and Disinfection Name: HNO WALES. peRKetA . core: Section: _YSN_ 4-0 Date: AgR W202 -———__ LEARNING OBJECTIVES Ld At the end of the laboratory period, the student should be able to: 1. interpret the result of each method of sterilization; 2. differentiate bacteriostatic from bactericidal agents; and 3. discuss the functions and uses of commonly used physical methods of sterilization and commonly used chemical agents. —————— | INTRODUCTION | a There are several methods that can be employed to kill organisms or inhibit their growth, These can be classified into physical and chemical methods (refer to Chapter 7). Of the physical methods, heating is the most reliable and whenever possible, it should be the method of choice Tt is also the method that is readily accessible and universally accepted. «used to achieve sterilization and disinfection, Disinfection is important in infection control, not only in hospitals but also at home. A wide variety of chemical agents belonging to several BrOUPS (c.g. detergents, heavy metals, alkylating agents, étc) can be used. Different modes of action have been ascribed to them. These chemical agents ™ay interfere with the functions of the cell membrane, denature proteins, or destroy or modify the functional groups of proteins. In this exercise, different physical methods of sterilization, as well as chemical agents for disinfection, will be evaluated. Chemical agents can also bIth Sciences H Microbiology and Parasitology: A Textbook and Laboratory Manual for the Physical Methods of Inactivation of Organisms Materials: + 4 broth cultures of Bacillus subtilis + 4 broth cultures of Escherichia coli + 8 tubes of nutrient broth 1. Treat the four culture tubes of each organism as follows + First tube — autoclave at 15 pounds per square inch (psi) for 15 minutes Second tube — stand in a pan of boiling water for 30 minutes + Third tube ~ stand in a pan of water at 60°C for one hour + Fourth tube — control tube 2. Transfer a loopful of the suspension of the microorganism from each tube to another tube of nutrient broth and incubate at 35°C for 24 hours. 3. Record the growth with sub-cultured tubes (no growth, minimal growth, moderate growth, or heavy growth) in the table below. "Growth Obtained Exposure to Heat ea : __[ Bacitus subtilis | Escherichia coll ‘Autoclave = 15 psifor 1S minutes | yyevere_tnt Wo Gu Boiling water - 30 minutes ey gown Voge cor, (ata Heating - 60°C for one hour Wrenn, egy (wom WRBE Chemical Method of Inactivation of Organisms Materials: + One broth culture with light suspension of Staphylococcus aureus + Four 1 mL water blanks + Nutrient agar plate Chemical agents: + 1:1000 Zephiran + 70% alcohol * Povidone-iodineSterilization and Disinfection 1, Using a glass pencil, divide the nutrient agar plate into four the bottom of the dish. Label the quadrants 1, 2, 3, and 4, disinfectant to be used. quadrants by marking , and the name of the 2. Transfer a loopful of the S. aureus suspension to each of the water blanks. Label each test tube as follows: + Test tube no. 1 — control (no chemical agent added) + Test tube no. 2 — 0.3 mL of 1:1000 Zephiran added + Test tube no. 3 — 0.3 mL 70% alcohol added + Test tube no. 4 - 0.3 mL povidone iodide added 3. Let stand for three minutes after addition of the different chemical agents. 4, After three minutes, transfer a loopfal of the suspension from the control test tube and streak on quadrant number 1 of the nutrient agar plate. From test tube number 2, get another loopful and streak on quadrant number 2, From test tube number 3, get a loopful and streak on quadrant number 3. Finally, from test tube number 4, get a loopful and streak on quadrant number 4. Remember to sterilize the wire loops in-between each step to avoid contamination. e. Incubate at 35°C for 24 hours. £, Observe growth and record results as follows: (+) — with growth; (-) - without growth of organisms. Chemical Agent ae Growth Observed onal Control + 11000 Zephiran * 70% alcohol * Povidone iodide Answer the following: 1. Differentiate sterilization from disinfection. 4) N Ww NOt inamisma’ UHMicrobiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences 2. Enumerate the factors that may influence the efficiency of chemical agents. nasi an smn nef Megane, UT Mm Me it, Ay ns. 3. What is thermal death time? At AN Hts. Aaspit Sofucis. Hn, tgiad yA. Yakag ing aula 4. Define the following terms: a. Bactericidal wating alt Poewag Speen a nd i igidal) b. Bacteriostatic Aegan gut gg Wag c. AntisepsisSterilization and Disinfection 5. Identify the method of sterilization/chemical 7 . space provided. ‘mical agent described. Write your answers on the a. Amethod of sterilization that involves the physi . the fluid. Ives the physical separation of microorganisms from FAR ETON b. This method is utilized to deprive the organisms of moisture. csv havo c. This method is used to destroy disease-producing microorganisms in milk, milk products, food, and beverages. Pectemga Zin w d. At 15 psi, the temperature reaches 121°C requiring only 15 to 20 minutes to sterilize the material. (ENE en TO e. The material to be sterilized is exposed to live steam for 30 minutes for 3 consecutive . The material to » exennte seats ee eeMicrobiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences f. This chemical agent is used as a standard for evaluating new chemical agents, pheno 8 This chemical agent is used as water disinfectant. CHU NE h. A phenol-derivative that is less toxic and more potent than phenol. (REGUL i. Considered the best antiseptic TOOL NT j. Used as prophylaxis for ophthalmia neonatorum Aver Common’