Methods to Control Microbial Growth Lab. 11 • 1. Physical • 2. Chemical The effect of disinfectant and antiseptics on the growth of bacteria • Objective: To understand the principle of disk diffusion test. • To be able to read a disk diffusion test result. • To understand the concept of zone of inhibition • Disinfectant vs. Antiseptic • Disinfectant – antimicrobial agent used on inanimate objects • Antiseptic – antimicrobial agent used on living tissue Controlling Bacteria Antiseptic vs. Disinfectants Antiseptic- chemicals that kill bacteria on living things •means – “against infection” Examples: iodine, hydrogen peroxide, alcohol, soap, mouthwash Disinfectants- stronger chemicals that destroy bacteria on objects or nonliving things Disinfectants Example: • Dettol • Clorox • Iodine • Alcohol • Flash • Jif • vim Antiseptics Example: • Dettol • Alcohol • Iodine • Tooth past • Mouth wash Procedure 1. 2. 3. 4. Take sterile nutrient agar plate and label Surface streak the plate with the given culture Make four holes in the agar using a cork borer Add 2-3 drops of the chemical agent in the labeled hole 5. Incubate the plate at 37C for 24-48 hours Cloro Dettol D C A M Cork borer Alcohol Mouth wa Result The bacteria will start to grow ,then it will stop growing when it is effected by the disinfectants, and will have a clear zone These results depend on : 1- the kind of bacteria . 2- the kind of disinfectant or antiseptic. • Conclusion: The larger the zone ,the stronger the effect of disinfectant The smaller the zone ,the weaker the effect of disinfectant No zone indicates no effect. Antibiotic Disk-Diffusion Method • Also called Kirby-Bauer method. Purpose: • It is used to detect the susceptibility and resistance of test bacteria to a given antibiotic with known concentration. Procedure • The suspension of tested bacteria is swabbed on the surface of a solid culture media, and disks of different antibiotics which contain known concentration of these antibiotics are placed on the swabbed culture and incubated 18-24hr. • The antibiotics will diffuse from the disk into the media and according to the sensitivity of the bacteria to a given antibiotic. The effect of antibiotics on the growth of bacteria • There are different antibiotics that have different effects on the growth of bacteria • This experiment also depends on: – The kind of bacteria – The kind of antibiotic • Example: penicillin ,Amoxilline ,Streptomycin ,chloramphenicol,Tetracycline The bacteria will start to grow ,but around the antibiotic disks , there is no growth making clear zones with different diameters in length. Results • If the bacteria is susceptible to the antibiotic (sensitive): the antibiotic kills or inhibits their growth and a zone of inhibition (no growth of the bacteria) will formed around the disk. • If the bacteria is resistant to the antibiotic: no zone of inhibition will formed around the disk (there will be a growth of bacteria all over around the disk). • The diameter of the zone is measured by roller, and the reading will be compared with the reference range of this bacteria to this antibiotic and then the one can judge wither the bacteria is susceptible or resistant conclusion The larger the zone the stronger the effect of antibiotics The smaller the zone the weaker the effect of antibiotic No zone indicates no effect Physical methods for controlling microbial growth Controlling microbial growth Heat Filtration cold Heat Protein denaturation By Heat Moist heat Pasteurization 72c for 15 Sec. Dry heat Boiling or steam flow & Autoclaving at 121c For 15 min. Milk, cream,& Alcoholic drinks Direct flaming Incineration Hot air 170c,2hr. Pasteurization Dry Heat • Direct flaming: for loops& needles Filtration • Separation of bacteria from the suspension or liquid. • For sterilizing Enzymes ,Vaccines ,heat sensitive media Filtration Cold: for controlling &preserving microbial growth cold Refrigeration Of food culture & drugs Deep freezing -50 to-95 Food, culture,& drug Lypholization Food, drugs,& culture Most effective for Short preservation. Most effective for Long preservation Refrigeration Deep Freeze Lypholization • Is the removal of water by high vacuum under very low temperature.