Micro Biology Lab Packet

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Week1Day2MicroscopySimpleStains1-18-18.ppt
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Lab 2

Ex 3-1 Introduction to Microscopy

Ex 3-5 Simple Stains

Housekeeping Items

  • Did you Lysol your benchtop yet?
  • My name is Professor Giambernardi
  • Pronounced “Gim-burr-nardi”
  • Please introduce yourselves at your tables
  • Seating chart & attendance
  • Is everyone finding things on D2L?
  • Remember to purchase sharpies & gloves

Today we will do 3 things:

Observe our cultures inoculated last lab

Become familiar with our microscopes

Perform some simple stains?

Let’s check our cultures from last lab

  • Nutrient agar plates
  • Did they grow? How can you tell?
  • Did you see a difference between unwashed and hand sanitizer?

  • When finished, discard cultures in biohazard trash.

  • Remember to answer questions in “data sheets” (back of lab section) germane to the sections we are working on

Microscopy

  • Light microscope – uses visible light as the energy source

  • Brightfield microscopy
  • Produces an image made from light that passes through a specimen
  • The background appears bright
  • Objects appear darker, or might lack contrast

Microscopy

  • Compound microscope – has two lenses that magnify the image

Ocular

Objectives

The Compound Light Microscope

Light source at bottom

3 lenses produce an image:

The condenser lens focuses & concentrates light to evenly illuminate the specimen

The objective lens collects light, magnifies, and forms an image that you see with your eyes.

The ocular lens further magnifies the image formed by the objective lens.

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Concept #1: Magnification

  • Magnification = the size of the image
  • Four different objective lenses:

scanning low power high power oil immersion

4X 10X 40X 100X

  • Ocular lens: 10X
  • Total magnification = (objective mag) x (ocular mag)
  • Parfocal = object in focus under 1 objective will be in focus under all objectives
  • Paracentered = each objective will successively focus on the center of the previous field of view

Magnification

  • Field diameter = the actual diameter of the area viewed, using those particular lenses
  • Units will be a distance (mm, µm)
  • As magnification increases, field diameter decreases

Objective Field diameter

scanning 4.5 mm

low power 1.8 mm

high power 0.45 mm

oil immersion 0.18 mm

Magnification

  • Depth of Focus = the amount of vertical space that appears in focus, using those particular lenses
  • Similar to depth of field in photography

  • As magnification increases, depth of focus _____?______

decreases

Concept #2: Resolution

  • Resolution – the clarity of the image

Resolution & Car Headlights

UNRESOLVED PARTIALLY RESOLVED RESOLVED

Concept #2: Resolution

  • Limit of resolution (resolving power)
  • How far apart 2 points must be to be distinguished as separate
  • Indicates the size of the smallest object that can be clearly observed with those lenses
  • The limit of resolution is decreased as wavelength is decreased.

  • High magnification without high resolution is “empty.”

Magnification & Resolution

*

Limit of Resolution

  • As magnification increases, the limit of resolution gets smaller

For example,

  • Eye 0.1 mm (millimeters)
  • Light Microscope 0.2 m (micrometers)
  • Scanning EM 3.0 nm (nanometers)
  • Transmission EM 0.2 nm (nanometers)

Question about Resolution:

  • We just said the limit of resolution (resolving power) of our light microscopes is 0.2 m

  • We want to see a bacterium that has a diameter of 1 m

Will we be able to see this bacterium using our microscope?

Why or why not?

Concept #3: Working Distance

  • The amount of clearance between the slide and the bottom of the objective lens
  • As magnification increases, working distance _________
  • Don’t crash your objective lens into the slide!

decreases

Concept #3: Working Distance

  • The amount of clearance between the slide and the bottom of the objective lens

4X

40X

10X

Concept #4: Refraction

  • Refraction – bending of light as it passes through an object
  • Refractive Index – a measure of the light-bending ability of a medium

Concept #4: Refraction

  • Oil minimizes refraction to capture more light with the objective lens

Your microscope

  • Let me demonstrate proper carrying of the microscope
  • Carrying your microscope to your bench
  • Your seat # = your microscope #
  • Let’s get our microscopes (located in cabinet next to you)

