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?

AIM: How do we prepare specimen for observation using a

compound light microscope?

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!

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)

How else might a scientist prepare specimen for observation

using a microscope?

Smears on a Slide

• Used to view killed organisms

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

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

• Touch loop to culture & remove bacteria

• Flame test tube & replace cap

• Spread bacteria in water on slide

• Make thin film

• Allow the slide to air dry.

Fix Bacteria to Slide

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

Why fix bacteria to a slide?

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

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

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

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

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

important task in smear preparation.

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

After preparing the bacterial smear, why should a

microbiologist stain bacteria?

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.

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.

In Groups:

Identify possible bacterial structures that can be targeted for

staining.

Bacterial Cellular Structures to Stain

• Cell Membrane

• Cell Wall

Fig. 4-3An Overview of Structure

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.

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

Types of Stains

Simple or Differential

Simple Stains

• Single dye

• Reveals basic cell shapes and arrangements

• Examples – Methylene blue– Safranin– Carbolfuchsin– Gentian violet

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

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

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