Lab 1 - Lab Safety, Ubiquity, Aseptic Technique

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. Examples: Ebola virus, Marburg virus

- Standard microbiological practices are followed. - Change clothing before entering lab. - A hands-free sink and eyewash must be available. - The laboratory must be in a separate building or in an isolated and restricted zone of the building. - All work with microorganisms must be performed in a Class III biosafety cabinet or by wearing a full body, positive pressure suit. - Decontaminate all materials before exiting lab (including clothes). - Shower upon exiting lab. - Change clothes before exiting lab. -Laboratory must have negative air-flow (air can not escape to surrounding rooms) and exhaust air cannot be recirculated (must be vented to outside of building after decontamination by ultra-filtration) - Medical surveillance of laboratory employees. Immunizations may be required.

aseptic Turn the burner off gas is off media, equipment, or bacterial cultures

Always practice __________ technique. Use extreme caution when working with Bunsen burners. The flame can be hard to see. __________ when not in use and make sure __________ before leaving the lab. Do not remove __________ from the laboratory. This is absolutely prohibited and unnecessary.

- remove the cap of the tube with the pinky finger of your dominant hand - pass it through the flame of the Bunsen burner. - insert the loop into the broth and twist the loop several times to deposit the bacteria in the broth drawing the loop up the surface of the slant with a zig-zag pattern until reaching the top of the slant. - flame the mouth of the tube again by briefly passing it through the flame of the Bunsen burner.

Culture Tube Flaming and Inoculation Before inserting a cooled loop or needle in to a culture tube, _____________. To kill any bacteria or fungi on the mouth of the tube, briefly __________ If the tube is a broth tube, ____________. If the tube is a slant tube, the surface of the slant is inoculated by _________ Before closing the tube, __________

disinfect your work area disinfectant. Lysol, 70% ethanol, 10% bleach, and quaternary ammonia sterilize

Disinfection of Your Work Area You should ________ first. All work surfaces must be wiped down with a __________. ____________ are all examples of popular disinfectants. Disinfectants can destroy vegetative cells and viruses but may not destroy endospores. Due to this limited ability, disinfectants can not be used to _________ surfaces or equipment.

waste and contaminated labware biohazard can (blue bag) sharps container discard cart. regular garbage cans paper towels disinfectant solution.

Dispose of all _________ and ________ appropriately i. All items that have come in contact with bacteria are disposed in the _________ ii. Dispose of slides, swabs, and pipettes in the _________ iii .Glassware (flasks, beakers, bottles, test tubes) are placed on the ___________ iv Gum, paper products not contaminated with microorganisms, and items brought in to the laboratory are disposed of in the __________ Notify instructor of all spills or broken glass. - Immediately cover spilled cultures or broken culture tubes with _________ and then saturate them with _________ Notify your GTA that there has been a spill.

mouth or eyes Eating and drinking cosmetics eye drops

Do not place anything in your __________ while in the lab. This includes pencils, food, and fingers. Keep your hands away from your face. _________ and _________ are prohibited in the lab at all times. This includes gum, cough drops, and candy. Do not apply _________ in the lab. This includes Chapstick. Blistex, etc Do not apply _______ in the lab

bench tops incineration sharps containers

Do not place contaminated instruments such as inoculating loops, needles, and pipettes on ____________ Loops and needles should be sterilized by ___________, and pipettes should be disposed of in designated ___________.

right front sink (facing front of lab). both front sinks and the sink in the back of the lab. left front of the lab near the instructor's desk exit shelter in place.

Emergency Procedures - The first aid kit is located next to the __________ - The eyewash station is located at _________ - The shower is located at the ___________ - The fire extinguishers are located at each ______ NOTIFY YOUR GTA IF THERE IS A FIRE. DO NOT USE THE FIRE EXTINGUISHER EXCEPT IN AN EMERGENCY SITUATION. - Severe Weather: The lab space is in a near perfect location for severe weather shelter. It is in the basement of the building with no windows. If there is severe weather, we will ____________

the needle or loop is flamed bench top treat the bench with disinfectant.

Final Flaming of the Loop or Needle - After inoculating the broth, slant, or plate, ____________ to destroy any remaining organisms and replaced to the wooden holding block Never place the loop or needle on the ___________. Final Disinfection of Your Work Area When you finish working for the day, _____________ This ensures that any microorganisms deposited on the bench are destroyed.

containers door test tube racks

Label all ___________ clearly. Keep ________ closed while the laboratory is in session. Individuals not enrolled in the course are not allowed in the laboratory. Use __________ when moving test tubes around the laboratory.

touch the loop or needle to any part of you, even your gloved hand, to determine if it is cool - If it is hot you will be burned. - Nitrile gloves will melt to your skin. - You will contaminate your loop or needle. blow air on or wave your loop or needle around to cool it - You will contaminate your loop or needle.

