LABORATORY EXERCISES to accompany MICROBIOLOGY LABORATORY BIO 209 Professor Susan C. Kavanaugh Bluegrass Community & Technical College Spring 2016 0 FUNDAMENTAL REQUIREMENTS FOR LABORATORY SAFETY Learn about the hazardous properties of all the materials used in your work and observe the applicable safe handling, storage, disposal, and emergency procedures. treat all substances are hazardous. confine drinking, eating, chewing gum or tobacco, smoking and the storage and disposal of food, beverages, and tobacco products to uncontaminated or designated areas outside of the laboratory. Absolutely no food or drink may be present in the laboratory. wear the appropriate personal protective equipment for the activities being conducted. As a minimum protection, wear a lab coat or gown, gloves, and safety glasses whenever chemicals, radiochemicals, or biohazards are being used in the laboratory. wear close-toed shoes providing full coverage, no open shoes such as sandals. keep exits, passageways, and access to safety equipment like emergency eyewash stations and showers or fire extinguishers free from obstruction. be familiar with the locations and operating procedures for safety and emergency equipment such as fire extinguishers, first aid kits, emergency eyewash stations and showers*, fire alarm pull stations, emergency telephones##, and emergency exits. wash hands before leaving the laboratory and remove lab coat or gown before leaving the laboratory and before entering other areas, particularly eating facilities. tie back or otherwise restrain long hair or loose articles of clothing or jewelry when working with chemicals, biohazards, radioactive material, flames, or moving machinery. use mechanical pipetting devices-never pipette by mouth. perform procedures involving the liberation of volatile materials or aerosols of a toxic or flammable nature in a fume hood. know the location of the laboratory safety manual and the Material Safety Data Sheets (MSDS) notebook. report all accidents, dangerous incidents, and suspected occupational diseases and seek preventative measures to avoid recurrences.** *(recommended duration = 15 minutes with lukewarm water) ##(the nearest phone is in Room 244A) **(report forms are on the instructor’s desk) 1 Microbiology Laboratory Specific Guidelines Your1. Your lab bench lab be areanch isa rtoea be is cleanedto be clea ned with Lysol disinfectant before lab begins, after each procedure, withand Lysol befor disinfectante you leav ebefore. lab begins, after each procedure, and before you leave. For2. liquidFor liqui cultured c ultspills,ure immediatelyspills, immedia tely saturate the area with Lysol and cover with paper towels. Allow saturatecontact thewit areah the with disi Lysolnfect aandnt forcover at least 10 minutes. Wipe away all liquid with clean paper towels. with paper towels. Allow contact with theDispose disinfectant of all for pa peat leastr tow 10els in the trash. minutes. Wipe away all liquid with clean paper towels. Dispose of all paper towelsFor3. surfacesFor in sur thef acontaminatedtrash.ces c ontamin byated inoculating by inocula ting loops, pipettes, swabs, test tube lids, Petri dishes or other loops,such itpipettes,ems, imm swabs,ediat testely tubespra ylids, the surface with Lysol, and cover with a paper towel. Allow contact with Petri dishes or other such items, immediatelythe disinfe cspraytant f theor a surfacet least 10with mi nutes. After this time, the area should be wiped with paper towels which Lysol,are then and disposed cover with into a paper the tra towel.sh. Allow contact with the disinfectant for at least 10 minutes. After this time, theAll4. broken areaAll brokeshould glasswaren beglasswa wiped shouldr withe sho be paper ulddisposed be disposed of into the sharps containers or cans with the biohazard towelsoflabe intols. thewhich Non sharps are-contaminat then containers disposeded or bro cansintoke nthe g lass must be swept up by the instructor with a broom and dustpan. trash.with the biohazard labels. Non- contaminatedContaminate brokend broke glassn gla mustss m ustbe sweptbe disi nfected first using the Lysol disinfectant and covered with paper uptowe by lsthe for instructor at least with 10 mia broomnutes beandfor e being swept up by the instructor as previously described. Glass dustpan.microsc Contaminatedope slides, sta brokenined sm glassears, must and cover slips should also be disposed of into the sharps containers. be disinfected first using the Lysol disinfectant and covered with paper Swabs,towels5. Swa for tonguebs, at tleastong depressors,ue 10 d minutesepres sors,and before disposable and disposa ble pipettes should be disposed of in the plastic trays containing pipettesbeingdisinfe swept cshouldtant up pr bebyovided thedisposed