1. Chemical Agents  Phenolics  Alcohols  Halogens  Heavy metals  Quaternary Ammonium Compounds  Aldehydes  Sterilizing Gases  Evaluating Effectiveness of Chemical Agents

2. Chemical Agents: Phenolics  Aromatic organic compounds with attached -OH  Denature protein & disrupt membranes  Phenol, orthocresol, orthophenylphenol, hexachlorophene  Commonly used as disinfectants (e.g. “Lysol”); are tuberculocidal, effective in presence of organic matter, remain on surfaces long after application  Disagreeable odour & skin irritation; hexachlorophene once used as an antiseptic but its use is limited as it causes brain damage

3. Chemical Agents: Alcohols  Ethanol; isopropanol; used at concentrations between 70 – 95%  Methanol less bactericidal than ethyl alcohol  Denature proteins; disrupt membranes  Kills vegetative cells of bacteria & fungi but not spores  Used in disinfecting surfaces; thermometers; “ethanol-flaming” technique used to sterilize glass plate spreaders or dissecting instruments at the lab bench

4. Chemical Agents: Halogens  Act as oxidizing agents; oxidize proteins & other cellular components. Fluorine, bromine, chlorine, and iodine  Chlorine compounds  Used in disinfecting municiple water supplies (as sodium hypochlorite, calcium hypochlorite, or chlorine gas)  Sodium Hypochlorite (Chlorine Bleach) used at 10 - 20% dilution as benchtop disinfectant  Chlorine + water HCl + HClO  HClO HCl + O (highly reactive nascent Oxygen)- destroys cellular constituents

5. Chemical Agents: Halogens  Iodine Compounds  Tinctures: solutions dissolved in pure alcohol or water-alcohol mixtures  Tincture of iodine (2%iodine + 2% Potassium iodide solution in alcohol )  Iodophors: Iodine complexed to an organic carrier like PVP (polyvinylpyrrolidine); e.g. Wescodyne, Betadyne  Used as antiseptics for cleansing skin surfaces and wounds  Possesses sporicidal activity,fungicidal and virucidal  Mode of action – iodination of tyrosine units of enzymes and cellular proteins

6. Chemical Agents: Heavy Metals  Mercury-mercuric chloride, Colloidal silver, zinc, arsenic, copper ions(copper sulfate)  Form precipitates with cell proteins  Mercuric chloride – the enzymes inactivated by reacting with sulfhydryl groups  Enzyme-(SH)2 + HgCl2 Enzyme-(S-Hg-S) + 2HCl  Examples: 1% silver nitrate was used as opthalmic drops in newborn infants to prevent gonorrhea; has been replaced by erythromycin or other antibiotics;  copper sulfate used as algicide in swimming pools

8. Chemical Agents: Quaternary Ammonium Compounds  Quaternary ammonium compounds are cationic detergents. Amphipathic molecules that act as emulsifying agents  Denature proteins and disrupt membranes  Used as disinfectants and skin antiseptics  Examples: cetylpyridinium chloride, benzalkonium chloride

9. Chemical Agents: Aldehydes  Formaldehyde (HCHO) and  Gluteraldehyde O=CH-CH 2 CH 2 CH 2 -HC=O  40% formaldehyde aqueous solution- Formalin  React chemically with nucleic acid and protein, inactivating them  Aqueous solutions can be used as disinfectants  Used in fumigation of cell culture and microbiological rooms for reducing contamination

10. Chemical Agents: Sterilizing Gases  Ethylene oxide (EtO)  Used to sterilize heat-sensitive equipment and plasticware  Explosive; supplied as a 10 – 20% mixture with either CO 2 or dichlorofluoromethane  Its use requires a special EtO sterilizer to carefully control sterilization conditions as well as extensive ventilation after sterilation because of toxicity of EtO  Much of the commercial use of EtO (for example, plastic petri dishes) has in recent years been replaced by gamma irradiation

11. Chemical Agents: Sterilizing Gases  Betapropiolactone (BPL)  In its liquid form has been used to sterilize vaccines and sera  Decomposes after several hours and is not as difficult to eliminate as EtO, but it doesn’t penetrate as well as EtO and may also be carcinogenic  Has not been used as extensively as EtO  Vapor-phase hydrogen peroxide  Has been used recently to decontaminate biological safety cabinets

12. Chemical Agents: Evaluating the Effectiveness  Phenol Coefficient Test  A series of dilutions of phenol and the experimental disinfectant are inoculated with Salmonella typhi and Staphylococcus aureus and incubated at either 20°C or 37°C  Samples are removed at 5 min intervals and inoculated into fresh broth  The cultures are incubated at 37°C for 2 days  The highest dilution that kills the bacteria after a 10 min exposure, but not after 5 min, is used to calculate the phenol coefficient

13. Chemical Agents: Evaluating the Effectiveness  Phenol Coefficient Test (cont.)  The reciprocal of the maximum effective dilution for the test disinfectant is divided by the reciprocal of the maximum effective dilution for phenol to get the phenol coefficient  For example: Suppose that, on the test with Salmonella typhi The maximum effective dilution for phenol is 1/90 The maximum effective dilution for “Disinfectant X” is 1/450 The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5

14. Chemical Agents: Evaluating the Effectiveness  Phenol Coefficient Test (cont.)  Phenol coefficients are useful as an initial screening and comparison, but can be misleading because they only compare two pure strains under specific controlled conditions  Use dilution tests and simulated in-use tests  Are tests designed to more closely approximate actual normal in-use conditions of a disinfectant

17. Preservation of Microbial Cultures  Periodic Transfer and Refrigeration  Mineral Oil Slant  Freezing in Growth Medium  Drying  Lyophilization  Ultracold Freezing

18. Preservation of Microbial Cultures: Periodic Transfer and Refrigeration  Stock cultures are aseptically transferred at appropriate intervals to fresh medium and incubated, then stored at 4°C until they are transferred again  Many labs use “agar slants;” care has to be taken to avoid contamination  Major problem with possible genetic changes in strains; most labs need a way to keep “long term” storage of original genetic stocks

19. Preservation of Microbial Cultures: Mineral Oil Slant  Sterile mineral oil placed over growth on agar slants to preserve cultures for longer period of time in the refrigerator  Contamination problems; messy; many organisms are sensitive to this; generally it is a poor technique and doesn’t work well

20. Preservation of Microbial Cultures: Freezing in Growth Medium  Used as a “long term” storage strategy  Broth cultures of the organisms are frozen at -20°C  Often, sterile glycerin (glycerol) is added at a 25 – 50% final concentration; this helps to prevent ice crystal formation and increases viability of many organisms

21. Preservation of Microbial Cultures: Drying  Suitable for some bacterial species  Samples are grown on sterile paper disks saturated with nutrient, then the disks are allowed to air dry and stored aseptically  Reconstituted by dropping disk into nutrient broth medium

22. Preservation of Microbial Cultures: Lyophilization  Suitable for many bacterial species as well as fungi and viruses  Broth cultures are placed in special ampules and attached to a vacuum pump; the vacuum removes all of the water from the cells leaving a “freeze- dried” powder  The culture is reconstituted by adding broth to the lyophilized powder and incubating it  Considered the best method of long-term storage for most bacterial species

23. Preservation of Microbial Cultures: Ultracold Freezing  Similar to freezing, but at very cold temperature  At about -70 to -80°C, in liquid nitrogen or in an ultracold freezer unit