L07 Microbial Growth Control

一、Principles of microbial control

Definition of Frequently Used Terms

1. Sterilization 杀菌

destruction or removal of all viable organisms

2. Disinfection 消毒

killing, inhibition, or removal of disease causing (pathogenic) organisms (not bacterial endospore)

disinfectants

usually chemical
usually used on inanimate (无生命的) objects

3. Sanitization 清洁

reduction of microbial population to levels deemed safe (based on public health standards)

4. Antisepsis 抗菌

prevention of infection of living tissue by microorganisms

antiseptics

chemical agents that kill or inhibit growth of microorganisms when applied to tissue

5. Comparisons of The Four Method

Antimicrobial Agents

1. Chemotherapy

use of chemicals to kill or inhibit growth of microorganisms within host tissue

Agents that kill microorganisms or inhibit their growth

cidal agents（杀菌剂） kill (bactericides)
static agents（抗菌剂） inhibit growth (bacteriostatic)

The Pattern of Microbial Death

Microorganisms are not killed instantly

Population death usually occurs exponentially （指数的,幂数的）

Measure of agent’s killing efficiency： decimal reduction time （D Value） – time to kill 90%

Conditions Influencing the Effectiveness of Antimicrobial Agent Activity

Population size
- larger populations take longer to kill than smaller populations
Population composition
- microorganisms differ markedly in their sensitivity to antimicrobial agents
Concentration or intensity of an antimicrobial agent
- usually higher concentrations kill more rapidly
Duration of exposure
- longer exposure → more organisms killed
Temperature
- higher temperatures usually increase killing
Local environment
- pH, viscosity, concentration of organic matter, etc. can profoundly impact effectiveness
- organisms in biofilms are less susceptible to many antimicrobial agents

二、Mechanical removal methods

Filtering Liquids

Membrane filters

porous（能渗透的；有气孔的；多孔渗水的） membranes with defined pore sizes

Filtering Air

Surgical masks

Cotton plugs on culture vessels

High-efficiency particulate air (HEPA) filters

used in laminar（薄片状的） flow biological safety cabinets

三、Physical Control Methods

Heat

1. Moist Heat 湿热

Destroys viruses, fungi, and bacteria

Boiling will not destroy spores and does not sterilize

Degrades nucleic acids, denatures proteins, and disrupts membranes

2. Steam Sterilization 蒸汽灭菌

Carried out above 100°C which requires saturated steam under pressure

Uses an autoclave （高压蒸气灭菌器）

Effective against all types of microorganisms (including endospores!)

3. Pasteurization 加热杀菌(法);巴斯德氏杀菌法

Controlled heating at temperatures well below boiling

Process does not sterilize but does kill pathogens present and slow spoilage by reducing the total load of organisms present

4. Dry Heat Sterilization 干热灭菌

Less effective than moist heat sterilization, requiring higher temperatures and longer exposure times

items subjected to 160–170°C for 2 to 3 hours

Oxidizes cell constituents and denatures proteins

5. Dry Heat Incineration 干热焚烧

Bench top incinerators are used to sterilize inoculating loops used in microbiology laboratories

Radiation

1. Ultraviolet (UV) Radiation

mutations — death
causes formation of thymine dimers in DNA
requires direct exposure on microbial surface

Wavelength of 260 is most bactericidal (DNA absorbs)

Causes thymine dimers preventing replication and transcription

UV limited to surface sterilization because it does not penetrate glass, dirt films, water, and other substances

2. Ionizing Radiation 电离辐射

x-rays and gamma rays
mutations — death (sterilization)
disrupts chemical structure of many molecules, including DNA and proteins

Gamma radiation penetrates deep into objects

Destroys bacterial endospores

Used for sterilization and pasteurization of antibiotics, hormones, plastic disposable supplies, and food

3. Visible Light

at high intensities generates singlet oxygen($^{1}O_{2}$)

powerful oxidizing agent

carotenoid （类胡萝卜素） pigments
protect many light-exposed microorganisms from photooxidation (光氧化（作用）)

四、Chemical Agents

Aldehydes 醛类

Commonly used agents are formaldehyde（甲醛） and glutaraldehyde（戊二醛）

Highly reactive molecules

Sporicidal（杀孢子的） and can be used as chemical sterilants

Combine with and inactivate nucleic acids and proteins

Sterilizing Gases 消毒气体

Used to sterilize heat-sensitive materials

Combine with and inactivate DNA and proteins

Microbicidal and sporicidal

Heavy Metals 重金属

e.g., ions of mercury, silver, arsenic, zinc, and copper

Combine with and inactivate proteins; may also precipitate proteins

Effective but usually toxic

五、Biological Control of Microorganisms

Emerging field showing great promise

Natural control mechanisms

viral-mediated lysis using pathogen specific bacteriophage
toxin-mediated killing using bacteriocins
predation by Bdellovibrio 蛭弧菌属

Biofilms

Biofilm are complex, slime-encased communities of microbes.