instructor at e aofc hin asst theude nt bench. plasticpreviously trays described. containing Glass disinfectant microscope providedslides, stained at each smears, student and bench. cover slips Usedshould6. Use gloves alsod g lovesbe should disposed should be disposed of be into disposed the of into of into the designated biohazard trash bag. Used gloves should not be sharps containers. thethrow designatedn into th biohazarde regular trashtrash. bag. Used gloves should not be thrown into the regular trash. Nothing7. Nothing should shoul be thrownd be thr intoown the into small the small sinks at each student bench. sinks at each student bench. Any8. Anclothingy clot thathing becomes that bec contaminatedomes contaminate d must be removed immediately, placed in a biohazard bag, mustand abeutocla removedved. immediately, Lab coats m placeday not in be a worn outside of the Microbiology Lab. biohazard bag, and autoclaved. Lab coats may not be worn outside of the UsedMicrobiology9. Use Petrid P dishe triLab. culturesdi sh cult mustures bemus t be discarded into designated buckets containing biohazard bags, to be discardedautoclave intod prio designatedr to dispos bucketsal. Glasswa re cannot be discarded into these containers. containing biohazard bags, to be autoclaved prior to disposal. Glassware Usedcannot10. Usetest be d tubesdiscarded test torube other sinto or specimens theseother spec otherimens other than Petri dishes should be placed in the designated area for thancontainers.used Petri suppl dishes ies in should the corn be eplacedr at the in end of the front counter of the lab. the designated area for used supplies in the corner at the end of the front counter of the lab. 2 INTRODUCTION TO CULTURING, MEDIA, AND ASEPTIC TECHNIQUES You may be unaware of the number and variety of microorganisms (microbes) found everywhere in our environment, including the human body. In this laboratory you will learn new techniques and make observations which relate to the concepts of microbiology. Most of the microorganisms that you will use in these laboratories are normal inhabitants of our environment and our bodies. These microbes are called normal microbiota for the environment in which they normally reside. Health professionals need this knowledge in order to be able to distinguish normal flora from a possible infectious agent when interpreting microbiological reports. They also need to understand how normal microbiota can occasionally cause an infection when they invade a different area of the body, or when the patient's immune responses have been compromised. Microorganisms are found almost everywhere. In these first laboratory exercises you will be introduced to aseptic techniques, the procedures followed by microbiologists and healthcare workers to prevent contamination from outside sources and to prevent introduction of potentially disease-causing microbes (pathogens) into the human body. The methods for handling previously sterilized materials, for taking samples, for handling cultures, and for disposal of contaminated materials are all designed to prevent the spread of microbes from one area to another. Pay close attention to the details in the written procedures and to the instructor's demonstrations to prevent contamination of your cultures, yourself, your environment, and the other people in your laboratory as well as prevention of infecting people outside of the laboratory, such as your friends and family. These techniques can be applied not only here in the microbiology laboratory, but also throughout your career, and in your daily life. Most of the laboratory exercises performed in this course will involve a two-step process. During the lab session you will set up the cultures and then after these cultures have incubated for the appropriate length of time (usually 24 to 48 hours) you will need to observe the growth and record your observations and results. Wait for the instructor to demonstrate the procedures described and to make the specific assignments. In the rest of the exercises in the course, you are dealing with living bacteria, so it is very important to follow the procedures exactly to avoid contamination or infection. The following precautions are especially important: 1. Always wash your hands with the antiseptic soap provided before you begin and after you have finished each procedure. 2. Always wipe off your work area with the disinfectant provided before you begin and after you have finished each procedure. 3. Always wear gloves when handling cultures or specimens. 4. Discard all used materials in the appropriate designated place after you are done. Put all used materials and cultures into the special containers for contaminated material. Never put any used materials back into the supply