MICROBIAL CONTROL 3.

Concentration of Reagents  For majority of culture media

 Prevention of microbial growth to stop  Higher conc. = higher rate of killing preparation
transmission of infectious diseases  Except…  Disadvantage
 Affected by...  Alcohol (70%) : Prions are not destroyed
 Microbicide  Sodium hypochlorite (10%) : Melting of plasticwares
 Carried out by “cidal” agents  Lysol (3-5%) : Breaking down of chemicals
 Killing microorganisms : Sharps instruments become dull
 e.g. fungicide, bactericide 4. Duration of Exposure : Oily substance not treated
 Cidal/static effects are not
 Microbiostasis instantaneous a.3. Tyndallization
 Inhibition of growth and  Dependent on other factors  Aka Fractional sterilization
reproduction  Equipment: Arnold’s sterilizer
 Carried out by “static” agents 5. Temperature  Culture media preparation:
 e.g. bacteriostatic agent  Direct relationship with rate of killing protein, high sugar, egg content
 Increased temp. = higher rate of killing  100oC for 30 min in 3 days incubation
Importance of microbial control Day 1: Vegetative cells are killed;
a. Prevent spread of infectious diseases 6. pH Endospores still live
b. Prevent contamination of specimen  Effect is dependent on agent and Day 2: Endospores transform to
c. Conserve specimen microbe vegetative cells (killed)
d. Neutralization of biohazards e.g. Gluteraldehyde-more effective in acid pH Day 3: Residual vegetative cells killed
Base effective in acid-loving microbe
DEFINITION OF TERMS a.4. Inspissation
1. Sterilization 7. Extraneous matter  Same with tyndallization
 Complete destruction of all viable cells  Its presence affect viability of microbe  Lower temp. for longer duration
 Including spores  Organic substances protect microbes  70oC – 80oC; 2 hrs. for 3 days
 Agent: Sterilants  e.g. Mucus on URTI specimens  For culture media preparation
especially for Egg-based culture
2. Disinfection MECHANISM OF ACTION medium
 Elimination of vegetative pathogens on (How control agents damage microbes)
non-living/immobile things a. Damage to cell wall a.5. Pasteurization
 Exclusion of endospores b. Damage to cell membrane  Louis Pasteur = 55oC
 Agent: Disinfectant c. Denaturation of microorganism’s  Disease and spoilage causing microbes
protein  E.g. Dairy products
3. Antisepsis d. Damage to nucleic acids  Variety of techniques:
 Microbicide/ microbiostasis in living  Classic method
tissues  Aka Bulk or Holding method
 Agent: Antiseptic METHODS OF MICROBIAL CONTROL
 Low temp = long time (LTLT)
 Aseptic I. Physical method  63oC for 30 min
 Complete absence of any viable
1. Heat  Flash method
pathogen
 Most commonly employed physical  High temp = short time (HTST)
 An environment free from pathogenic
microbes
method  71.6oC for 15 sec
 Fast, reliable, inexpensive  Used for high quality raw milk
 Mechanisms:
4. Sanitization  Ultrapasteurization method
 Denaturation of proteins
 Reduction of microbial population to  Ultra-high temperature (UHT)
 Driving off water
levels that is considered safe by public  2 methods
health standards  Oxidation
: 140oC for 3 sec
 Agent: Sanitizer  Factors:
: 149oC for 0.1 sec
 Thermal Death Point
FACTORS AFFECTING MICROBIAL CONTROL  Thermal Depth Time B. Dry heat
 Decimal Reduction Time  Mechanisms:
1. Microbial load
 Population size of microbe  Burning to Ashes
A. Moist heat
 Larger pop’n = slower rate of killing  Oxidation
 Mechanism: Denaturation of protein
 Methods:
 Penetrates materials rapidly
2. Microbial composition b.1. Direct Flaming
(shorter period of time)
 Type of microorganisms  Killing in open flame
 Lower temp. = shorter time
 Established relative difficulty of killing  Sterilization of inoculating loops and
*Water molecules - conducts heat
the microbe needles
a.1. Boiling / Flowing steam
*Most Resistant b.2. Incineration
 Only kill vegetative pathogens
Prions  Conversion of materials to ashes
 < 1x106 mL for 10 min (exposure time)
Endospores of bacteria  870-1200oC
 Endospores: 2 hrs more exposure time
Mycobacteria
Cysts of protozoa b.3. Hot air sterilization
a.2. Autoclaving
Vegetative protozoa  160-180oC for 2 hours
 Pressurized steam
Gram-negative bacteria  Quality Control:
 Increased pressure = increased temp.
