Microbiology Lab Quiz 1

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. - 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

BSL-2

- indigenous microorganisms that can lead to diseases of varying severity in healthy adults. - 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-3

- indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission - 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-1

- microorganisms not known to cause disease in healthy adults. - standard microbiological practices are followed - personal protective equipment (PPE) is not required - a sink must be available for handwashing

Personal Protection Requirements

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

Light microscopes:

1. brightfield microscopes 2. phase contrast microscopes 3. darkfield microscopes 4. fluorescent microscopes

In order to prevent these 3 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

brightfield microscope components:

1. framework 2. light source 3. stage 4. lens system 5. focusing knobs

- as compound microscopes, brightfield microscopes have 3 lens systems:

1. the oculars 2. the objectives 3. the condenser

We label the bottom (agar side) for 2 reasons:

1. you will always know the identity of the plate 2. you incubate agar plates bottom (agar side) up

the most common objectives have magnifications of...

10X (low power), 40X (high dry), and 100X (oil immersion)

The total magnification achieved when using the oil immersion objective would be calculated as follows:

10X x 100X = 1000X

The first functional electron microscope was constructed in...

1933

It must be emphasized that ___ microorganisms __ be handled as though they are capable of causing disease.

ALL MUST

This laboratory course will involve work with ____ and ____ microorganisms only

BSL1 and BSL2

BSL

Biosafety Levels

Bacillus subtilis, serratia marcescens, staphylococcus saprophyticus

Examples of BSL 1 Microorganisms

Staphylococcus aureus, Streptococcus pyogenes, Salmonella

Examples of BSL 2 Microorganisms

Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis

Examples of BSL 3 Microorganisms

Ebola virus, Marburg virus

Examples of BSL 4 Microorganisms

__________, best known for improving the telescope and investigating the laws of motion, made some of the earliest improvements in 1609. His work was followed by ______ in the 1660s, and ________ in the 1670s.

Galileo Galilei Hooke Leeuwonhoek

The first microscope was invented in...

Holland

Do you blow air on or wave your loop or needle around to cool it?

No

Do you touch the loop or needle to any part of you, even your gloved hand, to determine if it is cool?

No

Do you touch the loop to anything after flaming/sterilizing it?

No

refers to the concept that microorganisms are everywhere

Ubiquity of Microorganisms

emergency procedures

a. the first aid kit is located next to the right front sink (facing front of lab) b. the eyewash station is located at both front sinks and the sink in the back of the lab. c. the shower is located at the left front of the lab near the instructor's desk d. the fire extinguishers are located at each exit. notify your gta if there is a fire. do not use the fire extinguisher except in an emergency situation e. 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 shelter in place.

standard laboratory practices

a. wash your hands with soap and dry with paper towels when entering and leaving the lab. alcohol based hand sanitizer is also available, but it is not a replacement for hand washing b. never place books, backpacks, purses,etc., on bench tops. place them in the spaces assigned by your gta c. electronic devices can not be kept on the lab bench after the quiz is finished. this includes, but it not limited to cell phones, tablets, laptops, and calculators. d. report all cuts, scrapes, or burns to the gta upon entering the lab. bandages and antibiotic ointment are available in the first aid kit. e. tie back long hair. f. do not wear dangling jewelry. g. disinfect bench before and after the laboratory session with a disinfectant (Lysol in this lab) h. wear a lab coat and gloves at all times while working in the lab to prevent contamination and accidental staining of your clothing and hands 1. closed toe shoes are to be worn in the lab. 2. long hair must be tied back to prevent exposure to flame and contamination by microbial cultures i. do not place anything in your mouth or eyes while in the lab. this includes pencils, food and fingers. keep your hands away from your face. 1. eating and drinking are prohibited in the lab at all times. this includes gum, cough drops, and candy 2. do not apply cosmetics in the lab. 3. do not apply eye drops in the lab j. do not place unnecessary items on the bench top k. label all containers clearly l. keep door closed while the laboratory is in session. individuals not enrolled in the course are not allowed in the laboratory m. use test tube racks when moving test tubes around the laboratory n. dispose of all waste and contaminated labware appropriately: 1. all items that have come in contact with bacteria are disposed in the biohazard can (blue bag) 2. dispose of slides, swabs, and pipettes in the sharps container. 3. glassware (flasks, beakers, bottles, test tubes) are placed on the discard cart. 4. gum, paper products not contaminated with microorganisms, and items brought in to the laboratory are disposed of in the regular garbage cans. o. notify instructor of all spills or broken glass. 1. immediately cover spilled cultures or broken culture tubes with paper towels and then saturate them with disinfectant solution. Notify your GTA that there has been a spill. p. always practice aseptic technique q. use extreme caution when working with bunsen burners. the flame can be hard to see. turn the burner off when not in use and make sure gas is off before leaving the lab. r. do not remove media, equipment, or bacterial cultures from the laboratory. this is absolutely prohibited and unnecessary. s. do not place contaminated instruments such as inoculating loops, needles and pipettes on bench tops. loops and needles should be sterilized by. incineration, and pipettes should be disposed of in designated sharps containers t. immune compromised students and students living with or caring for an immune compromised individual should consult a physician to determine the appropriate level of participation in the laboratory

the frame is composed of the ___ and _____.

