Lab 1: Orientation, Laboratory Safety, and Aseptic Technique

Disinfectants

can destroy vegetative cells and viruses but may not destroy endospores cannot be used to sterilize surfaces or equipment ex: Lysol, 70% ethanol, 10% bleach, quaternary ammonia

Ubiquity of microorganisms

refers to the concept that microorganisms are everywhere -not entirely true, but they do occur an amazing range of environments ex: archer live in ocean floor vents where temps can exceed boiling and chemicals, poisonous to most life forms -others live in solid rock, others revived from amber suspensions and ice they are in the air we breath, the food we eat, and the water we drink

Why practice aseptic technique

to prevent the types of contamination with microorganisms

Loops and needles

used to transfer cultures must be sterilized

Three detrimental things prevented by aseptic technique

1. Contaminate others with microorganisms that could lead to infection 2. Contaminate themselves with microorganisms that could lead to infection 3. Contaminate media and/or the microorganisms being studied in the lab

Rules for loops

1. Do not touch the loop to anything after flaming/sterilizing it, or the loop will be contaminated and the process will have to be repeated 2. Do not touch the loop to any part of you, even gloved hands, to determine if it is cool -hand will be burned -nitrile gloves will melt to hand -you will contaminate the loop

Petri plate inoculation

1. Place plate lid down on table 2. Flame loop 3. Retrieve innocuous and lift the plate, leaving lib on bench 4. Turn ager toward you and gently streak the inoculum over the surface of the plate 5. Place plate back down on the lid

Tube inoculation

1. Remove the cap with pinky finger of dominant hand 2. Briefly pass the mouth of the tube through the flame of the Bunsen burner 3. Either swirl the loop inside the tube broth gently to remove inoculant or move up the slant form the bottom in a zig zag pattern to inoculate 4. Flame the mouth of the tube and replace the cap

Emergency Procedure

Locate and understand use of emergency equipment Report all injuries immediately to the GTA

Standard Laboratory Practice

Wash hands after entering and before exiting Tie back long hair Do not wear dangling jewelry Disinfect bench before and after the laboratory session with a disinfectant (Lysol) Do not bring food, gum, drinks, or water bottles into the laboratory Place personal items on appropriate shelves Do no place unnecessary items on the bench top Do not touch the face, apply cosmetics, adjust contact lenses, or bite nails Do not mouth pipette Label all containers clearly Keep door closed while the laboratory is in session Use test tube racks when moving test tubes around the lab Dispose of all waste and contaminated lab ware appropriately -All items that have come in contact with bacteria are disposed in the biohazard can (blue bag) -Dispose of slides, swabs, and pipettes in the sharps container -Glassware (flasks, beakers, bottles, test tubes) are placed on the discard cart -Gum, paper products not contaminated with microorganisms, and items brought in to the laboratory are disposed of in the regular garbage cans Notify instructor of all spies, broken glass, or injuries Always practice aseptic technique

Personal Protection Requirements

Wear closed-toe shoes that cover the top of the foot Wear gloves when handling microorganisms or handling chemicals -do not wear gloves outside the lab Wear lab coat while in the lab -do not wear lab coats outside the lab

Microorganism

a general term for living organisms that cannot be seen without a microscope some people include prions and viruses in the group

BSL 2

indigenous microorganisms that can lead to diseases of varying severity in healthy adults -ex: Staphylococcus aureus, Streptococcus pyogenes, Salmonella BSL 1 practice plus: -lab access is restricted -PPE must be worn including lab coats and gloves and eye protection and face shields wore when needed -work that may generate aerosols or splashes are performed in a biological safety cabinet -contaminated waste is autoclaved -the lab has self closing doors -a sink and eye wash are available

BSL 3

indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission -ex: Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis BSL 2 practice plus: -medical surveillance of lab employees. Immunizations may be required -locking, double door access separates lab work area from other parts of the facility -PPE must be worn including lab coats, gloves, eye protection, and respirators -all work with microorganisms must be performed in a biosafety cabinet -decontamination of all waste and lab clothing before laundering -laboratory must have negative air flow and exhaust air cannot be recirculated -a handsfree sink and eyewash must be available

Biosafety Level (BSL) 1

microorganisms not known to cause disease in healthy adults -ex: Bacillus subtilis, Staphylococcus saprophyticus standard microbiological practice are followed personal protective equipment (PPE) is not required a sink must be available for hand washing

BSL 4

microorganisms that are dangerous and exotic with high risk of aerosol transmitted infections rarely are there treatments or vaccines for these microorganisms, and the diseases they cause are frequently fatal -ex: Ebola virus, Marburg virus BSL 3 practice plus: -change clothing before entering lab -shower upon exiting the lab -decontaminate all material before exiting lab -all work with microorganisms must be performed in a class III biosafety cabinet or by wearing full body, protective pressure suits -laboratory must have negative airflow and exhaust cannot be recirculated -the lab must be in a separate building or in an isolated and restricted zone of the building

Flaming your loop

sterilization of loops/needles by placing the loop into the bunsen burner flame until it is red hot -the entire wire portion of the loop or needle must be heated this way any contaminant present on the loop will be incarcerated, leaving it sterile, but must let cool before transferring bacteria from a culture (15-20 seconds) make sure to flame the loop after inoculating anything to kill any microbes

Only place free of microbes

the places we intentionally exclude microorganisms ex: blood, cerebrospinal fluid, pharmaceutical plants, semi-conductor manufacturing plants, "clean rooms" designed to eliminate contamination, growth media