CONTROL OF MICROBIAL GROWTH

1. Controlling Microbial growth is necessary

Decline in Salmonella typhi deaths in the US from 1 in 1000 (1900) to 4 in 200 million (1970) due to control measures

Water chlorination (1908)
Milk pasteurization (1909)
Sewage treatment plant design improvements
Transmission control

Fly populations (vectors)
Detection of diseased persons

carriers in the milk industry
patients

preventive vaccination
antibiotic therapy

Lister (1827 - 1912)

Washing hands prior to surgery lead to decline in infection rates
heat sterilization of surgical instruments
application of phenol (carbolic acid) to wounds

Todays control measures are more sophisticated and include a wide array of methods but not all methods do not kill microbes eg filtration = removal

Terminology related to the control of microbial growth

TERM	DEFINITION	EXAMPLE
Sterilization	Complete destruction	121^oC/15 min; 170^oC/2h
Disinfection	Application of chemicals to objects	Chlorination of water; kill pathogens
Antisepsis	Application of chemicals to living tissue	Treatment of wounds
Bacteriostasis	Halts growth but not killed	Refrigeration, dyes in food
Asepsis	Absence of pathogens; aseptic techniques	Air filtration, uv light, gloves, gowns
Sanitization	Public health; mechanical / chemical cleansing	Palatability of food

2. Factors influencing the effectiveness of control methods

A number of factors affect the usefulness (efficacy) of control methods & all factors should be considered to devise control measures

Size of microbial population:

Death is exponential ie more microbes = more time to kill the population
Plot the log of the no. of surving microbes vs time = straight line, the slope = killing rate
Initial microbe concentrations one can predict the kill time

Exposure time of the agent:

Increasing exposure time increases kill rates; kill time used is usually well past the required minimal contact time
large volumes / vessels require more time for complete destruction

Container Size*	Liquid Volume (ml)	Sterilization time (min)
Test tubes / Erlenmeyer flask	10 - 900	15
Erlenmeyer flask	1000 - 2000	20
Bottle	6750	70

A container is not usually filled past 75% of its capacity.

Effect of concentration, temperature & pH:

Increasing concentration is similar to increasing exposure time
Temperature, pH & concentration at which the agent works best
Stability of the agent at various pHs & temepratures
Legionella pnuemophila (Legionnaires disease) - the effect of the microbe suspended in tap water & exposed to different temperatures, pH & chlorine concentrations

Protective features of the microbes (refer to microbial structures section)

Endospore formers such as C. tetani
Cysts -- Protozoa
Porins -- Pseudomonas
Cell wall -- Mycobacterium tuberculosis (opportunistic pathogen)
Active growth vs resting cells - antibiotics (penicillin - transpeptidase)

Interactions & protective features afforded by the environment:

sewage is rich in organics - polio virus protected
Disinfecting in hospitals - selection of ressistance
Lipids & fats in dairy industry - protect spoilage microbes
Remember that lab conditions used in testing are different to field conditions

Methods for controlling microbial growth

Many methods to choose from:

Physical

Heat (Dry, Moist, Pasteurization)
Filtration
Low Temperatures (fridge, freezer)
Desication & osmotic pressure
Radiation (ionizing and nonionizing)

Chemical

Phenol & phenolics
Halogens
Alcohol
Heavy metals & their compounds
Surface active agents ( anionic & cationic detergents)
organic acids
gaseous sterilants
oxidising agents
chemotherapeutic agents target

cell wall
protein synthesis
nucleic acids
cell membrane
essential metabolites (antimetabolites)

The method of choice depends on the situation & practicality of the approac

The material to be treated eg heat labile or heat stable???