Loop and Needles DO NOT ____________

closed-toe shoes gloves lab coat

Personal Protection Requirements Wear __________ that cover the top of the foot. Wear _______ when handling microorganisms or handling chemicals. Wear _______ while in the laboratory.

Loops bottom up (lid down) on the bench into the Bunsen burner flame until it is red hot; The entire wire portion of the loop or needle must be heated this way. lift the plate, leaving the lid on the bench. toward you and gently streak the inoculum over the surface of the plate. back down on the lid.

Petri Plate Inoculation ________ are used to streak or inoculate Perti plates. Place a petri plate _________ Then pass the loop ___________ After the loop is cool, retrieve your inoculum and _________ Turn the agar __________ and ___________ This takes practice; as it is very easy to gouge or slice the agar. Place the plate __________

before and after lab coat and gloves

Standard Laboratory Practices: Disinfect bench __________ the laboratory session with a disinfectant (Lysol in this lab). Wear a __________ and __________ at all times while working in the lab to prevent contamination and accidental staining of your clothing and hands.

cuts, scrapes, or burns long hair dangling jewelry

Standard Laboratory Practices: Report ALL _________ to the GTA upon entering the lab. Bandages and antibiotic ointment are available in the first aid kit. Tie back ________ Do not wear ___________.

entering AND leaving the lab. bench tops Electronic devices; cell phones, tablets, laptops, and calculators.

Standard Laboratory Practices: a. Wash your hands with soap and dry with paper towels when __________ Alcohol based hand sanitizer is also available, but it is not a replacement for hand washing b. Never place books, backpacks, purses, etc., on __________. Place them in the spaces assigned by your GTA. c. ___________ CAN NOT be kept on the lab bench after the quiz is finished. This includes, but is not limited to _____________

blood or cerebrospinal fluid.

The only places where microorganisms are not found are places where we intentionally exclude them. For example, microorganisms are usually not found in our ___________

1 and 2

This laboratory course will involve work with BSL ___ and BSL ___ microorganisms only.

BSL 3 - Indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission. Examples: Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis

What biosafety level is this: - Standard microbiological practices are followed. - A hands-free sink and eyewash must be available. - Locking, double door access separates the lab's 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/hood. - Decontamination of all waste and lab clothing before laundering. - Laboratory must have negative air-flow (air can not escape to surrounding rooms) and exhaust air cannot be recirculated (must be vented to outside of building after decontamination by ultra-filtration). - Medical surveillance of laboratory employees. Immunizations may be required.

BSL 2 - Indigenous microorganisms that can lead to diseases of varying severity in healthy adults. Examples: Staphylococcus aureus, Streptococcus pyogenes, Salmonella

What biosafety level is this: Standard microbiological practices are followed. A sink for handwashing and an eyewash must be available. Laboratory access is restricted. PPE must be worn including lab coats and gloves. Eye protection and face shields are worn when needed. Work that may generate aerosols or splashes are performed in a biological safety cabinet/hood. Contaminated waste is autoclaved. The laboratory has self-closing doors.

BSL 1 -Microorganisms not known to cause disease in healthy adults. Examples: Bacillus subtilis, Serratia marcescens, Staphylococcus saprophyticus

What biosafety level is this: Standard microbiological practices are followed. Personal protective equipment (PPE) is not required A sink must be available for hand washing.

to prevent these types of contamination, microbiologists use 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.

Why Practice Aseptic Technique?

Loops and needles placing the loop or needle into the Bunsen burner flame until it is red hot wire "flaming your loop/needle" sterile cool Fifteen to twenty

__________ are used to transfer cultures. Prior to use, these utensils must be _________. This is accomplished by _________. The entire ________ portion of the loop or needle must be heated this way. This technique is generally referred to as ____________. Any contamination present on the loop or needle will be incinerated, leaving it ________. It is absolutely necessary to allow the loop or needle to ______ before transferring an inoculum of bacteria. __________ seconds is usually enough time for the loop or needle to cool to a point that you will not kill the bacteria that you are transferring.

microorganism

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

ubiquity of microorganisms

refers to the concept that microorganisms are everywhere. It's not entirely true, but microorganisms (especially prokaryotes) do occupy an amazing range of environments. For example, some Archaea live in ocean floor vents where temperatures can exceed boiling and chemicals, poisonous to most forms of life, bubble from the Earth's crust. Other microbes live in solid rock.