings)

3. Hot Air Sterilization

Oven ( 170 C for 2 hours) used on substances that would be damaged by moist heat sterilization
gauzes, dressings or powders

Filtration

Removes microorganisms from solutions that might be damaged by heat

culture media enzymes vaccines antibiotics

Radiation

1. Ionizing Radiation
gamma rays & x-rays
penetrates most substances

Used on substances that could be damaged by heat

plastic petri dishes plastic syringes catheters surgical gloves

Radiation

2. Non-Ionizing Radiation
UV Light
does not penetrate plastic, glass or proteinaceous matter

Used to reduce microbial populations

hospital rooms nurseries operating rooms

Pasteurization

Disinfection - not sterilization (removes unwanted organisms) Mycobacterium tuberculosis 63 C for 30 minutes 72 C for 15 seconds

Methods used to control Microbial Growth

1. Heat
Moist Heat
Boiling Water Steam Heat (Autoclave)

Dry Heat
Direct Flaming Incineration Hot Air Sterilization (Oven)

2. Filtration 3. Radiation
Ionizing Radiation Non-Ionizing Radiation

4. Pasteurization (Heat)