arm base

never place the loop or needle on the ______.

bench top

microorganisms are usually not found in our _____ or ______

blood cerebrospinal fluid

- simplest form of microscopy where light is either passed through, or reflected off, a specimen - illumination is not altered by devices that change the properties of light (such as polarizers or filters) - requires the use of stains to visualize cells - the type of microscope with which most people are familiar

brightfield microscopes

Objects found on a slide are brought in to focus by first adjusting the ______ focus knob with the 10X objective. After moving to the 40X and 100X objectives, only the ______ focus knob is used for focusing.

coarse fine

the outer knob = the inner knob =

coarse focus knob fine focus knob

- collects and directs the light from the light source to the slide and is located under the stage - does not affect the magnification power of the microscope

condenser

it is absolutely necessary to allow the loop or needle to _____ before transferring an inoculum of bacteria (15-20 seconds)

cool

- contrast is created by a bright specimen on a dark background - it is ideal for revealing morphology and external structures, but does not provide a great deal of information about internal structure - stains are not required

darkfield microscopes

the amount of light that reaches the slide is controlled by a ______ located inside the condenser

diaphragm

all work surfaces must be wiped down with a _______.

disinfectant

when you finish working for the day, treat the bench with _______.

disinfectant

can destroy vegetative cells and viruses but may not destroy endospores - can not be used to sterilize surfaces or equipment

disinfectants

you should disinfect your work area...

first

- loop and needle sterilized by placing the loop or needle into the bunsen burner flame until it is red hot - the entire wire portion must be heated this way - any contamination present on the loop or needle will be incinerated, leaving it sterile

flaming your loop/needle

- uses high intensity illumination to excite fluorescent molecules (fluorophores) in the sample - when a molecule absorbs photons, electrons are excited to a higher energy level - as electrons "relax" back to the ground-state, vibrational energy is lost and, as a result, the emission spectrum is shifted to longer wavelengths - fluorescence emanates from the sample (and not the illuminating light) - requires use of fluorophores

fluorescent microscopes

- composed of the arm and base - structure to which all the other components are attached

framework

- oil with the same refractive index as glass - used with the 100X objective, it fills the gap between the objective and slide to form a continuous lens path which increases image resolution by reducing light refraction

immersion oil

- a light intensity control is usually found in the base as a knurled wheel or slide adjustment used to adjust voltage

light source

Most modern microscopes have either a halogen or LED ________.

light source

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

microorganism

a mathematical expression that describes how the condenser lens concentrates and focuses the light rays from the light source

numerical aperture

3 or more _______ are typically attached to a rotating _________.

objectives nosepiece

- the ability of a microscope to remain relatively in focus when changing from lower power objective to a higher power objective

parfocal

loops are used to streak or inoculate ______. Place a petri plate _______ on the bench.

petri plates bottom up (lid down)

- converts the differences in optical density (refractive index) of cells into different shades of brightness (contrast) - allows for the visualization of morphology, external structures, and some internal structures - stains are not required

phase contrast microscopes

Before inserting a cooled loop or needle in a culture tube, remove the cap of the tube with the _____ of your dominant hand. To kill any bacteria or fungi on the mouth of the tube, briefly pass it through the _______. Before closing the tube, ______ the mouth of the tube again by briefly passing it through the flame of the Bunsen burner.

pinky finger flame of the bunsen burner flame

the ability of a lens system to show 2 closely spaced objects as distinct and separate

resolving power

- the horizontal area that supports the microscope slide - has a slide holder and stage adjustment knobs that allow for the holding and moving of the slide around it

stage

The stage has a slide holder and ______ that allow for the holding and moving of the slide around the stage.

stage adjustment knobs

prior to use, loops and needles must be...

sterilized

determined by multiplying the ocular power by the objective power

total magnification

loops and needles are used to...

transfer cultures

'Ubiquity of Microorganisms" is not entirely ____, but microorganisms (especially prokaryotes) do occupy an amazing range of _______.

true environments

Microbes are ______ ubiquitous

virtually

the distance between the bottom of the objective lens and the slide

working distance