Lab Safety and Aseptic Technique Micro

What is appropriate dress for lab?

Closed toed shoes

Gloves

Lab coat

Hair tied back

No dangling jewelry

Can you wear gloves and lab coat outside the laboratory?

No

When should you wash your hands

Entering and leaving the lab

All injuries (even minor) must be reported (T/F)

True

What items go in biohazard

Plates

Gloves

Paper Towels

Anything that isn’t pointy/sharp that has come into contact with bacteria

What items go into the sharps container

Slides

Swabs

Pipettes

Where do you dispose of tubes

Discard cart

What should be done to start clean up of a spill

Cover with paper towels and saturate with disinfectant

BSL 1

Microorganisms that are not known to cause disease in healthy adults

PPE not required

Handwashing required

BSL 2

Indigenous microorganism that can lead to disease in healthy adults

PPE must be worn: coats and gloves

Lab is closed off

BSL 3

Indigenous or exotic microorganism that cause serious or lethal disease through respiratory transmission

PPE must be worn: lab coat, gloves, eye protection, and respirator

Lab must have negative air-flow

BSL 4

Microorganism that are dangerous and exotic with high risk of aerosol transmission; rarely have treatments or vaccines and are often fatal

Change clothes before lab

All work performed in Class III biosafety cabinet or wearing full body pressure suit

Shower exiting lab

Microorganism/microbe

General term for living organisms that cannot be seen without a microscope

Ubiquity of microorganisms

Concept that microorganisms are everywhere

Contamination

Undesired introduction of impurities like microbes into or onto an item

Agar

semi-solid surface with various nutrients that many microorganisms grow on

Angelina Heese

Started the use of agar in labs instead of gelatin

Who invented the first petri dish

Robert Koch

Pure culture

A growth of only one type of microorganism on agar or broth

Aseptic technique

Set of precautions taken when working in the lab to avoid contamination

What do you risk when you don’t use aseptic technique

Contamination of others with microorganisms

Contamination of yourself with microorganisms

Contamination of media within the lab

What should you always do to your workbench before starting work

Disinfect

Disinfectant

Destroy vegetative cells and viruses but not endospores

How do you sterilize loops and needles

Placing the loop/needle in the flame of a bunsen burner until the entire thing is white hot

What can you not do after sterilization of a loop/needle

Touch it with anything except the microorganism being transferred

Touch it to any part of you to determine if it is cool

Blow air onto it or wave it around

What steps must be taken when transferring from a broth

The mouth of the tube must be passed quickly through the flame 3 times both before collecting and before closing it

How many loopfuls of bacteria is taken from the broth for plate inoculation

2

Brightfield microscopes

Light is either passed through or reflected off a specimen

Requires the use of stains to visualize cells

Where was the first microscope invented

Middleburg, Holland

Phase contrast microscopes

Converts the differences in refractive index of cells into different shades of brightness

Can visualize morphology, external structures, and some internal structures

Stains not required

Darkfield microscopes

Specimen is illuminated on dark background

Ideal for revealing morphology and external structures

No stains or internal structures

Fluorescent microscopes

High intensity illumination to excite fluorescent molecules in the sample

Energy excites electrons to higher energy level, when they relax they emit longer wavelengths

Colored light comes from sample

Requires use of fluorophores

What are the components of brightfield microscopes

Framework

Light source

Stage

Lens system

Focusing knobs

Framework

Rigid structure that all other components of the microscope are connected to

Composed of arm and base

Light source

Source of light for microscopes

Often has light intensity control knob

Stage

Horizontal area that supports the microscope slide

Comes with adjustment knobs that allow for movement

Lens system

System of magnification that is composed of

oculars

objectives

condenser

Oculars

Part of the microscope where one would place their eyes and look

Objectives

Tubes connected to the rotating nosepiece of the microscope

Range in magnification of 10X, 40S, and 100X

Total magnification

Determined by multiplying ocular power by objective power

Condenser

Collects and directs light from light source to the slide

Located under the stage

Diaphragm

Controls how much light reaches the slide, located inside condenser

Focusing knobs

Consist of 2 knobs

Outer coarse focus knob

Inner fine focus knob

Immersion oil

Oil with same refractive index as glass

Only used with 100x objective

Forms continuous lens path and increases image resolution

Parfocal

Ability of a microscope to remain relatively in focus when changing from lower power objective to higher power objectives

Numerical aperture

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

Resolving power

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

Working distance

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

Who invented gram staining

Hans Christian Gram

How does gram staining work

By retaining stain within the more complex cell walls of gram positive and washing away (and adding different stain) to those with less complex cell walls (gram negative)

What is the compound that determines if a bacteria is gram negative or gram positive

Peptidoglycan

Gram positive cocci

Purple stained bacteria with spherical morphology

Examples of Gram positive cocci

Enterococcus

Micrococcus

Streptococcus

Staphylococcus

Gram negative rod

Pink to red stained bacteria with spherical to bean shaped morphology

Examples of gram negative cocci

Moraxella

Gram positive rods

Family bacilli

Purple stained bacteria with rod shaped morphology

Examples of gram positive rods

Bacillus subtilis (single)

Bacillus cereus (chains)

Gram negative straight rods

Family bacilli

Pink to red bacteria with rod-shaped morphology

Examples of gram negative rods

Pseudomonas

Gram negative curved/spiral rods

Family bacilli

Pink to red bacteria with rod shapes that are curved or spiral

Examples of gram negative curved/spiral rods

Vibrio (curved)

Campylobacter (spiral)