Temperature’s lethal effects on bacterial growth can be seen after subjecting various bacterial cultures to excessive heat in the range of 90 to 100 degrees C. Microorganisms are killed by elevated temperatures mainly because of the susceptibility of their macromolecules to heat. High temperatures cause proteins to denature and unfold which results in the loss of the tertiary structure and loss of overall function. Most proteins are enzymes which means that metabolic activity relies on their function. If these proteins are damaged the metabolic capabilities of the organism will be irreversibly damaged. Nucleic acids are also damaged by heat resulting in the loss of structure of DNA and RNA. This will lead directly to cell death. Smaller co-factors like NAD+ can also be damaged by heat and loss of these factors leads to cell damage and death. Some cells like that of Bacillus and Clostridium are more resistant to heat because of their endospores. These endospores contain calcium dipicolinate that protects the cells from heat. Endospore-specific proteins can bind to nucleic acids and prevent denaturation.
We use antiseptics everyday. An antiseptic is a chemical agent used to kill or control the growth of microorganisms. Hospitals use sodium hypochlorite which is identical to household bleach to control microorganisms. Medical professionals use alcohol to sterilize the skin before an injection and betadine (an organic form of iodine) on the skin before surgery. The betadine prevents Staphylococcus aureus from causing a post surgical infection. Cities add chlorine to the water supply to prevent the spread of pathogens such as Salmonella typhi from getting into the drinking water. Chemical agents are also added to food to slow down spoilage by microorganisms. This increases the shelf life of the packaged food. Clostridium botulinum causes a deadly form of food poisoning and is controlled by adding chemicals to canned food. Antiseptics are not to be confused with disinfectants which are used to control microbial activity on inanimate objects.
Antimicrobial Sensitivity Testing
As soon as the causative agent of an infection has been determined it is up to the physician to presribe the most effective antimicrobial agent that will kill the pathogen without harming the patient. Antibiotics vary in effectiveness against different bacteria. Some antibiotics are more effective against gram negative bacteria while some are referred to as broad spectrum and are effective against both gram negative and gram positive bacteria. Antibiotics must be tested against specific organisms to determine how effective they are. The Kirby-Bauer method of sensitivity testing is one of the most popular methods for testing antibiotics. This test is performed by uniformly streaking a medium with the test organism. Paper disks containing specific concentrations of an antibiotic are deposited on the agar surface. A chemical in the disk diffuses out and forms a concentration gradient. If the agent inhibits the growth of the bacteria there will be a zone around the disk where no growth occurs called the zone of inhibition.
Membrane Filter Technique
The Membrane Filter Technique is an effective, accepted technique for testing fluid samples for microbiological contamination. It involves less preparation than many traditional methods, and is one of a few methods that will allow the isolation and enumeration of microorganisms. The MF Technique also provides presence or absence information within 24 hours.
Antibiotic Sensitivity Testing
Antibiotic sensitivity testing (AST) aims to determine the susceptibility of an isolate to a range of potential therapeutic agents. This can be with a view to individualizing the antibiotic to be administered or to monitor resistance patterns developing in that environment, gathering this information is important for revising and updating the standard antibiotic prescribing policy for a particular population or institution.
Resistance to antibiotics can either be naturally occurring for a particular organism/drug combination or acquired resistance, where mis-use of anti-microbials results in a population being exposed to an environment in which organisms that have genes conferring resistance (either spontaneously mutated or through DNA transfer from other resistant cells) have been able to flourish and spread.
Identification of an organism normally goes hand in hand with the AST test, knowing what organism you have isolated together with knowledge of the isolation site, will give an indication of what type of antibiotics should be considered. The sensitivity of an isolate to a particular antibiotic is measured by establishing the Minimum Inhibitory Concentration (MIC) or breakpoint, this is the lowest concentration (conventionally tested in doubling dilutions) of antibiotic at which an isolate cannot produce visible growth after overnight incubation.