Lecture for microbiology manual INTRODUCTION TO T H E L A B O R ATO RY Design by: Abed Al Rahman Hamad M.sc. Biological science

O R I E N TAT I O N TO T H E L A B O R ATO RY: ● Welcome to the microbiology laboratory. The goal of the laboratory is to expose students to the wide variety of life in the microbial world. Although the study of microbiology includes bacteria, viruses, algae and protozoa, this lab will concentrate primarily on the bacteria. ● Microbiological techniques are important in preparing the students for the much harder task of identifying the pathogenic microorganisms in a clinical specimens. In this manual, Each experiment with a brief theoretical introduction revealing the theoretical basis on which the experiment is based on, so that there will be a strong conjunction between the practical and theoretical session. Also included in this manual, the safety precautions that each one in this field of microbiology should know and practice.

● Some of the laboratory experiments may be hazardous if you handle materials improperly or carry out procedures incorrectly. ● Safety precautions are necessary when you work with any microorganism, and with chemicals, glass test tubes, hot water baths, sharp instruments, and similar materials.

● This type of activity must be carried out with good aseptic technique (prevent contamination) ● In aseptic technique, all materials that are used have been sterilized to kill any microorganisms. ● Even if the microorganisms you are studying are not usually considered pathogenic (disease producing). ● It is important to prevent contamination of your hands, hair, and clothing with culture material and also to protect your neighbors from such contamination. In addition, you must not contaminate your work with microorganisms from the environment.

● An important problem in hospitals is the transmission of microorganisms between patients, especially by contaminated hands (nosocomial infections). ● In general, all safety procedures and precautions followed in the microbiology laboratory are designed to: ● 1) Restrict microorganisms present in specimens or cultures to the containers in which they are collected, grown, or studied. ● 2). Prevent environmental microorganisms

● Any student with a fresh, unhealed cut, scratch, burn, or other injury on either hand should notify the instructor before beginning or continuing with the laboratory work. If you have a personal health problem and are in doubt about participating in the laboratory session, check with your instructor before beginning the work. Careful attention to the principles of safety is required throughout any laboratory course in microbiology.

Laboratory Safety ● Following directions and preparation ● Lab coat ● Disinfecting bench tops ● Hand washing ● Eating and drinking ● Long hair ● Mouth-pipetting ● Labels ● Decontamination and waste disposal ● Broken glass ● Accidents ● Gloves ● Attire ● You will be working with opportunistic pathogens

Professional conduct ● Following safety guidelines ● Following directions ● Attendance ● Be on time ● Academic dishonesty ● Sterile technique

Laboratory Rules ● Always – Wear a lab coat – Label everything – Flame sterilize innoculating loops and needles before and after use – Tell me if something goes horribly awry – Place waste, contaminated materials, trash, and broken glass in the appropriate containers – Wash hands every time you enter or leave the lab – Notify me before class if you will be absent – Be nice to the microscope – Wear appropirate lab attire

Laboratory Rules Continued ● Never – Eat, drink, apply make-up, touch your face, put your hands in your mouth... while in the lab – Be rough with the microscope – Leave with your gloves on when you leave the lab – Remove lab cultures – Re-use slides

DISINFECTANTS, ANTISEPTICS, AND SANITIZERS ● Disinfection is the elimination of microorganisms from inanimate objects or surfaces, whereas decontamination is the treatment of an object or inanimate surface to make it safe to handle.

● a. The term disinfectant is used for an agent used to disinfect inanimate objects or surfaces but is generally to toxic to use on human tissues. ● b. The term antiseptic refers to an agent that kills or inhibits growth of microbes but is safe to use on human tissue. ● c. The term sanitizer describes an agent that reduces, but may not eliminate, microbial numbers to a safe level.

● disinfectants and antiseptics work slowly on some viruses, bacteria with an acid-fast cell wall, bacterial endospores. ● Sterilization consist of : 1) Physical Antimicrobial Agents 2) Chemical antimicrobial agents ● There are a number of factors which influence the antimicrobial action of disinfectants and antiseptics, including: 1. The concentration of the chemical agent. 2. The temperature 3. The kinds of microorganisms present 4. The number of microorganisms present 5. The nature of the material bearing the microorganisms

1) Physical Antimicrobial Agents ● The term sterilization is an absolute one; it means total, irreversible destruction of living cells. A number of physical environmental agents—such as ultraviolet or ionizing radiation, ultrasonic waves, or total dryness—exert stress on microorganisms and may kill them, but they cannot destroy large concentrations of microorganisms in a laboratory culture or a clinical specimen. ● It is an excellent sterilizing agent when applied at high enough temperatures for an adequate period of time, because it effectively stops cellular activities. ● Depending on whether it is moist or dry, heat can coagulate cellular proteins (think of a boiled egg) or oxidize cell components (think of a burned finger or a flaming piece of paper).

Moist and Dry Heat ● Moist heat coagulates microbial proteins (including protein enzymes), inactivating them irreversibly. ● In the dry state, protein structures are more stable; therefore, the temperature of dry heat must be raised much higher and maintained longer than that of moist heat. ● The autoclave: Only 15 minutes at 121°C, 15 to 20 lb of steam pressure may be needed. The choice of heat sterilization methods then depends on the heat sensitivity of materials to be sterilized.

There are 2 common antimicrobial modes of action for disinfectants, antiseptics, and sanitizers: ● 1. They may damage the semi-permeable cytoplasmic membrane ● 2. They may denature microbial enzymes and other proteins Such chemical agents are in common use: 1. Phenol and phenol derivatives 2. Soaps and detergents 3. Alcohols 4. Acids and alkalies 5. Heavy metals 6. Chlorine