Microbial Control - Reasons and Types

Lecture 08 [Notes] 



Reasons for Microbial Control         

          
   1. prevention of disease transmission and infection
   2. prevention of decomposition and spoilage
   3. prevention of contamination (communication of disease
      organisms through contact, dispersal, fomites)
          
Microbial control is necessary to maintain a balance between man
and microbes.
   The methods of control are: Physical and Chemical
   The results of control are: Stasality and Cidality
          
Growth in most organisms is represented as an orderly increase in
all cell components and activity within the cell as well as an
increase in the overall number of cells.
In microbiology, growth is usually defined more narrowly as an
increase in the total number of cells or an increase in microbial
mass (as in colony size).
          
GROWTH RATE is defined as the amount of growth/unit of time.
Multiplication usually takes place in a GEOMETRIC or EXPONENTIAL
fashion rather than arithmetically.
GENERATION TIME equals the length of time required for one
organism to become two organisms.
          
STASALITY (­stasis; ­static) ­ organisms do not reproduce; a
   permanent period of stasis is death, growth is inhibited;
   recovery is possible if cause of stasality is removed
          
CIDALITY (­cide; ­cidal) ­ absolute death, no possibility of
   reproduction, all metabolic functions eradicated


	
Mode of Action - How Control Agents Work

 
  Damage usually occurs at the level of:
     a) the cell wall
     b) the cell membrane
     c) the internal proteins and cytoplasm

a) Damage to the cell wall - maintains the integrity of the cell
   cell and is the site of various enzymatic reactions; porous
   but protective; some Gram + bacteria are attacked by LYSOZYME
   (enzyme in tears, mucous secretions, white blood cells) which
   helps control skin staph; penicillins and cephalosporins
   inhibit cell wall synthesis in rapidly growing cultures

b) Alteration of Cell Membrane Permeability - responsible for         
   selective transport; "leakage" is term used when cell membrane
   is destroyed; phenolic compounds, detergents, quaternary
   ammonium compounds, all destroy selective permeability and
   cause leakage; alcohol (dissolve lipids and denature proteins)
   and heat have similar effects
          
c) Alteration of Colloidal Nature of Cytoplasm - death is rapid  
   when proteins are denatured; alcohol and high temperatures,
   organometallics; denature proteins and coagulate proteins
          
d) Inhibition of Enzyme Action - cytochrome oxidase is inhibited      
   by cyanide, trivalent arsenic interferes with the Krebs cycle'
   glycolysis is inhibited by halogens (chlorine, fluorine, etc.)
   mercury, hydrogen peroxide, sulfanilamide, some antibiotics,
   usually stop protein synthesis; oxidative phosphorylation is
   stopped by the nitrophenols
e) Interference with Synthetic Processes - antimetabolites;           
   blockage of folic acid by sulfonilamides ­ needed for PABA;
   common TB treatment
          
	
Conditions Influencing Antimicrobial Action



1. Concentration & Kind of Agent ­ usually the more 
   concentrated the agent the more cidal the action; many products, 
   if used in low concentrations, will act as a stimulus to microbes
2. Intensity & Nature of the Physical Agent ­ the more intense
   the heat or radiation the more rapid the kill; consideration
   is needed as to what happens to other compounds present as
   result of action of agent
3. Time ­ the longer the time in contact, the greater the kill
4. Temperature ­ the higher the temperature the more effective
   the kill
5. Number of organisms ­ greater numbers require longer time
6. Kinds of organisms ­ Pseudomonas aeruginosa is one 
   of the toughest to kill, Mycobacterium with its tough waxy walls 
   and encapsulated forms are also difficult
7. Nature of the materials bearing the organism ­ the microenvironment; 
   organic matter inactivates certain chemicals; high protein concentrations 
   may clog filtration systems

Standards are developed by the Food and Drug Acts; the first ones
were developed by the Theodore Roosevelt administration. Over the
years the standards became tougher as the attitudes of industry
and laymen began to change and recognize the need for control. 
The biggest change occurred after 1969 with the adoption of the
AOAC Use­Dilution Test (Association of Official Analytical
Chemists). This test replaced the Phenol Coefficient Test because
it provided a means of determining effectiveness of the agent
under the conditions of its usage, rather than simply in vitro.

Eventually it became the province of the Public Health Administration 
to determine what would be considered safe levels or standards. This is 
was purely administrative bureau which used the results of testing done 
by industry to determine the standards. The Public Health Laboratories 
are required to do the testing to determine if these standards are 
being met.


The 1st Food and Drug Act contained the following information:
"the language used in the label is to be given the meaning
ordinarily conveyed by it to those to whom it is addressed" This
was the first attempt to require a label which would indicate the
contents of the product, and use common language to explain it. 
This brought about a dearth of ambiguous terms. The following
terms were finally agreed to by the FDA and Industry.