1. Forming Process

Substratum preconditioning by ambient molecules
Cell deposition 细胞沉积
Cell adsorptipn
Desorption
Cell-to-cell signaling and onset of exopolymer production 细胞间的信号传导和外聚物的产生
Convective and diffusive transport of O2 and nutrients
Replication and growth
Secretion of polysaccharide matrix
Detachment, erosion, and sloughing 分离，腐蚀和脱落

2. Biofilm Heterogeneity 生物膜异质性

3. Cell-to-Cell Communication – Quorum Sensing (群体感应)

Introduction

Bacterial cells in biofilms communicate in a density-dependent manner called quorum sensing

Produce small proteins that increase in concentration as microbes replicate and convert a microbe to a competent state

DNA uptake occurs, bacteriocins are released

Acylhomoserine lactone (AHL) is an autoinducer molecule produced by many gram-negative organisms

diffuses across plasma membrane
once inside the cell, induces expression of target genes regulating a variety of functions

Quorum Sensing Systems

Processes regulated by quorum sensing involve host-microbe interactions

symbiosis – Vibrio fischeri and bioluminescence in squid
pathogenicity and increased virulence factor production
DNA uptake for antibiotic resistance genes

六、Laboratory culture of cellular microbes

Types of Culture Media

Basis for Classification	Types
Chemical composition	Defined (synthetic), complex
Physical nature	Liquid, semisolid, solid
Function	Supportive (general purpose), enriched, selective, differential

1. Classified by Chemical Composition

Defined or Synthetic Media

Complex Media

2. Classidied by Functions

Supportive or General Purpose Media

Supportive or general purpose media (e.g. TSA)

support the growth of many microorganisms

Enriched Media

Enriched media (e.g. blood agar)

general purpose media supplemented by blood or other special nutrients

Selective Media

favor the growth of some microorganisms and inhibit growth of others

• e.g., MacConkey agar

selects for gram-negative bacteria

Differential Media

Distinguish between different groups of microorganisms based on their biological characteristics

e.g., blood agar

hemolytic versus nonhemolytic bacteria

• e.g., MacConkey agar

lactose fermenters versus nonfermenters

Isolation of Pure Cultures

Population of cells arising from a single cell developed by Robert Koch

Streak plate, spread plate, and pour plate are techniques used to isolate pure cultures

1. Streak Plate

Spreading a mixture of cells on an agar surface so that individual cells are well separated from each other

involves use of bacteriological loop

Each cell can reproduce to form a separate colony

2. Spread Plate

small volume of diluted mixture containing approximately 30–300 cells is transferred

spread evenly over surface with a sterile bent rod

3. Pour Plate

sample is serially diluted

diluted samples are mixed with liquid agar

mixture of cells and agar are poured into sterile culture dishes

Both spread and pour plate may be used to determine the number of viable microorganisms in an original sample

Microbial Growth on Solid Surfaces

Colony characteristics that develop when microorganisms are grown on agar surfaces aid in identification

Differences in growth rate from edges to center is due to

oxygen, nutrients, and toxic products
cells may be dead in some areas

The Growth Curve

Growth refers to population growth rather than growth of individual cells

Usually plotted as logarithm of cell number versus time and has four/five distinct phases

1. Lag Phase

Cell synthesizing new components

e.g., to replenish spent materials
e.g., to adapt to new medium or other conditions

Varies in length

in some cases can be very short or even absent

2. Exponential (Log) Phase

Also called log phase

Rate of growth and division is constant and maximal (balanced growth)

Population is most uniform in terms of chemical and physical properties during this phase

3. Unbalanced Growth

Rates of synthesis vary relative to each other

Occurs under a variety of conditions

change in environmental conditions
change in nutrient levels

4. Stationary Phase

Closed system population growth eventually ceases, total number of viable cells remains constant

active cells stop reproducing or reproductive rate is balanced by death rate

Possible Reasons For Stationary Phase

Nutrient limitation

Limited oxygen availability

Toxic waste accumulation

Critical population density reached

5. Prolonged Decline in Growth 增长持续下降

Process marked by successive waves (连续波) of genetically distinct variants

Bacterial population continually evolves
Natural selection occurs

6. Growth In A Closed System

Mathematics of Growth

Generation (doubling) time

time required for the population to double in size
varies depending on species of microorganism and environmental conditions
range is from 10 minutes for some bacteria to several days for some eukaryotic microorganisms