Laboratory conditons differ from field conditions

Factors affecting growth eg growing or resting cells, ressistance factors

Physical Methods of controlling microbial growth

Method	Mechanism of Action	Comments	Preferred Use
1. Moist Heat^(A)
a. Boiling	Denaturation	Kills vegetative cells but not spores	Equipment, dishes
b. Autoclaving	Denaturation	Sterilization (autoclaves, pressure cookers, retorts)	Media, linens, equipment, dressings
c. UHT	Denaturation	Sterilization; 141^oC / 2secs	Milk falls in a thin film thro a chamber of superheated steam
2. Dry Heat^(A)
a. Flaming	Burning to ashes	Sterilization	Inoculating loops
b. Incineration	Burning to ashes	Sterilization	animal cacasses, dressings, wipes
c. Hot-air sterilization	Oxidation	170^oC / 2hrs	Glassware, needles, glass syringes
3 Pasteurization^(A)
a. Low temp long time (LTLT)	Denaturation	63^oC/30mins	Milk: batch process in tanks
b. High temp, short time (HTST)	Denaturation	72^oC / 15secs	Milk: Flash method thro continous winding pipe
4. Filtration^(B)	Separation	Liquid thro screen	Heat labile material
5. Low Temp
a. Fridge	Growth slows	Bacteriostatic	Drug, Food
b. Deep Freezing	Growth slows	Preservation -70^oC	Drug, food & culture
6. Desiccation^(C)
a. Lyophilization	Growth arrested	Long term preservation of microbes	Food, Drug & culture
b. Osmotic Pressure	Plasmolysis	Loss of water	Food preservation
8. Radiation^(D)
a. Ionizing	DNA destruction	Not commonly used	Sterilizing medical & dental supplies
b. Nonionizing	DNA py-py dimers (eg UV)	Not very penetrating radiation	UV (germicidal) lamp

^(A)Moist Heat is used extensively by the food (canning & milk) industry

Remember: microbe properties are important & so are the environmental conditions
Improve shelf life (sporeformers, anaerobes - hermetic sealing, thermodurics are a problem why?)
Kill pathogens to break the route of transmission

Non-spore formers in milk (M. bovis - TB, Coxiella burnetii - Q fever, Brucella - brucellosis); S. typhi & carriers eg the milk industry
C. botulinum in canning industry

Thermal Death Point (TDP): lowest temp required to kill all microbes in liquid suspension in 10 mins.
Thermal Death Time (TDT): Minimal time required to kill all microbes keeping temperature (or bacteriocidal agent) ) constant.
Decimal Reduction Time (DRT or D value): Used for heat ressistant bacteria; Time required to kill 90% of the population at a given temp. (Log growth and log death concept)

B. stearothermophilus & / or C. sporogenes is used to determine acceptable D values
C. botulinum endspore D value = 0.21 min at 121^oC (heating food at 121^oC for 2.52 mins means 10^-12 chances of endospore survival ie 1 in trillion chance
Accidic canned foods (tomatoe, pineapples, orange juices) require lower temps. Eg 100^oC for 10mins. Why?
Milk industry uses lower temps. Eg pasteurization preferred. Why?

^(B) Filtration is used for sterilizing heat-labile liquids and to sterilize air (gas) for creating aseptic environment (physical removal of microbes)

(i) Liquids:

Filters for removing pathogens come in different types:

Cellulose acetate
Celulose nitrate
Polycarbonate
Teflon

Filters vary in pore size (0.22um, 0.45um , 0.8um) but 0.2um are usually used but Mycoplasma and viruses may pass thro. Why?

(ii) Air:

To create an aseptic environment for microbiological procedures in semi contained areas. Eg laminar flow hood

Directs sterile air from the outside into the confined space via a 0.3um HEPA filter (High-efficiency Particulate Air) & stops contaminants coming into the space

Cotton wool used to "plug" test tubes, flasks, pipettes for growing ro working with cultures, surgical masks act in a similar way

^(C) Cells require water for metabolic activities. Desiccation is a process by which water is removed and therefore growth is affected

Desciccation prevents microbial reproduction but is dependent on

Cell structure. Waxy material of cell wall protects M. tuberculosis, fungal spores (plant pathogens) disemmination
Environment. Nasal secretions can protect influenza virus to survive

Lyophilization (Freeze-Drying): Preserving microbial cultures

American Type Culture Collection (ATCC)
Australian Culture of Microorganisms (ACM)

Drying food prevents spoilage:

Aw < 0.9 inhibits bacteria
Aw < 0.65 inhibits fungi & most microbes
Examples: prunes, dates, figs, rasins - natural, Evaporated milk - removal of 60% water from milk, powdered milk - removal of 85% water

^(D) Electromagnetic radiation causes cellular damage

Chemical methods of controlling microbial growth

Microbiology laboratory standards, design and safety

Author and HTML'd by: Dr Bharat Patel <bharat@trishul.sci.gu.edu.au>

[Created 25 Sept 1995]
[Modified 29 Aug 1997]