Sterilization ­ the act or process of destroying all forms of
life on an object or in a material; this includes the destruction
of endospores; sterilization is absolute, there are no degrees of
sterilization ­ HOWEVER, the literature is ambiguous about
viruses; if viruses are considered life forms then they can be
killed, if not then it is possible, at least semantically, for a
solution to be sterile and contain viruses
          
Disinfectant ­ usually chemicals which kill growing forms
(vegetative) of pathogens but not necessarily spore forms or
encapsulated forms or viruses; they are commonly applied to
inanimate objects and can be used as sprays or fogs; dependent on
concentration they can be static or cidal; a minor consideration
is the fact that our understanding of what constitutes a pathogen
has changed since the original term was defined; the bottom line
is simply to READ ALL LABELS CAREFULLY
          
Antiseptic ­ a chemical agent that arrests or prevents the growth
or action of microbes; it controls by destruction or inhibition ­
dependent upon concentration; works on cuts, scrapes, abrasions,
wounds on or in the body; ERGO ­ chemical disinfection of living
tissue
          
Sanitizers - chemical agents that reduce microbial populations     
to safe levels (as determined by the PHS); usually destroys 80­90
percent; normally used on inanimate objects such as in dairy
industry; more importantly to the public ­ sanitizers imply
cleanliness as well as disinfection

	PHYSICAL METHODS OF MICROBIAL CONTROL
	
A. Temperature ­ must take into account what is being subjected
   to high or low temperature
	Thermal Death Point (TDP) ­ lowest temperature required to
     kill ALL microorganisms in a liquid suspension within 10
     minutes (time is held constant, temperature varies)
	Thermal Death Time (TDT) ­ minimal length of time in which
     all bacteria in a liquid culture will be killed at a given
     temperature (temperature is held constant, time varies) ­
     useful in situations such as pasteurization where certain
     products will be rendered unusable if temperature becomes
     too high
          
   1. Flame or incineration ­ used for inoculating loops and
      needles and for contaminated paper cups, bags, dressings.
   2. Dry heat sterilization ­ normally used for drying glassware
      requires minimum temperature of 1700 C for two hours to
      ensure sterilization
   3. Boiling ­ need a minimum of 10 continuous minutes of
      boiling to destroy spores of fungi, many viruses, and most
      bacterial pathogens; endospores and some viruses may
      require up to 25 HOURS for destruction
   4. Autoclaving ­ moist heat, greater penetration, steam under
      pressure; normally requires 1210 C at 1 atmosphere of
      pressure beyond normal atmospheric pressure; length of time
      varies with type of material being autoclaved
   5. Low temperature ­ normally used to restrict reproduction;
      may slow down enzymatic action but does not stop it;
      freezing or cold normally does not kill unless done slowly
      so that ice crystals may develop within the cells

B. Filtration ­ difficult to use in high protein content
   materials; will cause clogs in filter; cannot be trusted to
   remove all viruses; best used to determine water quality

C. Dessication ­ reproduction affected but viability of many
   organisms is unchanged; ineffective against endospores and
   some viruses; used in preservation certain foods

D. Osmotic Pressure ­ increase in sugar or salt concentration
   will lead to dessication and is common method of food preser-
   vation; may not be effective against all fungi, endospores,
   or some viruses

E. Electricity ­ poor method; hard to electrocute bacteria;
   really a combination of heat and dissociation of gases; ozone
   is formed; still used in milk and juice industries

F. Sonic & Ultra Sonic - causes cavitation (thunder following         
   lightning); molecules are moved out leaving a cone; as the
   vacuum becomes filled the molecules smash together; causes
   cracking or bursting ­ more effective against rods than cocci;
   Gram + more resistant, acid fast and spores very resistant; no
   assurance of total sterility; used most frequently for
   disruption of eukaryotic cells
          
G. Radiation ­ ionizing radiation such as X­rays or gamma rays
   are most effective; X­rays are very penetrating but expensive;
   Gamma radiation are extremely penetrating
   Non­ionizing radiation such as ultraviolet radiation causes
   changes in DNA molecules by causing thymine dimers to form and
   makes separation at the time of replication difficult if not
   impossible; UV rays at 260 nm or 2650 A is considered germi-
   cidal; UV converts oxygen into ozone which is an effective
   oxidant; poor penetration, usually only on the surface ­
   effects may be reversible; UV is adsorbed to protein and
   destroys enzyme systems;potentially damaging to eyes and skin
          

	CHEMICAL METHODS OF MICROBIAL CONTROL
	
Important considerations for chemical killing agents include:
   1. spectrum of kill (types of organisms)
   2. effective dilution

	Ideal Disinfectant - Characteristics Looked for by FDA
 1. Toxicity to microbes ­ broad spectrum, effective against
    spores, waxy coats; lowest concentration still effective
 2. Solubility ­ in water, in tissue fluids; aesthetic
    qualities ­ clear or cloudy
 3. Stability ­ shelf life ­water soluble products tend to last
    long; alcohol based products have reduced shelf life
 4. Toxicity to man, other animals and plants ­ skin tests,
    kidneys will usually show first; conjunctiva test and
    respiratory tests; if substance goes in or on the body it is
    under the control of FDA and the National Institutes of
    Health studies the protocol aspects
 5. Homogeneity of mixture ­ will it separate on standing
 6. Combining power with organic substances ­ want low affinity
    or will cause dilution ­ test with organisms coated with
    mucin; does it combine with lipids in the body, toxic to man
 7. Temperature ­ toxicity at room temperature or body
    temperature; materials must not change at very high or very
    low temperature on the shelf
 8. Penetrating power - few compounds penetrate skin (ex. DMSO);
    usually refers to cut, wounds, abrasions
 9. Non­corroding and non­staining
10. Deodorizing quality ­ organometallics are especially good;
    usually the result of washing out of the atmosphere
11. Detergent action ­ removal of dead tissue from wounded area
12. Availability in large quantities
13. Affordable
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