Fungi with fungal spores Bacillus subtilis var. niger
 121oC at 15 psi for atleast 15 min to
Gram-positive bacteria
achieve optimum conditions
Viruses with lipid envelopes
 Quality control: Bacillus
*Least Resistant
stearothermophilus
2. Filtration 6. Radiation C. Phenol Coefficient
 Mechanism of Action: Separation of A. Ionizing Radiation  Sets phenol as a standard chemical
bacteria from suspending liquid  Wavelength shorter that non-ionizing agent
 Quality Control: radiation  Number comparing agent’s disinfecting
Pseudomonas diminuta  Can stimulate the production of ions ability to that of phenol (Identical
conditions)
Commonly Used Filters:  < 1nm
 Includes  Results and interpretation:
A. Inorganic filters 1 : Comparable to phenol
: Gamma rays
: X-rays >1 : More effective than phenol
a.1. Seitz filter <1 : Less effective than phenol
 Ground glass/ porcelain mounted in filter : High-Energy electron beams
flasks D. Commonly Used Chemical Agents
B. Non-ionizing Radiation
a.2. Porcelain Filter (Chamberland)  Wavelength longer than non-ionizing 1. Phenol and Phenolics
 Separates viruses from other microbes radiation - Phenol: 1st antiseptic used
 > 1nm - Phenolics:
a.3. Sand/Petri Filter - A molecule of phenol with a soap or
 Separates bacteria from other microbes C. Sterilizing Radiation detergent
 Penetrates cells, causing damage to the - Phenol chemically altered (reduce its
DNA then lead to eventual death of irritating qualities or increase its
B. Organic filter antibacterial activity)
bacteria
b.1. Berkefield Filter
 Commonly used - Injures lipid-containing plasma
 Attacks organic components of membranes then results to leakage of
: UV light
bacterial cell cellular contents
: X-rays and Gamma rays
- Ionizing - O-phenylphenol : major component of
b.2. Membrane Filter Pads Lysol
- Ions react with water content of the
 Made up of: Cellulose acetate (aka bacteria
nitrocellulose pad) or - Mechanism: Destruction of DNA 2. Bisphenols
polycarbonate : Microwave - Derivatives of phenol
- High speed motion generates heat of - Contain 2 phenolic groups connected by
C. HEPA filter friction a bridge
 High Efficiency Particulate Filter - Then absorbed by water molecules e.g. Hexachlorophene
 Pleated borosilicate fibers with of the microbe
aluminum separators - Then eventual death of microbe 3. Biguanides
 Pore Size: 0.3 micrometer - Broad spectrum of activity
 Removes 99% of all particles II. Chemical method : Gram (+)Bacteria
 Utilizes chemical reagents : Gram (-)Bacteria except pseudomonads
3. Cold method  Most commonly used: - Primarily affects bacterial cell
 Mechanism Disinfectants and Antiseptics membranes
: Decreased chemical reactions - Not sporicidal (not capable of destroying
: Possible changes in protein leading to  Disinfectants spores)
denaturation - Used against infections e.g Clorhexidine, Alexidine
 Antiseptics
A. Refrigeration - To have an aseptic environment 4. Halogens
 Bacteriostatic effect; not capable of
 Value of an Agent: a. Chlorine
killing microbes
1. Should not separate on standing - Sodium hypochlorite (Chlorox)
 Preservation of food, drugs, and culture - Calcium hypochlorite
- No disintegration of the active
B. Deep Freezing component
2. Should penetrate cell wall b. Iodine
 Quick freezing at -50oC to -95oC
3. Should not corrode instrument  Tincture of Iodine – iodine in sol’n
 For storage of stock cultures with aqueous alcohol
4. Does not combine with organic matter
 Iodophor – combination of iodine and
C. Lyophilization an organic molecule (wetting agent
 Removing water by high vacuum under A. Parameters
 Temperature – each chem’l agent has and reservoir of free iodine)
low temperature e.g. Povidone-Iodine / Betadine
 Most effective in long-term storage of optimum temp
microbial culture  pH - each chem’l agent has optimum pH
 Solubility – should be soluble enough to 5. Alcohol
penetrate and kill microoganisms - Bactericidal and fungicidal
4. Desiccation - Not effective against bacterial spores
 Process of adding salt or sugar to or non-enveloped viruses
remove water from microbes B. Disinfectants/ Antiseptics
 Kill/ slow the growth of microbes e.g. 70% isopropanol ; 70% ethanol
 Mechanism of action: Disruption of
metabolism  Non-toxic to animal/human
 Soluble in water
5. Osmotic Pressure  Useful in much diluted forms
 Mechanism of action: Plasmolysis (licing  Able to perform its job in a short time
of plasma membrane)
 Damage the cell wall and cell
membrane
 By removal or influx of water from the
cell