Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron...

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Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving objects smaller than 0.2 μm. 3.Why is microscopy and important technique to employ in microbiology?

Transcript of Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron...

Page 1: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Do Now

1. Discuss the primary function of a microscope.

2. Define resolution and justify why electron microscopes are the best tools to use when resolving objects smaller than 0.2 μm.

3. Why is microscopy and important technique to employ in microbiology?

Page 2: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

AIM: How do we prepare specimen for observation using a

compound light microscope?

Page 3: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

IN GROUPS:

How would you prepare a slide to observe protists (e.g.

paramecia, amoeba, etc) using a microscope??

HINT: You did this in Regents Biology!

Page 4: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Wet Mount

• Used to observe living organism– A drop of broth with the organism is placed on

the slide or– A drop of water is placed on the slide and a

sample of bacteria from agar is added

• 2% carboxymethyl cellulose slows them down (if mobile)

Page 5: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

How else might a scientist prepare specimen for observation

using a microscope?

Page 6: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Smears on a Slide

• Used to view killed organisms

• Organisms are living when placed on the slide but the preparation techniques kill them

Page 7: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Smear Procedure

• Sterilize loop & place loopful of water on each slide if culture is from agar slant.

• Flame loop

• Allow to cool

• Remove cap from test tube & flame mouth

Page 8: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

• Touch loop to culture & remove bacteria

• Flame test tube & replace cap

Page 9: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

• Spread bacteria in water on slide

• Make thin film

Page 10: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

• Allow the slide to air dry.

Page 11: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Fix Bacteria to Slide

• After dry, pass through flame – film side up – about three times- and touch bottom – should feel just warm

Page 12: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Why fix bacteria to a slide?

Page 13: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Why Fix Bacteria to Slide?

• Prevents washing off during staining– Bacteria contain albumins, a type of material

like blood, which causes them to cling to the glass slide

• Cultures used are 24 hours old– Prevents distortion of cells

Page 14: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Analyze the procedure for making smears and discuss possible technical pitfalls.

Page 15: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Problems with a Smear

• Too thick – can’t see through• Too thin – can’t find any organisms• Preparation may disrupt structural chains or other

arrangements• Too much heat fixing may destroy organisms beyond

observation

Page 16: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Hints for Preparing Bacterial Smears

• Use clean slides– Wash with soap and water– Towel dry – polish– Flame side to be used to remove grease– Handle only on edges

Page 17: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Though it may seem trivial, why is labeling arguably the most

important task in smear preparation.

Page 18: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Label Slide

• Make a circle on each slide the size of a dime using a wax pencil

• Include Group names and date

• Label each slide in the upper left– Example

• Ec = Escherichia coli

• Bs = Bacillus subtilis

• Sl = Sarcinia lutea

Page 19: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

After preparing the bacterial smear, why should a

microbiologist stain bacteria?

Page 20: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Why Stain Bacteria?

• Unstained bacteria are transparent with the light microscope.

• Staining aids in the identification of bacteria.

• Bacterial stains are made from synthetic dyes.

Page 21: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.
Page 22: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Principles of Staining

• A stain or dye is a salt composed of a positive and negative ion.

• One ion has color and is called the chromophore.

Page 23: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

In Groups:

Identify possible bacterial structures that can be targeted for

staining.

Page 24: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Bacterial Cellular Structures to Stain

• Cell Membrane

• Cell Wall

Page 25: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Fig. 4-3An Overview of Structure

Page 26: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Basic Dyes

• Positively charged ion has color.• Good for bacterial cell membranes which

are negatively charged at a ph of 7.• Examples:

– Methylene blue– Crystal violet– Safranin– Malachite green

• More commonly used than acidic dyes.

Page 27: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Acidic Dyes

• Negatively charged ion has color

• Repelled by negatively charged bacterial membranes

• Used for negative staining to see shape, size and capsules

• Examples– Eosin– Nigrosin

Page 28: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Types of Stains

Simple or Differential

Page 30: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Differential Stain

• Uses two or more dyes• Distinguishes between two kinds of

organisms or two parts of an organism• Examples

– Gram stain (most frequently used)– Ziehl-Nielsen Acid Fast Stain (vivid red color)

• For leprosy and TB

– Schaeffer-Fulton spore stain

Page 31: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

A Simple Stain

• E. coli - Eosin (Red) - 2 minutes

or Carbolfuchsin- 30 seconds

• S. lutea - Methylene Blue- 2 minutes

• B. subtilis- Crystal Violet - 30 seconds

Page 32: Do Now 1.Discuss the primary function of a microscope. 2.Define resolution and justify why electron microscopes are the best tools to use when resolving.

Procedure for Staining

• Cover smear with stain

• Allow to stand for required time

• Wash off stain in coffee can using water bottle

• Dry wet slide – use bibulous paper

• Examine under microscope