Microscope parts and their function

  • Ocular – provides magnification
  • Objective – provides magnification
  • Stage – holds the specimen; enables movement of the specimen
  • Condenser – concentrates or focuses light to illuminate the specimen
  • Iris diaphragm – changes the size of the cone of light, thus adjusting the amount of light
  • Coarse focus knob – raises & lowers the stage in larger increments, to adjust the focus
  • Use ONLY with scanning & low power objectives
  • Fine focus knob – raises & lowers the stage in smaller increments, to adjust the focus
  • Stage – holds the specimen; enables movement of the specimen

Microscope parts and their function

Stage

Stage clip

Tips for Today – Using Immersion Oil

  • Use oil only with the oil immersion objective (100X)
  • Get the specimen in focus under any lower power (4X or 10X) objective
  • Swing that objective out of the way, add a small drop of oil right on the specimen, and swing the oil immersion objective into place-oil will contact 100X objective
  • Don’t move the stage down to add oil
  • Use only the fine focus knob with oil immersion
  • Clean up all oil with lens paper
  • Objective, stage, specimen, etc.

Tips for Today - Drawings

  • Purpose of drawings is to cause you to carefully observe what you’re seeing
  • Draw a representative sample
  • This is not art class

  • Every drawing must be labeled with the name of the specimen and the total magnification
  • For example: yeast suspension, 100X

Tips for Today – Returning your microscope to the cabinet

  • Remove slide
  • Clean off all oil, using lens paper
  • Engage the lowest power objective lens (4X)
  • Wrap cord neatly around the base
  • Replace dust cover
  • Place scope carefully on the shelf, with oculars spun around and arm outward

Expectations: work as individuals

  • Examine a slide of letter “e”
  • Mount so “e” appears correctly as you see the slide on the stage

  • Examine prepared slide of colored threads
  • Examine using low power (10X) & high power (40X) objectives

  • Examine a prepared slide of bacteria
  • Examine using scanning, low power, & high power objectives

e

Why make smears and stain bacteria?

  • Looking at a bacterium under the microscope may be one of the first methods used to identify the cause of an infection
  • Direct exam of specimen
  • After culture of specimen

  • What’s the limitation of brightfield microscopy?
  • Bacterial cells are very small and lack contrast.
  • With brightfield microscopy, a stain is used to add contrast and make them visible.

Preparing Specimens for
Light Microscopy – The Smear

  • A smear is a thin film of microbes on a slide.
  • From broth: A loopful of broth culture
  • From slant/plate: A small amount of a colony is added to a very small drop of water
  • Let the slide air-dry
  • A smear is heat-fixed in order to:
  • attach the microbes to the slide
  • kill the microbes
  • increase stain penetration.

Types of Stains used by Microbiologists

  • Simple Stains – a single dye is used to add contrast, so microbes can be viewed microscopically
  • Positive stain
  • Negative stain
  • Differential Stains
  • Gram stain
  • Acid-fast stain
  • Special Structure Stains
  • Capsule stain
  • Spore stain
  • Flagellar stain

Simple Stains

  • Positive stain
  • Basic (positively-charged) dye interacts with negatively-charged bacterium.
  • Colored bacterium appears in a bright field.
  • Negative stain
  • Acidic (negatively-charged) dye is repelled by negatively-charged bacterium.
  • Stains background and leaves cells unstained.

Positive and Negative Staining

Positive stain

Negative stain

Making a Bacterial Smear (Ex. 3.5, pg. 186-187)

  • When observing a stained smear, you should always observe & record:
  • Shape
  • Arrangement
  • Size (a comment, if you can)
  • Don’t forget to label every drawing

What information in the label?

  • Specimen, total mag, staining procedure
  • Reminder: total magnification =

ocular (10X) X objective (4X or 10X or 40X or 100X)

Simple stains

coccus bacillus spirillum

Bacterial Shape

  • Singly
  • Pairs: diplococci, diplobacilli
  • Clusters: staphylococci
  • Chains: streptococci, streptobacilli

Arrangement

  • Stay organized with your label & observations

Tip for Today: Drawings

Name of bacterium

1000X

Simple stain with safranin

bacillus in
singles

singles

bacillus

1000X

Simple stain

Name of bacterium

safranin

For Next Lab:

  • Read: Ex 2-2 Colony Morphology and

Ex 1-3 Aseptic Transfers & Inoculation Methods (pgs. 29 - 48)